Énfasis en Sistemas de Producción Agrícola (SPA)

Tesis de Doctorado

Inducción de variabilidad genética en una variedad

comercial de arroz (Oryza sativa) mediante el uso de
radiación gama a partir de la selección in vitro de
mutantes tolerantes a ariloxifenoxipropionatos para su
uso en el fitomejoramiento del cultivo

ALEJANDRO HERNANDEZ SOTO

Dra Ana Abdelnour Esquivel Dra Marta Valdez Melara

Director de Tesis Asesor de Tesis

Dr. Fabián Echeverría Beirute

Asesor de Tesis

Cartago, Costa Rica, enero 2023

MIEMBROS DEL TRIBUNAL EXAMINADOR

TEODOLITO Firmado digitalmente

por TEODOLITO GUILLEN
GUILLEN GIRON (FIRMA)
Fecha: 2023.01.26
GIRON (FIRMA) 17:42:24 -06'00'
Dr. Teodolito Guillén Girón
Dirección de Posgrados TEC

ANA MARIA Firmado digitalmente por ANA

MARIA ABDELNOUR ESQUIVEL
ABDELNOUR ESQUIVEL (FIRMA)
(FIRMA) Fecha: 2023.02.01 19:06:00 -06'00'

Dra Ana Abdelnour Esquivel

Director de tesis

Marta Valdez Firmado digitalmente por

Marta Valdez Melara

Melara Fecha: 2023.01.26 14:55:20

-06'00'

Dra Marta Valdez Melara

Asesora de Tesis

FABIAN ECHEVERRIA Firmado digitalmente por FABIAN

ECHEVERRIA BEIRUTE (FIRMA)
BEIRUTE (FIRMA) Fecha: 2023.01.26 09:44:43 -06'00'

Dr. Fabián Echeverría Beirute

Asesor de tesis

Firmado por GIOVANNI SAENZ ARCE (FIRMA)

PERSONA FISICA, CPF-01-1153-0569.
Fecha declarada: 07/02/2023 11:50 PM

Dr. Giovanni Sáenz Arce

Coordinador General DOCINADE
Tabla de contenidos
ÍNDICE DE FIGURAS 4

ÍNDICE DE TABLAS 7

DECLARACIÓN DE AUTENTICIDAD 8

DEDICATORIA 10

RESUMEN 11

PALABRAS CLAVES 11

ABSTRACT 12

KEYWORDS 12

1. INTRODUCCIÓN 13

2. OBJETIVOS 23
2.1. OBJETIVO GENERAL 23
2.2. OBJETIVOS ESPECÍFICOS 23

3. SÍNTESIS 24

4. ARTÍCULO 1 RICE BREEDING IN THE NEW ERA: COMPARISON OF USEFUL AGRONOMIC TRAITS 27

5. ARTÍCULO 2, A TEMPORARY IMMERSION SYSTEM IMPROVES REGENERATION OF IN VITRO

IRRADIATED RECALCITRANT INDICA RICE (ORYZA SATIVA L.) EMBRYOGENIC CALLI 41

6. ARTÍCULO 3. TOLERANCE TO ARYLOXY-PHENOXY-PROPIONATE (APP) AS A MODEL FOR LAZARROZ

FL RICE IN VITRO GAMMA IRRADIATION VARIABILITY SELECTION 53

7. ARTÍCULO 4, NTH2 1271_1272DELTA GENE DISRUPTION RESULTS IN SALT TOLERANCE IN

SACCHAROMYCES CEREVISIAE 64

8. CONCLUSIONES Y RECOMENDACIONES 80

9. REFERENCIAS 81

10. ANEXO 1. OTROS ARTÍCULOS PUBLICADOS DURANTE EL DOCTORADO (4) 89

3
Índice de figuras

Figure 1. Representation of biotic and abiotic stress factors that affect rice production. .......... 28
Figure 2. Schematic representation of different systems used for breeding rice: natural variability,
mutation breeding, tissue culture mutation, and new breeding techniques.. .............................. 28
Figure 3. Representation of salt tolerance traits mediated by three different methods: 1)
overexpression, 2) knockout of specific genes, and 3) particular sodium channels. Note that the
first corresponds to transcription factors that trigger adaptive responses
labeled MSL37, NAC2, NAP, and P5CS. The second is a knockout of those that result in salt
sensitivity: OsRR2, STL1, DST; and the sodium channel SKC1 in rice. The third is the sodium
channel SKC1 containing amino acid V395. .............................................................................. 30
Figure 4. Representation of five rice genes and the corresponding mutation that results
in herbicide tolerance. The genes are shown organized by their Mode of Action (MoA). Note the
name of the gene in orange circles, the exons in blue filled boxes and the corresponding
untranslated exon regions in the blue empty boxes. Created with BioRender.com (For
interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)................................................................................................................. 32
Figure 5. Representation of traits such as grain number, quality, weight and plant structure and
gene relationships in rice. Note that heading and flowering are positively influenced by Se5,
Hd2, and Hd1 knockout; structure by DEP1, HTD1, IPA1, LPA1, Pin1a, and Pin15b; grain size
by Gn1a, and Ep3; grain size by GS3, GW6a, GW5, and GW5L; and grain starch by ISA1,
NAC20-26, and WX1. Created with BioRender.com. ................................................................. 34
Figure 6. DNA markers used to identify the matK and rbcL genes in non-irradiated Lazarroz FL
rice variety. Note in green the synonymous SNP (C/T) and the biallelic synonymous mutations
A/G and G/T (circled). None of the mutations had biological importance but helped in the genetic
characterization of the cultivars. ................................................................................................. 44
Figure 7. Calli induction of Lazarroz FL cultivar on MS medium with 2 mg L−1 2,4-D after 15 days
of culture under darkness. (A) Embryogenic calli obtained from the scutellum of mature zygotic
embryos (arrow) (B) the compact and friable calli as observed under the stereoscope.. .......... 45
Figure 8. Correlation comparison of radiation dose influence on regeneration (A), sprouting (B)
and browning rates (C) of calli of Lazarroz Fl cultivar, after 30 days of culture on regeneration
medium. Letters a, b, bc, c and d represent significant differences for Tukey’s test (p ≤ 0.05). All

4
treatments had 12 replicates of 7 calli each replicate (n = 84). Data were compiled at 15 days
post-radiation. ............................................................................................................................. 46
Figure 9. Response of 60 Gy irradiated calli Lazarroz FL cultivar after (A) 45 days and (B) 60
days of culture on regeneration medium. ................................................................................... 47
Figure 10. Regeneration in MS medium with 0.5 mg L−1 of NAA + 3 mg L−1 BA of an induced calli
with 2 mg L−1of 2,4-D after 15 days of induction. Regeneration in (A) semisolid medium and (B)
RITA® (Saint-Mathieu-de-Tréviers, France). .............................................................................. 47
Figure 11. Regeneration of cultivars CR-5272 and recalcitrant CR-1821 and CR-1113 in MS
medium with 0.5 mg L−1 of NAA + 3 mg L−1 of BA from calli induced with 2 mg L−1 of 2,4-D. (A)
CR-5272 regeneration in RITA with immersion for 30 s or 60 s, and the semisolid medium control.
(B) CR-1821 regeneration in RITA with immersion for 30 s or 60 s and the semisolid medium
control. (C) CR-1113 regeneration in RITA with immersion for 30 s or 60 s and the semisolid
medium control. Note that all RITA treatments contain calli with green areas that regenerate into
plants. The absence of regeneration of the recalcitrant cultivars CR-1821 and CR-1113 was
observed in the semisolid media. ............................................................................................... 48
Figure 12. Regenerated calli of vitroplants of the Lazarroz FL cultivar on MS medium with 3 mg
L−1 BA under fluazifop-P-butyl stress after 21 days of culture. A) 5 mg L−1 fluazifop-P-butyl, (B) 4
mg L−1 fluazifop-P-butyl, (C) 1 mg L−1 fluazifop-P-butyl, (D) necrotic vitroplants subcultured in fresh
me-dium for 21 days to verify mortality after exposure to 5 mg L−1 and 4 mg L−1 fluazifop-P-butyl
stress, as shown in (A) and (B). Note that all remained necrotic and had no sprouting or green
spots. .......................................................................................................................................... 56
Figure 13. Tolerant vitroplant of the Lazarroz FL cultivar obtained by gamma mutagenesis on calli
at 60 Gy on MS medium with two rounds of selections, the first at 5 mg L−1 of fluazifop-P-butyl
stress af-ter 21 days of culture, 21 days of recovery with no stress agent, and another round of
21 days of 10 mg L−1 of fluazifop-P-butyl stress. Note that nontolerant plants became necrotic at
5 mg L−1 fluazifop-P-butyl stress after 21 days of culture, while only 1 out of 100 putative tolerant
vitro-plants remained green during the second round of selection at 10 mg L−1 fluazifop-P-butyl
stress. ......................................................................................................................................... 57
Figure 14. DNA markers used to identify exon 32 of the ACC2 gene in the nonirradiated Lazarroz
FL rice variety. Note in (A) the sequence of exon 32 is shown in green, and the locations of the
primers used for PCR and sequencing are shown in black and blue boxes. In the red box is the
site corresponding to the biallelic mutation. (B) Sequencing electropherogram and corresponding
amino acid mutation putative changes. ...................................................................................... 58

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Figure 15. DNA markers (A) matK and (B) rbcL genes of the mutants in comparison with the
control reported sequences MZ558335 and MZ558334. Note in green the biallelic mutations G/T
in matK resulting in isoleucine change to methionine and GT/AG coding for tyrosine that may
change into serine in a mutation in rbcL. .................................................................................... 59
Figure 16. Gamma radiation optimization using Lazzaroz FL seeds as a starting material and
further calli induction, regeneration, and selection in two rounds of fluazifop-P-butyl selection at 5
and 10 mg L−1. Note the optimization window of gamma exposure ranges from 50 to 350 Gy,
allowing predictable calli induction and regeneration, as well as putative tolerant vitroplants. .. 59
Figure 17. Confirmation of NTH2 gene disruption in S. cerevisiae by CRISPR/Cas9. (A).
Amplification of the segment containing the expected mutation site on a 1% m/v agarose gel; (B).
Sequencing of the S. cerevisiae NTH2 gene. In the box, it can be observed how the deletion
1271_1272delTA caused the Y424X mutation in the nth2 1271_1272delTA strain. Image created
with BioRender.com (accessed on March 30th, 2022). ............................................................... 69
Figure 18. Confirmation of NTH1 gene disruption in S. cerevisiae by CRISPR/Cas9. (A).
Amplification of the segment containing the expected mutation site on a 1% m/v agarose gel; (B).
Sequencing of the S. cerevisiae NTH1 gene revealed the insertion of a T in position 893 resulting
in a truncated 301 AA protein with SWW* instead of PGGR. Image created with BioRender.com
(accessed on March 30th, 2022). ................................................................................................ 70
Figure 19. Analysis of the sizes of the wild-type and nth2 1271_1272delTA yeasts. (A). Scanning
electron microscopy views of the wild-type CEN.PK2-1C strain and mutated strain nth2
1271_1272delTA grown in 0 and 0.85 M NaCl; (B). Box plots representing the sizes of the wild-
type and nth2 1271_1272delTA strains of yeast under nonstress and stress (NaCl) conditions. No
significant difference was observed (p <0.05). ........................................................................... 72
Figure 20. Growth curves of S. cerevisiae nth2 1271_1272delTA strains in the presence and
absence of osmotic stress (0.85 M NaCl) by indirect measurements of optical density at 600 nm
after 20 h of growth under stress. The curve was built with the mean of four independent samples
per hour for each condition. The growth rate was calculated for the wild type in non-stress
conditions of 0.2255 ± 0.0037 h−1 versus 0.1580 ± 0.0009 h−1 in 0.85M NaCl stress, and nth2
1271_1272delTA 0.2327 ± 0.0057 h−1 versus 0.2179 ± 0.0061 h−1 in 0.85M NaCl stress. ........ 73
Figure 21. Agar plate comparison of yeast strains: nth1 893_894insT, nth2 1271_1272delTA, and
wild-type after two weeks of growth under 0, 0.85 M and 1.2M NaCl stress. Note serial dilutions
of yeast starting in OD600 = 1. ..................................................................................................... 74

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Índice de tablas

Table 1. Rice genes and mutations involved in stress tolerance or sensitivity traits. ................ 31
Table 2. Rice genes and mutations in herbicide resistance traits. ............................................. 33
Table 3. Rice genes and mutations with pathogen-resistant traits. ........................................... 33
Table 4. Rice genes and mutations involved in grain quality, quantity, weight, and plant structural
traits. ........................................................................................................................................... 35
Table 5. Rice genes and mutations in traits such as oleic acid, color, fragrancy, and nitrogen use.
.................................................................................................................................................... 37
Table 6. Genome Editing related norms and links. .................................................................... 38
Table 7. Rice calli induction and browning rate from different induction treatments .................. 44
Table 8. Rice calli response of Lazarroz FL cultivar after 4 weeks of culture on different
regeneration media..................................................................................................................... 45
Table 9. Lethal effect of gamma radiation on the embryogenic rice calli of Lazarroz FL cultivar
after 30 days of culture on regeneration medium, determined by probit model of survival (%) . 46
Table 10. Immersion regeneration, sprouting and browning rates ............................................. 48
Table 11. Non irradiated Lazarroz FL calli toxicity to Fluazifop-P-butyl ..................................... 55
Table 12. Non irradiated Lazarroz FL rice vitro plants toxicity to Fluazifop-P-butyl ................... 56
Table 13. Predicted Open Reading Frames (ORF) of mutants in comparison with the wild-type
NTH1 and NTH2 genes. ............................................................................................................. 71
Table 14. Intracellular trehalose content of yeast cells under non-stress and stress conditions.
.................................................................................................................................................... 74

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Declaración de autenticidad

Yo Alejandro Hernández Soto, estudiante del Doctorado en Ciencias Naturales para el

Desarrollo, declaro que la Tesis Doctoral que presento para su exposición y defensa titulada
“Inducción de variabilidad genética en una variedad comercial de arroz (Oryza sativa) mediante
el uso de radiación gama a partir de la selección in vitro de mutantes tolerantes a
ariloxifenoxipropionatos para su uso en el fitomejoramiento del cultivo” y comité asesor de tesis
son la Dra Ana Abdelnour Esquivel (directora de tesis), Dra Marta Valdez Melara (asesor) y el
Dr. Fabián Echeverría Beirute (asesor), es original y que todas las fuentes utilizadas para su
realización han sido debidamente citadas en el mismo. Este material no lo he presentado, en
forma parcial o total, como una tesis en esta u otra institución.

Cartago, Costa Rica, 25 de enero de 2023.

ALEJANDRO HERNANDEZ SOTO (FIRMA)

PERSONA FISICA, CPF-01-1100-0510.
Fecha declarada: 13/02/2023 01:56:05 PM
Lugar: San Jose, Costa Rica Contacto: [email protected]

Firma
Alejandro Hernández Soto

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Agradecimientos

Jason Pérez por toda su colaboración en el desarrollo del trabajo en laboratorio, dedicación,
colaboración y gestión oportuna que hicieron posible la obtención de resultados de laboratorio
que culminaron en artículos científicos.
Al Doctor Andrés Gatica por permitir colaborar en múltiples proyectos y facilitar el acceso a
infraestructura, aporte de contenidos técnicos en la redacción de artículos y apoyo para la
pasantía.

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Dedicatoria

A mi esposa Elena Ortiz, a mis hijos Elena Hernández Ortiz y José Gabriel Hernández Ortiz a
quienes motivaron e hicieron posible este trabajo.
A mis padres por su confianza, apoyo.
A Dios por darme salud y bendición para culminar esta etapa.

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Resumen

El principio de mejoramiento genético basado en mutaciones consiste en generar cambios

hereditarios en el ADN mediante agentes externos. La mutagénesis inducida mediante radiación
gamma ofrece una alternativa para desarrollar líneas de arroz resistentes al estrés biótico y
abiótico al acelerar el proceso de mutación espontánea y aumentar el conjunto de variantes
alélicas disponibles para la mejora genética. En el presente trabajo se exploran las bases
técnicas para lograr la mutagénesis in vitro mediante radiación gamma (60Co) con el rasgo de
tolerancia al ariloxifenoxipropionato Fluazifop-P-Butil como modelo para crear variabilidad en una
variedad local de arroz Lazarroz FL. El primer parámetro estandarizado que se logró fue la
radiosensibilidad de los callos embriogénicos. La dosis letal 30 se calculó a partir de 1680 callos
con exposición de gradiente de 0-120 Gy en 81,8 Gy (77,507-86,403). El segundo parámetro fue
la sensibilidad a los ariloxifenoxipropionatos como agente de selección. La dosis letal media fue
de 6,93 mg / L (0,425 mg / L - 15,743 mg / L, R2 = 0,402, 1000n) para callos y la dosis letal media
de 3.771mg / L (R2 = 1, 290n) para plantas in vitro. La secuenciación del gen diana ACCasa2 y
las secuencias de control matK, rbcL con el pre-identificador NCBI MZ558337, MZ558334,
MZ558335, demostraron que la variedad comercial utilizada tiene ausencia de mutaciones. El
sistema se ejecutó con 60Gy en callo embriogénico, regeneración, preselección a 5mg/L y
selección final a 5mg/L de Fluazifop-P-Butil. Lo anterior resultó en un único mutante putativo
tolerante a partir de 8000 vitroplantas regeneradas. La línea tolerante presentó una tasa de
mutación aproximada de 1/1000pb en el gen blanco ACC2 (T2222I/T2222M) y en los controles
matK, rbcL. Es posible que la mutación en el gen blanco ACC2 esté vinculada a la tolerancia, sin
embargo, no se puede descartar otros mecanismos no evaluados. El trabajo exploró el uso de
semilla como tejido de partida para la radiación in vitro, lo que permitió una exposición de 50-
350Gy, y un incremento en el número de mutantes putativos de 31. Un método alterno para
generar mutaciones con mayor precisión es mediante CRISPR-Cas9, el proyecto exploró la
técnica en levaduras de un gen homólogo de arroz como modelo para proveer tolerancia a
salinidad. Se logró una mutación en el gen NTH2, nth2 1271_1272delTA, lo que incrementó la
tolerancia de la levadura al estrés salino con capacidad de crecimiento a 0.8M NaCl a una tasa
de 0.2179 ± 0.0061 h−1 en comparación con la silvestre de 0.1580 ± 0.0009 h−1.

Palabras claves

Mutagénesis; Cobalto-60; cultivo in vitro; mejoramiento genético; CRISPR-Cas9; levadura.

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Abstract

The mutation breeding principle is to generate heritable changes in the DNA by external agents.
Induced mutagenesis by gamma rays offers a promising alternative for developing rice varieties
resistant to biotic and abiotic stresses. It could accelerate the spontaneous mutation process and
increase the pool of allelic variants available for genetic improvement. Here we present the
technical bases to achieve in vitro Gamma radiation (60Co) mutagenesis with Fluazifop P-Butyl
tolerant trait as a model to create rice variability in a local variety used by farmers, Lazarroz FL.
The first standardized parameter achieved was the radiosensitivity of embryogenic calli. The dose
was 81.8Gy (77.507-86.403) for LD30, obtained from 1680 callus tested on a 0-120Gy gradient.
The second parameter was the sensibility to the aryloxyphenoxypropionates as a selection agent.
The dose was 6.93mg/L (0.425 mg/L- 15.743 mg/L, R2= 0.402, 1000 callus) for callus LD50, and
3.771mg/L (R2= 1, 290n) LD50 for Vitro plants. Target gene sequencing ACCasa2, and control
sequences matK, rbcL summited to the NCBI id MZ558337, MZ558334, MZ558335,
demonstrated that the commercial variety used had an absence of mutations. The mutation
system was done at 60Gy on embryogenic calli, regeneration, preselection on 5mg/L and final
selection on 10mg/L Fluazyfop P-Butyl resulting in 1/8000 putative tolerant vitro plant. The tolerant
line had a mutation rate of approximately 1/1000bp in the control genes matK, rbcL as well as the
target gene ACC2 (T2222I/T2222M). The mutation may be linked to the tolerance; however, it
could also be the result of other non-evaluated detoxification mechanism. The in vitro system was
improved by using seeds as a starting point for irradiation, allowing an exposition window of 50-
350Gy and 31 putative mutants. An alternative method to generate precise mutations is CRISPR-
CAS9. The project explored the technique in a yeast gene homologous to rice as a model to
provide salt tolerance. We achieved a mutation on gene NTH2, nth2 1271_1272delTA, resulting
in increased salt tolerance of the yeast capable of better growing under 0.8M NaCl at a rate of
0.2179 ± 0.0061 h−1 versus wild type 0.1580 ± 0.0009 h−1.

Keywords

Mutagenesis; Cobalt-60; plant tissue culture; plant breeding; CRISPR-Cas9; yeast.

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1. Introducción

El arroz (Oryza sativa) es uno de los cereales de mayor consumo mundial, responsable del 20%
de las calorías de la dieta de al menos 3500 millones de personas particularmente de Africa, Asia
y América (Gutaker et al., 2020). En Costa Rica se consume 406,599 toneladas métricas de
arroz, que se abastecen en un 35% con producción nacional de cantones de alta vulnerabilidad
económica, particularmente la región Chorotega, Brunca y Pacífico Central. Los agricultores de
Costa Rica tienen una preferencia por cuatro cultivares, Lazarroz FL con un 45%, Senumisa 20FL
(18%), Palmar18 (11%) y Puita INTA (11%) (CONARROZ, 2021; Morales et al., 2018; Orozco &
Carrodeguas, 2022). Los programas de fitomejoramiento de arroz en Costa Rica deben
considerar estos materiales de partida para añadir variabilidad en caracteres que mejoren el
cultivo.
Las mutaciones son útiles para el mejoramiento de cultivos como el arroz, sin embargo, la tasa
de mutaciones espontáneas es baja, lo que impide el avance de programas de fitomejoramiento
(Oladosu et al., 2016). Las mutaciones con métodos físicos, químicos o biológicos permiten
desarrollar variabilidad mediante cambios en el material genético (Nakagawa & Kato, 2017). La
radiación gamma en semillas es capaz de generar mutaciones con una dosis de 250Gy tales
como sustituciones únicas (57), deleciones (17.7), inserciones (5.9) (F. Li et al., 2019). Un método
alternativo que disminuye el trabajo y selección en campo es la inducción de mutaciones in vitro
a partir de callo, suspensiones celulares y embriones inmaduros, para lo cual se requiere la
optimización a nivel de laboratorio según el material de partida (Viana et al., 2019).
Las mutaciones en arroz permiten el desarrollo de características de interés agronómico y de
importancia para la sostenibilidad del cultivo, algunas de las cuales se describen a continuación.
El incremento en el número de granos está asociado a mutaciones en el gen Os01g0197700
(Gn1a, OsCKX2), el tamaño del grano al gen Os03g0407400 (GS3); mutaciones en el gen
Os08g0509600 (IPA1) están relacionadas con un mayor número de granos y alta frecuencia;
mientras que la floración en día corto o largo en el gen Os06g0275000 (Hd1/ SE1) (Fan & Li,
2019; M. Li et al., 2016; Shen et al., 2017; Song et al., 2022; Tanaka et al., 2020; Yang et al.,
2019). El rendimiento puede aumentar de 41.3 a un 68.3% mediante la sobre expresión del gen
DREB1C(Wei et al., 2022). El aroma está relacionado a mutaciones en el gen Os08g0424500
(Shen et al., 2017). El genotipo de planta erecta y corta por mutaciones en el gen Os09g0441900
(DEP1) (M. Li et al., 2016). Existen genes relacionados a la domesticación que ocurrió por
mutación en los genes como el Os06g0133000 (WX1) (Chen et al., 2019). Otros genes de interés

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son la tolerancia a estrés salino relacionados con el gen Os06g0183100 (A. Zhang et al., 2019).
Además, existe resistencia a bacterias relacionada con los genes Os11t0508600-01 y
Os08g0535200 (H. Li et al., 2020; Xu et al., 2020). En el caso de resistencia a moléculas químicas
comúnmente llamados herbicidas, se conocen mutaciones relacionadas con trifluralina en el gen
Os11g14220 (L. Liu et al., 2021), quizalofop Os05g22940.1 (ACC2) (de Andrade et al., 2018; X.
Liu et al., 2020; Xu et al., 2020), así como las moléculas químicas que actúan sobre las enzimas
Acetolactato sintasa en el gen Os02g0510200 (Bzour et al., 2018; Q. Lin et al., 2020; Sagare et
al., 2020; Shimatani et al., 2017; Xu et al., 2020) y sobre la enzima EPSPS (J. Li et al., 2016). Es
posible lograr mutaciones inducidas mediante radiación gamma en los genes descritos con
anterioridad para lograr las características deseadas según se detalla a continuación (F. Li et al.,
2019).
Las radiaciones ionizantes producen daños directos o indirectos en el ADN. Estos daños pueden
ser de dos tipos, puntuales lo que generan mutaciones en el material genético o bien afectan de
manera letal al organismo (Nakagawa & Kato, 2017). Las mutaciones ocurren de forma aleatoria
a lo largo del genoma y son fuente de diversidad útil para el fitomejoramiento. Una manera de
optimizar el proceso de mutación es utilizando marcadores que correlacionen con estos cambios
en el genoma como la resistencia a un agente químico (Murphy & Tranel, 2019; Tan & Li, 2022).
El uso de un agente de selección químico permite el establecimiento y optimización de los
protocolos in vitro de manera predecible, en este caso a partir de una variedad comercial de
mayor uso por el agricultor costarricense Lazarroz FL. Los ariloxifenoxipropionatos son
recomendados a nivel mundial para el control de gramíneas, este tipo de moléculas actúan a
nivel de la síntesis de ácidos grasos y la resistencia está dada por mutaciones puntuales en el
gen ACC2 que cataliza la caboxilación de acetil-CoA a Malonil-CoA (de Andrade et al., 2018;
Takano et al., 2020; R. Zhang et al., 2019). Lo anterior permite las bases teóricas para determinar
el equilibrio entre la dosis correcta de radiación gamma, muerte celular, protocolos in vitro y
mutaciones puntuales. La presente tesis plantea el sistema de creación de variabilidad genética
in vitro al utilizar como agente de selección una molécula química estrechamente relacionada
con mutaciones puntuales en el gen ACC2 (de Andrade et al., 2018; Murphy & Tranel, 2019;
Pereira et al., 2019).
El objetivo de esta investigación es establecer un modelo de inducción de variabilidad genética
de Arroz (Oryza sativa) Lazarroz FL mediante radiación gamma usando como agente de
selección los ariloxifenoxipropionatos, particularmente la molécula Fluazifop, para su uso como
una fuente de genes para el mejoramiento genético. Mutaciones en el gen blanco ACCasa

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(ACC2, Os05g0295300, LOC_Os05g22940 B9FK36) están directamente relacionadas a la
resistencia a ariloxifenoxipropionatos. Para ello, el proyecto planteó el establecimiento in vitro de
callo embriogénicos del escutelo de semillas de arroz, la determinación de la dosis letal media
(DL50) del agente de selección, así como de la dosis de la irradiación gama, para generar los
procesos posteriores de irradiación y selección de células tolerantes. Finalmente, las líneas
tolerantes se regeneraron, multiplicaron y fueron transferidas al invernadero para la obtención de
la M2. Adicionalmente, estas plantas de la M1 y M2 con tolerancia a ariloxifenoxipropionatos se
analizaron mediante secuenciación de los genes blanco ACCasa, mediante herramientas
bioinformáticas para determinar el tipo de mutación y la heredabilidad de la resistencia.

Pregunta de investigación

El presente trabajo tiene como pregunta de investigación determinar condiciones óptimas para
generar mutaciones en arroz in vitro de una variedad comercial (Lazarroz FL) mediante
radiaciones gamma usando como modelo las mutaciones que otorgan resistencia a los
ariloxifenoxipropionatos.

Marco teórico:

Mutación. El mejoramiento de cultivos se basa en la variabilidad genética de las plantas. Las

mutaciones son la mayor fuente de esta variabilidad, que es posteriormente amplificada por
recombinación genética de cromosomas homólogos durante la meiosis (Viana et al., 2019). El
término mutación fue acuñado a finales del siglo XIX por Hugo de Vries en referencia a la
ocurrencia de cambios o “saltos” en características que llevan al desarrollo de variaciones y de
nuevas especies, como resultado de redescubrir las leyes de la herencia mendeliana (Oladosu
et al., 2016). La radiación ionizante es el agente mutagénico de mayor uso en el mejoramiento
genético de cultivos vegetales (F. Li et al., 2019). La base conceptual de la capacidad de las
radiaciones ionizantes de producir mutaciones en organismos superiores fue demostrada por
Muller en 1927 en mosca de la fruta (Drosophila) (Muller, 1927) y en cultivos vegetales por Stadler
en 1928 en maíz (Zea mays) mediante rayos X (Stadler, 1928).
La mayoría de las variedades que se utilizan en agricultura fueron producidas por mutagénesis
inducida por métodos físicos, particularmente por radiación gamma cuyo origen es el cobalto
radiactivo (60Co) y/o Cesio (137Cs). La preferencia de este sistema se debe a su capacidad de
penetración de los tejidos vegetales. Existen otros agentes físicos que pueden ser utilizados para
la mutagénesis física tales como los rayos X; los neutrones producidos por 235U; las partículas
beta producidos por núcleos de Helio; partículas alfa producidos por 32P y 14C; protones

15
producidos por núcleos de Hidrógeno; y los iones producidos por aceleración de partículas (en
inglés ion beam) (Oladosu et al., 2016).
La base de datos de la FAO/IAEA registra más de 3200 variedades de cultivos liberadas en todo
el mundo, incluyendo 830 registros que corresponden a arroz obtenidos por este método en su
mayoría a partir de semillas (FAO/IEAE 20191). En suma, en Costa Rica, existen antecedentes
del uso exitoso de estas técnicas de radiaciones ionizantes, en la década de los noventa se
desarrolló la variedad CAMAGO-8, que fue utilizada comercialmente por su tolerancia a
Pyricularia oryzae. La UNA, UCR y TEC se unieron en un proyecto FEES-CONARE durante el
2016-2018 para buscar variantes de arroz tolerantes a sequía y salinidad (Abdelnour-Esquivel et
al., 2020).
Existen otros métodos como inducción con Etil Metano Sulfonato (EMS) con el inconveniente de
que las mutaciones no se distribuyen uniformemente en el cromosoma, tiene regiones preferidas
con contenidos altos de GC, elementos transponibles y regiones con niveles bajos de expresión
génica y heterocromatina y tienen poca capacidad de penetración del tejido (Hu et al., 2020; Yan
et al., 2021). Este método no debe ser descartado como opción, pero en el presente trabajo no
se utilizó como primera opción debido al riesgo del manejo del químico EMS en condiciones de
laboratorio.
Avance en el mejoramiento de cultivos. El mejoramiento genético de cultivos se encuentra en
evolución e integración de métodos no convencionales. En el mejoramiento convencional la
introducción de nuevos caracteres ocurre por mutación inducida, así como por introgresión por
medio de cruzas intergenéricas e interespecíficas. Lo anterior permite introducir rasgos como el
incremento en rendimiento, resistencia a enfermedades y mejora de la calidad de grano
(Hernández-Soto et al., 2021; Oladosu et al., 2019).
Los métodos no convencionales, tales como los Nuevos Métodos de Mejora Genética (en inglés
NBTs, New Breeding Techniques) son el producto del avance en el entendimiento a nivel
molecular de los cultivos mediante las ómicas, la fisiología y los métodos automatizados de
caracterización fenotípica(Singer et al., 2021). Este entendimiento no sólo permite una mayor
precisión en la mejora del cultivo, sino el abordaje de caracteres más complejos controlados por
múltiples genes mediante la introducción de genes por cisgénesis, transgénesis y ARN de
interferencia para un gen funcional o bien para factores de transcripción que interactúan con
múltiples genes (Anders et al., 2021; Mohd Saad et al., 2022; Oladosu et al., 2019). Lo mismo es

1
FAO/IAEA (2019) Mutant Variety Database. http://mvd.iaea.org/. Visitado el 12 de junio de 2019.

16
posible también sin la introducción de nuevo material genético mediante técnicas como la edición
de genomas mediada por meganucleasas como las nucleasas de dedos de zinc (ZFN) y las
nucleasas efectoras similares a factores de transcripción (TALEN), así como el sistema CRISPR-
Cas9 que permiten la corrección o la introducción de mutaciones de manera precisa (Karmakar
et al., 2022; Singer et al., 2021; Smyth, 2022).
La ventaja de la alta integración de las herramientas no convencionales es ahorro en tiempo
(Karmakar et al., 2022; Smyth, 2022). El análisis de fenotipo, el conocimiento genómico de
parentales silvestres, el procesamiento de datos por aprendizaje automático (en inglés machine
learning) y posterior uso de ese conocimiento para generar correcciones puntuales mediante
edición de genomas tiene el potencial de aumentar la precisión y reducir los tiempos de desarrollo
de cultivos más resilientes y sostenibles (Mohd Saad et al., 2022). La presente tesis aborda
métodos in vitro útiles para el uso de estas técnicas con métodos convencionales que son útiles
para el mejoramiento no convencional y explora de manera práctica el uso de CRISPR-CAS9
con un modelo de levadura estos conceptos para la mejora de arroz.
¿Por qué utilizar la resistencia a ariloxifenoxipropionato como modelo para generar
mutaciones?
Los agentes de selección permiten optimizar los procedimientos, existen mutaciones específicas
relacionadas con la resistencia ariloxifenoxipropionato en el gen ACC2 por lo que los resultados
se correlacionan. Adicionalmente, la tolerancia a herbicidas es una característica que podría
servir para el fitomejoramiento del cultivo. A continuación, se detallan los tres argumentos
indicados con anterioridad.
1. Los procedimientos de generación de variabilidad mediante radiación gamma son posibles,
pero requieren optimización. Un agente de selección como la resistencia a
ariloxifenoxipropionatos permitiría esta optimización. Los procedimientos de inducción de
mutaciones mediante la radiación gamma son posibles gracias a que el ITCR cuenta con un
irradiador de cobalto Gamma-Cell, las facilidades en cultivo de tejidos y equipo en el Centro de
Investigación en Biotecnología y la Escuela de Física. La inducción de mutación es un
procedimiento común y se realiza con un irradiador de cobalto Gamma-Cell. Lo anterior es la
premisa para lograr la inducción de mutaciones mediante radiación gamma y posteriormente
seleccionar mediante la exposición in vitro a dosis correctas de radiación y herbicida como agente
de selección. Las células que sobrevivan generarán líneas que adquirieron la tolerancia como
resultado de la mutación.

17
2. Mutaciones específicas en el gen ACC2 están relacionadas con la tolerancia a herbicidas
ariloxifenoxipropionatos por lo que son un indicador para determinar si el sistema de generación
de mutaciones es funcional. La enzima es la Acetil-CoA carboxilasa ACC2 (EC 6.4.1.2) ubicado
en el cromosoma 5: 14,067,726-14,079,652 en la hebra reversa (ACC2, BGIOSGA018366-TA,
A2Y2U1). Específicamente, mutaciones en el dominio caboxiltransferasa entre los aminoácidos
1,781- 2,078 y 2,027- 2,096 relacionadas a la tolerancia de herbicidas ariloxifenoxipropionatos
(Hernández-Soto et al., 2021). En esta región, se conocen de mutaciones como Ile1781-Leu,
Trp-1999-Cys, Trp-2027-Cys, Ile-2041-Asn, Ile-2041-Val, Asp-2078-Gly, Cys-2088-Arg, Gly-
2096-Ala (Powles & Yu, 2010). La mayoría de estas mutaciones son del tipo transversiones por
ejemplo Gln-1756-Glu C/G (CAG por GAG), Ile1781-Leu/Val/Ala/Thr (ATA por CTA Leu, GTA
Val), y algunas pueden ser transiciones como A/G Ile1781-Val (ATA por GTA). La mutación
reportada en trigo de A2004V en la accesión AIQ78380.1 por transición de C por T confirmada
por Zhang (R. Zhang et al., 2019), le otorga tolerancia a quizalofop a 10gramos de ia por ha−1,
mientras que esta molécula en leguminosas como soya y lentejas es de 31-92g ia por Ha (Ostlie
et al., 2014; R. Zhang et al., 2019). Mutaciones en Oryza japonica de la ACCasa 2
(LOC_Os05g22940) correspondientes a I1781V, C2088R y W2027C proveen tolerancia a
herbicidas (X. Liu et al., 2020). Existe el antecedente de un arroz comercial con la mutación por
transversión A/T que ocasiona un cambio de aminoácido I1781L en el gen ACCasa de arroz de
herencia mendeliana y está disponible en el mercado estadounidense desde el 2018 (Camacho
et al., 2019). Otros sitios de mutación por transversión en malezas resistentes a estos herbicidas
están disponibles en la accesión AJ310767.1 de la maleza resistente Alopecurus myosuroides
(Hernández-Soto et al., 2021).
El gen ACC2 se puede caracterizar mediante secuenciación con lo que se dispondría de un
método visible y cuantitativo para determinar las condiciones óptimas que resultan en los
fenotipos esperados.
3. La tolerancia a moléculas químicas es una característica potencialmente útil para el
fitomejoramiento. La práctica de usar suelos inundados y cultivo de arroz mediante transplante
es eficiente y ampliamente usada en países asiáticos particularmente para el control de arvenses
(Wang et al., 2017) pero el impacto ambiental por la producción de metano (Basavalingaiah et
al., 2020), así como la huella hídrica es muy elevado con un promedio de 1325 m3/t (48% verde,
44% azul y 8% gris) (Lovarelli et al., 2016). La práctica puede resultar en degradación química y
fitotoxicidad en suelos ultisoles ricos en hierro, que se solubilizan al acidificarse por efecto del
agua, lo ocasiona también mayor compactación del suelo. La degradación puede ser también a

18
nivel de pérdida de materia orgánica (Wang et al., 2017). Alternativamente el arroz se cultiva de
manera directa (en secano), con la ventaja de menos uso de agua, menor producción de metano,
maduración en menor tiempo, pero la desventaja de la presencia y manejo de arvenses (Sagare
et al., 2020). Una estrategia para solventar esta problemática es el desarrollo por
fitomejoramiento de variedades que muestren resistencia a moléculas químicas (herbicidas) o a
condiciones que limitan la producción (Reddy et al. 2017). En el caso específico del arroz, uno
de los mayores problemas en el control de arvenses es el arroz maleza (Oryza sativa f.
spontanea) (Nadir et al., 2017). El arroz tolerante a herbicidas es una solución ya adoptada en el
pasado con tolerancia a herbicidas imidazolinonas como resultado de las mutaciones S653N,
A122T y G654E en el gen Acetolactato Sintasa (ALS) de nombre comercial Clearfield (tales como
CL121, CL141 contienen G654E; CL161 y CLXL8 contienen S653N con 32% más resistencia;
INTA Puitá-CL contiene A122T). Sin embargo, el gen responsable de la resistencia se cruza
eventualmente con el arroz maleza y por tanto adquiere la resistencia que bajo una constante
presión de selección puede llegar a niveles del 20-60% (Singh et al., 2017). Una molécula
alternativa es la tolerancia a herbicidas de distinto modo de acción como los
ariloxifenoxipropionatos como el Fluazifop.
El Fluazifop cuyo nombre es Fluazifop-p-butyl, peso molecular de 327,25g mole-1 es un herbicida
que pertenece a los Ariloxifenoxipropionato que actúan bloqueando el dominio funcional de la
enzima Acetil-CoA carboxilasa. La enzima Acetil-CoA carboxilasa (EC 6.4.1.2) es una enzima
multifuncional para la síntesis de ácidos grasos primarios en cloroplastos, mitocondrias, así como
en la síntesis de ácidos grasos de cadena larga en el citoplasma. El arroz convencional es
susceptible al herbicida Fluazifop a concentraciones de 210g ia/Ha y 420g ia/Ha en aplicación en
suelo que afectan en un 13% y 15% de daño respectivamente y proveen de poco efecto residual
de arvenses al aplicarse como pre- emergente (Lancaster et al., 2018). Existen mutaciones que
confieren resistencia a herbicidas en trigo A2004V ariloxifenoxipropionato, específicamente a
quizalofop a 10gramos de ia por ha−1, mientras que esta molécula en leguminosas como soya y
lentejas es de 31-92g ia por Ha (Ostlie et al., 2014; R. Zhang et al., 2019). A nivel de selección
por mutación, existe un reporte de dosis de selección de 1uM y 2uM por Litro (PM 327,25g mol-
1
) es decir de 0,327mg y 0,654 mg respectivamente en callo embriogénico, así como selección
en invernadero con aspersión de 150mg/L de las plantas resistentes (X. Liu et al., 2020).

El presente proyecto plantea realizar la inducción por mutación en etapas in vitro mediante callos
embriogénicos y selección mediada por ariloxifenoxipropionatos. Las vitroplantas que sobrevivan

19
generarían líneas que adquirieron la tolerancia potencialmente relacionada con la mutación en el
gen ACCasa2. Los genes se procederán a caracterizar mediante secuenciación de los genes
blanco de las líneas mutantes regeneradas M1 con tolerancia al herbicida.

Marco metodológico:

La investigación es cuantitativa en su diseño (McCusker & Gunaydin, 2015) y utiliza dos factores
en etapas independientes. El primer factor es la radiosensibilidad de los callos de arroz para el
cálculo de la dosis letal media (LD50), en dosis de 0, 20, 40, 60, 80, 100 y 120 Gy, con 5-10
repeticiones, y 20 unidades (callos) para un total de 1500 callos. La segunda es la dosis letal
media del herbicida que se realiza en dosis seriadas del herbicida, mediante una primera
aproximación de dosis letal media de 0,10,100mg de herbicida 10 repeticiones, y 20 unidades
(callos o semillas germinadas in vitro) cada uno y posteriores ensayos de una mejor aproximación
a la dosis letal media. Con esta información se generan los datos de línea base para generar
mutantes tolerantes y someterlos a selección con el agente de selección con el fin de identificar
las líneas resistentes. Posteriormente se analizarían a nivel genético las plantas tolerantes para
entender la resistencia, se llevarían al invernadero y eventualmente a campo para un análisis de
comportamiento. La metodología específica se describe en el apartado de procedimientos.
OE 1 Establecer el cultivo in vitro de callo embriogénico de una variedad comercial de
arroz y la radiosensibilidad de estos a radiaciones gamma (Co-60).
Preparación de material. Semilla de arroz (Oryza sativa subsp. indica) Lazarroz FL en granza,
facilitada por productores de arroz, se utilizaron como material de partida para la introducción e
inducción de callo embriogénico según se detalla a continuación. En la desinfección y el
establecimiento in vitro se utilizaron 6000 semillas. La palea y la lemma se eliminaron utilizando
una lija No. 100 y posteriormente se utilizó una doble incubación en una solución de hipoclorito
de sodio (NaOCl) al 5,25 % (v/v) con 5 μl de Tween 20 ® por cada 10 ml, en agitación en orbital
a 200 rpm por 20 minutos. En la cámara de transferencia de flujo laminar, se realizó cinco lavados
con agua destilada estéril, seguido de un enjuague con la solución biocida (Methylisothiazolone
(2-methyl-4-isothiazolin-3-one, MIT) y Methylchloroisothiazolinone (5-chloro-2-methyl-4-
isothiazolin-3-one, CIT)), reactivos Químicos Gamma, Laboratorios ARVI S.A) al 40% (v/v).
Inducción del callo embriogénico. Se colocaron 15 semillas por plato Petri con 20ml de medio de
inducción de callos según lo descrito por Bai et al. (2014), el cual consiste de las sales minerales
y vitaminas de Murashige y Skoog (MS, 1962) suplementado con 2,5 mg.L-1 ácido 2,4-
diclorofenoxiacético (2,4-D), 30 g.L-1 sacarosa, 0,3 g.L-1 caseína hidrolizada y 0,5 g.L-1 prolina

20
(Bai et al., 2014; Murashige & Skoog, 1962; Pawar et al., 2015). El pH se ajustó a 5,8 y el medio
se esterilizará en autoclave (presión de 1,2 ATM.cm-2 y 121 °C de temperatura por 25 min). Los
cultivos se mantuvieron en condiciones de oscuridad durante la inducción de callo embriogénico
por 20 días. La inducción se tuvo de optimizar lo que resultó en un medio MS con 2 mg L−1 2,4-
D, en lugar de 2.5mg L.
Regeneración de los embriones somáticos. Se evaluó la regeneración de 200 callos en medio
semisólido con sales minerales MS, 30 g.L-1 maltosa, 0,5 mg.L-1 ácido α-naftalenacético (ANA),
3 mg.L-1 6-bencilaminopurina (BAP), gelificado con 4 g.L-1 Phytagel ®, y el pH fue ajustado a 5,8
(Sudhakar et al., 1998). Las condiciones de cultivo serán de luz directa con una
intensidad lumínica de 72 µmol·s-1·m-2, un fotoperiodo de 16h y una temperatura de 26 ± 2 °C.
La metodología de regeneración se tuvo que optimizar. Se propuso el uso de kinetina y tidiazurón
como fitohormonas alternativas para la regeneración en caso de ser necesario, por ejemplo
(Mohd Din et al., 2016) reporta el uso del medio MS con 0.5 mg.L-1 6-bencilaminopurina (BAP),
1.5 mg.L-1 Kinetina, 0,5 mg.L-1 ácido α-naftalenacético (ANA), y 0,5 mg.L-1 tidiazurón (TDZ). Lo
anterior se debe a Tidiazurón ocasiona formacion de brotes y específicamente en arroz genera
un cambio de expresión genética asociado a la regeneración a concentraciones de 1mg/L
(Chakrabarty et al., 2010; Indoliya et al., 2016). La regeneración mejoró mediante el uso de
inmersión temporal en un 100%.
Determinación de la radiosensibilidad. Los callos embriogénicos se irradiaron en el sistema de
irradiación tipo Ob-Servo Ignis (Institute of Isotopes Co, Ltd., Budapest, Hungary), con 24 fuentes
de cobalto 60 (tipo CoS 44HH-N). La radio sensibilidad se calculó al exponer grupos de 20 callos
a dosis de 0, 20, 40, 60, 80, 100 y 120 Gy. Se irradiaron en promedio 180 callos por dosis, para
un total aproximado de 1500 callos evaluados. Cada ensayo se analizó en un diseño
completamente aleatorio y con un análisis de regresión lineal de Probit y Logit con el paquete
estadístico IBM SPSS versión 26. Los callos se colocaron en medio de multiplicación con el fin
de evaluar exclusivamente la variable de radiosensibilidad.
OE 2. Determinar la concentración correcta de dosis letal, que permita el posterior ensayo
de inducción y selección in vitro de líneas de arroz con tolerancia al herbicida.

Determinar la sensibilidad a los herbicidas de callos embriogénicos en regeneración. Callos

embriogénicos no irradiados se cultivaron en el medio de multiplicación de callo según lo descrito
por Bai et al. (2014) con dosis de 0, 10 y 100mg de herbicida ariloxifenoxipropionato por Litro,
para un total de 2000callos por herbicida. Los cultivos se mantuvieron con un fotoperiodo de 16h

21
luz con intensidad de 72 µmol·s-1·m-2 y una temperatura de 26 ± 2 °C. Después de seis semanas
de cultivo, se evaluó el porcentaje de callos que sobrevivieron y regeneraron plantas. Cada
ensayo se analizará en un diseño completamente aleatorio y con un análisis de regresión lineal
de Probit y Logit con el paquete estadístico IBM SPSS versión 26. Una vez determinado una
aproximación de la dosis letal media, se realizarán ajustes en las dosis para repetir el ensayo
con las dosis que se aproximen a la dosis calculada.
Alternativamente se realizó un análisis de toxicidad en plántulas germinadas in vitro. Esto debido
al acceso limitado al laboratorio por la pandemia. El análisis se realizará en iguales condiciones,
esto es, con dosis de 0, 10 y 100mg de herbicida ariloxifenoxipropionato por Litro. El número de
individuos disminuiría a 40 plántulas por dosis de herbicida en medio de regeneración semisólido
con sales minerales MS, 30 g.L-1 maltosa, 0,5 mg.L-1 ácido α-naftalenacético (ANA), 3 mg.L-1 6-
bencilaminopurina (BAP), gelificado con 4 g.L-1 Phytagel ®, con pH fue ajustado a 5,7(Sudhakar
et al., 1998). Después de seis semanas de cultivo, se evaluará el número de plantas que
sobrevivan. Cada ensayo se analizará en un diseño completamente aleatorio y con un análisis
de regresión lineal de Probit y Logit con el paquete estadístico IBM SPSS versión 26. Una vez
determinado una aproximación de la dosis letal media, se realizarán ajustes en las dosis para
repetir el ensayo con las dosis que se aproximen a la dosis calculada.
Identificar líneas promisorias de arroz con tolerancia a los herbicidas.
Los callos embriogénicos de arroz (2000 callos) de 21 días de inducción en el medio optimizado,
se sometieron a la dosis letal media calculada para la radiación gamma (60Gy) y se regeneraron
en medio de regeneración mencionado con anterioridad por un periodo de 2 meses (Sudhakar
et al., 1998). Las plántulas regeneradas se sometieron a la dosis letal del agente de selección
(5mg/L de Fusilade) en el medio de regeneración con un fotoperiodo de 16h luz e intensidad
lumínica de 72 μmol·s-1·m-2 y una temperatura de 26 ± 2 °C. Después 8 semanas se evaluó la
sobrevivencia y las plantas sólo se seleccionaron las tolerantes (100 plantas). Posteriormente se
incrementó a la dosis del agente de selección (10mg/L de Fusilade) para identificar potenciales
líneas resistentes. La planta resultante se mantuvo en multiplicación en los medios de cultivo
respectivos.

OE 3. Verificar los cambios genéticos asociados a la resistencia de al menos una de las

líneas promisorias de arroz con tolerancia a herbicida.

22
La determinación de la mutación presente en líneas tolerantes a los herbicidas se basó en el
estudio del gen reportado en la literatura como responsables de la tolerancia, ACCasa. Las
plantas con tolerancia serán cultivadas hasta llegar a invernadero donde serán analizadas
mediante secuenciación del gen ACCasa. La extracción de ADN se realizará a partir de hojas de
vitroplantas resistentes al herbicida, mediante el Kit NucleoSpinTM Tissue (Macherey-Nagel),
según las condiciones del fabricante. El ADN se visualizó en un gel de agarosa al 1,5% con
buffer TAE 1X. La corrida se realizó durante 45 min a 70 V en una cámara de electroforesis
horizontal. La secuenciación se realizará mediante el método de sanger. El diseño molecular de
para el diseño de los imprimadores se realizó con Primer3 Plus, sobre la base de las secuencias
disponibles en línea ACCasa (ACC1 Os10g0363300, LOC_Os10g21910 Q8S6N5; ACC2,
Os05g0295300, LOC_Os05g22940, B9FK36). Para ello se realizó un análisis bioinformático de
las secuencias disponibles en las bases de datos para el diseño, amplificación y secuenciación
de los genes de interés tanto en el control, como en al menos un arroz maleza. Los resultados
fueron procesados y analizados por herramientas bioinformáticas para entender el tipo de
mutación, su cambio a nivel de aminoácidos y su relación con la resistencia al herbicida. Una vez
obtenidas las plantas en el invernadero, se analizó para verificar su tolerancia al herbicida al
exponerlas la línea a 150mg/L.

2. Objetivos

2.1. Objetivo general

Establecer un sistema de inducción de variabilidad genética de arroz (Oryza sativa) in vitro

mediante radiación gamma usando como agente de selección ariloxifenoxipropionatos para su
uso como una fuente de genes para el mejoramiento genético del cultivo.

2.2. Objetivos específicos

1. Establecer el cultivo in vitro de callo embriogénico de una variedad comercial de arroz y

la radiosensibilidad de estos a radiaciones gamma (Co-60).
2. Determinar la concentración correcta de dosis letal de ariloxifenoxipropionatos, que
permita el posterior ensayo de inducción y selección in vitro de líneas de arroz con
tolerancia al herbicida.
3. Verificar los cambios genéticos asociados a la resistencia de al menos una de las líneas
promisorias de arroz con tolerancia a herbicida.

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3. Síntesis

Los artículos científicos de la presente tesis se presentan bajo el siguiente orden.

El primer artículo (Hernández-Soto et al., 2021) es una revisión de literatura donde se exploran
potenciales genes blanco para las técnicas de mutación. Las mutaciones son una herramienta
para crear variabilidad genética, sin embargo, las dos preguntas que motivaron esta revisión de
literatura fueron las siguientes. La primera si a nivel exploratorio existían fenotipos asociados a
uno o dos genes específicos. La importancia para el fitomejoramiento radica en que la mutación
pueda ser seleccionada en el proceso de manera más expedita, identificarse a nivel genético y
validarlo en la descendencia para efectos de la heredabilidad. Efectivamente se lograron
identificar genes relacionados con rendimiento, número de granos, floración, aroma, tolerancia a
estrés biótico y abiótico. La segunda pregunta era si el agente de selección, tolerancia a
ariloxifenoxipropionatos era efectivamente útil para correlacionarla con mutaciones de interés en
genes blanco y qué otros genes o caracteres podrían ser útiles para el mismo fin. La revisión de
literatura demostró que el gen Os05g22940.1 (ACC2) es un excelente candidato dado que
mutaciones en el extremo carboxi terminal se relacionan directamente con tolerancia. De igual
manera la revisión de literatura permitió identificar otros genes útiles que se detallan a
continuación. Los genes de tolerancia a agentes químicos como la trifluralina en el gen
Os11g14220, las moléculas químicas que actúan sobre las enzimas Acetolactato sintasa en el
gen Os02g0510200, y sobre la enzima EPSPS. Existen además genes de tolerancia a salinidad
que podrían ser útiles durante la etapa de exposición a un agente de selección.

El segundo artículo (Hernández Soto et al., 2022) consolida los primeros resultados
experimentales. El artículo explica la herramienta de creación de variabilidad genética mediante
radiación gamma in vitro de arroz y presenta las conclusiones de los objetivos 1 y 2. El artículo
explora la identidad genética del material comercial que se utilizó, Lazarroz FL mediante
marcadores moleculares estándar matK y rbcL, presenta el establecimiento y optimización de los
protocolos in vitro de manera predecible y precisa. La optimización de los protocolos de cultivos
de tejidos, de la radiación a 60Gy y regeneración requirió de buscar formas alternativas para
alcanzar los objetivos según se detalla a continuación. La línea Lazarroz FL era recalcitrante para

24
la regeneración y presentaba porcentajes de regeneración muy bajos (30%) o nulos. Se requirió
de optimización de los protocolos y se logró identificar a la inmersión temporal como una solución
sumamente eficiente. Lo anterior hizo que se validasen los protocolos de inmersión temporal con
un control (CR-5272), la variedad en estudio Lazarroz FL y los materiales recalcitrantes CR-1821,
CR-1113, que mejoraron su regeneración.

En el tercer artículo (Hernández-Soto et al., 2022) corresponde un pre-print del segundo y tercer
objetivo de la tesis y presenta la consolidación del trabajo de estandarización previamente hecho
en los objetivos 1 y 2. El sistema se ejecutó con base en lo aprendido, para ello se calculó la
dosis letal del agente de selección (Fluazifop-p-Butil) y se re-evaluó el tejido in vitro a irradiar, a
saber, callo y semilla. La dosis letal media en callo fue de 6,93 mg/L (0,425 mg/L - 15,743 mg/L,
R2 = 0,402, 1000n); mientras que fue 3.771mg / L (R2 = 1, 290n) en plantas in vitro regeneradas
a partir de callo. Se seleccionó la dosis de 5 mg/L (DL100) y una segunda selección con 10 mg/L
para evitar falsos positivos. Se obtuvo solamente un mutante putativo de 8000 plantas irradiadas,
regeneradas y seleccionadas. El mutante putativo disponía de la mutación T2222I/T2222M en el
gen blanco y de mutaciones en los genes control matK y rbcL, en una proporción aproximada de
1/1000 pares de bases. La evaluación de lo obtenido llevó a un cambio en el tipo de tejido a
irradiar de callo embriogénico a semilla. Esto permitió disponer de una mayor diversidad de dosis
gamma dado que la semilla fue más tolerante, dispuso además de una etapa de inducción de
callo y regeneración más predecible, así como un incremento de 1 a 31 mutantes putativos que
serán evaluados en el futuro.

El cuarto artículo (Hernández-Soto et al., 2022) corresponde a la pasantía realizada en el

Laboratorio del Dr. Gatica, Universidad de Costa Rica en el marco del Proyecto UCREA “Edición
del genoma de arroz: alternativa para contribuir a la mitigación del cambio climático y una
contribución al logro de la seguridad alimentaria”. El trabajo es una exploración de técnicas de
mutación de mayor precisión mediante CRISPR-Cas9 en levaduras como un modelo para arroz,
en búsqueda del rasgo de tolerancia a estrés salino mediado por trehalosa. La trehalosa en un
disacárido de reserva con capacidad de funcionar con un osmoprotector tanto en arroz como en
levadura (Fichtner & Lunn, 2021; Rapoport et al., 2019; X. Zhang et al., 2020). En arroz existe un
solo gen OsTRE Os10g0521000 que codifica para la enzima que degrada la trehalosa. En
levadura existen tres enzimas una vacuolar ATH1 y dos neutras NTH1, NTH2 que son homólogas
a la de arroz. Estas últimas dos enzimas son responsables del 75 y 25% de la actividad

25
enzimática respectivamente de la hidrólisis intracelular. Originalmente se planteó usar como
pregunta de investigación si disminuir un 25% de la actividad enzimática mediante la disrupción
del NTH2 resultaba en tolerancia en levadura de tal manera que se tuviese al menos un
acercamiento sobre el potencial de lograr algo similar en arroz. Se realizó una mutación en el
gen NTH2 que resulta en una mayor tolerancia de la levadura a condiciones salinas. El artículo
se enfoca en el diseño y prueba de concepto para la levadura exclusivamente, y demuestra el
potencial de la creación de variabilidad genética, en este caso mediante una técnica de mayor
precisión.

26
 Current Plant Biology 27 (2021) 100211

Contents lists available at ScienceDirect

Current Plant Biology

journal homepage: www.elsevier.com/locate/cpb

Review article

Rice breeding in the new era: Comparison of useful agronomic traits

Alejandro Hernández-Soto a, *, Fabián Echeverría-Beirute b, Ana Abdelnour-Esquivel a,
Marta Valdez-Melara c, Jens Boch d, Andres Gatica-Arias c
a
Technologic Institute of Costa Rica, DOCINADE, Biology School, Biotechnology Research Center, Costa Rica, P.O. Box 159-7050, Cartago, Costa Rica
b
Technologic Institute of Costa Rica, DOCINADE, APDO, 223-21001, Alajuela, San Carlos, Ciudad Quesada, Costa Rica
c
University of Costa Rica, School of Biology, Plant Biotechnology Laboratory, P.O. Box 2060, San José, Costa Rica
d
Institute of Plant Genetics, Leibniz Universität Hannover, 30419, Hannover, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Understanding agronomic traits at a genetic level enables the leveraging of this knowledge to produce crops that
Mutagenesis are more productive and resilient, have better quality and are adjusted for consumer preferences. In the last
Domestication decade, rice has become a model to validate the function of specific genes, resulting in valuable but scattered
CRISPR
information. Here, we aimed to identify particular genes in rice related to traits that can be targeted by different
Gamma radiation
Chemical mutagen
mutation techniques in the breeding of crops. We selected gain of function, misfunction, and specific mutations
Biotic stress associated with phenotypes of agronomic interest. The review includes specific trait-related genes involved in
Abiotic stress domestication, stress, herbicide tolerance, pathogen resistance, grain number/quality/weight, plant structure,
nitrogen use, and others. The information presented can be used for rice, other cereals, and orphan crops to
achieve a superior and sustainable production in challenging farming conditions.

1. Introduction by mutagenesis of rice lines and related crops to produce predictable

changes in gains or losses of function. We present traits that could result
Induced mutagenesis is a valuable tool to support functional geno­ from the use of different techniques; remarkably, genome editing rep­
mics studies and the development of new genotypes. Rice serves as an resents an exciting opportunity to transfer the information about
outstanding model because of its impact on the worldwide food supply gene-trait relationships to other crops to improve their traits. Conse­
chain and the availability of genomic and agronomic resources to utilize. quently, they represent a challenge from a regulatory point of view for
Rice was the first crop sequenced in 2004 [1], biotechnological tech­ countries that have established a different regulation for genome-edited
niques are available, and the genomic information is available to search plants in contrast to other mutagenesis techniques.
for specific target mutations, such as from the Rice Genome Annotation
Project and Oryza Genome which can contribute to the precise engi­ 2. Methods
neering of the crop [2–4]. Biological, chemical, and physical agents can
induce mutagenesis. Typical methods are radiation (first used on vege­ The methodology applied a search based on PubMed articles and
tables in 1928), ethyl methanesulfonate (EMS) (which produces 2–10 keywords: rice, traits, stress tolerance, resistance, breeding; selection of
mutations per Mb), and new breeding techniques to introduce specific papers with agronomic traits linked to specific genes described within
mutations via genetic engineering [5–8]. In this review, we present rice 2010–2021. Finally, verification of each gene, trait, and mutation was
traits that have emerged or have been validated in the last decade performed using specialized web servers such as Gramene, Ensembl­
(2010–2021) and were derived from technological advances in geno­ Plants, Rice Diversity, FunRiceGenes, Rice Genome Annotation Project,
mics [9]. This paper is focused on characteristics that could be targeted Oryza Base, and Rice Information GateWay [10]. The search resulted in

Abbreviations: EMS, Ethyl methanesulfonate; PTR, Puddled Transplanted Rice; DSR, Direct Seeded Rice; DAS, Days After Sowing; HRAC, The Herbicide Resistance
Action Committee; WSSA, Weed Science Society of America; MoA, Mode of Action; BLS, Bacterial Blight Streak; AC, Amylose Content; ALS, Acetolactate Synthase;
NUE, nitrogen use efficiency.
* Corresponding author.
E-mail addresses: [email protected] (A. Hernández-Soto), [email protected] (F. Echeverría-Beirute), [email protected] (A. Abdelnour-Esquivel),
[email protected] (M. Valdez-Melara), [email protected] (J. Boch), [email protected] (A. Gatica-Arias).

https://doi.org/10.1016/j.cpb.2021.100211
Received 11 May 2021; Received in revised form 15 June 2021; Accepted 17 June 2021
Available online 18 June 2021
2214-6628/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Fig. 1. Representation of biotic and abiotic stress factors that affect rice production. Created with BioRender.com.

Fig. 2. Schematic representation of different systems used for breeding rice: natural variability, mutation breeding, tissue culture mutation, and new breeding
techniques. Created with BioRender.com.

a selection of 117 papers out of 500.

(continued on next column)

2
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

(continued ) 4.3. New breeding techniques

Webserver Link

Webserver Link
4.3.1. CRISPR/Cas9
The clustered regularly interspaced short palindromic repeats
Gramene https://ensembl.gramene.org/genome
(CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) system from
_browser/index.html
EnsemblPlants http://plants.ensembl.org/index.html Streptococcus pyogenes targets a specific genomic sequence using an
Rice Diversity http://www.ricediversity.org/data/ engineered 20 base pair (bp) RNA guide sequence that binds to matching
index.cfm DNA and the Cas9 protein, upon recognition of an additional 3’ localized
FunRiceGenes http://funricegenes.ncpgr.cn/
PAM sequence 5′ -NGG-3′ , generates a double-strand break at a desired
Rice Genome Annotation Project, http://rice.plantbiology.msu.edu
Michigan State University
location in the genome. This genome editing method allows the inser­
Oryza Base https://shigen.nig.ac.jp/rice/o tion, deletion, or modification of DNA with high specificity and effi­
ryzabase/ ciency [5].
Rice Information GateWay http://rice.hzau.edu.cn/
4.3.2. CRISPR/Cpf1 system
The nuclease Cas12a requires a small crRNA for inducing double
strand breaks with efficiencies similar to those of CRISPR/Cas9. More­
3. Importance for breeding rice over, this nuclease uses a 18–23 nt spacer for its maximum efficiency
and specificity and identifies a T-rich PAM located 5’ upstream of the
Rice, such as many other tropical crops, is susceptible to a large set of guide and generates staggered ends with 5’ overhangs [19].
biotic (fungi, bacteria, nematodes, insects, and viruses) and abiotic
(salinity, drought, heat, and cold) stresses that cause yield and economic 4.3.3. Base editing
losses (Fig. 1). In general, biotic stress cause losses worldwide up to 35 % This system allows the conversion of nucleotides without inducing
of the total food production [11]. As an example, losses in rice due to double-stranded DNA breaks or using donor templates. It is based on
insects can account for over 40 %. Moreover, losses caused by the fungal Cas9 nickase fusions to a nucleotide deaminase domain and has been
pathogens Magnaporthe grisea, Thanatephorus cucumeris, and used for changing a C–G base pairs into T-A (cytidine deaminase base
C. miyabeanus have been estimated worldwide at 35 %, 24 %, and 16 %, editor), or A–T into G-C (adenosine deaminase base editor) [20].
respectively [12]. On the other hand, abiotic stress represents the pri­
mary cause of crop losses worldwide, and yield losses can be as high as 4.3.4. Prime editing
50 % of crop production [128]. This system uses a catalytically impaired Cas9 endonuclease fused to
In this regard, the generation of rice-resistant varieties to biotic and a reverse transcriptase enzyme, and a prime editing guide RNA
abiotic conditions represents one of the major challenges that breeders (pegRNA). This complex is capable of identifying a target site and
face. For decades, breeding strategies include selection, hybridization, replace the target DNA nucleotides without double-stranded DNA
mutation induction using chemical and physical agents, and somaclonal breaks or using donor templates [21,22].
variation. More recently, the availability of genome editing technolo­
gies, genome sequences, efficient tissue culture, and transformation 5. Agronomic traits of interest
methodologies could remarkably facilitate the breeding of rice (Fig. 2).
5.1. Domestication genes
4. Rice breeding systems
The Oryza genus is composed of species with a variety of genome
Several methods are available for breeding rice with natural or structures, including six diploids (n = 12; named AA, BB, CC, ee, ff, gg)
induced mutagenesis; among them, we can mention mutation breeding, and five polyploids (n = 24, named BBCC, CCDD, HHJJ, HHKK, and
tissue culture, and new breeding techniques (CRISPR mutagenesis, base KKLL) [23–26]. Only two diploid (2n = 24) species of rice have been
editing, and prime editing) (Fig. 2). domesticated and used for cultivation: Oryza sativa and African
O. glaberrima. Rice domestication favored the selection of specific loss of
4.1. Mutation breeding function alleles. Wild relatives typically have functional versions of
these genes such as sh4, waxy, BH4, qSH1, AN1, brown pericarp, PROG1,
The mutation breeding principle is to generate heritable changes in and OsG1. The sh4 gene is related to reduced seed shattering
the DNA by external agents. The changes result by exposing plant cells to (Os04g0670900). The waxy gene controls the amylose content
physical (UV, X-ray, gamma radiation) or chemical (sodium azide and (Os06g0133000). BH4 is related to the hull color of the seeds
ethyl methanesulfonate) agents [13]. Induced mutagenesis offers a (Os04g0460200). The gene qSH1 is involved in seed shattering
promising alternative for developing rice varieties resistant to biotic and (Os01g0848400). The AN1 gene is related to seeds, morphology, and
abiotic stresses since it could accelerate the spontaneous mutation grain shape (Os04g0350700). RC Brown pericarp is involved in the seed
process and increase the pool of allelic variants available for genetic coat (Os07g0211500). PROG1 is related to an erect plant structure
improvement [14,15,8]. (Os07g0153600). OsLG1 is related to a closed-panicle structure
(Os04g0656500) [27]. The importance of such genes and their domes­
4.2. Tissue culture ticated alleles is critical in understanding how de novo domestication can
be achieved from wild Oryza varieties and how such genes can be further
Totipotency, a distinguishable characteristic of plant cells, in prin­ used for breeding of rice and other crops.
ciple allows each cell to regenerate an entire plant. This process involves This concept was demonstrated in polyploid O. alta (CCDD) by Yu
the culture of plant tissue fragments or individual cells on special growth et al. [28], targeting SD1, GS3, IPA1, Ghd7, Gn1a, Wx, Bh4, TAC1, An-1
media enabling the cells to grow, divide, and differentiate into organs homologs, as well as African landraces of Oryza glaberrima by disrupting
[16]. Among the techniques available, somaclonal variation are spon­ the HTD1 (O. sativa Os04g0550600), GS3 (O. sativa Os03g0407400),
taneous changes in the DNA leading to genetic and phenotypic varia­ GW2 (O. sativa Os01g0197700) and GN1A (O. sativa Os02g0244100)
tions among clonally propagated plants. The somaclonal variants genes [29]. For plant breeding, the use of non-domesticated, more
obtained could be detected using in vitro selection by applying selective genetically diverse rice species that better adapt to stress conditions,
pressure in culture conditions [17,18]. such as African landraces O. glaberrima, O. barthii, O. meridionalis (AA),

3
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Fig. 3. Representation of salt tolerance traits mediated by

three different methods: 1) overexpression, 2) knockout of
specific genes, and 3) particular sodium channels. Note that the
first corresponds to transcription factors that trigger adaptive
responses labeled MSL37, NAC2, NAP, and P5CS. The second is
a knockout of those that result in salt sensitivity: OsRR2, STL1,
DST; and the sodium channel SKC1 in rice. The third is the
sodium channel SKC1 containing amino acid V395. Created
with BioRender.com.

Australian landraces O. longistaminata (AA), O. australiensis (EE), and transplanted-rice (PTR) and direct-seeded-rice (DSR). The first is the
Asian landraces O. rufipogon (AA) or Porteresia coarctata (O.coarctata) conventional method, which requires water flooding and represents a
(KKLL), harbors a valuable potential of developing more sustainable rice sustainability issue because of water scarcity, methane production, and
crops [30,31]. the consumption of nonrenewable energy [63]. DSR, on the other hand,
represents opportunities for efficient water and nitrogen use, and a
5.2. Stress tolerance reduction of both greenhouse gas emissions and labor demand, espe­
cially in countries such as China, where 90 % of rice is currently pro­
Rice susceptibility to salt is evidenced by a yield decrease due to duced under PTR [64]. However, weed management is a challenge in
delays in heading and panicle sterility especially in salt-sensitive vari­ DSR, specifically during the first 41 days after sowing (DAS). This in­
eties like MI48, IR29 [31–33]. In contrast, salt tolerance in varieties like cludes complication by weedy rice (O. sativa f. spontanea), which is a
Pokkali, Cheriviroppu, FL478, IR651, CSR27, FL30, Fontan, SR86, variety of rice that is morphologically similar to cultivated rice, but
IR9884− 54-3 results from ion exclusion, osmotic and tissue tolerance grows as a weed. It produces far fewer grains than cultivated rice, and
with multiple genes involved in the process, which confers agronomic can result in rice yield losses of up to 50 % [25]. Weedy rice usually has
stability of this trait [31–40]. The orchestrated stress system can be increased seed longevity, seed shattering and stress tolerance which
targeted for achieving salt tolerance by mutating genes encoding key makes it difficult to control [65]. The use of chemical control represents
transcription factors, specifically OsRR22 (Os06g0183100), STL1 a tool to manage weeds including weedy rice, but poses additional
(Os04g0110600), and the zinc finger transcription factor encoded by challenges.
DST (Os03g0786400) [38,41–44]. Other transcription factors are critical The Herbicide Resistance Action Committee (HRAC) and the Weed
in stress adaptation, which results in stress sensitivity when inactivated. Science Society of America (WSSA) classify herbicides into 34 groups
This is the case for MSL37 (Os11g0163500) which encodes a positive salt and one unknown group based on their "mode of action" (MoA) at the
stress transcription factor response by regulating ion transporters, P5CS biochemical level [66–68]. The discovery of a new mode of action has
(Os05g0455500), which causes accumulation of the osmoprotectant been rare in the last 30 years. A good example is leptospermone, and its
proline, the transcription factor SNAC2 (Os01g0884300), which is key in analogous inhibitors that act as hydroxyphenylpyruvate inhibitors of
root adaptation, and OsNAP (Os03g0327800), which triggers a stress dioxygenase (HPPD) [69]. Different modes of herbicide use, such as
response mediated by ABA [45–49]. For details, see Fig. 3 and Table 1. rotations, delay the emergence of herbicide-resistant weeds. However,
Osmoprotection by accumulating molecules such as trehalose or weeds are evolving to resist multiple MoA types of herbicides. For
proline is a possible pathway leading to salt tolerance, as proven example, Chloris radiata is found in Colombian rice fields with dual
currently in plants like rice and Arabidopsis [59,47,60,61]. Other indi­ resistance to glyphosate (mode of action 9) and the acetolactate syn­
vidual genes can confer osmoprotection, such as the Na + transporter thase (ALS) inhibitor imazomox (mode of action 2) [70]. Weedy rice
SKC1 (Os01g0307500) with a V395 L that provides salt tolerance [50]. infestation in the USA resulted in 5.7 million tons of harvest lost and
Knocking out an independent gene, OsEPFL9 (Os01g0824500), results in $457 million in environmental costs between 2002–2014 [71]. To
increased water use efficiency under stress because of the reduced sto­ control weedy rice, herbicide tolerance was introduced into cultivated
matal count [51,52]. rice 20 years ago based on an acetohydroxy acid synthase AHAS/ALS
Other stress tolerance pathways can be modified by specific alleles, (Os02g0510200) gene mutation, providing tolerance to the mode of
too. Low cadmium accumulation occurs after mutating the metal action 2 [72]. Currently, rice herbicide tolerant varieties are used in the
transporter genes OsNramp5 (LC196140; japonica homologue: USA (700,000 Ha), Brazil (600,000 Ha), Uruguay (70,000 Ha),
Os07g0257200) and OsNramp1 (LC196122; japonica homologue: Argentina (32,000 Ha), Malaysia (95,000 Ha), and Italy (60,000 Ha), as
Os07g0258400). Plants are able to resist heat stress only when the gene well as in many Central America countries, such as Costa Rica,
OsNTL3 (Os01g0261200) is functioning correctly, whereas cold toler­ Honduras, Panamá, and the Dominican Republic [73]. The incorrect use
ance can result from mutation of the OsMYB30 (Os02g0624300) gene. of this variety allowed introgression and outcrossing of the resistance
Finally, more cuticle wax is deposited when the gene DHS into weedy rice, which means that weed herbicide control requires
(Os02g0682300) is mutated [54–58,62] stricter farming practices, such as herbicide rotation [74]. Alternatives
such as aryloxphenoxy propionate-resistant rice (mode of action 1),
which is the result of mutations in the ACCase2 (Os5g0295300) gene,
5.3. Herbicide resistance monogenic traits
already exist and will allow for herbicide rotation [75,76].
According to the literature, at least five target genes have the
Rice is usually cultivated under two agronomical systems: paddy-

4
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 1
Rice genes and mutations involved in stress tolerance or sensitivity traits.
Gene Position Protein Obtained mutation Method Trait details Reference

OsRR22 Two-component response regulator ORR22. Salt

Chr 6 Q5SML5 Knockout CRISPR/Cas9 [38]
Os06g0183100 tolerance 0.75 % NaCl.
STL1 hap1 tolerance 0.9 % salt, the gene is the
Os04g0110600 homolog of Arabidopsis salt tolerance gene SRP1
Chr 4 Q7XXF2 SNP None (Stress associated RNA-binding protein 1, [44]
Salt tolerance Level 1, Stress
AT2G17975). Knock-out mutation in the srp1
repressive zinc finger protein 4
allele reduced sensitivity to ABA and salt stress.
MSL37 Knock-out results in salt sensitivity. The
Spontaneous
Os11g0163500 Natural variability- transcription factor is a positive salt stress
Chr 11 Q53PP7 mutation- [42]
Knockout regulator, and binds to promoters of OsHKT2; 1,
OsGTgamma-2, OsGTγ-2 CRISPR/Cas9
OsNHX1 and OsHKT1.
Knockdown improved the tolerance to stress, as
Os03g0786400 OsDST, DLN102,
Mutant/ CRISPR/ also observed in the dst mutant. C2H2 zinc finger
OsDLN102, Negative regulation Chr 3 Q10CE2 Knockdown [41,43]
Cas9 transcription factor, drought and salt tolerance,
of response to salt stress
stomatal aperture control
P5C Natural: cultivar LPT123 is
The enzyme increases the proline accumulation
Chr 5 O04226 salt-susceptible versus salt- None [48]
Os05g0455500 and salt resistance mediated by ABA application.
tolerant line LPT123-TC171
SKC1
Os01g0307500 Variant V395 (is salt tolerant), while L395 is
Chr 1 Q0JNB6 Wild relatives None [50]
OsHKT1;5, sensitive.
OsHKT8
Os10g0521000 Mutation of domain WDS to replicate Selaginella
Based on Z.mays moellendoffii WDT. The enzyme may be less
Chr 10 Q9FWC1 Substitutio S163T CRISPR/Cas9 [47]
GRMZM2G162690 and A. efficient in allowing the accumulation of
thaliana AT4G24040 trehalose.
OsEPFL9
Os01g0824500 Increased water use efficiency under stress
Chr 1 Q5JN76 Knockout CRISPR/Cpf1 [51,52]
Epidermal Patterning Factor because of reduced stomatal count
Like-9
DHS Knockout results in more cuticular wax.
Os02g0682300 Overexpression (DHS OE) plantlets grew more
CRISPR/Cas9-
Chr 2 Q6EU38 Knockout-Overexpression slowly. The enzyme is a ubiquitin that degrades [53]
gene transfer
Drought hypersensitive ROC4 that positively regulates cuticular wax
biosynthesis
RCS1
Os12g0625000 Tolerates 20 μM As (III). The mutation increases
O-acetylserine (thiol) lyase, Chr 12 Q9XEA6 S189N EMS As tolerance/decreased accumulation in the [141]
Cysteine synthase. arsenite grain/increase Se accumulation in the grain.
tolerant 1
OsNramp5 B8B4U0
LC196140 (indica rice) (indica rice)
Os07g0257200 (japonica rice) Q8H4H5 [54,55,
Chr 7 Knockout CRISPR/Cas9 Low Cd accumulation
I7GYG6 56]
Manganese and Cadmium
(japonica
transporter, Mn and Cd uptake,
rice)
OsNramp1 A2YK11
Chr 7 Low Cd accumulation. It works as a plasma
LC196122 (indica rice) (indica rice)
membrane-localized transporter/uptake for Mn
Os07g0258400 Q0D7E4 Knockout CRISPR/Cas9 [56]
and Cd; it is complementary to OsNRAMP5 in
(japonica
Metal transporter (japonica rice) the uptake of Mn and Cd.
rice)
OsNTL3 OsNTL3 is required for heat stress tolerance in
Os01g0261200 rice. Loss-of-function mutation of OsNTL3
Chr 1 Q7GCL7 Natural variability- None confers heat sensitivity. It regulates the [57]
Thermotolerance expression of genes involved in ER protein
folding.
OsMYB30 The protein OsMYB30 is a nuclear protein that
Os02g0624300, Cold tolerance Chr 2 Q6K1S6 Knockout CRISPR/Cas9 acts as a negative regulator of cold tolerance. [58]
gene Mutant shows increased cold tolerance.

potential to develop herbicide-resistant rice varieties with a different (GGAACCAAAAGAATTAGAGACGATATCA) in the fourth exon. Finally,
mode of action. Two of those have already been described above: the double mutation known as “TIPS” (T102I + P106S) in the OsEPSPS
ACCase2 on aryloxphenoxy propionates (MoA-1) and AHAS/ALS on ALS (Os02g0510200) gene provides tolerance to MoA-9 (glyphosate). For
(MoA-2). For ACCase2, mutations such as I1781 L, S1866 F, I1879 V, details, see Fig. 4 and Table 2.
A1884 P, W2027C, W2125S, D2176 G, and C2186R/P1927 F/G2201A/ Weeds that are tolerant to the inhibition of photosynthesis at PSII by
W2125C in exon 32 provide herbicide tolerance at a different level. herbicides can also provide insights to generate herbicide tolerant crops.
AHAS/ALS alleles cause ALS (MoA-2) resistance when carrying the The S264 G mutation in psbA increases tolerance more than 50-fold to
following mutations: A96 V/A122 T/P171 H/P171S/P197S/C287 T and triazine in herbicide-tolerant radish (MoA-5). However, it can also
W548 L/W574 L/S627I/S653I/S653 N/G654E. OsTubA2 compromise fitness because of less efficient photosynthesis [77]. Other
(Os11g0247300) provides tolerance to dinitroanilines (MoA-3) with a mutations, such as Val219Ile, Asn266Thr, Phe255Ile, and Ala251Val,
mutation in the fourth exon, M268 T. HPPD (Os02g0280700), provides can also provide tolerance [68]. It is important to note that the psbA
tolerance to triketones with a natural insertion mutation Val-219-Ile provides tolerance to the amide propanil MoA-5 on

5
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Fig. 4. Representation of five rice genes and the corresponding mutation that results in herbicide tolerance. The genes are shown organized by their Mode of Action
(MoA). Note the name of the gene in orange circles, the exons in blue filled boxes and the corresponding untranslated exon regions in the blue empty boxes. Created
with BioRender.com (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Cyperus difformis [79]. Propanil is widely used in rice cultivation for infection by bacterial Xanthomonas oryzae pv. oryzae pathogens
because the crop is naturally capable of degrading the molecule by a causing bacterial leaf blight (BLB) [85–87]. The pathogen emerges by
putative enzyme located in the mitochondria, and an additional breaking the resistance of varieties planted in approximately 80 % of the
pathway could increase its tolerance [80,81]. The described mutations total crop cultivation area carrying the resistance gene Xa4 on chro­
could also result in herbicide tolerance in rice when targeting the ho­ mosome 11 introduced in the 60 s [88]. Some Xanthomonas oryzae
mologous gene AAS46167, encoding protein P0C434, to address an pathovars can also infect wild grasses and could become an emergent
additional MoA. pathogen that is difficult to control [89].
Rice is also known to be resistant to Bentazon (MoA-6), as it is A gene to target for fungal resistance is the transcription factor IPA1
degraded by cytochrome P450 CYP81A6 [82]. Additionally, the P450 (Os08g0509600); higher expression levels of IPA1 result in increased
gene CYP72A31 is responsible for conferring tolerance to bispyribac yield and immunity when tested against the fungal pathogen Magna­
sodium (BS) in Oryza sativa cv. indica, while its absence in japonica rice porthe oryzae. Resistance relies on time- and pathogen-specific phos­
varieties results in BS-sensitivity [83,84]. phorylation and activation of the transcription factor at Ser163.
Subsequently, phosphorylated IPA1 activates the WRKY45 promoter
and following basal resistant gene expression within 48 h after infection,
5.4. Bacteria, fungi and virus resistance
while the nonphosphorylated IPA1 protein binds to the DEP1 promoter
related to yield (Jing [53]). A different way to achieve M. oryzae resis­
Rice breeding of pathogen resistance is possible by mutation of
tance is by mutation of OsERF922 ethylene response factor 922
specific promoter regions of the Sweet 14,11,13 genes (Os11g0508600,
(Os01g0752500) [90]. Another important trait is tungro spherical virus
Os08g0535200, Os12g0476200), respectively, since they are required

6
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 2
Rice genes and mutations in herbicide resistance traits.
Gene (*) Position Protein Obtained mutation Method Trait details References

OsTubA2 CRISPR/Cas9-Base In vitro trifluralin 4 mg/,

Chr 11 Q53M51 M268T [140]
Os11g0247300 editor pendimethaline 6.6 mg/L
Seeds-Gamma Rays quizalofop-p-ethyl =75 g/ha;
ACCase2 W2027C [76]
280Gy haloxyfop-p-methyl =62.35 g/ha
I1879V CRISPR/Cas9-Base haloxyfop-R-methyl, 1 and 2 μM in
[142,42]
W2125S editor vitro.
Tissue culture
I1781L quizalofop-p-ethyl = 235 g ai ha-1 [75]
mutation
Chr 5 B9FK36
D2176G CRISPR-Prime
Os05g0295300 Herbicide resistance [139]
G2201A Editing
C2186R CRISPR-Base editor Herbicide resistance [134,42]
Herbicide resistance 34 g/Ha. High
P1927 F, W2125C,
CRISPR-Base editor tolerance P1927 F, W2125C versus low [133,42]
S1866 F and A1884 P
tolerance S1866 F and A1884P
psbA
Wild radish,
AAS46167 Atrazine > 50-fold (4000(187 g a.i. ha-
Chloroplast P0C434 S264G Spontaneous [77]
(Photosystem II 1 atrazine), (S) Bromoxynil
mutation-
protein D1, psbA)
HPPD Fe(II)/2- wild Nipponbare b-Triketone herbicides, HIS1detoxifies
28-bp deletion allele
Os02g0280700 Chr 2 oxoglutarate–dependent lacked deletion b-triketone herbicides by [135]
(his1).
Inhibitor Sensitive 1 oxygenase (HIS1) hydroxylation.
W548L CRISPR-Prime
AHAS, ALS Herbicide tolerance [22,139]
P171S Editing
CRISPR/Cas9 Base
Os02g0510200 A96V (C287 T) Imazamox (quantity not reported) [137]
editor
Clearfield 121
G654E Chemical mutation Clearfield 141 [73,71]
IRGA422
Named CL161 and CLXL8 increased
Chr 2 S653N Chemical mutation [73]
herbicide tolerance
Recombinant
Q6K2E8 W548 L or P171S Herbicide tolerance [131]
protein
Acetohydroxy acid W548 CRISPR-Prime
Herbicide tolerance [139]
synthase E549 Editing
A122T Sodium Azide IMINTA1, IMINTA4 [136]
W548L
CRISPR Herbicide tolerance [138]
S627I
Arabidopsis P171 H/W548 L 100 mM IQ
W574 L 100 mM CS/BM/IQ/IP/PS
Recombinant [131]
Modeling P197S 100 mM BM
S653I 100 mM IP
OsEPSPS T169I CRISPR-Prime
NA [132]
A170 V P173S Editing
Chr 6 A0A0N7KLH2
Os06g0133900 In vitro resistance 1 mg l–1 glyphosate,
T102I + P106S CRISPR [78]
400x dilution Greenhouse.

(S)= Susceptible, genomic.

(*) Additional information at The Rice Annotation Project (RAP). [85–87].
CS, chlorsulfuron; BM, bensulfuron-methyl; IQ, imazaquin; IP, imazapyr; PM, pyriminobac; PS, pyrithiobac-sodium; BS, bispyribac- sodium.

Table 3
Rice genes and mutations with pathogen-resistant traits.
Gene Position Protein Obtained Mutation Method Trait details Reference

Os11g0508600 Xanthomonas oryzae pv. oryzae resistance,

TALEN /
Chr 11 Q2R3P9 promoter edited probably by avoiding sugar access for the [86,87]
Sweet 14 CRISPR-Cas9
pathogen growth
Os08g0535200 Xanthomonas oryzae pv. oryzae resistance,
Chr 8 Q6YZF3 promoter edited CRISPR-Cas9 probably by avoiding sugar access for the [86,87]
Sweet11
pathogen growth
Os12g0476200 Xanthomonas oryzae pv. oryzae resistance,
Chr 12 Q2QR07 promoter edited CRISPR-Cas9 probably by avoiding sugar access for the [86,87]
Sweet13
pathogen growth
Os07g0555200 translation initiation factor Knockout and mutations
Chr 7 B9FXV5 CRISPR-Cas9 Resistance to rice tungro spherical virus (RTSV) [91]
4 gamma gene (eIF4G) on SVLFPNLAGKS
Os01g0752500, ethylene response factor
Chr 1 Q5JMX7 Knockout CRISPR Magnaporthe oryzae, Blast resistance [90]
922 OsERF922, LOC_Os01g54890.1

resistance which results by mutation of gene eIF4G (Os07g0555200) 5.5. Grain number, quality, weight and plant structure
coding a translational factor that is key in the initiation of the virus
mRNA [91]. For details, see Table 3. Rice quality traits are essential to achieve a better yield, consumer
preference, and growth efficiency. The genes involved in grain number

7
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Fig. 5. Representation of traits such as grain number, quality, weight and plant structure and gene relationships in rice. Note that heading and flowering are
positively influenced by Se5, Hd2, and Hd1 knockout; structure by DEP1, HTD1, IPA1, LPA1, Pin1a, and Pin15b; grain size by Gn1a, and Ep3; grain size by GS3, GW6a,
GW5, and GW5L; and grain starch by ISA1, NAC20-26, and WX1. Created with BioRender.com.

and size, plant density, structure, panicles, and flowering have complex 5.5.3. Grain starch
interactions. However, recent findings and key mutations now provide Grain starch quality is an essential trait, which depends on the
insight into their regulatory mechanisms and greater predictability in relative content of amylose and protein. The global starch content relies
achieving the desired phenotype (for details, see Fig. 5 and Table 4). on the gene ISA1 (Os08g0520900) and the protein content relies on
NAC20− 26 (Os01g0104500, Os01g0393100) [101,102]. The waxy gene
5.5.1. Grain size WX1 (Os06g0133000) controls the grain amylose content (AC). Muta­
The GS3 Grain Size3 gene (Os03g0407400) is responsible for nega­ tions in this gene correlate with a phenotype that ranges from opaque
tively controlling the grain length. Its mutation can result in better or (8%), semitranslucent (8–12 %), and transparent (12 % or more) grains
worse weight and size that correlates with the composition of its do­ [97–100,39].
mains: organ size regulation (OSR), a transmembrane necrosis factor
receptor/nerve growth factor receptor (TNFR/NGFR), and a von Wille­ 5.5.4. Flowering
brand factor type C (VWFC) (Meiru [78,94–96,58]). The wild type allele Flowering and photoperiodic insensitivity results from over­
contains all of the domains and results in medium grains [95]. Loss of expression of OsMeCP (Os12g0620400 [110] or by knocking out several
function results in long-grain varieties; for example, Minghui 63 has a genes. For example, Se5, Hd2 and Hd1 [4,94,111,112]. Another critical
stop mutation C165A at the second exon, resulting in a loss of function regulator of heading date and grain weight seems to be HGW, but its
and a long-grain phenotype (Meiru [78,58]). In contrast, a mutation or homozygous null mutant is embryonic lethal [113].
deletion in the fifth exon creates a truncated protein with no VWFC
domain and a short seed phenotype [95,114]. Grain size, in general, is 5.5.5. Structure
controlled by several additional genes: higher expression of GW6a Farmers prefer smaller plants with many panicles and fewer tillering
(Os06g0650300), and knockout of GW5 (Os05g0187500), GW6 traits. Knockout of the DEP1 (Os09g0441900) gene, as well as the loss of
(Os06g0623700), and GW5L (Os01g0190500) results in increased grain function of the HTD1 (Os04g0550600) gene introgressed from landraces
size [104–107,103]. produces short, dense, erect panicles [29,78,108].
The transcription factor IPA1 Ideal Plant Architecture1
5.5.2. Grain number (Os08g0509600), is related to fungal resistance and yield as mention
Malfunction of the gene Os01g0197700 (GN1a) produces an incre­ previously, and its specific mutations between bases 854 and 876 can
ment of grain per panicle number and flowering because of a lower increase the production of the transcription factor protein because they
degradation of cytokines produced by the corresponding cytokinin interrupt transcript cleavage due to the micro RNA OsmiR156. For
oxidation enzyme [92,78,94]. Another gene that correlates with example, C874A in the third exon (leucine to isoleucine) generates a rice
increased production and downregulates cytokine level regulation is plant with a reduced tiller number, increased lodging resistance, and an
EP3 Erect Panicle 3 (Os02g0260200) [94,115]. enhanced grain yield [78.53].
The number of panicles and consequently the yield can be increased
by mutating the genes Pin1A and Pin15b or indirectly blocking their

8
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 4
Rice genes and mutations involved in grain quality, quantity, weight, and plant structural traits.
Gene Position Protein Obtained mutation Method Trait details Reference

OsDEP1 More expression, yield increase 15 %. The

interaction between DEP1 and LPA1
suppresses PIN1a expression, leading to an
Spontaneous mutation [92,93,
Chr 2 Q67UU9 Mutation, promoter increase in planting density. The panicle
Os09g0441900 -CRISPR /Cas9 116]
number per plant was the main contributor to
the increase in grains per rice plant in the
DEP1 mutants.
Gn1a Catalyzes the oxidation of cytokinin,
Os01g0197700 enhanced the grain yield by increasing the
Chr 1 Q4ADV8 Knockout CRISPR/Cas9 [78,92,94]
grain number per panicle. Twice flowering
OsCKX2
relative to the wild type.
GS3 δ subunit of G protein. Regulator of grain size
and organ size. Produces a longer grain
length. Knockout and deletions produce short
seeds, such as 320 bp and 13 bp deletions in
CRISPR/Cas9 -Spontaneous [94,95,96]
Chr 3 C6L686 Knockout the fifth exon of GS3 that occurred in a
Os03g0407400 mutation b
japonica-like ancestor. The 4 bp and 1 + 3 bp
deletions occurred in an indica-like ancestor.
Farmers and early breeders imposed artificial
selection favoring short seeds
IPA1 Squamosa promoter-binding-like protein 14.
Os08g0509600 Specific mutations between bases 854 to 876
result in more protein and produce less
tillering, more grains and a higher frequency
Chr 8 Q7EXZ2 Knockout CRISPR/Cas9 [78,62]
Transcription factor Ideal of seed set. It reduces unproductive tillers
Plant Architecture 1 and increases the number of grains per
panicle, while higher IPA1 levels enhance
immunity.
WX1 CRISPR/Cas9 Modulate the synthesis of amylose in the
P124 F, R125W endosperm. Amylose contents change the
appearance of the rice endosperm >12 %
results in transparent endosperm/
semitranslucent (8–12 %)/or opaque (<8%).
Favorable rice palatability usually requires
Os06g0133000 granule- T178I, T178S, R158H, [39,97,98,
Chr 6 Q0DEV5 Knockout, mutations low to intermediate AC (10–20 %). The null
bound starch synthase I Y191H, R158H, G159A, 99,100]
wax results in an absence of amylose,
GBSSI, OsGBSS1, waxy D161 N, G159 K, G159A,
resulting in starch granules with 100%
G159E, V160 F, S415 P
amylopectin production, referred to as waxy
or glutenous starch. S415 P changes
phosphorylation, resulting in moderate
enzyme activity and a content of amylose.
ISA1 Decreased endosperm contents of total
starch, amylose and amylopectin. Increased
Os08g0520900 isoamylase Chr 8 D0TZF0 Knockout CRISPR/Cas9 [101]
soluble sugar content and starch gel
1
consistency.
OsNAC20 Q9FTY0 Double knockout osnac20/26 displayed a
Os01g0104500 (OsNAC20) floury grain caused by decreased starch and
OsNAC26 storage protein content. Both proteins
transactivate the expression of SSI, Pul,
Chr 1 Knockout CRISPR/Cas9 [102]
Q5VNK1 GluA1, GluB4/5, α-globulin and 16 kD
Os01g0393100 (OsNAC26) prolamin and indirectly influence DPE1
expression to regulate starch and storage
protein synthesis.
GW5 Q75KY5 GW5 could function as a key regulator to
Os05g0187500 coordinate the performance of the other
Chr 5 Knockout Spontaneous mutation grain size genes. gw5 contributes to an [103]
Grain Size on Chromosome A0A1D8GZC0
increased grain width and weight. Positive
5, qSW5/GW5, GSE5
regulator of brassinosteroid signaling.
GW5L Knockout results in shorter and wider grains.
Overexpression could confer salt stress
Os01g0190500 GW5L Chr 1 B8ADP5 Knockout Spontaneous mutation [104]
resistance through an association with
homologue of GW5
calmodulin protein OsCaM1− 1.
GW6a Histone H4 acetyltransferase, regulation of
Os06g0650300 grain weight, yield, and plant biomass.
Chr 6 Q67UR2 Over expression Spontaneous mutation Elevated OsglHAT1 expression enhances the [105,106]
OsglHAT1, Grain weight
grain weight and yield. Increases global
on chromosome 6
acetylation levels of histone H4.
GW6 Loss of function of the Kasalath allele
Os06g0623700 enhances the grain weight through
pleiotropic effects on source organs and leads
Chr 6 Q69U01 Loss of function Spontaneous mutation [107]
TOTAL GRAIN WEIGHT6, to significant yield increases. Encodes a
total grain weight6, protein with indole-3-acetic acid (IAA)-
glucose hydrolase activity.
OsPIN5b Chr 8 Q6ZIB5 Knockout CRISPR Increased panicle length in the mutant. [58]
(continued on next page)

9
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 4 (continued )
Gene Position Protein Obtained mutation Method Trait details Reference

Os08g0529000
a panicle length gene
Hd1/ SE1 Zinc finger protein, Heading date. Under long
CRISPR-Cas9/ Spontaneous day conditions suppresses HD3A/FT
Chr 6 Q9FDX8 Knockout [94,4]
Os06g0275000 mutation expression, causing the suppression of
flowering.
HTD1 Landraces contain HTD1, while domesticated
Os04g0550600 rice have htd1. The defect in HTD1 is
responsible for both high-tillering and dwarf
Spontaneous mutation/ phenotypes in the htd1 mutant. Auxin
Chr 4 Q7XU29 Loss of function [108,29]
CRISPR induces HTD1 expression. The protein
High-Tillering Dwarf 1
negatively regulates the outgrowth of
axillary buds and is related to strigolactones
biosynthesis
LPA1 Plant architecture. Related to lamina
Os03g0237250 inclination by suppressing auxin signaling.
LPA1 is an active transcriptional repressor.
Negatively controls the tiller and lamina joint
angle in an expression level-dependent
Overexpression/ manner. LPA1 overexpressors contain higher
Chr 3 L7PBL4 Spontaneous mutation [109]
Knockout levels of IAA, increases planting density and
Loose Plant Architecture1
resistance to sheath blight disease via
activation of PIN-FORMED 1a. Exaggerated
lamina angles observed in knockout mutants
(lpa1). lpa1 mutants might exhibit less
efficient auxin flux.
OsMeCP
Os12g0620400
Overexpression/ CRISPR/Cas9 knockout, Overexpression of OsMBD707 results in
methyl-CpG binding
Chr 12 Q0ILV0 RNAi/ CRISPR Gene transfer overexpression larger tiller angles and reduced photoperiod [110]
domain protein, Methyl-
Knockout and RNAi sensitivity.
CpG binding domain
containing protein
Hd2
2− 8bp deletion in Early flowering/low photosensitivity. Plants
Os07g0695100 Chr 7 Q0D3B6 Hap_3 and Hap_6 mutants [111]
Hd2 can be planted at any time of year
Heading date 2
Ep3 60Co Irradiated japonica Increased panicle size. Mutants modulate
Mutation (knockout,
Os02g0260200 ERECT Chr 2 G3CKN6 cultivar Zhonghua 11, cytokinin level in plant tissues by down [60,94]
recesive)
PANICLE 3, CRISPR/Cas9 knockout regulating cytokinin oxidase ⁄dehydrogenase
Se5 Identified in a gamma-irradiated Bahia
Os06g0603000 collection, displays early flowering and
Chr 6 Q69XJ4 Gamma rays s73 mutant [112]
photoperiodic insensitivity due to a null
Photosensitivity5
mutation.
Is a key regulator of heading date and grain
HGW
weight.
Os06g0160400 heading and
Chr 6 B6TN35 Natural Spontaneous mutation [113]
grain weight, heading Encodes a protein with a UBA domain.
date- and grain weight- Homozygous null mutant is embryonic lethal.
related protein

expression. The indirect mechanism results in higher expression of DEP1 6. Regulatory approaches
and LPA1, which interact to suppress PIN1a expression [93,92,116].
LPA1 is also important in the erect phenotype, and its mutation results in The traits presented in this article can result from the application of
lamina inclination [109,117]. conventional or new breeding techniques, such as genome editing. It is
important to note that the advance in sequencing technologies allow for
5.6. Other traits a detection of mutations; however, it is unfeasible to identify the specific
technique or natural cause that resulted in a mutation like a single
Other rice traits provide value for breeding and for satisfying con­ nucleotide polymorphism or a few nucleotide variations [123,129,130].
sumer preferences, such as nitrogen use, fragrance, oleic acid content, Trying to create a legal system that differentiates between genome
and color. Regarding nitrogen provision, there is a better efficiency with editing and other mutagenesis approaches or natural variations repre­
a higher expression of the nitrate transporter OsNPF6.1 and the two sents a challenge, given that detection is not achievable under realistic
transcription factors OsNAC42 and OsNLP4 [118,119]. Mutation of the circumstances [123]. It is a challenge to regulate a product that cannot
FAD2 gene results in an oleic acid increment [120,121]. Furthermore, a be practically distinguished once in the market, but that falls under a
mutation in the Osor (Os02g0651300) gene results in potential norm that requests such a differentiation. Such a legal norm is currently
orange-colored rice [122], and the fragrance can be increased or applied in Europe. A supreme court resolution on case C-528/16
decreased by modulating the BADH2 gene, which prevents the forma­ enforced that the genetically modified organism (GMO) norm (Directive
tion of the aromatic compound 2AP (2-acetyl-1-pyrroline) [94]. For 2001/18/EC) is applied on genome-edited plants [124]. A recent study
details, check Table 5. of the European Commission delivered to the Council of the European
Union in April 2021 has collected opinions from different stakeholders
and concluded that "similar products with similar risk profiles can be ob­
tained with conventional breeding techniques, certain genome editing

10
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 5
Rice genes and mutations in traits such as oleic acid, color, fragrancy, and nitrogen use.
Gene Position Protein Obtained Mutation Method Trait details Reference

FAD2
Increased oleic acid (twice) and decreased [120,
Os02g0716500 fatty acid Chr 2 Q6ZGW6 Knockout CRISPR/Cas9- RNAi
linoleic acid content. 121]
desaturase 2
Osor β-carotene accumulation resulting in orange-
Chr 2 Q6H3Y3 Knockout CRISPR/Cas9 [122]
Os02g0651300 colored calli.
BADH2 Betaine aldehyde dehydrogenase 2, prevents
the formation of 2-acetyl-1-pyrroline (2AP),
Chr 8 A0A0P0XG36 Knockout CRISPR/Cas9 [94]
Os08g0424500 which gives fragrant rice its aromatic
properties. Change in fragrance.
OsNPF6.1 HapB, 160 Gly to Asp
and two additional Natural, validation with
Nitrate transporter OsNPF6.1 is more
Os01g0103100 Nitrate Chr 1 Q9FTZ3 CACG motifs at the CRISPR/CAS9 Knockout- [118]
efficient and has increased expression.
transporter promoter -0.5Kb and Gene transfer
-1kb
OsNAC42 Natural, validation with
Transcription factor OsNAC42 related to the
Os09g0493700 CRISPR/CAS9 Knockout-
expression of the nitrate transporter
NUE (nitrogen use Chr 9 Q0J0L8 Natural- Knockout lost-of- function SNP [118]
OsNPF6.1. Loss of function decreased
efficiency)-related mutation (Pro51 changed to
expression of nitrate transporter OsNPF6.1
transcription factor Leu, P51 L)
Natural, HapB distributed in
OsNLP4 South China, India and The gene is upregulated by nitrogen
South-East Asia starvation. OsNLP4 binds to the NRE motif
Os09g0549450 Chr 9 A0A0P0XQL5 Natural 131 T (UTR), 181 T (UTR), and promotes the expression of OsNiR that [119]
transcriptional factor, 614A, 842 T, 2889C, 4662 T encodes a critical nitrite reductase in
Promotion of nitrogen (UTR), 4674 T(UTR), 4888C nitrogen assimilation.
use efficiency (NUE) (UTR)

techniques and cisgenesis. It may not be justified to apply different levels of wild relatives and the results can be extrapolated to other crops with
regulatory oversight to similar products with similar levels of risk" [125]. An homologous traits. Farmers urgently require advanced breeding to
adjustment of the GMO norms should be endorsed to correspond with respond to the challenges of climate change, consumer demands, water
such a conclusion. scarcity, nitrogen usage, and sustainable production.
The legal status of a genome editing product depends on norms
established at a country level based on a discriminate process to deter­ Ethics approval and consent to participate
mine whether the final product is a Living Modified Organism (LMO) or
not. For countries like Argentina, Australia, Colombia, Brazil, and the Not applicable.
United States, a variety is equivalent to conventional in the absence of a
foreign DNA [126,127]. For details, see Table 6. Consent for publication
The legal frameworks dealing with genome editing plants currently
are country-specific. Still, there is some common background in the Not applicable.
international definitions of a Living Modified Organism (LMO) given in
the Cartagena Protocol on Biosafety as ”any living organism that possesses Availability of data and material
a novel combination of genetic material obtained through the use of modern
biotechnology”. The keyword in defining such differentiation is "novel Not applicable.
combination of genetic material," which is usually explained in legal terms
as the presence of foreign DNA, as described previously. Funding
There is an international Central America norm RT 65.06.01:18
approved by Resolution 60− 2019 that provides a legally binding defi­ This study was financed by the Project “Producción de mutantes de
nition on Article 4.6 for "novel combination of genetic material," arroz (Oryza sativa) tolerantes a herbicidas utilizando rayos gamma para
currently applied in Honduras and Guatemala. The definition states in contribuir con el manejo sostenible del cultivo” (1510-1022, Research
simple words, that a new combination of genetic material means a stable Vice-Rectory of TEC, Costa Rica), and is part of the Doctoral Thesis of the
insertion of DNA that could not be obtained by conventional breeding or first author, Doctorado en Ciencia Naturales para el Desarrollo (DOC­
available in nature. The procedures and information requested in both INADE), Instituto Tecnológico de Costa Rica, Universidad Nacional,
countries are aligned with the international definition and are available Universidad Estatal a Distancia, Cartago, Costa Rica.
in decree CD-008-SENASA-2019 for Honduras and 271-MAGA chapter
VI for Guatemala. This legal antecedent provides a background for Authors’ contributions
comparative laws within countries with norms still in discussion. The
latter is interesting because the Supreme Court of Guatemala endorsed A.H.-S conceived the paper, designed and coordinated the inputs,
the international standard in Case Resolution 6767− 2019. For details, analyzed the data, and wrote the manuscript; F.E.-B reviewed, discussed
see Table 6. the content and edited the paper; A. A-E discussed the results and edited
the paper; A.G.-A. wrote, reviewed, discussed the results and edited the
7. Conclusion paper; M.V.-M. discussed the results and edited the paper; J.B. reviewed,
discussed the content and edited the paper. All authors read and
Induced mutations targeting specific genes associated with known approved the final manuscript.
phenotypes, as described in this review, will allow for advances in more
precise rice breeding to improve varieties that farmers are currently
using. It can also result in new varieties and de novo domestication from

11
A. Hernández-Soto et al. Current Plant Biology 27 (2021) 100211

Table 6 Table 6 (continued )

Genome Editing related norms and links. Norm Country Link (Visited on May,
Norm Country Link (Visited on May, 2021)
2021)
https://docs.wto.org/d
Court of Justice’s judgment The EU https://eur-lex.europa. South America Ministries Argentina, Brazil, Chile, ol2fe/Pages/SS/directd
in Case C-528/16. eu/legal-content/en/ of Agriculture Paraguay and Uruguay oc.aspx?filename=q
TXT/?uri=CELEX:62 :/G/SPS/GEN1699.pdf
016CJ0528
EC study on new genomic The EU https://ec.europa.eu/
techniques food/plant/gmo/mode
rn_biotech/new-genom Declaration of Competing Interest
ic-techniques_en
Food Hygiene Handling Japan https://www.mhlw.go.
The authors report no declarations of interest.
Procedures for Food and jp/content/000550824.
Additives Derived from pdf
Genome Editing Acknowledgments
Technology
RESOL-2021-21-APN- Argentine https://www.boletinof Not applicable.
SABYDR#MAGYP icial.gob.ar/detalle
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15
 plants
Article
A Temporary Immersion System Improves Regeneration of In
Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.)
Embryogenic Calli
Alejandro Hernández-Soto 1,2, * , Jason Pérez 2 , Rebeca Fait-Zúñiga 2 , Randall Rojas-Vásquez 3,4,5 ,
Andrés Gatica-Arias 3,6 , Walter Vargas-Segura 7 and Ana Abdelnour-Esquivel 2

1 Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica,
Universidad Nacional, Universidad Estatal a Distancia, Cartago P.O. Box 159-7050, Costa Rica
2 Biotechnology Research Center, Biology School, Costa Rica Institute of Technology, Cartago P.O. Box 159-7050,
Costa Rica; [email protected] (J.P.); [email protected] (R.F.-Z.); [email protected] (A.A.-E.)
3 Plant Biotechnology Laboratory, School of Biology, University of Costa Rica, San José P.O. Box 2060,
Costa Rica; [email protected] (R.R.-V.); [email protected] (A.G.-A.)
4 Programa de Posgrado en Ciencias Agrícolas y Recursos Naturales (PPCARN), School of Agronomy,
University of Costa Rica, San José P.O. Box 2060, Costa Rica
5 Vitroflora Labs S.A., Alajuela 20701, Costa Rica
6 Programa de Posgrado en Biología (PPB), School of Biology, University of Costa Rica,
San José P.O. Box 2060, Costa Rica
7 Gamma Irradiation Laboratory, School of Physics, Costa Rica Institute of Technology,
Cartago P.O. Box 159-7050, Costa Rica; [email protected]
* Correspondence: [email protected]






Citation: Hernández-Soto, A.; Pérez, Abstract: The development of gamma ray-mutated rice lines is a solution for introducing genetic
J.; Fait-Zúñiga, R.; Rojas-Vásquez, R.; variability in indica rice varieties already being used by farmers. In vitro gamma ray (60 Co) mutagen-
Gatica-Arias, A.; Vargas-Segura, W.; esis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization
Abdelnour-Esquivel, A. A Temporary of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL,
Immersion System Improves the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity
Regeneration of In Vitro Irradiated
to gamma radiation, and optimization of the generation procedure. All sequenced genes matched
Recalcitrant Indica Rice (Oryza sativa
perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction
L.) Embryogenic Calli. Plants 2022,
improved using MS medium containing 2 mg L−1 2,4-D, and regeneration was achieved with MS
11, 375. https://doi.org/10.3390/
medium with 3 mg L−1 BA and 0.5 mg L−1 NAA. The optimized radiation condition was 60 Gy,
plants11030375
(LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA® , Saint-Mathieu-de-Tréviers,
Academic Editor: France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the
Iyyakkannu Sivanesan
recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved
Received: 23 December 2021 regeneration in the immersion system. To our knowledge, this is the first study reporting the use of
Accepted: 26 January 2022 an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica
Published: 29 January 2022 rice materials.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Keywords: somatic embryogenesis; Cobalt-60; radiation-induced mutagenesis; temporary immersion
published maps and institutional affil- systems (TIS)
iations.

1. Introduction
Copyright: © 2022 by the authors.
Rice is an important cereal that provides 20% of the world’s energy, particularly in
Licensee MDPI, Basel, Switzerland.
Asia, Africa, and Latin America [1]. The Oryza genus consists of 22 species, but only two
This article is an open access article
are commonly planted, namely, O. sativa and O. glaberrima [2–6]. Farmers prefer only a
distributed under the terms and
few cultivars, depending on the country. The limited genetic variability of commercial
conditions of the Creative Commons
materials can become an obstacle in increasing productivity given emerging conditions
Attribution (CC BY) license (https://
such as heat, salt stress, soil acidification, plague sensitivity, and weeds. Introducing
creativecommons.org/licenses/by/
4.0/).
variability with crossbreeding is slow and can result in the introduction of undesired traits.

Plants 2022, 11, 375. https://doi.org/10.3390/plants11030375 https://www.mdpi.com/journal/plants

Plants 2022, 11, 375 2 of 12

Radiation methods to generate variability in seeds are standard techniques used since 1928
on vegetables, while other methods exist, such as ethyl methanesulfonate (EMS), and new
breeding techniques to introduce specific genetically engineered mutations [7–12].
Plant-tissue culture represents an opportunity to overcome time limitations, land
requirements, and the selection of the desired variability that results when using gamma-
ray mutagenesis in seeds. Once irradiated, the seeds must be planted several times from
M0 = seeds prior to mutagenic treatment; M1 = plants produced from material treated
with the mutagen, to subsequent generations termed M2, M3, M4, to avoid chimeras
and heterogenicity until exposed to stress-selection conditions, as has been done in the
past [6,8]. In contrast, rice-tissue culture can produce a primitive cell aggregate or calli with
embryogenic potential and consequently mutate and regenerate from one or a few cells
with stressor selection from the beginning, such as NaCl or herbicide [11,13,14].
The latter is possible because plant cells are totipotent, which means that whole plants
can develop from single cells [15]. We previously reported embryogenic calli mutation
using gamma radiation for the Costa Rican cultivar CR-5272 for salt and drought tolerance;
however, farmers no longer use this cultivar and instead use modern materials recalcitrant
to tissue culture [14]. The establishment of embryogenic rice calli is influenced by the
germplasm of origin and the 2,4-D concentration [15,16]. Embryogenic cells result when
exposed to Murashige and Skoog (MS) medium supplemented with 2 to 2.5 mg L−1 2,4-D,
resulting in pro-embryos and somatic embryos [17–19]. Costa Rican cultivars such as CR-
201, CR-1707, CR-1821, CR-8334, and CR-8341 have unpredictable and variable behavior,
while CR-1113 and CR-5272 have predictable induction and regeneration in 2.5 mg L−1
2,4-D [20].
Here, we faced three challenges. First, the identification of Lazarroz FL rice line with
molecular markers, the chloroplast maturase gene K (matK) and ribulose-1,5-bisphosphate
carboxylase small/large subunit (rbcL). Second, the improvement of our plant-tissue culture
methods. Third, determining the radiosensitivity of embryogenic calli and further plant
regeneration. We present a simple method to induce mutations using gamma rays in em-
bryogenic calli of a recalcitrant cultivar, with an alternative immersion method that allowed
our material to generate homogeneous and predictable in vitro plants after irradiation.

2. Results
2.1. Molecular Markers Used for the Identification of the Rice Cultivars
The chloroplast maturase gene K (matK) and ribulose-1,5-bisphosphate carboxylase
small/large subunit (rbcL), MZ558335 and MZ558334 sequences of Lazarroz FL showed
perfect matches with the already published NCBI Oryza sativa indica demonstrated the iden-
tity of the non-irradiated material as expected. We detected three synonymous mutations
in the rbcL sequence that are important for the characterization of the variety (Figure 1).
Specifically, one synonymous SNP on the sixth glutamic acid triplet (GAA/GAG) indicates
a putative origin of the germplasm ancestors from the Southeast Asia region because of
its unique presence and matches with three cultivars of the region: the Pakistan culti-
var NARC 17958 (GenBank KP827660.1), the Indonesia cultivar Pandak Kembang (Gen-
Bank MZ198248) and the Vietnam cultivar “Lua Khau Ky” isolate GBVN15800 (GenBank
KR073275.1). We also detected two synonymous biallelic mutations at glycine 82 and 150
codifying triplets (GGC/GGA; GGT/GGC), which helped characterize and further identify
the material. None of the mutations suggested biological importance since the open reading
frame remained unaltered.
Plants
Plants 2022,
2022, 11,
11, x375
FOR PEER REVIEW 3 3ofof 12
12

Figure 1.
Figure DNAmarkers
1. DNA markersused usedtoto identify
identify thethe matK
matK andand
rbcLrbcL genes
genes in non-irradiated
in non-irradiated Lazarroz
Lazarroz FL
FL rice
variety. Note Note
rice variety. in green the synonymous
in green SNP (C/T)
the synonymous SNPand the and
(C/T) biallelic
the synonymous mutationsmutations
biallelic synonymous A/G and
G/T
A/G (circled). None of None
and G/T (circled). the mutations had biological
of the mutations importance
had biological importancebut but
helped
helpedininthe
the genetic
genetic
characterization
characterization ofof the
the cultivars.
cultivars.

2.2.Embryogenic
2.2 EmbryogenicCalli
CalliInduction
Induction
The
The embryogenic
embryogenic calli calliinduction
inductionininLazarroz
LazarrozFLFL was
was affected
affected byby
thethe combination
combination of
of the
the plant
plant growth
growth regulators
regulators used.used. Thus,
Thus, induction
induction percentages
percentages ranging
ranging fromfrom
12.7712.77 to 71.44
to 71.44 were
were obtained,
obtained, with significant
with significant differences
differences betweenbetween all treatments.
all treatments. The higherThe higher
calli calli
induction
induction percentage (71.44) was achieved with −21 mg L −1 2,4-D alone (Table 1). In our case,
percentage (71.44) was achieved with 2 mg L 2,4-D alone (Table 1). In our case, the
the positive
positive induction
induction at 2 mg −1 ofL−1
at 2Lmg of 2,4-D
2,4-D contrasted
contrasted with awith
highera higher
brown brown callus
callus rate rate
(3.55%)
(3.55%) and although
and although it isvalue,
it is a small a smallforvalue, for steps
the next the next steps of regeneration
of regeneration represented represented
a challenge a
challenge and particularly
and particularly in our finalin our finalgoal,
irradiation irradiation goal,triggers
which also which browning
also triggers
(Tablebrowning
1).
(Table 1).
Table 1. Rice calli induction and browning rate from different induction treatments.
Table 1. Rice calli induction and browning rate from different induction treatments.
Treatment 1 id Embryogenic Calli (%) Browning Rate (%)
Treatment
− 1
1 id Embryogenic Calli (%) Browning Rate (%)
2.5 mg L −12,4-D i 21.44 b 2.00 b
2,5 mg −L1 2,4-D i 21.44 b 2.00 b
2.0 mg L −12,4-D ii 71.44 a 3.55 a
2.0 mg L 2,4-D ii 71.44 a 3.55 a
1.0 mg L−1 2,4-D + 1.0 mg L−1 BA iii 12.77 d 0.66 c
1.0 mg − L1
−1 2,4-D + 1.0 mg L−1 BA
2.0 mg L 2,4-D + 1.0 mg L BA − 1 iii
iv 12.77 d
16.77 c 0.66cc
0.21
2.0 mg− 1L −1 2,4-D + 1.0 mg L−1 BA
2.0 mg L 2,4-D + 0.25 mg L TDZ − 1 iv
v 16.77 c
23.00 b 0.66 cc
0.21
2.0 mg L −1 2,4-D + 0.25 mg L−1 TDZ v 23.00 b 0.66 c
1 All treatments had 30 replicates of 30 seeds per replicate (n = 900). Letters represent a significant difference
1 All treatments had 30 replicates of 30 seeds per replicate (n = 900). Letters represent a significant
(p ≤ 0.05).
difference (p ≤ 0.05).
Rice embryogenic calli obtained from induction medium supplemented with 2 mg L−1
Rice embryogenic calli obtained from induction medium supplemented with 2 mg
were produced after 15 days of culture from the scutellum of mature zygotic embryos
L −1 were produced after 15 days of culture from the scutellum of mature zygotic embryos
(Figure 2A) and were composed of yellow friable aggregates (Figure 2B).
(Figure 2A) and were composed of yellow friable aggregates (Figure 2B).
Plants 2022, 11, x FOR PEER REVIEW 4 of 12
Plants 2022, 11, 375 4 of 12

Calliinduction
Figure2.2.Calli
Figure inductionofofLazarroz
LazarrozFL
FLcultivar
cultivarononMSMSmedium
mediumwith mg LL−1−12,4-D
with 22 mg 2,4-Dafter
after1515days
days
ofofculture
cultureunder
underdarkness.
darkness.(A)(A)Embryogenic
Embryogeniccalli
calliobtained
obtainedfrom
fromthe
thescutellum
scutellumofofmature
maturezygotic
zygotic
embryos
embryos(arrow)
(arrow)(B)
(B)the
thecompact
compactand
andfriable
friablecalli
callias
asobserved
observedunder
underthe
thestereoscope.
stereoscope.

2.3.Regeneration
2.2. RegenerationononSemisolid
SemisolidMedium
Medium
Thebest
The bestregeneration
regenerationrate
rateofofapproximately
approximately70% 70%resulted
resultedfrom
from0.50.5mg
mgofofNAA+
NAA+33mg mg
of 6-BA, with sprouting of 7.14% and browning of only 9.52%, for calli induced on 2 mg L −1
of 6-BA, with sprouting of 7.14% and browning of only 9.52%, for calli induced on 2 mg
L2,4-D
−1 2,4-D(Table 2). 2).
(Table Other regeneration
Other regeneration medium
mediumrecipes alsoalso
recipes resulted in regeneration
resulted but with
in regeneration but
a higher
with browning
a higher and and
browning lower sprouting
lower rate,rate,
sprouting and and
werewere
consequently
consequently useless for our
useless next
for our
step, gamma radiation mutagenesis.
next step, gamma radiation mutagenesis.
Ricecalli
Table2.2.Rice
Table calli response
response of of Lazarroz
Lazarroz FLFL cultivar
cultivar after
after 4 weeks
4 weeks of culture
of culture on different
on different regenera-
regeneration
tion
media.media.

Induction
Induction Regeneration Sprouting
Sprouting Browning
Browning
Regeneration Treatment
Regeneration Treatment Regeneration (%)
Treatment 11 (%) (%) (%)
Treatment (%) (%)
0.5 mg NAA + 3 mg BA 69.04 a 7.14 ab 9.52 d
0.5 mg NAA + 3 mg BA 69.04 a 7.14 ab 9.52 d
0.5 mg NAA + 0.5 mg TDZ 38.09 c 2.38 b 61.90 a
2 mg 2,4-D 0.5 mg NAA + 0.5 mg TDZ 38.09 c 2.38 b 61.90
2 mg 2,4-D 0.5 mg NAA + 0.5 mg Kinetin 47.61 b 9.52 a 23.80 ca
0.50.5mgmgNAA
NAA++0.5 0.5mg
mg Kinetin
BA 47.61
28.57 d b 2.389.52
b a 23.80
54.76 bc
0.5
0.5mg
mg NAA
NAA ++30.5 mgmgBABA 28.57
28.29 b d 02.38
a b 54.76
58.43 bb
1 mg BA + 2 0.50.5
mgmg NAANAA+ 0.25
+ 3mgmgTDZ
BA 58.82 a b
28.29 0 a0 a 100 ab
58.43
1 mgmg BA2,4-D
+ 2 mg 0.5
0.5mgmgNAANAA + 0.5 mg mg
+ 0.25 Kinetin
TDZ 9.22 c a
58.82 0 a0 a 56.81
100 ab
0.5 mg NAA + 0.5 mg BA 12.82 c 0a 44.26 c
2,4-D 0.5 mg NAA + 0.5 mg Kinetin 9.22 c 0a 56.81 b
0.5 mg de NAA + 3 mg BA 58.45 ab 16.38 a 18. 69 bc
0.5 mg NAA + 0.5 mg BA 12.82 c 0a 44.26 c
0.5 mg de NAA + 0.5 mg TDZ 61.75 a 10.71 ab 27.93 a
1 mg BA + 1
0.50.5
mgmg de NAA
de NAA + 0.5+mg 3 mg BA
Kinetin 58.45
56.31 ab ab 16.38
15.92 a a 18. 69 ab
20.01 bc
mg 2,4-D
0.5mg
0.5 mg de
de NAA
NAA++0.5 0.5mg
mgBATDZ 49.88 b a
61.75 4.16
10.71 b ab 11.66 ca
27.93
1 mg BA + 1 mg
0.5 0.5
mgmg dede NAA+ +0.5
NAA 3 mg
mgBA
Kinetin 58.45 ab ab
56.31 16.38
15.92 a a 18. 69 ab
20.01 bc
2,4-D
0.5 mg NAA + 3 mg
0.5 mg de NAA + 0.5 mg BA BA 34.64 b
49.88 b 0 b
4.16 b 9.20
11.66 cc
0.5 mg NAA + 0.5 mg TDZ 51.41 a 9.61 a 22.96 a
2.5 mg 2,4-D 0.5 mg de NAA + 3 mg BA 58.45 ab 16.38 a 18. 69 bc
0.5 mg NAA + 0.5 mg Kinetin 43.62 ab 0b 15.73 b
0.5 mg NAA + 3
0.5 mg NAA + 0.5 mg BA mg BA 34.64
18.00 c b 0b 0 b 15.19 cb
9.20
0.50.5mg NAA + 0.5
mg NAA + 3 mg BA
mg TDZ 51.41
77.27 a
a 9.61
2.27 a
a 22.96
96.59 a
a
2,5 mg 2,4-D
2 mg 2,4-D + 0.50.5mgmgNAA
NAA ++ 0.5 mgTDZ
0.5 mg Kinetin 43.62
50.25 b ab 0 a0 b 15.73
86.36 bb
0.25 mg TDZ 0.50.5
mgmgNAANAA+ 0.5+ mg
0.5 Kinetin
mg BA 73.86 a c
18.00 3.400ab 82.95 bb
15.19
0.5 mg NAA + 0.5 mg BA 44.29 b 0a 72.81 c
0.5 mg NAA + 3 mg BA 77.27 a 2.27 a 96.59 a
1 All treatments had 6 replicates of 7 calli each replicate (n = 42). Letters represent significant differences for
2 mg 2,4-D + 0.25 0.5 mg NAA + 0.5 mg TDZ 50.25 b 0a 86.36 b
Tukey’s test (p ≤ 0.05) and the comparisons were made between treatments from same induction medium.
mg TDZ 0.5 mg NAA + 0.5 mg Kinetin 73.86 a 3.40 a 82.95 b
0.5 mg NAA + 0.5 mg BA 44.29 b 0a 72.81 c
2.4. Gamma Radiation Mutagenesis
1 All treatments had 6 replicates of 7 calli each replicate (n = 42). Letters represent significant

The effect of cobalt-60 60 Co) gamma radiation on embryogenic indica rice calli was
differences for Tukey’s test (p ≤( 0.05) and the comparisons were made between treatments from
evaluated.
same inductionA lethal
medium. dose (LD50) of the embryogenic calli was found to be 110 Gy, while the
Plants 2022, 11, x FOR PEER REVIEW 5 of 12

2.3. Gamma Radiation Mutagenesis

Plants 2022, 11, 375 The effect of cobalt-60 (60Co) gamma radiation on embryogenic indica rice calli was 5 of 12
evaluated. A lethal dose (LD50) of the embryogenic calli was found to be 110 Gy, while
the 20% lethal dose was 64 Gy, resulting in a 0 to 120 gray gradient exposure, with 200
calli
20%per exposure
lethal (Table
dose was 3). resulting in a 0 to 120 gray gradient exposure, with 200 calli per
64 Gy,
exposure (Table 3).
Table 3. Lethal effect of gamma radiation on the embryogenic rice calli of Lazarroz FL cultivar after
30Table
days 3.
of Lethal
cultureeffect
on regeneration medium,on
of gamma radiation determined by probit
the embryogenic ricemodel ofLazarroz
calli of survival FL
(%)cultivar
1.
after
30 days of culture on regeneration medium, determined by probit model of survival (%) 1.
Lethal Gamma Rays
Dose (Gy) Lower Limit (Gy) Upper Limit (Gy)
Dose Model
Lethal Gamma Rays
LD10 Dose
41.145(Gy) Lower Limit (Gy)
34.552 Upper Limit (Gy)
46.708
Dose Model
LD20 64.799 60.083 69.20
LD10
LD25 41.145
73.785 34.552
69.388 46.708
78.139
LD20 64.799 60.083 69.20
LD30 81.855 77.507 86.403
LD25 73.785 69.388 78.139
LD40 96.429 91.649 101.85
LD30 81.855 77.507 86.403
LD50
LD40 110.050
96.429 104.435
91.649 116.720
101.85
1 All treatments had 10 replicates with 20 calli each (n = 200), p ≤ 0.05. Data were compiled at 30 days
LD50 110.050 104.435 116.720
post-radiation.
1 All treatments had 10 replicates with 20 calli each (n = 200), p ≤ 0.05. Data were compiled at 30 days post-

radiation.
The best radiation/regeneration ratio was achieved at 60 Gy with 83% regeneration,
allowing
Thefor
besta radiation/regeneration
balance between gamma ratioradiation at lethal
was achieved dose
at 60 Gy 2083%
with andregeneration,
regenerational-
(Figure
lowing3).
forThe increased
a balance regeneration
between achievedatwith
gamma radiation 40dose
lethal Gy (75.00%) and 60 Gy (83.85%)
20 and regeneration (Figure 3).
versus the control
The increased (69.04%) (Figure
regeneration 3A) with
achieved is a hormetic behavior
40 Gy (75.00%) previously
and reported
60 Gy (83.85%) in our
versus the
lab [14]. (69.04%) (Figure 3A) is a hormetic behavior previously reported in our lab [14].
control

Figure
Figure 3. 3. Correlation
Correlation comparison
comparison ofof radiation
radiation dose
dose influence
influence onon regeneration
regeneration (A),
(A), sprouting
sprouting (B)
(B) and
and
browning
browning rates
rates(C)
(C)ofof
calli ofof
calli Lazarroz
LazarrozFlFl
cultivar, after
cultivar, 3030
after days
daysofof
culture
cultureononregeneration
regeneration medium.
medium.
Letters
Lettersa,a,b,b,bc,
bc,ccand
and dd represent significant
significantdifferences
differencesfor
forTukey’s
Tukey’s test
test (p (p ≤ 0.05).
≤ 0.05). AllAll treatments
treatments had
had 12 replicates of 7 calli each replicate (n = 84). Data were compiled at 15 days
12 replicates of 7 calli each replicate (n = 84). Data were compiled at 15 days post-radiation.post-radiation.

Compared
Comparedtotothe thenon-irradiated
non-irradiatedcontrol
control(0(0Gy),
Gy),a significant
a significantdecrease
decreaseininthe
thesprouting
sprouting
ofofembryogenic
embryogeniccalli
calliwas
wasobserved,
observed,asasthethedose
doseincreased
increasedfrom
from0 0toto8080Gy.
Gy.Sprouting
Sproutingofof
embryogeniccalli
calliirradiated
irradiatedwithwithgamma
gammarays 60 Co) decreased significantly (p < 0.05)
rays(60(Co)
embryogenic decreased significantly (p < 0.05)
occurredatatdoses
occurred doseshigher
higherthan
than 6060
GyGy (Figure
(Figure 3B).
3B). Moreover,Gamma-ray
Moreover, Gamma-raydoses doseshigher
higherthan
than
6060Gy
Gyseverely
severelyaffected
affectedbrowning
browningraterate(Figure
(Figure3C).
3C).
Gamma-irradiatedcalli
Gamma-irradiated calliproduced
producedplants
plantsafter
after4545days
daysininthe
theregeneration
regenerationmedium
medium
(Figure 4A) and fully in vitro plants (Figure 4B) at 60 days post-irradiation
(Figure 4A) and fully in vitro plants (Figure 4B) at 60 days post-irradiation at 60 Gy. at 60 Gy.
Plants2022,
Plants 2022,11,
11,375
x FOR PEER REVIEW 66 of 12
of 12

Figure 4. Response
Figure 4. Responseofof60
60Gy
Gyirradiated
irradiatedcalli
calliLazarroz
LazarrozFLFL
cultivar after
cultivar (A)(A)
after 4545
days and
days (B)(B)
and 60 60
days of
days
culture on on
of culture regeneration medium.
regeneration medium.

2.5.
2.4. Regeneration
Regeneration in
in Recipient
Recipient for
for Automated
Automated Temporary
TemporaryImmersion
Immersion (RITA® Saint-Mathieu-de-
(RITA ® Saint-Mathieu-de-Tréviers, France)
Tréviers, France)
Regeneration
Regeneration inin the
the MS
MS semisolid
semisolid medium
medium was
was irregular
irregular and
and not
not homogenous
homogenous
(Figure ® ®
(Figure 5A).
5A). Instead,
Instead, the
the RITA
RITA® immersion
immersion system
system (RITA
(RITA®,, Saint-Mathieu-de-Tréviers,
Saint-Mathieu-de-Tréviers,
France)
France) provided
provided predictable
predictableand
andhomogeneous
homogeneousregeneration
regeneration(Figure
(Figure5B).
5B).

Figure 5. Regeneration in MS medium with 0.5 mg L−1−1 of NAA + 3 mg−1 L−1 BA of an induced
Figure 5. Regeneration in MS medium with 0.5 mg L of NAA + 3 mg L BA of an induced calli
calli with 2 mg L−1 of 2,4-D after 15 days of induction. Regeneration in (A) semisolid medium and
with 2 mg L−1 of 2,4-D after 15 days of induction. Regeneration in (A) semisolid medium and (B)
(B) RITA ® (Saint-Mathieu-de-Tréviers, France).
RITA ® (Saint-Mathieu-de-Tréviers, France).

The potential for temporary immersion system to regenerate recalcitrant materials

The potential for temporary immersion system to regenerate recalcitrant materials
(CR-5272, CR-1821,
Figure 5. Regeneration CR-1113
in MS medium withand Lazarroz
0.5 mg FL) was
L−1 of NAA evaluated.
+ 3 mg Our
L−1 BA of an resultscalli
induced showed no
(CR-5272, CR-1821, CR-1113 and Lazarroz FL) was evaluated. Our results showed no
with 2 mg significant
L of 2,4-D differences
−1 after 15 daysbetween
of induction.
the Regeneration in (A)(60
immersion time semisolid
vs. 120medium andregeneration
s) on the (B)
significant differences between the immersion time (60 vs. 120 s) on the regeneration
RITA® (Saint-Mathieu-de-Tréviers,
capacity, sprouting andFrance).
browning (Table 4). Nevertheless, a higher regeneration rate was
capacity, sprouting and browning (Table 4). Nevertheless, a higher regeneration rate was
obtained using the RITA®® (Saint-Mathieu-de-Tréviers, France) in 60 s (100%) compared to
obtained using the RITA (Saint-Mathieu-de-Tréviers, France) in 60 s (100%) compared to
regeneration on the best semi-solid medium (70%). Similarly, the sprouting rate was higher
regeneration on the best semi-solid medium (70%). Similarly, the sprouting rate was
using the RITA® system independently of the immersion time compared to that obtained
higher using the RITA® system independently of the immersion time compared to that
using the selected semi-solid medium (Tables 2 and 4).
obtained using the
Although, the immersion
selected semi-solid
time (60 medium
vs. 120 s)(Tables
did not2significantly
and 4). affect the browning
Although, the immersion time (60 vs. 120 s) did not significantly
rate, and it was higher using an immersion of 120 s (97.56%) compared to affect
60 sthe browning
(60.00%) and
rate, and it was higher using an immersion
a semi-solid medium (9.52%) (Tables 2 and 4). of 120 s (97.56%) compared to 60 s (60.00%)
and a semi-solid medium (9.52%) (Tables 2 and 4).
Plants 2022, 11, x FOR PEER REVIEW 7 of 12
Plants 2022, 11, 375 7 of 12

Table 4. Immersion regeneration, sprouting and browning rates 1.

Table 4. Immersion regeneration, sprouting and browning rates 1 .
Immersion Time Regeneration Sprouting Browning Rate
Immersion
60 s Time Regeneration
100.00 a Sprouting
25.00 a Browning
60.00 a Rate
12060s s 100.00
97.56 aa 25.00
31.71 a a 97.5660.00
a a
1 All treatments120hads 4 replicates of 10 calli
97.56 a replicate (n = 40), 31.71
per a Data were compiled
p ≤ 0.05. 97.56at
a 15
days 1post-radiation.
All treatments had 4 replicates of 10 calli per replicate (n = 40), p ≤ 0.05. Data were compiled at 15 days
post-radiation.
The potential for a temporary immersion system to regenerate recalcitrant materials
The potential for a temporary immersion system to regenerate recalcitrant materials
was also validated for the well-known recalcitrant cultivars CR-5272, CR-1821 and CR-
was also validated for the well-known recalcitrant cultivars CR-5272, CR-1821 and CR-1113
1113 (Figure
(Figure6).
6).

Figure 6. Regeneration of cultivars CR-5272 and recalcitrant CR-1821 and CR-1113 in MS medium
Figure
with 0.5 mg L−1 ofof
6. Regeneration cultivars
NAA + 3 mg L−1 of and
CR-5272 BA fromrecalcitrant CR-1821
calli induced and
with L−1 ofin2,4-D.
CR-1113
2 mg MS medium
(A) CR-5272
with regeneration
0.5 mg L−1 ofinNAA + 3 mg L −1 of BA from calli induced with 2 mg L−1 of 2,4-D. (A) CR-5272
RITA with immersion for 30 s or 60 s, and the semisolid medium control. (B) CR-1821
regeneration in RITA
regeneration withwith
in RITA immersion
immersionfor 30
for s30ors 60 s, and
or 60 s andthe
thesemisolid
semisolidmedium
mediumcontrol.
control.(B)
(C)CR-
CR-1113
1821 regeneration in RITA with immersion for 30 s or 60 s and the semisolid medium control. (C)
regeneration in RITA with immersion for 30 s or 60 s and the semisolid medium control. Note that all
CR-1113 regeneration in RITA with immersion for 30 s or 60 s and the semisolid medium control.
RITA treatments contain calli with green areas that regenerate into plants. The absence of regeneration
Note that all RITA treatments contain calli with green areas that regenerate into plants. The absence
of the recalcitrant
of regeneration cultivars CR-1821
of the recalcitrant andCR-1821
cultivars CR-1113and wasCR-1113
observedwas
in the semisolid
observed in media.
the semisolid
media.
3. Discussion
The present work optimized in vitro gamma ray (60 Co) mutagenesis in an embryogenic
calli of a recalcitrant Costa Rican Lazarroz FL cultivar. The cultivar seems to be related to
Southeast Asian rice cultivars based on its rbcL sequence pattern.
Plants 2022, 11, 375 8 of 12

The best calli induction was observed for MS with 2mg of 2,4-D and regeneration
with MS with 0.5 mg ANA + 3 mg BA. The calli induction step with a 2 mg L−1 of 2,4-D
concentration was initially not expected because of previous local cultivar reports. Local
cultivars CR-5272 and CR-1113 had a positive response at 2.5 mg L−1 of 2.4 D but a low
performance at 2 mg L−1 of 2,4-D, while CR-201, CR-1707, CR-1821, CR-8334, and CR-8341
had recalcitrant and unpredictable in vitro behaviors [17,20]. Our result is similar to that
obtained by other authors on Southeast Asian cultivars such as Malaysia MR219, where
2,4-D is critical and performs the best as an inducer at 2 mg L−1 [21,22]. On the contrary, a
better calli induction occurred with higher concentrations of 2,4-D (from 2.5 mg to 3 mg L−1 )
with other cultivars, such as MR220, GNY-53, and JP-5 [23,24].
Regeneration was improved using a temporal immersion system (RITA® , Saint-
Mathieu-de-Tréviers, France) with either 60 or 120 s of immersion every 8 h, which achieved
a predictable and more homogeneous regeneration response. The radiation dose with which
to start mutagenesis was proposed as a lethal dose 20 at 60 Gy, as regeneration was not
affected but remained as high as 80%. At 80 Gy, the regeneration fell to 30%, consistent
with the oxidative damage provoked by radiation and corresponding to the lethal dose 30
of the embryogenic calli. We believe 60 Gy is an excellent condition to start mutagenesis,
considering that in other in vitro plants, such as pineapple, potato, and banana, a 5–40 Gy
dose of gamma irradiation was shown to be sufficient to produce variability [25]. The
calculated radiation dose of the Lazarroz FL cultivar at 60 Gy corresponded to the LD20
and was different from our previous data achieved for CR-5272 with an LD50 of 60Gy [14].
Optimization of embryogenic calli in our recalcitrant Lazarroz FL cultivar became a
challenge while at the same time an opportunity compared to our previous results from
CR-5272 for the following reasons First, the alternative immersion method allowed our
material to generate homogeneous and predictable in vitro plants after irradiation. The
technique enabled regeneration for our Lazarroz FL cultivar and other recalcitrant rice lines
CR-1821 and CR-1113, and consequently, is a great potential tool for regenerating gamma
radiation mutated materials. The constant liquid and airflow can dilute oxidative com-
pounds and facilitate more homogeneous exposure to nutrients. We validated the results
with recalcitrant cultivars CR-1821 and CR-1113, which showed a total regeneration rate
in contrast to the null regeneration in the conventional semisolid method. Plant cultivars’
nonhomogeneous in vitro behavior is not fully understood, but recent discoveries provide
insights into the genetic basis of the sucrose metabolism and calli browning. External
phytohormones seem to trigger rice sucrose metabolism required for regeneration. The
system appears to rely on the expression of endogenous cytokinin, auxin, and ABA sig-
naling genes: ORYZA SATIVA RESPONSE REGULATOR 1 (ORR1), PIN-formed 1 (PIN1),
and late embryogenesis-abundant 1(LEA-1) [26]. The expression of OsSRO1c, a regulator
of oxidative stress, seems to be vital for avoiding calli browning in indica cultivars [27].
Tissue culture and gamma radiation produce oxidation via reactive oxygen species (ROS),
usually contained in chloroplasts, peroxisomes, and mitochondria. ROS include superoxide
(O2 − ), hydroxyl (OH− ) radicals, and hydrogen peroxide (H2 O2 ) [28]. Tissue culture and
gamma rays trigger ROS, consequently damaging the DNA by oxidation of the molecule
into 8-oxo-7-hydroxyguanosine (8-oxo-dG) and further transversions of C/G and T/A [29].
Second, we can develop the desired mutations in a cultivar that farmers are already
using, leading to faster breeding and adoption of a derived improved cultivar. Rice traits
associated with specific genes are well known, which paves the way for producing novel
cultivars based on mutation of desired or required conditions, such as biotic and abiotic
stress tolerance [11]. We foresee the development of new traits such as NaCl, herbicide, and
pH tolerance based on the corresponding selection agents and not limited by recalcitrant
cultivars, which used to be our bottleneck to bring innovation.
Plants 2022, 11, 375 9 of 12

4. Materials and Methods

4.1. Molecular Markers
A NucleoSpinTM Tissue Kit Macherey-Nagel (Düren, Germany) was used for DNA
extraction from 1 mg of on non-irradiated lyophilized leaf tissue of Lazarroz FL. Thermo
Fisher K1071 (Vilnius, Lithuania) was used for the subsequent PCR following the recommen-
dations of the manufacturer. The primers used in this study are as follows: for rbcL, rbcLaf
50 ATGTCACCACAAACAGAGACTAAAGC30 and rbcLar 50 GTAAAATCAAGTCCACCR
CG-30 or rbcLaf 50 ATGTCACCACAAACAGAGACTAAAGC30 and rbcLr590 50 AGTCCAC
CGCGTAGACATTCAT-30 ; for matK, matK-xf 50 TAATTTACGATCAATTCATTC-30 and
matKr 50 ACAAGAAAGTCGAAGTAT-30 . Briefly, the PCR master mix consisted of a mix-
ture (50 µL) containing 1X Dream Taq Master mix Thermo Fisher (Vilnius, Lithuania),
20 µM of each primer, and 5 µL of DNA (50 ng/uL). The thermocycling program was 95 ◦ C
for 5 min, 40 cycles at 95 ◦ C for 45 s, 55 ◦ C for 45 s and 72 ◦ C for 1 min, and a final cycle of
72 ◦ C for 7 min.

4.2. Embryogenic Calli Induction

For initial assays, the palea and lemma of rice caryopsis of a commercial local indica
cultivar were removed with No. 80 grit sandpaper. The obtained seeds were surface-
sterilized as previously reported [14] through two incubations in 4% (v/v) NaOCl for 10 min
each with constant agitation, using 10 mL of disinfectant solution for every gram of seeds.
After the first and final incubation, the seeds were washed six to seven times with distilled
sterilized water and were cultured in media composed of mineral salts and vitamins as
described by Murashige and Skoog (MS), with 20 g L−1 sucrose and 0.1 g L−1 hydrolyzed
casein. Treatments consisted of supplementing the basal medium with one of the following
combinations of plant growth regulators: (i) 2.5 mg L−1 2,4-dichlorophenoxyacetic acid
(2,4-D) was used as a control, (ii) 2.0 mg L−1 2,4-D, (iii) 1.0 mg L−1 2,4-D + 1.0 mg L−1 6-
benzyladenine (6-BA), (iv) 2.0 mg L−1 2,4-D + 1 mg L−1 6-BA, and (v) 2.0 mg L−1 2,4-D
+ 0.25 mg L−1 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea (thidiazuron, TDZ) (Table 1). After
adding plant growth regulators, the media volumes were adjusted as required, the pH
was adjusted to 5.8 with 1 N NaOH or 1 N HCl, and 5.4 g L−1 Gelzan® (Phytotechnology
Laboratories® , Shawnee Mission, KS, USA) were added as a gelling agent. All previously
mentioned chemicals were supplied by Phytotechnology Laboratories® (Shawnee Mission,
KS, USA). An autoclave (1.2 ATM. cm−2 and 121 ◦ C for 30 min) was used for the sterilizing
medium and further dispensed on 94 × 16 mm vented polystyrene Petri dishes (Greiner Bio-
One, Fisher-Scientific, Waltham, MA, USA) in a laminar flow chamber. For each treatment,
at least 900 seeds were cultured after surface sterilization. Cultures were maintained in
the dark at 26 ± 2 ◦ C. Calli induction and browning rates (brown or necrotic/total calli)
were recorded as response variables and analyzed in a completely randomized design with
a generalized linear model with a Poisson distribution and logit link function. Post hoc
analysis consisted of an Honest Significant Difference test on IBM SPSS version 27 [30] and
differences between each combination of factors were recognized.

4.3. Regeneration on Semisolid Medium

Embryogenic calli obtained from each induction medium were transferred to different
regeneration treatments. Basal regeneration medium was similar to that described by
Sudhakar et al. [31], and was constituted by MS mineral salts and vitamins, 20 g L− 1
sucrose and 0.3 g L− 1 hydrolyzed casein. Treatments consisting of supplementing the basal
medium with variations of growth regulators, and the control was supplemented with
0.5 mg L− 1 α-Naphthaleneacetic Acid (NAA) + 3 mg L− 1 6-BA, while the second treatment
was supplemented with 0.5 mg L− 1 NAA + 0.5 mg L− 1 6-BA and the third treatment was
supplemented with 0.5 mg L− 1 NAA + 1.5 mg L− 1 Kinetin (KIN) and a fourth treatment
consisted of supplementing basal medium with 0.5 mg L− 1 NAA + 0.5 mg L− 1 TDZ. After
adding growth regulators, pH was adjusted to 5.8 with 1 N NaOH or 1 N HCl and 5.4 g L− 1
Gelzan ® (Phytotechnology Laboratories® , Shawnee Mission, KS, USA) were added as
Plants 2022, 11, 375 10 of 12

gelling agent. All previously mentioned reagents were supplied by Phytotechnology

Laboratories® (Shawnee Mission, KS, USA). After a properly dissolving of gelling agent,
60 mL of media were dispensed on 475 mL polypropylene WNA Deli Containers, and
afterwards, media were sterilized at 1.2 ATM.cm− 2 and 121 ◦ C for 30 min. For each
treatment, 6 replicates of 7 calli were cultured on a factorial design. Factor 1 consisted of
induction media and factor 2 was regeneration media. Cultures were maintained at an
irradiance of 72 µmol s− 1 m− 2 , a 16 h light/8 h dark photoperiod was used and 26 ± 2 ◦ C
for a period of 4 weeks. Calli showing mature coleoptilar germinated embryos were
determined as regenerated, calli with completely differentiated plantlets greater than 1 cm
were categorized as sprouted, and necrotic calli with a dark coloration were identified as
browning calli. These response variables were analyzed with a generalized linear model
with a Poisson distribution and logit link function. Post hoc analysis consisted of an
Honest Significant Difference test on IBM SPSS version 27 [31] and differences between
each combination of factors were recognized.

4.4. Gamma Irradiation

Embryogenic calli irradiation was achieved with a gamma irradiator Ob-Servo Ignis
type with 24 cobalt 60 source pencils (Institute of Isotopes Co, Ltd., Budapest, Hungary).
To determine radiosensitivity and the median lethal dose (LD50), calli were irradiated at 0,
40, 60, 80, 100, 120 Gy. Ten repetitions, and 20 embryogenic calli per exposure were used.
The survival rate was recorded after calli were transferred to the regeneration medium
composed by basal regeneration medium supplemented with 0.5 mg L−1 NAA + 3 mg L−1
6-BA, selected after previous experiments were analyzed. Culture conditions were the
same indicated above and after 4 weeks lethal doses were calculated using probit analysis
on IBM SPSS version 27 [31] based on calli death rates.

4.5. Regeneration in Recipient for Automated Temporary Immersion (RITA® ,

Saint-Mathieu-de-Tréviers, France)
The RITA® (Saint-Mathieu-de-Tréviers, France) temporary immersion system regener-
ation of the embryogenic calli consisted of 200 mL regeneration media previously described
in 4.3, with 23 four-week-old calli per unit, different rice cultivars (Lazarroz FL, CR-5272,
CR-1821, and CR-1113). Light and temperature culture conditions remained unchanged,
with 60 or 120 s immersion used as treatments, every eight hours. After four weeks of
culture, the variables evaluated were regeneration, sprouting, and browning, green area
calculations with ImageJ version 1.52p [32].

5. Conclusions
Rice tissue culture is a tool for conventional and modern breeding, but is limited
to the genotype response, particularly during the regeneration of recalcitrant varieties.
Our results collected using an immersion system helped to overcome such difficulties and
allowed for the induction of gamma-ray mutants. A temporary immersion system seems
to help overcome calli browning while allowing the tissue to recover and consequently
presenting a more efficient method. We foresee that having access to such methods could
diminish the time to trigger innovation and focus on selecting mutants with desired traits
in commercially used varieties.

Author Contributions: Conceptualization, A.H.-S., J.P., R.F.-Z., R.R.-V. and A.G.-A.; writing—original
draft preparation, A.H.-S., J.P. and A.G.-A.; writing—review and editing, A.H.-S., J.P., A.G.-A. and
A.A.-E.; visualization, A.H.-S. and J.P; investigation W.V.-S., R.F.-Z., R.R.-V. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the Research Vice-Rectory of TEC, Costa Rica, project number
1510-1022.
Institutional Review Board Statement: Not applicable.
Plants 2022, 11, 375 11 of 12

Informed Consent Statement: Not applicable.

Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.

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 This is a preprint; it has not been peer reviewed
by a journal

Research Article

Tolerance to aryloxy-phenoxy-propionate (APP) as a model for

Lazarroz FL rice in vitro gamma irradiation variability selection
Alejandro Hernández-Soto 1,2,*, Daniela Méndez-Navarro 2, Jason Pérez 2, Andrés Gatica-Arias 3, Walter Vargas-
Segura 4 Fabián Echeverria-Beirute1, and Ana Abdelnour-Esquivel 2

1 Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica,
Universidad Nacional, Universidad Estatal a Distancia, Cartago P.O. Box 159-7050, Costa Rica; fecheve-
[email protected] (F.E.B)
2 Biotechnology Research Center, Biology School, Costa Rica Institute of Technology, Costa Rica

PO Box 159-7050 Cartago, Costa Rica; [email protected] (D.M.-N); [email protected] (J.P.);

[email protected] (A.A.-E.)
3 Plant Biotechnology Laboratory, School of Biology, University of Costa Rica, P.O. Box 2060,

San José, Costa Rica; [email protected] (A.G.-A.)

4 Gamma Irradiation Laboratory, School of Physics, Costa Rica Institute of Technology, Costa Rica, PO Box 159-

7050 Cartago, Costa Rica; [email protected]

* Correspondence: [email protected]

Abstract: In vitro gamma ray (60Co) mutagenesis is a powerful tool to achieve variability in commer-
cial rice lines used by farmers, such as Lazarroz FL. We previously reported the optimized in vitro
gamma mutagenesis system for Lazarroz FL Indica callus. As a continuation, in the present study,
we targeted the ACC2 gene mutagenesis that provides tolerance to aryloxy-phenoxy-propionate
Citation: Hernández-Soto, A.; Mén- (APP) fluazifop-P-butyl as a model to show the system's potential to create variability while provid-
dez-Navarro, D.; Pérez, J.; Rojas- ing a solution for weed management. The DL50 of fluazifop-P-butyl was calculated in calli as DL50=
Vásquez, R.; Gatica-Arias, A.; Var- 6,93 mg/L (0,425 mg/L - 15,743 mg/L, R2 = 0,402, 1000n) and regenerated vitroplants at an LD50 of
gas, W.; Echeverria-Beirute,F; Ab- 3.771 mg/L (R2 = 1, 290n). We used 5 mg/L fluazifop-P-butyl as a selection agent and the second
delnour-Esquivel, A. Tolerance to ar-
round of selection of 10 mg/L (3000 vitroplants) resulted in one survivor plant when using calli as a
yloxy-phenoxy-propionate (APP) as
starting material. The putative tolerant plant also tolerated 150 mg/L in the greenhouse. The ACC2
a model for Lazarroz FL rice in vitro
gene was sequenced, and a heteroecious mutation, T2222I/T2222M, was discovered that may be
gamma irradiation variability selec-
linked to tolerance. We improved the in vitro system by using seeds as a gamma irradiation starting
tion. Research Square, 2022.
https://doi.org/10.21203/rs.3.rs-
point instead of embryogenic calli, followed by calli induction, regeneration, and exposure to the
1950230/v1 selection agent. The modification allowed higher gamma doses with an LD50 of 350 Gy and one to
thirty-one putative tolerant plants. The in vitro model showed that gamma-ray mutants from recal-
Posted: 11 August 2022 citrant indica rice materials are possible, and the use of selection agents such APP can help create
variability useful for breeding a more resilient rice.

License: This work is licensed under Keywords: Mutagenesis, Biotechnology, Plant Tissue Culture, Fluazifop-P-butyl
a Creative Commons Attribution 4.0
International License.

© 2022 Research Square Platform,

1. Introduction
LLC. All rights reserved.

Rice (Oryza sativa L.) is a crucial crop responsible for 20% of calories of half the human
population and is facing constraints of yield related to biotic factors, such as pathogens,
and abiotic stresses, such as salinity, drought, heat or cold [1-2]. Breeding can introduce
desired traits based on genetic variability related to heat, salt, soil acidification stress, and
plague tolerance to the few cultivars preferred by farmers in specific geographic areas [3-
6]. Rice-induced genetic variability obtained by seed irradiation is well known and has
been used since it was proven in 1928 on vegetables; however, it is slow and requires land,
resources, and human capabilities to find desired traits [7-10]. Plant tissue culture pro-
vides a platform to produce gamma mutations from primitive embryogenic cells [11-13].

Research Square, 2022. https://doi.org/10.21203/rs.3.rs-1950230/v1 https://www.researchsquare.com/

Research Square, 2022. Preprint 2 of 11

We previously reported a system to create variability using in vitro gamma radiation at an

optimized dose of 60 Gy in embryogenic calli of Lazarroz indica rice [14]. The proposed
model required a method to narrow the in vitro selection while correlating with desired
mutations. In this paper, we suggest resistance to stressors such as the herbicide aryloxy-
phenoxy-propionate (APP) to regenerate complete plants from tolerant mutated cells as a
model for such challenges. The herbicide is commonly used to control weeds in rice by
acting on the ACC2 enzyme, which is related to fatty acid synthesis, while mutations in
the carboxyl transferase domain of ACC2 correlate with APP tolerance, as described below
[15-21].
The herbicides acting on the ACC2 enzyme include at least two main groups, ar-
yloxy-phenoxy-propionates (APP) and cyclohexanediones [15]. In grasses, there are two
types of ACCases, cytosolic and plastidic. The second ACC2 is affected by APPs in rice.
The APPs include clodinafop-propargyl, cyhalo-fop-butyl, fenoxapropethyl, metamifop,
diclofop-meth fenthiaprop, quizalofop-ethyl, haloxyfop-R-methyl, and fluazifop-P-butyl.
Fluazifop is a molecule of 327.25 g mol-1 that can control grasses at a dose of 210 g ai Ha−1
and 420 g ai Ha−1 preemergent with low residual effects [15-16]. Specific mutations in
ACC2 enzymes correlate with APP tolerance, as described below.
The enzyme acetyl-CoA carboxylase 2 ACC2 (EC 6.4.1.2, UniProt: A2Y2U1) is located
on chromosome 5 at amino acids 14,067,726-14,079,652 and is delivered to the plastid. Mu-
tations in the carboxyl transferase domain of ACC2 between amino acids 1,781-2,078 and
2,027-2,096 are related to APPS tolerance, such as Ile1781-Leu, Trp-1999-Cys, Trp-2027-
Cys, Ile-2041-Asn, Ile-2041-Val, Asp-2078-Gly, Cys-2088-Arg, and Gly-2096-Ala [16-19].
Mutations such as A2004 V provide wheat with tolerance to 10 g ai Ha-1 of quizalofop
[16,17]. Similarly, Oryza japonica ACCase 2 (LOC_Os05g22940), mutations on I1781 V,
C2088R, and W2027C, also provide tolerance to APPs [21]. Finally, an indica rice with a
mutation tolerant to APPs with the mutation I1781 L has been available in the USA market
since 2018 [22].
This paper aims to improve our previous system and obtain more predictable traits
that correlate with specific mutations. Here, we present a nonreported mutation,
T2222I/T2222M, that may be linked to tolerance to APPs and present an improvement of
our earlier in vitro system by using seeds instead of calli during gamma irradiation. We
also show aryloxy-phenoxy-propionate (APP) fluazifop-P-butyl tolerance as a model to
demonstrate the system's potential for incorporating features that correlate with muta-
tions. The obtained mutant can be incorporated into the breeding program to look for
functional variability.

2. Results
2.1 Fluazifop-P-butyl toxicity to embryogenic calli

The toxicity of fluazifop-P-butyl to Lazarroz FL embryogenic calli was calculated based

on calli browning to be LD50=6.93 mg -L R2=0.402 (Table 1). A higher brown callus rate of
79% resulted in over 10 mg -L of the selection APP agent corresponding with the calcu-
lated LD75=17.8 mg (Table 1). We decided to test regenerated vitroplants from calli instead
of directly exposed calli to fluazifop-P-butyl because of the absence of regeneration of
nonbrowned surviving calli (139n) and the variability of the results (Table 2).

Table 1. Nonirradiated Lazarroz FL calli toxicity to fluazifop-P-butyl

Non Browning LD501 LD751

Treatment1 Total Brown
brown rate (%)2 (mg L−1) (mg L−1)
0 mg L−1 Fluazifop-P-butyl 200 120 80 40
1 mg L−1 Fluazifop-P-butyl 200 97 103 48 6.93 17.8
10 mg L−1 Fluazifop-P-butyl 200 42 158 79 (0.42-15.74) (4.1-35.2)
100 mg L−1 Fluazifop-P-butyl 200 0 200 100
Research Square, 2022. Preprint 3 of 11

1 Probit statistical analysis resulted in R2=0.402, y=-0.42+0.6x

2 Brown or necrotic/total calli

2.2 Vitroplant toxicity to fluazifop-P-butyl

The Fluazifop-P-butyl toxicity to Lazarroz FL vitroplants regenerated from calli was
calculated based on necrosis of plants at 21 days and resulted in LD50=3.771 mg -L and
LD75= 4.287 mg -L, R2=1 (Table 2, Figure 1A, 1B, 1C).

Table 2. Nonirradiated Lazarroz FL rice vitroplant toxicity to fluazifop-P-butyl

Mortality LD501 LD751

Treatment1 Total Alive Necrotic
Rate (%) (mg L−1) (mg L−1)
0 mg L−1 Fluazifop-P-butyl 55 55 0 0
1 mg L−1 Fluazifop-P-butyl 40 40 0 0
4 mg L−1 Fluazifop-P-butyl 71 36 35* 49
5 mg L−1 Fluazifop-P-butyl 58 0 58* 100 3.771 4.287
10 mg L−1 Fluazifop-P-butyl 88 0 88* 100
25 mg L−1 Fluazifop-P-butyl 57 0 57* 100
100 mg L−1 Fluazifop-P-butyl 70 0 70* 100
1 Probit statistical analysis resulted in R2=1, y=-4.59+7.6x

* Necrotic vitroplants were subcultured into fresh medium to verify mortality

The necrotic plants were subcultured into fresh medium for 21 days to verify mortal-
ity, resulting in null sprouting, regeneration, or green spots but remaining necrotic (Figure
1D).

Figure 1. Regenerated calli of vitroplants of the Lazarroz FL cultivar on MS medium with 3 mg L−1
BA under fluazifop-P-butyl stress after 21 days of culture. A) 5 mg L−1 fluazifop-P-butyl, (B) 4 mg
L−1 fluazifop-P-butyl, (C) 1 mg L−1 fluazifop-P-butyl, (D) necrotic vitroplants subcultured in fresh
medium for 21 days to verify mortality after exposure to 5 mg L−1 and 4 mg L−1 fluazifop-P-butyl
stress, as shown in (A) and (B). Note that all remained necrotic and had no sprouting or green spots.
Research Square, 2022. Preprint 4 of 11

2.3. Gamma radiation mutagenesis and plant regeneration

We decided to use 5 mg L-1 as the fluazifop-P-butyl dose to select putative tolerant mu-
tants to avoid false-positives. We selected 100 putative resistant lines out of 8000 vitro-
plants regenerated from embryogenic calli irradiated at 60 Gy (60Co) that survived when
exposed to 5 mg L−1 fluazifop-P-butyl stress (Figure 2). The plants grew normally and put
through a second selection round to validate their tolerance. The second round of selection
consisted of a higher dose of 10 mg-L fluazifop-P-butyl with the survival of only one pu-
tative tolerant plant out of 100 (Figure 2).

Figure 2. Tolerant vitroplant of the Lazarroz FL cultivar obtained by gamma mutagenesis on calli at
60 Gy on MS medium with two rounds of selections, the first at 5 mg L−1 of fluazifop-P-butyl stress
after 21 days of culture, 21 days of recovery with no stress agent, and another round of 21 days of
10 mg L−1 of fluazifop-P-butyl stress. Note that nontolerant plants became necrotic at 5 mg L−1 flua-
zifop-P-butyl stress after 21 days of culture, while only 1 out of 100 putative tolerant vitroplants
remained green during the second round of selection at 10 mg L−1 fluazifop-P-butyl stress.

2.4. Molecular markers used for the identification of the rice cultivars
We sequenced and published exon 32 of the acetyl-CoA carboxylase 2 gene (ACCase2)
MZ558337 and compared it with nonirradiated Lazarroz FL plants to validate perfect
matches with our control. The control sequence perfectly matched our accession, and the
reference genes of Oryza sativa indica at chromosome five demonstrated the identity and
absence of mutations as expected.
Research Square, 2022. Preprint 5 of 11

Figure 3. DNA markers used to identify exon 32 of the ACC2 gene in the nonirradiated Lazarroz FL
rice variety. Note in (A) the sequence of exon 32 is shown in green, and the locations of the primers
used for PCR and sequencing are shown in black and blue boxes. In the red box is the site corre-
sponding to the biallelic mutation. (B) Sequencing electropherogram and corresponding amino acid
mutation putative changes.

The sequence of exon 32 of the acetyl-CoA carboxylase 2 gene (ACCase2) of the fluazifop-
P-butyl tolerant vitroplant, showed one mutation in the expected domain related to toler-
ance. The mutation may have resulted in a change in the amino acid T2222I/T2222M (Fig-
ure 3). The plant remained tolerant in a greenhouse at a dose of 150 mg/L.

We also sequenced the matK and rbcL genes of the mutants that matched almost perfectly
with the control reported sequences MZ558335 and MZ558334, respectively. We noted
that a biallelic G/T in matK may change the amino acid isoleucine into another nonpolar
amino acid or methionine. We also noted a biallelic rbcL GT/AG mutation that could
change the amino acid tyrosine into another uncharged polar amino acid serine. The re-
sults are consistent with our mutation in the ACC2 gene of 1 per 1000 bp (Figure 4).
Research Square, 2022. Preprint 6 of 11

Figure 4. DNA markers (A) matK and (B) rbcL genes of the mutants in comparison with the control
reported sequences MZ558335 and MZ558334. Note in green the biallelic mutations G/T in matK
resulting in isoleucine change to methionine and GT/AG coding for tyrosine that may change into
serine in a mutation in rbcL.

Our results were positive, but we still had room for improvement since the number of
putative mutants was low. We tried a different approach that allowed us to increase the
gamma dose and, consequently, the mutation rate. We used seeds instead of calli for
gamma exposure and kept the same tissue culture methods as previously reported [14].
The system improved in terms of having a larger window for optimization that allows for
using a range of gamma rays from 50 to 350 Gy with 31 tolerant vitroplants after the two
rounds of fluazifop-P-butyl selection at 5 and 10 mg L−1 (Figure 5).

Figure 5. Gamma radiation optimization using Lazzaroz FL seeds as a starting material

and further calli induction, regeneration, and selection in two rounds of fluazifop-P-butyl
selection at 5 and 10 mg L−1. Note the optimization window of gamma exposure ranges
Research Square, 2022. Preprint 7 of 11

from 50 to 350 Gy, allowing predictable calli induction and regeneration, as well as puta-
tive tolerant vitroplants.

3. Discussion

In previous experiences, gamma radiation allowed the development of salt and

drought tolerance in cultivar CR-5272 [9]. Nevertheless, such a trait was useless for farm-
ers and breeders who no longer use that variety for planting or breeding programs. It was
also not a good trait for optimization because salt tolerance can be a multigenic trait. We
investigated Lazarroz and developed its protocols for gamma radiation variability [14].
Lazarroz is a commercial cultivar used and preferred by farmers, representing 45% of
planted rice in Costa Rica [23]. The present work resulted in an optimized in vitro gamma-
ray (60Co) on the ACC2 mutagenesis system based on APPs selection from a recalcitrant
Costa Rican Lazarroz FL cultivar. This trait will help breeding programs to manage
weeds.
The selected tissue and fluazifop-P-butyl dose were vital in optimizing the mutation
system. We were aware that selection on calli reduces time, but we preferred using regen-
erated vitroplants for optimization because of the following. We used calli browning to
calculate mortality, but the interval was not as accurate as vitroplants. The LD50 on calli
resulted in 6,93 mg L−1 R2 = 0,402, 1000 n, compared with a more precise LD50 of 3.771
mg for regenerated vitroplants. We believe browning triggered by radiation and tissue cul-
ture can have an oxidative background noise that masks the accurate lethal dose [24,25].
Our second issue was that calli regeneration was problematic, having nonbrown calli un-
able to regenerate after irradiation and stress selection; 139 calli that survived at 1 mg (97n)
and 10 mg exposition (42n) did not regenerate (Table 1).
For the first attempt we used 60 Gy irradiation on calli and direct selection with 5
mg L−1 fluazifop-P-butyl but had no regeneration after 21 d (500 n, data not presented).
Direct calli selection is an option with a recovery time after irradiation and a low selection
agent dose to allow regeneration. Other authors used such rationale in rice but with the
CRISPR/Cas9 technique at a maximum amount of 2 µM of a similar molecule haloxyfop-
R-methyl (0,654 mg L−1) for calli selection and further validation in a greenhouse of 150
mg L−1 [21,26]. In maize (Zea mays L.) and other grass, such as Paspalum vaginatum, selec-
tion was possible on calli with a different molecule acting on ACC2 at 10 µM of sethox-
ydim [27-29].
In our case, a recovery step was preferred on regeneration medium for 21 days when
having green calli and subsequent selection with fluazifop-P-butyl at 5 mg L−1. The ra-
tionale is that investing 21 days in this previous step guaranteed predictable regeneration.
The putative mutants had a second selection round that helped us better select stable mu-
tants linked to tolerance. We considered a mutation stable when micropropagation re-
sulted in 100% clonal offspring with tolerance to a higher concentration of the herbicide,
10 mg L−1. We had 100 mutants, but only one had clonal offspring resistance. The plant
had one mutation in the expected domain that has never been reported to be linked with
APPs tolerance, T2222I/T2222M. It seems that such a putative mutation can potentially be
a real linked mutation because of the nature of the mutation and the dose used. Threo-
nine's uncharged polar nature differs from the hydrophobic nature of isoleucine or methi-
onine. The tolerance at 150 mg/L is the dose of selection used by other authors linked to
ACCase mutations [21]. New mutations are being discovered, such as I1879S, P1927Y and
W2097G, very near our site of mutation [30]. However, we still cannot fully validate such
a hypothesis because tolerance could also result from another detoxification mechanism,
and we have not yet studied detoxification mediated by cytochrome P450, glutathione S-
transferase, reduced absorption or reduced xylem or phloem translocation [31,32].
The proposed system of using APP tolerance could allow the selection of small ge-
nomic mutations of approximately 1 per 1000 bp, which seems to be valid and a starting
point for working with cultivars that farmers are using. We foresee using whole genome
Research Square, 2022. Preprint 8 of 11

sequencing and analysis of the offspring of our mutant to accurately understand the mu-
tation percentage and potential to create variability for the breeding program.
Our finding of using seeds instead of calli, which improved our method from 1 pu-
tative plant tolerant to APPs to 31 plants, could help create variability for even more
difficult tissue culture cultivars for rice and other plants. The tissue was consistently dam-
aged as the gamma dose increased, having brown nonembryogenic calli, which is com-
mon in stressed indica rice cells [33]. Having a window between 50 and 350 Gy also allows
flexibility in the kind of mutations resulting from the DNA damage response (DDR) as
well as the desired or required dose for a given trait [34].

4. Materials and Methods

4.1. Fluazifop-P-butyl toxicity to embryogenic calli

Embryogenic calli induction was achieved as previously described starting with
seeds followed by disinfection with 4% (v/v) NaOCl and water [14]. The calli induction
media was composed of mineral salts and vitamins as described by Murashige and Skoog
(MS), with 20 gL−1 sucrose, 0.1 gL−1 hydrolyzed casein and 2.5 mgL−1 2,4-dichlorophenox-
yacetic acid (2,4-D). The pH was adjusted to 5.8 with 1 N NaOH or 1 N HCl, and 5.4 gL−1
Gelzan® was added as a gelling agent.
To determine the median lethal dose (LD50) of fluazifop-P-butyl, 200 calli per treat-
ment were subcultured into 0, 1, 10, and 100 mgL−1 fluazifop-P-butyl in basal regeneration
medium supplemented with 0.5 mg L−1 NAA + 3 mg L−1 6-BA. All previously mentioned
chemicals were supplied by Phytotechnology Laboratories® (Shawnee Mission, KS, USA).
An autoclave (1.2 ATM. cm−2 and 121 °C for 30 min) was used for the sterilizing medium
and further dispensed on 94 × 16 mm vented polystyrene Petri dishes (Greiner Bio-One,
Fisher Scientific, Waltham, MA, USA) in a laminar flow chamber. Calli browning rates
(brown or necrotic/total calli) were recorded as response variables and analyzed in a com-
pletely randomized design with a generalized linear model with a Poisson distribution
and logit link function. Lethal doses were calculated using probit analysis on IBM SPSS
version 27 [35] based on calli browning rates.

4.2. Fluazifop-P-butyl toxicity to vitroplants

The determination of the toxicity of fluazifop-P-buty for vitroplants was calculated
based on regenerated vitroplants from embryogenic calli as described next. Embryogenic
calli obtained as described in 4.1 were transferred to regeneration medium as described
by Sudhakar et al. [36] and were constituted by MS mineral salts and vitamins, 20 gL−1
sucrose and 0.3 gL−1 hydrolyzed casein supplemented with 0.5 mg L−1 NAA + 3 mg L−1 6-
BA. To determine the median lethal dose (LD50), the 42-day regenerated vitroplants were
subcultured into regenerated plants containing 0, 1, 4, 5, 10, 25 and 100 mg L−1 fluazifop-
P-butyl. After adding the growth regulators NAA and BA, the pH was adjusted to 5.8
with 1 N NaOH or 1 N HCl, and 5.4 g L−1 Gelzan® (Phytotechnology Laboratories®, Shaw-
nee Mission, KS, USA) was added as a gelling agent. All previously mentioned reagents
were supplied by Phytotechnology Laboratories® (Shawnee Mission, KS, USA). After
properly dissolving the gelling agent, 60 mL of media was dispensed on 475 mL polypro-
pylene WNA Deli Containers supplied by Phytotechnology Laboratories® (Shawnee Mis-
sion, KS, USA), and afterward, media was sterilized at 1.2 ATM.cm−2 and 121 °C for 30
min. Necrotic plants were considered dead after transplanting into fresh medium with no
fluazifop-P-butyl for 100 days and remained necrotic. Lethal doses were calculated using
probit analysis on IBM SPSS version 27 [35] based on calli browning rates.

4.3. Gamma irradiation of embryogenic calli

Embryogenic calli irradiation was achieved at 60 Gy with an Ob-Servo Ignis type
gamma irradiator with 24 cobalt 60 source pencils (Institute of Isotopes Co, Ltd., Budapest,
Research Square, 2022. Preprint 9 of 11

Hungary). We tested 1000 embryogenic calli exposed to 60 Gy and transferred them to

basal regeneration medium supplemented with 0.5 mg L−1 NAA + 3 mg L−1 6-BA. A total
of 8000 regenerated vitroplants were exposed to the same medium containing 5 mg L−1
fluazifop-P-butyl for 21 days. The putative tolerant vitroplants had 21 days of recovery in
medium with no selection agent, followed by another round of selection of a higher dose
of 10 mg-L fluazifop-P-butyl. The tolerant plant was taken to the greenhouse for acclima-
tization, grown until the 3-5 leaf stage and exposed to 150 ml/L spray fluazifop-P-butyl to
validate tolerance, similar to Liu et al. [21].

4.4. Gamma irradiation of seeds

Seed irradiation was achieved at 0 to 500 Gy with an Ob-Servo Ignis type gamma
irradiator as described above. We tested 100 seeds per dose, followed by callus induction
and regeneration as described previously. Selection of the putative mutants was per-
formed as described in 4.3.

4.5. Molecular markers

A NucleoSpinTM Tissue Kit Macherey-Nagel (Düren, Germany) was used for DNA
extraction from 1 mg of nonirradiated lyophilized leaf tissue of Lazarroz FL. Thermo
Fisher K1071 (Vilnius, Lithuania) was used for the subsequent PCR following the recom-
mendations of the manufacturer. The primers used in this study are as follows: for 1844
bp amplification of ACC2 Exon 32, ACC2-DF 5′-GGATCATTTGGCCCAAGGGA-3′ and
ACC2-DR 5′-AGGGCTTGCAAATCTGAGCT-3′; for 1465 bp amplification of ACC2 Exon
32, ACC2-F 5′-GTGCTCGAATTGGCATAGCAG-3′ and ACC2-R 5′-CGTGAT-
TCTTCCCAGTCCACA-3′. The PCR master mix consisted of a mixture (50 µL) containing
1X Dream Taq Master mix Thermo Fisher (Vilnius, Lithuania), 20 µM of each primer, and
5 µL of DNA (50 ng/µL). The thermocycling program was 95 °C for 5 min, 40 cycles at 95
°C for 45 s, 55 °C for 100 s and 72 °C for 1 min, and a final cycle of 72 °C for 7 min.
PCR and sequencing of the matK and rbcL genes of the mutants were performed as
reported for the control sequences MZ558335 and MZ558334 [14].

5. Conclusions
In our previous work [14], we suggested the potential of gamma radiation to trigger
innovation of rice lines used by farmers. Here, we proved the model with a specific trait
based on chemical selection and showed the potential way to start searching for other
mutations of interest on desired traits for commercially used varieties.

Author Contributions: Conceptualization, A.H.-S., D.M-N., J.P., and A.G.-A.; writing—original

draft preparation, A.H.-S., J.P. and A.G.-A.; writing—review and editing, A.H.-S., J.P., A.G.-A.,
F.E.B, and A.A.-E.; visualization, A.H.-S. D.M-N. and J. P; investigation W.V.-S. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the Research Vice-Rectory of TEC, Costa Rica, project num-
ber 1510-1022.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.Ins
Conflicts of Interest: The authors declare no conflicts of interest.

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Article

NTH2 1271_1272delTA Gene Disruption Results in Salt

Tolerance in Saccharomyces cerevisiae
Alejandro Hernández‐Soto 1,*, José Pablo Delgado‐Navarro 2, Miguel Benavides‐Acevedo 3, Sergio A. Paniagua 4
and Andres Gatica‐Arias 3,5,6

1 Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE), Instituto Tecnoló gico de Costa Rica,
Universidad Nacional, Universidad Estatal a Distancia, Cartago P.O. Box 159‐7050, Costa Rica
2 Costa Rica Institute of Technology, Biology School, Biotechnology Research Center (CIB), Cartago P.O. Box

159‐7050, Costa Rica; [email protected]

3 Laboratory of Plant Biotechnology, School of Biology, University of Costa Rica,

San José P.O. Box 11501‐2060, Costa Rica; [email protected] (M.B.‐A.);

[email protected] (A.G.‐A.)
4 Centro Nacional de Alta Tecnología CeNAT, Laboratorio Nacional de Nanotecnología LANOTEC,

San José P.O. Box 1174‐1200, Costa Rica; [email protected]

5 Research Center in Cellular and Molecular Biology (CIBCM), University of Costa Rica, San José P.O. Box

11501‐2060, Costa Rica

6 Programa de Posgrado en Biología (PPB), School of Biology, University of Costa Rica, San José P.O. Box

11501‐2060, Costa Rica

* Correspondence: [email protected]

Abstract: Trehalose is a common energy reservoir, and its accumulation results in osmotic protec‐
tion. This sugar can accumulate through its synthesis or slow degradation of the reservoir by treha‐
lase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible
Citation: Hernández‐Soto, A.;
for 75% and 25% of the enzymatic metabolism. We were interested in the loss‐of‐function of both
Delgado‐Navarro, J.‐P.;
enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for mi‐
Benavides‐Acevedo, M.; Paniagua,
S.‐A.; Gatica‐Arias, A. NTH2
nor metabolic degradation of this sugar. It was believed that losing its functionality results in lim‐
1271_1272delTA Gene Disruption ited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85
Results in Salt Tolerance in M NaCl after independent nth2 1271_1272delTA mutation by CRISPR/Cas9 technology, compared
Saccharomyces cerevisiae. Fermentation with nth1 893_894insT and wild type. We suggest that this yeast model could give clues to breeding
2022, 8, 166. https://doi.org/10.3390/ commercial yeast resulting in non‐GMO salinity‐tolerant strains.
fermentation8040166
Keywords: mutation; precision biotechnology; stress tolerance; osmotolerance
Academic Editor: Alessandro
Robertiello

Received: 17 January 2022

Accepted: 30 March 2022
1. Introduction
Published: 5 April 2022
Trehalose (α‐D‐glucopyranosyl‐α‐D‐glucopyranoside) is a stress‐tolerance‐related
Publisher’s Note: MDPI stays neu‐ sugar [1]. This nonreductive disaccharide, composed of two molecules of D‐glucose, is
tral with regard to jurisdictional
also common in the metabolism of bacteria, fungi, plants, and some invertebrate animals
claims in published maps and institu‐
[2–4]. Trehalose protects against structural disorders during water removal. It has been
tional affiliations.
suggested that the hydroxyl groups of trehalose form hydrogen bonds with polar lipids
and proteins instead of water, protecting these hydroxyl groups from structural disorder
during water removal [5]. Trehalose can also work indirectly by stabilizing heat shock
proteins during the refolding of damaged proteins [6]. Trehalose acts as a cryoprotectant
Copyright: © 2022 by the authors. Li‐
censee MDPI, Basel, Switzerland.
to avoid aggregation by stabilizing proteins in a chaperone‐like manner [5,7–10].
This article is an open access article
The intracellular accumulation of this disaccharide is due to the trehalose synthesis
distributed under the terms and con‐ complex: TPS1, TPS2, TPS3, and TSL1 [11]. However, the NTH1, NTH2, and ATH1 genes
ditions of the Creative Commons At‐ are responsible for degrading trehalose and are also positively regulated under stress con‐
tribution (CC BY) license (https://cre‐ ditions, creating a metabolic cycle of carbohydrate regulation whose understanding is still
ativecommons.org/licenses/by/4.0/). controversial [12,13]. A high intracellular concentration of this molecule results in

Fermentation 2022, 8, 166. https://doi.org/10.3390/fermentation8040166 www.mdpi.com/journal/fermentation

Fermentation 2022, 8, 166 2 of 15

protecting the structures of biomolecules and the blockage of proteases while allowing
better recovery under stress [12,14,15]. Otherwise, the excessive accumulation of trehalose
obstructs the active transport of glucose and inhibits growth in yeast [1,16].
Saccharomyces cerevisiae was used as our model organism for a proof‐of‐concept study
to achieve salt‐tolerant phenotypic traits using CRISPR/Cas9 editing and independent dis‐
ruption of NTH2 compared to wild type and NTH1 disruption. A CRISPR‐derived mutant
is considered conventional in many legal frameworks like Brazil, allowing further breed‐
ing of commercial strains with no regulatory constraints [17]. We avoided other tech‐
niques such as homolog recombination because it could result in a genetically modified
organism (GMO) limiting its industrial use.
Three enzymes are responsible for trehalose degradation in yeast: NTH1 and NTH2
are cytosolic neutral pH orthologs, while the third ortholog, ATH1, is a vacuolar acid pH
enzyme [12,18]. The neutral trehalases NTH1 and NTH2 are responsible for 75% and 25%
of the intracellular hydrolysis of carbohydrates, respectively [19]. We selected salt toler‐
ance since yeast tends to decrease in viability under sodium chloride osmotic stress con‐
ditions. The viability of S. boulardii decreased at 0.4 M NaCl [20], and S. cerevisiae showed
decreased viability at 0.68 M NaCl (4% m/v) [21].
Previous research on trehalases has dismissed the importance of NTH2 because of its
low expression and has instead focused on NTH1 [7,22,23]. It was proposed that NTH1 is
the only protein that performs hydrolysis activity but that it works together with NTH2
for hydrolysis recovery after exposure to high temperatures [14,15,24]. The NTH1 and
NTH2 genes encode functional trehalases, with NTH2 playing a protagonist role in the
stationary phase [18]. It has also been proposed that eliminating the NTH1 gene and over‐
expressing tps1 (trehalose‐6‐phosphate synthase) increases trehalose and improves sur‐
vival in high ethanol concentrations [7,22]. The deletion of all three of these enzymes re‐
sults in intracellular trehalose accumulation and improved salt stress recovery [12]. The
triple deletion of trehalases in yeast leads to tolerance of high concentrations of ethanol
and high temperatures and higher viability after freezing [25]. Eliminating the NTH1 and
NTH2 genes together in S. cerevisiae results in up to 180 days of resistance under stress
conditions [26]. However, it seemed that eliminating the NTH2 gene resulted in no salt
tolerance [13]. Finally, accumulating trehalose resulting from neutral trehalases being
knocked out then affects the growth of yeast [16].
In this article, we report a CRISPR/Cas9‐specific NTH2 1271_1272delTA disruption in
S. cerevisiae that results in the capacity of the yeast to grow in 0.85 M NaCl and tolerate
1.2M NaCl in comparison with non‐mutant and mutant NTH1 893_894insT strains. We
propose that disrupting NTH2 alone with our design in yeast can trigger stress tolerance,
although the mechanism remains unknown. To our knowledge, there is no formal report
linking the disruption of NTH2 alone to stress tolerance in S. cerevisiae. The knowledge
generated herein is potentially valuable to improve industrial yeast or serve as a model to
develop osmotic stress tolerance in other organisms using a similar rationale.

2. Materials and Methods

2.1. The Strain, Medium and Growth Conditions
S. cerevisiae CEN.PK2‐1C (MATa; his3D1; leu2‐3_112; ura3‐52; trp1‐289; MAL2‐8c;
SUC2) was used for all experiments. Cultures were grown in YPD medium (2% yeast ex‐
tract, 1% peptone, and 2% dextrose), YPD agar (2% yeast extract, 1% peptone, 2% dextrose,
and 2.2% agar) or YPAD (2% yeast extract, 1% peptone, 2% dextrose, and 40 mg/L adenine
hemisulfate) at 30 °C at 30 °C at 200 rpm on an orbital shaker (Digisystem Laboratory
Instruments Inc.). All reagents were purchased from Thermo Fisher Scientific® (Thermo
Fisher, Carlsbad, CA, USA).
Fermentation 2022, 8, 166 3 of 15

2.2. Preparation of the CRISPR Plasmid

Plasmid bRA89, containing Streptococcus pyogenes Cas9, a single guide scaffold, and
hygromycin B resistance, was purchased from Addgene [27]. The single guide targeting
the gene NTH1 (YDR001C, strain ATCC 204508 / S288c) and NTH2 (YBR001C, strain R64‐
1‐1.80) was designed using the CRISPRdirect Platform [28]. Recognition of the single
guide insertion for the NTH2 gene (5′‐TGCTATTAAAGAATATAAAG [AGG]‐3′) and
NTH1 (5′‐GGTTACCCTTATGCTGTTCC [TGG]‐3′) were cloned into bRA89 at Genscript,
just below the RNA scaffold section, using BplI restriction sites. Sanger sequencing con‐
firmed the insertion of the single guide section next to the 5′ end of the RNA scaffold.
More details are provided in the Supplementary Materials (Figure S1, Figure S2).

2.3. S. cerevisiae Competent Cell Preparation and Transformation

Competent cells were prepared and transformed using the lithium acetate (LiAc)
method with the following modifications [29]. An aliquot of approximately 1×107 cells/mL
(16 h culture) was inoculated in 50 mL of YPD medium and incubated until reaching an
OD (600 nm) of 0.5. Then, the biomass was harvested by centrifugation (4000 rpm) for 10
min at room temperature, washed twice in 10 mL of sterile distilled water, and resus‐
pended in 1.5 mL of sterile lithium acetate buffer (1 volume of TE 10X buffer, pH 7.5; 1
volume of 1 M 10X LiAc, pH 7.5 and 8 volumes of distilled water). Freshly prepared com‐
petent yeast cells (200 μL) were mixed with 1μg of the final plasmid and combined with
200 μg of denatured Salmon Sperm DNA (Thermo Fisher, Carlsbad, CA, USA) and 1 mL
of fresh PEG buffer (8 volumes of PEG 3350 50% and 1 volume of TE 10X buffer, pH 7.5; 1
volume of 1 M LiAc 10X, pH 7.5). The solution was incubated at 150 rpm for 30 min at 25
°C and then heated to 42 °C for exactly 15 min. Subsequently, the solution was resus‐
pended in 200 μL of YPAD medium and incubated for another 45 min at 25 °C. Finally,
the cells were cultured on YPD agar plates supplemented with hygromycin B Phytotech‐
nology Laboratories® (Shawnee Mission, Kansas, USA) at 500 μg mL−1 for 72 h at 28 °C in an
incubator.

2.4. Selection and Sequencing Confirmation of Mutants

Thirty (30) randomly selected strains were grown on YPD overnight media. Subse‐
quently, the manufacturer’s instructions obtained genomic DNA using the ReliaPrepTM
gDNA Tissue Miniprep System (Promega, USA). Specific primers flanking the target
sgRNA site of the NTH2 gene were designed and named nth2f: 5’‐GCAA‐
GAGGTATGGTGGAGCA‐3’ and nth2r: 5’‐TTCAGCTAGCTCCTCCCAGT‐3′ (Tm 55 °C;
539 bp); while NTH1 primers flanking the target sgRNA site were also designed and
named nth1f: 5’‐ACCCCCGGTTTACTAGCATTG‐3’ and nth1r: 5’‐TAAGGTAAC‐
GCCGTGTTTCGA‐3’ (Tm 55 °C; 528 bp). Sanger sequencing of PCR products was per‐
formed in Macrogen at Rockville, MD, USA, to confirm the mutation and absence of off‐
targets. We selected two isolates with the same mutation on the NTH2‐disrupted gene and
intact NTH1 gene, named nth2 1271_1272delTA. Similarly, we selected two isolates with
the NTH1‐disrupted gene and intact NTH2 gene, named nth1 893_894insT.

2.5. nth1 893_894insT and nth2 1271_1272delTA Strain Phenotypes

The methylene blue staining technique allowed examination of the viability of the
wild‐type and mutated yeast strains in 0.85 M NaCl versus the control (0 M NaCl) on
Potato Dextrose (PD) (20%Potato, 2% dextrose) for 48 h at 28 °C. Growth curves of the
samples were generated with 0 and 0.85 M NaCl in the YPD medium. The test was carried
out with a SPECTROstarNANO plate reader from BMG LABTECH at 200 rpm and 30 °C
for 24 h, with four repetitions per variant. The growth curve comparisons resulted from
T‐test analysis with and without saline stress conditions of the wild‐type and mutated
strains. The comparison also consisted of seriated dilutions on agar PD plate‐based com‐
parison after two weeks of growth under stress (0.85 M and 1.2M NaCl). Trehalose content
Fermentation 2022, 8, 166 4 of 15

was extracted following the protocol described by Divate et al. [22] with the following
variations. The cells were grown after 0.85 M NaCl versus the control (0 M NaCl) on PD
for 48 h at 28 °C, collected by centrifugation at 7000× g for 5 min and dried at 100°C for 12
h. A pellet of 50mg was mixed with 1 mL ethanol (99.5%) and incubated in a boiling water
bath for 1 h. HPLC‐RID determined the content, P‐SA‐MQ‐006 provided by CITA‐UCR,
as follows: The ethanolic extract was centrifuged and filtered through a 0.20 μm regener‐
ated cellulose micropore (17761‐Q, Minisart‐RC15®, Sartorius AG, Göttingen, Germany),
the filtrate was collected in a 2 mL vial for HPLC. AC Chromatographic separation was
performed using Agilent Technologies 1260 Infinity liquid chromatograph equipped with
Suplecogel 8Ca high resolution column (300 mm × 7.8 mm, 8 μm, PN 59247‐U), quaternary
pump (G1311B), column compartment (G1316A), automatic liquid sampling module
(ALS, G7129A) and refractive index detector (G1362A) (Agilent Technologies, Santa Clara,
CA, USA). The mobile phase consisted of ultrapure water (type I, 0.055 μS cm−1 at 25 ◦C,
5 μg L−1 TOC) obtained using A10 Milli‐Q Advantage and Elix 35 purification system
(Merck KgaA, Darmstadt, Germany). Solvent flow, column compartment temperature,
detector cell temperature, and injection volume were constant during the elution at 0.40
mL min−1, 80 °C, 40 °C, and 10 μL, respectively. The area under the curve (AUC) of the
trehalose signal in the samples was interpolated in the calibration curve of the certified
reference standard (Sigma‐Aldrich, PHR1344‐500 mg), in a concentration range of 0.025
to 0.25 g/100mL.

2.6. Statistical Software

Minitab v.19.1.1 supported statistical analysis and RStudio v.1.1.423 was used for vis‐
ualization.

2.7. Scanning Electron Microscopy (SEM)

Wild‐type and mutated yeast grown in 0.00 and 0.85M NaCl and fixed with Kar‐
novsky fixative (2.5% w/v of glutaraldehyde, 0.1 M of paraformaldehyde in phosphate
buffer pH 7.4, during 48 h at 4 °C) were mounted on carbon tape and sputtered with gold
using a Denton Vacuum Desk V sputter system at 20 mA for 300 s. Images were taken
using a JSM‐6390LV (JEOL, Tokyo, Japan) scanning electron microscope with an acceler‐
ating voltage of 15 kV under high vacuum. Scanning electron microscopy images at dif‐
ferent resolutions of the wild‐type and NTH2‐mutated yeasts and subsequent cell area
calculations were analyzed with ImageJ version 1.52p.

2.8. Transmission Electron Microscopy (TEM)

Wild‐type and mutated yeast grown with and without NaCl were treated with Kar‐
novsky fixative for six days. The yeast was then centrifuged at 3000 rpm for 5 min and
rinsed with 0.1 M phosphate buffer solution (pH 7.2) for three 15 min wash/centrifugation
cycles. The yeast samples were then stirred for one hour in 2% w/v OsO4 in 0.1 M phos‐
phate buffer solution, followed by three wash cycles as per the previous step but with
type 3 water instead of buffer. The yeasts were finally isolated via centrifugation at 3000
rpm for 10 min and subsequently embedded in a solution of agar/agarose 4% w/v in a hot
water bath (50 °C). After cooling to 37 °C, one drop of the solution was added to an Ep‐
pendorf tube containing 100 μL of the yeast sediment and mixed thoroughly. After cool‐
ing, the solids were removed and cut into 3 mm3 segments. Dehydration was performed
by rinsing in an ascending gradient of acetone (30% to 100% v/v) followed by infiltration
with Spurr resin: acetone 50:50 overnight and three successive infiltrations with pure
Spurr resin for two hours each. The resulting solids were transferred to BEEM embedding
capsules. Polymerization was achieved in an oven at 70 °C for 24 h. Ultrafine segments
(approximately 70 nm) were cut with a Leica EM UC7 ultramicrotome with a diamond
knife and supported on Cu TEM grids (200 mesh). The sections were stained with 4% m/v
uranyl acetate in 50% v/v ethanol for 15 min and washed five times in DI water, followed
Fermentation 2022, 8, 166 5 of 15

by soaking for 10 min in Reynold’s stain and rinsing five times in water. Once dry, the
grids were mounted in a JEOL JEM 2011 TEM and observed at 120 kV at magnifications
of 3000–20,000×.

3. Results
3.1. Confirmation of NTH1‐NTH2 Gene‐Mutated Cells
NTH1 and NTH2 disruption were confirmed in the randomly selected strains. We
selected two isolates of NTH2 mutant strains named nth2 1271_1272delTA containing the
same mutation, a deletion of two bases, TA, located at positions 1271‐1272, three bases
downstream from the PAM site expected. A PCR of the target sequence (Figure 1A) and
Sanger sequencing confirmed that the deletion was present in only the strains treated with
the CRISPR/Cas9 technique directed through the single guide RNA complementary to
section 1258 to 1276 of the NTH2 compared with the chromosome 2 sequence. Sequencing
also confirmed that the open reading frame disruption results from forming the TAA tri‐
plet in the respective open reading frame, creating a stop codon at amino acid 424 (Figure
1B). Additionally, the integrity of the NTH1 gene was verified in the NTH2‐mutated strain
by PCR and sequencing of the specific mutation zone. The S. cerevisiae NTH1 gene was not
altered due to CRISPR/Cas9 genome editing in the strain nth2 1271_1272delTA.

Figure 1. Confirmation of NTH2 gene disruption in S. cerevisiae by CRISPR/Cas9. (A). Amplification

of the segment containing the expected mutation site on a 1% m/v agarose gel; (B). Sequencing of
the S. cerevisiae NTH2 gene. In the box, it can be observed how the deletion 1271_1272delTA caused
the Y424X mutation in the nth2 1271_1272delTA strain. Image created with BioRender.com (accessed
on March 30th, 2022).

The NTH1 mutant strains named nth1 893_894insT, consisted of three strains contain‐
ing an insertion of one base, T, located at a position three bases downstream from the
PAM site, as expected. The insertion results in disruption of the open reading frame three
triplets downstream TAG, resulting in a truncated 301 AA protein with SWW* instead of
PGGR (Figure 2).
Fermentation 2022, 8, 166 6 of 15

Figure 2. Confirmation of NTH1 gene disruption in S. cerevisiae by CRISPR/Cas9. (A). Amplification

of the segment containing the expected mutation site on a 1% m/v agarose gel; (B). Sequencing of
the S. cerevisiae NTH1 gene revealed the insertion of a T in position 893 resulting in a truncated 301
AA protein with SWW* instead of PGGR. Image created with BioRender.com (accessed on March
30th, 2022).

Mutation on the NTH1 and NTH2 genes showed disruption and absence of the active
sites for a putative first ORF protein translated, specifically amino acid residues D478,
E674 for NTH1, and D507 and E703 for NTH2. The nth2 1271_1272delTA may produce a
small truncated 423 amino acid protein instead of the complete enzyme, lacking biochem‐
ical function. Similarly, the insertion in NTH1 results in a knockout of three triplets down‐
stream of TAG, resulting in a truncated 301 AA with no disaccharide binding site. The
truncated sequence of mutants nth1 893_894insT and nth2 1271_1272delTA may still con‐
tain the Ca2+ binding domain if the three‐dimensional conformation is unchanged.
When comparing the resulting putative Open Reading Frames, we noted that phos‐
phorylation activation of the enzymes remains in the amino terminal that corresponds to
the first disrupted ORF of both nth1 893_894insT and nth2 1271_1272delTA. A putative,
predicted second ORF corresponding to the carboxyterminal end is different for each mu‐
tant. In the case of nth1 893_894insT, the second ORF may have all the binding sites (R302,
N346, E424, R473, G476), active sites (D507, E703), and substrate binding sites (338‐
339WD, 384‐386RSQ) in the second ORF. In the case of nth2 1271_1272delTA, the sites may
be spliced by having one binding site (G505) and the active sites in the second ORF (D478,
E674), but most binding sites (R331, N375, R384) and all substrate binding sites (309‐
310WD, 355‐357RSQ) in the first ORF (Table 1).
Fermentation 2022, 8, 166 7 of 15

Table 1. Predicted Open Reading Frames (ORF) of mutants in comparison with the wild‐type NTH1
and NTH2 genes.
Gene Open Reading Frames
NTH1 MSQVNTSQGPVAQGRQRRLSSLSEFNDPFSNAEVYYGPPTDPRKQKQAK‐
wild type PAKINRTRTMSVFDNVSPFKKTGFGKLQQTRRGSEDDTYSSSQGNRRFFIEDVDKTLNELLAAEDTDKNYQITIEDTGPKVLKVGTANSY
GYKHINIRGTYMLSNLLQELTIAKSFGRHQIFLDEAR‐
INENPVNRLSRLINTQFWNSLTRRVDLNNVGEIAKDTKIDTPGAKNPRIYVPYDCPEQYEFYVQASQMHPSLKLEVEYLPKKITAEYVKS
VNDTPGLLALAMEEHFNPSTGEKTLIGYPYAVPGGRFNELYGWDSYMMALGLLE‐
ANKTDVARGMVEHFIFEINHYGKILNANRSYYLCRSQPPFLTEMALVVFKKLGGRSNPDAVDLLKRAFQASIKEYKTVWTASPRLDPET
GLSRYHPNGLGIPPETESDHFDTVLLPYASKHGVTLDEFKQLYNDGKIKEPKLDEFFLH‐
DRGVRESGHDTTYRFEGVCAYLATIDLNSLLYKYEIDIADFIKEFCDDKYEDPLDHSITTSAMWKEMAKIRQEKITKYMWDDESGFFFDY
NTKIKHRTSYESATTFWALWAGLATKEQAQKMVEKALPKLEMLGGLAACTERSRGPISIS‐
RPIRQWDYPFGWAPHQILAWEGLRSYGYLTVTNRLAYRWLFMMTKAFVDYNGIVVEKYDVTRGTDPHRVEAEYGNQGADFKGAAT
EGFGWVNASYILGLKYMNSHARRALGACIPPISFFSSLRPQERNLYGL
nth1
MSQVNTSQGPVAQGRQRRLSSLSEFNDPFSNAEVYYGPPTDPRKQKQAK‐
893_894in
sT, ORF1 PAKINRTRTMSVFDNVSPFKKTGFGKLQQTRRGSEDDTYSSSQGNRRFFIEDVDKTLNELLAAEDTDKNYQITIEDTGPKVLKVGTANSY
GYKHINIRGTYMLSNLLQELTIAKSFGRHQIFLDEAR‐

INENPVNRLSRLINTQFWNSLTRRVDLNNVGEIAKDTKIDTPGAKNPRIYVPYDCPEQYEFYVQASQMHPSLKLEVEYLPKKITAEYVKS

VNDTPGLLALAMEEHFNPSTGEKTLIGYPYAVSWW
nth1
MLFPGGRFNELYGWDSYMMALGLLEANKTDVARGMVEHFIFEINHYG‐
893_894in
sT, ORF2 KILNANRSYYLCRSQPPFLTEMALVVFKKLGGRSNPDAVDLLKRAFQASIKEYKTVWTASPRLDPETGLSRYHPNGLGIPPETESDHFDT
VLLPYASKHGVTLDEFKQLYNDGKIKEPKLDEFFLHDRGVRESGHDTTYRFEGVCAYLATID‐

LNSLLYKYEIDIADFIKEFCDDKYEDPLDHSITTSAMWKEMAKIRQEKITKYMWDDESGFFFDYNTKIKHRTSYESATTFWALWAGLAT

KEQAQKMVEKALPKLEMLGGLAACTERSRGPISISRPIRQWDYPFGWAPHQILAWEGLRSY‐

GYLTVTNRLAYRWLFMMTKAFVDYNGIVVEKYDVTRGTDPHRVEAEYGNQGADFKGAATEGFGWVNASYILGLKYMNSHARRALG

ACIPPISFFSSLRPQERNLYGL
NTH2
MVDFLPKVTEINPPSEGNDGEDNIKPLSSGSEQRPLKEEGQQG‐
wild type
GRRHHRRLSSMHEYFDPFSNAEVYYGPITDPRKQSKIHRLNRTRTMSVFNKVSDFKNGMKDYTLKRRGSEDDSFLSSQGNRRFYIDNVD

LALDELLASEDTDKNHQITIEDTGPKVIKVGTANSNGFKHVNVRGTYMLSNLLQELTIAKS‐

FGRHQIFLDEARINENPVDRLSRLITTQFWTSLTRRVDLYNIAEIARDSKIDTPGAKNPRIYVPYNCPEQYEFYIQASQMNPSLKLEVEYLP

KDITAEYVKSLNDTPGLLALAMEEHVNPSTGERSLVGYPYAVPGGRFNE‐

LYGWDSYLMALGLIESNKVDVARGMVEHFIFEIDHYSKILNANRSYYLCRSQPPFLTDMALLVFEKIGGKNNPNAIQLLKRAFRAAIKE

YKEVWMSSPRLDSLTGLSCYHSDGIGIPPETEPDHFDTILLPYAEKYNVTLEKLRYLYNEG‐

MIKEPKLDAFFLHDRAVRESGHDTTYRFEGVCAYLATIDLNSLLYKYEKDIAFVIKEYFGNEYKDENDGTVTDSEHWEELAELRKTRIN

KYMWDEDSGFFFYYNTKLKCRTSYESATTFWSLWAGLATEEQAKITVEKALPQLEMLG‐

GLVACTEKSRGPISIDRPIRQWDYPFGWAPHQILAWKGLSAYGYQQVATRLAYRWLYMITKSFVDYNGMVVEKYDVTRGTDPHRVDA

EYGNQGADFKGVATEGFGWVNTSYLLGLKYMNNHARRALAACSPPLPFFNSLKPSEKKLYYL
nth2
MVDFLPKVTEINPPSEGNDGEDNIKPLSSGSEQRPLKEEGQQG‐
1271_1272
delTA, GRRHHRRLSSMHEYFDPFSNAEVYYGPITDPRKQSKIHRLNRTRTMSVFNKVSDFKNGMKDYTLKRRGSEDDSFLSSQGNRRFYIDNVD
ORF1 LALDELLASEDTDKNHQITIEDTGPKVIKVGTANSNGFKHVNVRGTYMLSNLLQELTIAKS‐

FGRHQIFLDEARINENPVDRLSRLITTQFWTSLTRRVDLYNIAEIARDSKIDTPGAKNPRIYVPYNCPEQYEFYIQASQMNPSLKLEVEYLP

KDITAEYVKSLNDTPGLLALAMEEHVNPSTGERSLVGYPYAVPGGRFNE‐

LYGWDSYLMALGLIESNKVDVARGMVEHFIFEIDHYSKILNANRSYYLCRSQPPFLTDMALLVFEKIGGKNNPNAIQLLKRAFRAAIKE
nth2
MSSPRLDSLTGLSCYHSDGIGIPPETEPDHFDTILLPYAEKYNVTLEKLRYLYNEG‐
1271_1272
delTA, MIKEPKLDAFFLHDRAVRESGHDTTYRFEGVCAYLATIDLNSLLYKYEKDIAFVIKEYFGNEYKDENDGTVTDSEHWEELAELRKTRIN
ORF2 KYMWDEDSGFFFYYNTKLKCRTSYESATTFWSLWAGLATEEQAKITVEKALPQLEMLG‐

GLVACTEKSRGPISIDRPIRQWDYPFGWAPHQILAWKGLSAYGYQQVATRLAYRWLYMITKSFVDYNGMVVEKYDVTRGTDPHRVDA

EYGNQGADFKGVATEGFGWVNTSYLLGLKYMNNHARRALAACSPPLPFFNSLKPSEKKLYYL
*Binding site, NTH1 in red R302, N346, E424, R473, G476; NTH2 R331, N375, R384, G505; Active site
in green NTH1: D478, E674; NTH2:D507, E703; Substrate binding underline NTH1: 338‐339WD, 384‐
386RSQ; NTH2 309‐310WD, 355‐357RSQ; Phosphorylation site of activation NTH1 S20, S21, S60, S83,
NTH2 R49, S52, R109, S112.

3.2. Behavior of the nth2 1271_1272delTA Strain under Salinity Stress

The nth2 1271_1272delTA strain has increased tolerance and can survive in high con‐
centrations of NaCl (0.85M NaCl). We noted that the nth2 1271_1272delTA strains were
slightly smaller than the control under the light microscopy, although we were not able
Fermentation 2022, 8, 166 8 of 15

to detect any statistical difference. We validated that the cells remained the same and had
no statistical differences from the control under high osmolarity conditions and also with
the scanning electron microscopy. Yeast dimensions were determined from the scanning
electron microscopy images (Figure 3A). The sizes of the cells remained statistically and
phenotypically identical to the wild‐type strain and were not collapsed, although we
noted that they were slightly smaller (Figure 3B).

Figure 3. Analysis of the sizes of the wild‐type and nth2 1271_1272delTA yeasts. (A). Scanning elec‐
tron microscopy views of the wild‐type CEN.PK2‐1C strain and mutated strain nth2 1271_1272delTA
grown in 0 and 0.85 M NaCl; (B). Box plots representing the sizes of the wild‐type and nth2
1271_1272delTA strains of yeast under nonstress and stress (NaCl) conditions. No significant differ‐
ence was observed (p <0.05).

The nth2 1271_1272delTA strain cells were also not different under transmission elec‐
tron microscopy analysis (data not shown). Organelles and structures such as vacuoles,
nucleus, mitochondrion, cell membrane, and cell wall had no differences compared to the
Fermentation 2022, 8, 166 9 of 15

wild‐type CEN.PK2‐1C strain. We noted no organelle disruption nor structural changes

in the tolerant strain (for more details check the Supplementary Materials).
The nth2 1271_1272delTA strain was viable in a maximum concentration of 0.85 M
NaCl and gave a standard growth curve and an average growth rate of 0.2327 ± 0.0057 h−1
(Figure 4). When statistically analyzing the specific growth rates (p<0.05), the nth2
1271_1272delTA strain under stress conditions had a growth rate of 0.2179 ± 0.0061 h−1 and
behaved in the same way as the wild‐type strain under normal conditions 0.2255 ± 0.0037
h−1. The behavior of the wild‐type strain in NaCl solution presented a significant decrease
of 0.1580 ± 0.0009 h−1.

Figure 4. Growth curves of S. cerevisiae nth2 1271_1272delTA strains in the presence and absence of
osmotic stress (0.85 M NaCl) by indirect measurements of optical density at 600 nm after 20 h of
growth under stress. The curve was built with the mean of four independent samples per hour for
each condition. The growth rate was calculated for the wild type in non‐stress conditions of 0.2255
± 0.0037 h−1 versus 0.1580 ± 0.0009 h−1 in 0.85M NaCl stress, and nth2 1271_1272delTA 0.2327 ± 0.0057
h−1 versus 0.2179 ± 0.0061 h−1 in 0.85M NaCl stress.

We expected a slight salt tolerance because of the neglected reported activity of

NTH2. The latter was reasonable because the strain still had functional NTH1 enzymes
that metabolize trehalose. However, the data obtained showed that the nth2
1271_1272delTA strains were superiorly tolerant. The nth2 1271_1272delTA strains had an
average growth curve in a 0.85M NaCl liquid medium, with two isolates having the same
mutation and behavior. Instead, the wild type had a slower growth curve (Figure 4).
We mutated the homolog gene NTH1 resulting in nth1 893_894insT strains to com‐
pare it with nth2 1271_1272delTA. The nth1 893_894insT strains showed no tolerance to
0.85 and 1.2M NaCl when grown on agar plate‐based comparison after two weeks (Figure
5). Growth on 0.85M and 1.2M was detected after three days for nth2 1271_1272delTA,
mutants but it took a week for the control and nth1 893_894insT mutants. We could not
detect nth1 893_894insT salt tolerance when growing the mutant in 0.85 M NaCl liquid
media (data not presented).
Fermentation 2022, 8, 166 10 of 15

Figure 5. Agar plate comparison of yeast strains: nth1 893_894insT, nth2 1271_1272delTA, and wild‐
type after two weeks of growth under 0, 0.85 M and 1.2M NaCl stress. Note serial dilutions of yeast
starting in OD600 = 1.

We noted no difference in the intracellular content of trehalose in nth2 1271_1272delTA

in comparison with the control in the stationary phase after 48h of growth with or with‐
out stress. However, in nth1 893_894insT, the trehalose content was low with or without
NaCl. (Table 2).

Table 2. Intracellular trehalose content of yeast cells under non‐stress and stress conditions.

Strain1 Intracellular Content of Trehalose

0 M NaCl 0.85 M NaCl
S. cerevisiae CENPK2 (control) (150 ± 22) mg 100 mL−1 (118 ± 18) mg/100 mL
S. cerevisiae CENPK2 nth2
(139 ± 21) mg 100 mL−1 (107 ± 16) mg/100 mL
1271_1272delTA
S. cerevisiae CENPK2 nth1 893_894insT (34.8 ± 5,2) mg 100 mL−1 (33.8 ± 5,1) mg/100 mL
1 All cells grew with the same conditions, trehalose content determined by HPLC.
Fermentation 2022, 8, 166 11 of 15

4. Discussion
The salinity tolerance of the S. cerevisiae strain CENPK2 increased when the NTH2
gene was disrupted using CRISPR/Cas9‐mediated genome editing compared with wild
type and NTH1 disruption. The nth2 1271_1272delTA yeast strains grew in 0.85 M NaCl
with no detectable changes in behavior other than stress tolerance. Although deletion of
NTH1 and NTH1‐NTH2 together was known to result in stress tolerance, to our
knowledge, this is the first report of using the CRISPR/Cas9 technique to disrupt NTH2
alone that results in remarkable stress tolerance, as confirmed by an automated measure‐
ment system. The result is similar to a predictive model suggesting such tolerance for
NTH2 kanMX4 deletion and a neglectable tolerance for NTH1‐disrupted strains [30].
The use of CRISPR/Cas9 resulted in the expected specific mutations of the S. cerevisiae
of both NTH2 and NTH1 independent genes, three bases downstream of the PAM section
(NGG) with the S. pyogenes Cas9 enzyme [31,32]. In the case of NTH2, the double‐strand
break resulted in the deletion of two nucleotides after nonhomologous end joining
(NHEJ), introducing the “TAA” stop codon in the respective open reading frame (Figure
1). The nth2 1271_1272delTA strain had a deletion of two nucleotides, TA, located at posi‐
tions 1271‐1272, which resulted in a stop codon Y424X mutation of the NTH2 gene; nota‐
bly, no changes in the NTH1 gene were observed. The anticipated tridimensional structure
of the putative 423 amino acid Open Reading Frame (ORF) of nth2 1271_1272delTA indi‐
cated that this modified enzyme should be inactive due to the absence of the active resi‐
dues ASP507 and GLU703. Similarly, the NTH1 mutant, nth1 893_894insT contained an
insertion of one T base three bases downstream of the PAM site as expected and resulted
in an early stop codon three triplets downstream.
In this study, nth2 1271_1272delTA strains remained viable and had better tolerance
to 0.85 M NaCl than the wild‐type and nth1 893_894insT strains. The behavior and size of
S. cerevisiae nth2 1271_1272delTA did not change compared to the wild type and had nor‐
mal variability depending on the generations and growth stage. Yeast tends to shrinkage
and collapse in NaCl osmotic stress without plasmolysis, but its primary difference is in
mitochondrial fragmentation [33]. We validate that nth2 1271_1272delTA did not collapse
under stress and was identical to the control with no stress and had no fragmentation of
its organelles using transmission electron microscopy.
The nth2 1271_1272delTA strain showed exponential growth under NaCl stress con‐
ditions (0.2179 ± 0.0061 h−1) very similar to the wild‐type strain without the presence of
the osmotic agent (0.2255 ± 0.0037 h−1). The results indicate that this mutation provides the
yeast with a greater tolerance to saline conditions without significantly affecting its spe‐
cific growth rate than the wild‐type strain under salinity stress (0.1580 ± 0.0009 h−1).
Our data differ from previous reports that have proposed that eliminating the NTH2
gene resulted in no salt tolerance [13]. However, the results are not comparable due to
methodological differences of complete deletion of the gene versus a point mutation. We
used 0.85M NaCl stress from the beginning, an automatic growing system under constant
salt stress sampling every 15 min beginning at time 0 (Figure 4) and validated the toler‐
ance in the semisolid plate (Figure 5). The growth rate is also not comparable with our
control strain CENPK2 having half the growth rate in non‐stress conditions of 0.2255 ±
0.0037 h−1 versus 0.1580 ± 0.0009 h−1 in stress, and nth2 1271_1272delTA 0.2327 ± 0.0057 h−1
versus 0.2179 ± 0.0061 h−1 in stress. We believe that these automatic results and visual col‐
ony growth can capture the behavior of the mutation while reducing human error [33].
We foresee the disruption of NTH2 to provide stress tolerance in an industrial strain,
because undisrupted NTH1 can provide the metabolic equilibrium as described next.
NTH1 and NTH2 are required and regulated for fueling growth. NTH1 is phosphorylated
by Cdk1(S66) and PKA1 (S20, S21, S60, S83) to be activated, and is required for fueling
biosynthesis during S, G2, and M [34]. NTH2 contains an N terminal phosphorylation re‐
gion (R49, S52, R109, S112) and is expressed at a high level in the stationary phase after
glucose exhaustion [15]. NTH2 and NTH1 are downregulated at the exponential phase
and have a higher expression at the stationary phase [19,35]. The presence of salinity stress
Fermentation 2022, 8, 166 12 of 15

causes trehalose accumulation in S. cerevisiae and higher ethanol osmotolerance [13,25].

Heat stress (40C), CuSO4, NaAsO2, H2O2, cycloheximide (CHX) but not NaCl (1.5M) trig‐
ger the expression of NTH1 and, in practice, its disruption is unrequired for salt tolerance
[36]. It is also known that strains with NTH2 disruption, previously named YBR0106, grow
normally in YEP glycerol and were associated with increased sensitivity against heat
shock at 50°C [14], while Δnth1 grows poorly in YEP glycerol and cannot mobilize endog‐
enous trehalose [24]. NTH1 disruption may provide some stress tolerance but not related
to NaCl, as previously reported [22]. However, disruption of NTH1 may not be useful for
industry because these mutants cannot hydrolyze trehalose after returning from a heat
stress temperature of 40 °C to an average growth temperature of 30 °C [15].
We do not fully understand the tolerance since there were no detectable changes in
trehalose in the stationary phase of nth2 1271_1272delTA and the control as described next.
We also did not note differences between the mutant nth2 1271_1272delTA (107 ± 16)
mg/100 mL and the control (118 ± 18mg/100 mL) under stress conditions (0.85M NaCl).
Instead, nth1 893_894insT mutants had a stable low concentration of trehalose in stress
(33.8 ± 5.1mg 100 mL−1) and non‐stress (34.8 ± 5.2 mg 100 mL−1) conditions in comparison
with the control in stress (118 ± 18mg 100 mL−1) and non‐stress conditions (150 ± 22 mg
100 mL−1). We expected no improvement of nth1 893_894insT mutants in salt as previously
reported [12]. In addition, no important change of intracellular trehalose was previously
reported when NTH2 is eliminated under osmotic NaCl stress [12,37]. However, in previ‐
ous reports testing the relationship of NTH1 and NTH2 with pressure tolerance, the tre‐
halose content was slightly high but not statistically different in Δnth2 in stationary phase
(Δnth2=316 ± 66 μg/mg of protein, wt = 257 ± 47 μg mL−1, Δnth1=519 ± 80 μg mL−1). Notably,
Δnth2 acquired a barotolerance dismissed by the authors (Δnth2=5.0 ± 2.0, wild type = 3.4
± 1.0, Δnth1 = 0.3 ± 0.09). Instead, the authors focused on Δnth1 sensitivity although having
a higher concentration of trehalose [37]. High trehalose concentration can protect from
pressure but requires hydrolysis mediated by NTH1 because it interferes with the reacti‐
vation of the cell [37,38]. A high trehalose concentration is insufficient for stress tolerance,
but its correct use as an energy reservoir seems essential. Yeast cells subjected to 50 MPa
of pressure results in the immediate induction of the TPS1 gene (at 0′,5′,10′,15′ was 2.41,
3.92, 4.15, 4.16) triggering trehalose synthesis, while NTH1 and NTH2 are induced primar‐
ily post‐pressurization (at 0′,5′,10′,15′ was NTH1=0.41, 2.07, 2.78, 3.14; NTH2=1.07, 2.23,
3.21, 3.73) [39].
NTH2‐disrupted mutants can mobilize and use trehalose. Its mutation results in an
increased acid trehalase activity [19], meaning that the metabolic stability of the strain is
not compromised. In addition, no significant change in intracellular trehalose occurs
when NTH2 is eliminated under osmotic NaCl stress such as in our results [12,22]. The
latter also means that trehalose negatively affects growth, for overaccumulation is unfea‐
sible [16].
Interestingly, in Cryptococcus neoformans, the disruption of NTH2 alone increased the
survival ability of the yeast, but the deletion of NTH1‐NTH2 was negative for the micro‐
organism [40]. Similarly, a database of yeast mutants growth modeling completed with
kanMX4 interrupting NTH2 in haploid BY4741 background predicts tolerance to salt
stress, such as our results [30].
In our NTH2 mutation model, an alternative explanation is that nth2 1271_1272delTA
translates the gene into two ORFs considering that yeast produces alternative ORFs [41–
43]. In that case, the ORFs from nth2 1271_1272delTA may not be active but may be able to
bind the substrate. The protein fragments could transitorily protect trehalose from catab‐
olism. The first ORF, containing binding sites R331, N375, R384, 309–10WD, and 355–
357RSQ but not the active sites, could bind to the trehalose and protect the molecule from
the enzymatic activity of NTH1.
Breeding industrial yeast can result in cost‐effectiveness or reductions in fermenta‐
tion. In the case of stress tolerance traits, yeast is constantly exposed to ethanol toxicity,
oxidative stress, temperature stress, and osmotic stress, diminishing its capacity to
Fermentation 2022, 8, 166 13 of 15

produce ethanol [44,45]. Our data also show that the osmotic tolerance of the nth2
1271_1272delTA disruption strain mediated by CRISPR is superior and could represent a
solution for the fermentation industry without compromising its metabolism, phenotype,
or behavior [46,47].

5. Conclusions
The S. cerevisiae NTH2 gene was disrupted with the CRISPR/Cas9 technique, result‐
ing in a nth2 1271_1272delTA phenotypically normal strain that could grow under osmotic
stress (0.85 M sodium chloride).

Supplementary Materials: The following supporting information can be downloaded at:

www.mdpi.com/article/10.3390/fermentation8040166/s1, Figure S1: TEM images of the CEN.PK2‐
1C wild‐type strain and mutated Δnth2 strain of S. cerevisiae grown in 0 M and 0.85 M NaCl. The
scale bar represents 0.5 μm in all cases (5000× magnification). N: nucleus, V: vacuole, M: mitochon‐
drion, CM: cell membrane, CW: cell wall.; Figure S2. DNA alignment of the sequences of the NTH1
and NTH2 genes, including the gRNA position and primers used in this study. sgNTH1= single
guide NTH1, sgNTH2= single guide NTH2, nth1f=forward NTH1 primer, nth2f =forward NTH2
primer, nth1r = reverse NTH1 primer, nth2r =reverse NTH2 primer; Figure S3. Representation of
the gRNA and scaffold. A. The bRA89 plasmid with the corresponding Bpl1 sites used for replace‐
ment of gRNA. B. The final NTH2‐sgRNA with the scaffold. C. The final NTH1 sgRNA with the
scaffold.

Author Contributions: Conceptualization, A.H.‐S., J.P.D.‐N. and A.G.‐A.; writing—original draft

preparation, A.H.‐S., J.P.D.‐N., M.B.‐A., S.A.P. and A.G.‐A.; writing—review and editing, A.H.‐S.,
J.P.D.‐N., M.B.‐A., S.A.P. and A.G.‐A.; visualization, A.H.‐S., J.P.D.‐N., M.B.‐A., S.A.P. and A.G.‐A.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was financed by the Espacio de Estudios Avanzados de la Universidad de
Costa Rica (Space for Advanced Studies at the University of Costa Rica), grant number 801‐B7‐294,
UCREA.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: This article is part of the Doctoral Thesis of the first author, Doctorado en Cien‐
cia Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica (TEC), Univer‐
sidad Nacional, Universidad Estatal a Distancia, Cartago, Costa Rica. We thank Fabián Echeverría‐
Beirute of DOCINADE‐ITCR for the assistance in reviewing and editing the manuscript. We also
thank Ethel Sánchez‐Chacón for TEM sample preparation and the Laboratorio Institucional de Mi‐
croscopía of Tecnológico de Costa Rica for ultramicrotome cuts.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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8. Conclusiones y recomendaciones

- El proyecto logró establecer un sistema de inducción de variabilidad genética in vitro para la

variedad comercial de arroz Lazarroz FL utilizando Fluazifop-P-Butil como un agente de selección
en la optimización del sistema según se había planteado en el objetivo general (OG). Los detalles
del sistema se detallan a continuación.
- El medio óptimo para la inducción de callo embriogénico consiste de medio MS con 2 mg L−1 2,4-
D. La regeneración consiste en un medio MS con 3 mg L−1 BAP y 0.5 mg L−1 NAA. La primera
condición de selección de la dosis de irradiación fue 60 Gy (DL20 = 64 Gy). El uso de un sistema
de inmersión temporal (RITA®) de 120segundos cada 8h permite una regeneración homogénea
y predecible. (OE1, OE2)
- La dosis correcta del cálculo de la dosis letal de Fluazifop-P-Butil en callo fue de DL50= 6,93
mg / L (0,425 mg / L - 15,743 mg / L, R2 = 0,402, 1000n); y en vitro plantas fue DL50 3.771mg /
L (R2 = 1, 290n). (OE2)
- El sistema se optimizó mediante dos etapas de selección que consisten en una primera
exposición de pre-selección a 5 mg/L y una segunda etapa de 10 mg/L Fluazifop-P-butil. Un lote
de 8000 vitro plantas regeneradas a partir de callo irradiado a 60Gy tuvo con un único
sobreviviente. El mutante dispone de una mutación bialélica en el dominio carboxiterminal del
gen blanco ACC2 T2222I/T2222M que podría estar relacionado con la tolerancia. El mutante
también dispone de mutaciones bialélicas en los genes control G/T matK y GT/AG en rbcL. Lo
anterior es consistente con la tasa de mutación en los tres genes analizados y corresponde
aproximadamente a una mutación por cada 1000 pb. En invernadero, una planta de tres hojas
aclimatada del mutante T2222I/T2222M tolera 150mg/L de Fluazifop-P-butil (OE3).
- El sistema se mejoró mediante el uso de semillas en lugar de callo como tejido inicial in vitro
para la irradiación, lo que permitió el uso de ventanas de aplicación de rayos gama entre 50 a
350Gy. La semilla irradiada responde a la inducción de callo, regeneración y tiene un incremento
en el resultado con respecto a la versión original del sistema, con un total de 31 mutantes
putativos a partir de 4000 vitro plantas. (OG)
- En la pasantía del proyecto de Doctorado se evaluó CRISPR-CAS9 en genes homólogos de
arroz en levadura relacionados con tolerancia a salinidad. La mutación nth2 1271_1272delTA
resultó en levaduras capaces de tolerar 0.85 M de Cloruro de Sodio. El modelo es útil en la
búsqueda de mutaciones dirigidas en arroz.

80
- Se recomienda el análisis de la M2 de la planta tolerante con la mutación ACC2 T2222I/T2222M
para evaluar si la descendencia dispone o no de tolerancia, así como la secuenciación de
genoma completo de la planta mutante en M1 y/o al menos una planta M2 según sea posible.
- Se recomienda el análisis de 31 plantas putativas nuevas en invernadero y su descendencia
M2.

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10. Anexo 1. Otros artículos publicados durante el Doctorado (4)

10.1 Rojas-Vásquez, R., Zuñiga-Umaña, J. M., Abdelnour-Esquivel, A., Hernández-Soto, A., &
Gatica-Arias, A. (2022). Development of synthetic seeds in Arabica coffee embryos under
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Stuart J. Smyth. 2020. “Genetically Modified Maize Impacts in Honduras: Production and
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00221-y

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Vegetos
https://doi.org/10.1007/s42535-022-00364-9

SHORT COMMUNICATIONS

Development of synthetic seeds in Arabica coffee embryos

under aseptic and non-aseptic conditions
Randall Rojas-Vásquez1,2 · Juan M. Zuñiga-Umaña1 · Ana Abdelnour-Esquivel3 · Alejandro Hernández-Soto3 ·
Andres Gatica-Arias1

Received: 20 April 2021 / Revised: 4 February 2022 / Accepted: 7 February 2022

© The Author(s) under exclusive licence to Society for Plant Research 2022

Abstract
In many tropical countries, Coffea arabica L. is a crop of great commercial and social importance. Synthetic seeds are an
effective technique to propagate elite, hybrids, and genetically modified plant material. However, they are highly susceptible
to microbial contamination when cultivated in non-aseptic conditions which results in limiting their applications. Here we
aimed to evaluate different strategies under both aseptic and non-aseptic conditions to culture encapsulated coffee zygotic
embryos with and without organic compounds (vitamins and sucrose). For both methods, encapsulated zygotic embryos were
cultured in peat moss, soil with rice husk, and germination paper under greenhouse or plant growth culture room conditions.
Growth room conditions resulted in better germination and capsules conservation compared with the greenhouse. The cap-
sules with organic compounds cultivated in germination paper, under growth room conditions and previously sprayed with
1.5 mgl−1 carbendazim, allowed better germination percentages (93%) after 1 month of culture. On the other hand, zygotic
embryos encapsulated without organic compounds, under the same conditions, stopped their development and died with the
course of the days until obtaining 100% of their mortality.

Keywords Calcium alginate · Synthetic seeds · Hydrogels technique · Greenhouse · Growth culture room

Introduction Synthetic seeds have tremendous potential for producing

and propagating elite, hybrid, or genetically modified plants
The taste, fragrance, and stimulating properties of caffeine (Ravi and Anand 2012). An artificial or synthetic seed is
make the non-alcoholic coffee as one of the most appreciated an artificially encapsulated embryo or tissue that can form
beverage by consumers worldwide (Ivamoto et al. 2017). an entire plant, under both in-vitro and in-vivo conditions
In Costa Rica, the production and export of coffee plays an (Islam and Bari 2012). Several gelling agents help in syn-
essential role in the economy. According to ICAFE (2021) thetic seed preparation, such as ethylene glycol, DMSO,
data, the national production of coffee (fruit) was 1,769,152 Polyox, agar, and agarose. Nevertheless, the most accepted
bushels between 2020 and 2021. agent is calcium alginate due to its low toxicity, low cost,
efficient gelation, and compatibility feature (Chandrasekhara
et al. 2012).
Although synthetic seeds provide significant advantages,
* Andres Gatica-Arias such as facilitating the handling of small explants and facili-
[email protected] tate cryopreservation, synthetic seeds are highly susceptible
1
Escuela de Biología, Laboratorio de Biotecnología de to microbial contamination when cultivated in non-aseptic
Plantas, San Jose Costa Rica, Universidad de Costa Rica, conditions. Therefore, synthetic seed technology involves
P.O. Box 11501-2060, San José, Costa Rica cultivation in presence of aseptic conditions and substrates.
2
Programa de Posgrado en Ciencias Agrícolas y Recursos However, it is possible to grow these artificial seeds in non-
Naturales (PPCARN), Escuela de Agronomía, Universidad sterile substrates and/or in vivo. Currently, there are few
de Costa Rica, San José, Costa Rica studies demonstrating the culture and germination of cap-
3
Centro de Investigación en Biotecnología (CIB), Instituto sules under non-sterile conditions. For example, this method
Tecnológico de Costa Rica, P.O Box 159-7050, Cartago,
Costa Rica

13
Vol.:(0123456789)
 International Journal of
Molecular Sciences

Review
RNAi Crop Protection Advances
Alejandro Hernández-Soto 1,2, * and Randall Chacón-Cerdas 2

1 Doctorado en Ciencia Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica,
Universidad Nacional, Universidad Estatal a Distancia, Cartago P.O. Box 159-7050, Costa Rica
2 Costa Rica Institute of Technology, Biology School, Biotechnology Research Center,
Cartago P.O. Box 159-7050, Costa Rica; [email protected]
* Correspondence: [email protected]

Abstract: RNAi technology is a versatile, effective, safe, and eco-friendly alternative for crop pro-
tection. There is plenty of evidence of its use through host-induced gene silencing (HIGS) and
emerging evidence that spray-induced gene silencing (SIGS) techniques can work as well to control
viruses, bacteria, fungi, insects, and nematodes. For SIGS, its most significant challenge is achieving
stability and avoiding premature degradation of RNAi in the environment or during its absorption
by the target organism. One alternative is encapsulation in liposomes, virus-like particles, polyplex
nanoparticles, and bioclay, which can be obtained through the recombinant production of RNAi in
vectors, transgenesis, and micro/nanoencapsulation. The materials must be safe, biodegradable,
and stable in multiple chemical environments, favoring the controlled release of RNAi. Most of
the current research on encapsulated RNAi focuses primarily on oral delivery to control insects
by silencing essential genes. The regulation of RNAi technology focuses on risk assessment using
different approaches; however, this technology has positive economic, environmental, and human
health implications for its use in agriculture. The emergence of alternatives combining RNAi gene
silencing with the induction of resistance in crops by elicitation and metabolic control is expected, as
!"#!$%&'(!
!"#$%&' well as multiple silencing and biotechnological optimization of its large-scale production.
Citation: Hernández-Soto, A.;
Chacón-Cerdas, R. RNAi Crop
Keywords: RNAi; dsRNA; silencing; encapsulation; liposomes; virus-like particles; polyplex nanopar-
Protection Advances. Int. J. Mol. Sci. ticles; bioclay; regulatory
2021, 22, 12148. https://doi.org/
10.3390/ijms222212148

Academic Editor: Tomer Ventura 1. Introduction

The world is moving toward a more sustainable crop production system that requires
Received: 12 October 2021
specific and efficient tools to battle plant pathogens. RNAi can be used for such purposes.
Accepted: 4 November 2021
The molecule is used in nature, degrades quickly, can disrupt the pathogen at a genetically
Published: 10 November 2021
specific level, and can complement the current agronomic crop protection practices used
for organic, conventional, ecological, or technological production [1]. The reader may be
Publisher’s Note: MDPI stays neutral
familiar with the concept of DNA and genes located in the nucleus of eukaryote cells,
with regard to jurisdictional claims in
containing the instructions to create organic molecules, mainly proteins. RNA messenger
published maps and institutional affil-
works as an intermediator, carrying the nucleus’s message to the cytoplasm to be read
iations.
by the ribosomes to assemble the protein. The RNAi eukaryotic machinery is a complex
system for virus defense and gene expression control, sometimes called post transcriptional
gene silencing (PTGS). The system can be triggered by external specific dsRNA, resulting in
its RNA messenger being blocked before it gets to the ribosome, leaving the organism, such
Copyright: © 2021 by the authors.
as a pathogen, disarmed [2]. The extravesical delivery of dsRNA to disarm the expression
Licensee MDPI, Basel, Switzerland.
system was proven to be natural and bidirectional from plant to fungal pathogens and vice
This article is an open access article
versa cross-kingdom communication [3–8].
distributed under the terms and
Consequently, RNAi represents an opportunity to emulate or improve the natural
conditions of the Creative Commons
plant pathogen control system by providing well-designed external dsRNA [9]. Here,
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
we aimed to present advantages in crop protection mediated by RNAi. There are two
4.0/).
RNAi plant-based technologies: host-induced gene silencing (HIGS) since the 1990s and

Int. J. Mol. Sci. 2021, 22, 12148. https://doi.org/10.3390/ijms222212148 https://www.mdpi.com/journal/ijms

 Agronomía Mesoamericana
Literature review
Volumen 32(1):326-337. Enero-abril, 2021
e-ISSN 2215-3608, doi:10.15517/am.v32i1.40896
http://www.revistas.ucr.ac.cr/index.php/agromeso

AGRONOMÍA
MESOAMERICANA

The RNAi as a tool to control tropical pathogens1

El ARNi como una herramienta para el control de patógenos tropicales

Alejandro Hernández-Soto2, Fabián Echeverría-Beirute3, Tomás Guzmán-Hernández3

1
Reception: 4 de marzo, 2020. Acceptance: 20 de julio, 2020. This work was part of the Doctoral Thesis of the first author, Doctorado en
Ciencia Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica, Universidad Nacional, Universidad Estatal a Dis-
tancia, Cartago, Costa Rica, and financed by the Project “Producción de mutantes de arroz (Oryza sativa) tolerantes a herbicidas utilizando
rayos gamma para contribuir con el manejo sostenible del cultivo”. Vice-Rectory for Research of thel Instituto Tecnológico de Costa Rica.
2
Instituto Tecnológico de Costa Rica, Biology School, Biotechnology Research Center. Costa Rica. [email protected] (corresponding
author, https://orcid.org/0000-0001-9435-5117).
3
Instituto Tecnológico de Costa Rica, sede San Carlos. Apartado postal 159-7050, Florencia, Alajuela, Costa Rica. Tel: (506) 2475-5310.
[email protected] (http://orcid.org/0000-0002-7238-220X); [email protected] (https://orcid.org/0000-0002-2719-8550).

Abstract
Introduction. Sustainable farming requires new tools for the control of pathogens, since there is a constant
evolution to overcome the current biological and chemical strategies. The information provided by the transcriptomics
allows creating new possibilities to tackle the pathogens. It is possible to interrupt the genetic expression of a pathogen
and disable it using RNA interference (RNAi). Objective. To perform an analysis of an emerging technology useful
for pest control, based on RNA interference. Development. Sustainable farming is measured based through social,
economic, and environmental indicators. A key indicator of agriculture is the decrease in inputs for the control
of pathogens and the increase in their specificity. Pest control mechanisms based on RNA interference meet both
parameters. RNAi is known to have at least two functions, first for gene expression regulation, and secondly as
a defense mechanism against pathogens. Consequently, RNAi can be used to protect crops from pathogens by
developing genetically modified plants, or by the external application form of an aerosol. The RNAi aerosol is a tool
that relies on inactivating the pathogen genes and can complement other agronomic tools available for this purpose.
It is possible to design RNAi against tropical pests based on published transcriptomes, although it is necessary to
overcome limitations regarding design, degradation, and stability. Conclusion. Interference RNA methods have the
potential to be useful tools to control tropical pathogens as an alternative to achieve sustainable farming.

Keywords: sustainable farming, biotechnology, aerosol RNAi.

Resumen
Introducción. La agricultura sostenible requiere de nuevas herramientas para el control de patógenos, dado
que existe una constante evolución para sobrepasar las estrategias biológicas y químicas que se usan actualmente.
La información derivada de los transcriptomas permite crear nuevas posibilidades para controlar a los patógenos.
Es posible interrumpir la expresión genética de un patógeno e inhabilitarlo mediante ARN de interferencia (ARNi).

© 2020 Agronomía Mesoamericana es desarrollada en la Universidad de Costa Rica bajo una licencia Creative Commons
Atribución-NoComercial-SinDerivar 4.0 Internacional. Para más información escriba a [email protected] o [email protected]
Transgenic Res (2020) 29:575–586
https://doi.org/10.1007/s11248-020-00221-y ( 01234567
(0123456789().,-volV) 89().,-volV)

ORIGINAL PAPER

Genetically modified maize impacts in Honduras:

production and social issues
Diego Maximiliano Macall . Carlos Rogelio Trabanino . Alejandro Hernández Soto .
Stuart J. Smyth

Received: 20 June 2020 / Accepted: 27 October 2020 / Published online: 11 November 2020
! Springer Nature Switzerland AG 2020

Abstract Maize production is one of the most concepts and GM maize. Overall, producers have a
important activities for the Honduran economy, both positive opinion about GM maize because yields are
in terms of area cultivated and food security provided. higher than conventional maize, and adopting farmers
This article reports the results of a survey undertaken have higher incomes. A significant finding was the
to gauge knowledge, perceptions, opinions, and atti- reduction in the number of necessary pesticide appli-
tudes of Honduran farmers towards genetically mod- cations, 84% of interviewees who used GM maize did
ified (GM) maize. Data were collected from 32 maize not apply any pesticides. Farmers indicate the two
producers in 2018–19, of both conventional and GM, main reasons for using GM maize are higher incomes
in five different departments (regions) of Honduras. (48%) and ease of use of the crop (33%). Overall, GM
Results show that over 75% of interviewed farmers maize impacts in Honduras could be greater if the
have significant knowledge of basic biotechnology federal government took on a more proactive role in
knowledge dissemination and facilitation of credit
access.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s11248-020-00221-y) con- Keywords Adoption benefits ! Chemical use !
tains supplementary material, which is available to authorised
users. Economic impacts ! Farm-level evidence ! Yield
increases
D. M. Macall (&) ! S. J. Smyth
Department of Agricultural and Resource Economics,
University of Saskatchewan, 51 Campus Drive,
Saskatoon, SK S7N 5A8, Canada
e-mail: [email protected] Introduction
S. J. Smyth
e-mail: [email protected] In 2002, Honduras became the first Latin American
country to authorize the commercial cultivation of
C. R. Trabanino
Panamerican Agricultural School (Zamorano University),
genetically modified (GM) maize. Production of
Zamorano, Honduras maize is an important activity for the Honduran
e-mail: [email protected] economy (Hintze 2003); it is also responsible for
providing 26% of the calories consumed by urban
A. H. Soto
Instituto Tecnológico de Costa Rica (ITCR), Cartago,
dwelling Hondurans, and 48% of the calories con-
Costa Rica sumed by those residing in rural areas (Cruz 2013).
e-mail: [email protected]

123

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