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Obtaining double haploid (DH) populations in crossbreeding

RESEARCH
of rice cultivars (Oryza sativa L.) with indica and japonic
subspecies and evaluation of their tolerance to drought
 Elizabeth Cristo-Valdés1, María C González2, Elsa Ventura3, Aida T Rodríguez1
Unidad Científico Tecnológica de Base, Los Palacios, Instituto Nacional de Ciencias Agrícola, INCA
1

Gaveta Postal 1, San José de las Lajas, CP 32700, La Habana, Cuba

2
Departamento de Genética y Mejoramiento Vegetal, INCA, Cuba
2
Centro de Desarrollo de Productos Bióticos del Instituto Politécnico Nacional, carretera Yautepec-Jojutla
km 8, Calle CEPROBI, No. 8, Col. San Isidro, Yautepec, Morelos, CP 62731, México
[email protected]
ABSTRACT
The drought causes negative effects on the productivity of the plants and constitutes a potential danger for the food
sustainability of the population. One of the most difficult tasks for rice breeders is to increase tolerance to salinity
and drought, in addition to achieving high and stable yields. In the genetic improvement program of this precious
grain, different improvement methods are used, where biotechnological techniques have been used, using the in
vitro anther culture to shorten the time for obtaining haploid lines, which will be subsequently evaluated in condi-
tions of water deficit. For these reasons, this work was aimed to obtain double haploid lines through in vitro anther
culture of F2 hybrid populations, and evaluate them under water deficit conditions. Crosses were made between five
drought-resistant cultivars and six cultivars of good agronomic performance. Anthers of F2 plants were cultured in
vitro to evaluate callus formation in three liquid media: N6m, NL and N6-1. Then, agronomic characters and tol-
erance to water deficit were evaluated in the first generation of isogenic lines obtained. Double haploid lines that
combined drought resistance and good agronomic traits were evaluated under field conditions without watering,
only with water passes as the plant required. The use of the in vitro anther culture technique showed the high de-
pendence on genotype and culture medium. The highest values of callus formation were achieved in the NL culture
medium. New double haploid lines resistant to water deficit and showing high agricultural yield were obtained.
Keywords: rice, anther culture, genetic improvement, double haploid variants
Biotecnología Aplicada 2018;35:1501-1503
RESUMEN
Obtención de poblaciones doble haploides (DH) en cruzamiento de cultivares de arroz (Oryza sativa L.)
con subespecies indica y japónica, y evaluación de su tolerancia al déficit hídrico. Una de las tareas más
difíciles para los mejoradores de los cultivos de arroz es lograr incrementar la tolerancia a la salinidad y la sequía,
además de lograr mayores producciones. En el programa de mejoramiento genético del arroz se emplean diferentes
métodos, y el uso de técnicas biotecnológicas mediante el cultivo in vitro de anteras, el cual acorta los tiempos para
la obtención de líneas haploides, las cuales se evalúan en condiciones de déficit hídrico. Por lo tanto, el propósito
de este trabajo fue la obtención de poblaciones hibridas F2 mediante la aplicación de este método, y su posterior
evaluación en condiciones de sequía. Se efectuaron cruzamientos entre cinco cultivares resistentes a la sequía y
seis de buen comportamiento agronómico, y las anteras de las plantas F2 fueron cultivadas in vitro para evaluar
la formación de callos en tres medios líquidos: N6m, NL y N6-1. A la primera generación de las líneas isogénicas
obtenidas se le evaluó los caracteres agronómicos y su tolerancia al déficit hídrico. Las líneas dobles haploides con
valores positivos en ambos parámetros fueron evaluadas en condiciones de campo sin aniego, solo con pases de
agua según la planta lo requería. La utilización de la técnica de cultivo in vitro de anteras mostró la alta depen-
dencia del genotipo y del medio de cultivo empleado. Con el medio NL se alcanzaron los valores más altos para la
formación de callos. De esta forma, se obtuvieron nuevas líneas dobles haploides resistentes al déficit hídrico y con
alto rendimiento agrícola.
Palabras clave: arroz, cultivo de anteras, mejoramiento genético, variantes dobles haploides

How to cite (Vancouver style):

Cristo-Valdés E, González MC, Ventura E, Rodríguez AT. Obtaining double haploid (DH) populations in crossbreeding of
rice cultivars (Oryza sativa L.) with indica and japonic subspecies and evaluation of their tolerance to drought. Biotecnol
Apl. 2018;35(4):4201-6.

Introduction
The world rice production has reached 701 million tons demand, obligating to import significant amounts to
of husk rice in average yearly during the last ten years, satisfy the dietary needs [2]. Despite the rice crop na-
and a stable 150-162 million ha plantation area [1]. tional variety policy comprising more than ten cul-
It is among the top consumed food in Latin Amer- tivars of the species, all of them show limitations to
ica and an essential food with 60 kg consumed per reached their expected production potential. In fact, 1. MINAG. Instructivo Técnico Cultivo
de Arroz. Instituto de Investigaciones
capita [1] in Cuba. However, the national produc- crop yields reach 3.6 ton/ha, below the world marks de Granos. La Habana: Ministerio de la
tion in this country does not surpass the 50 % of the [3]. It is mainly caused, for instance, by the influence Agricultura; 2014.

Publicación libre de costo

para el autor
No article processing charges Biotecnología Aplicada 2018; Vol.35, No.4
Cristo-Valdés E, et al. Drought-resistant rice double haploids

of the climate, the insufficient exploitation of the opti- Table 1. Hybridization made with progenitor cultivars to generate drought-resistant
mal seeding season, deficient crop management prac- cultivars
tices, the exhausting of soil components, the impact of No.
Crossbreeding
No.
Crossbreeding
pests, soil salinity and dryness, among others [1]. Dry- (female/male) (female/male)
ness is one of the most significant since there are more 1 Amistad’82/C4 153 13 C4 153/Amistad’82
than 100 000 areas under cultivation without enough 2 Amistad’82/IR 20 14 IR 20/Amistad’82
irrigation sources [4, 5]. 3 Amistad’82/IR 36 15 IR 36/Amistad’82
Currently, nearly 80 % of all the water available 4 Amistad’82/Perla de Cuba 16 Perla de Cuba/Amistad’82
in the country are used for agriculture, and 40 % of 5 INCA LP 10/C4 153 17 C4 153/INCA LP 10
produced foods worldwide are cultured in irrigated 6 INCA LP 10/IR 20 18 IR 20/INCA LP 10
7 INCA LP 10/IR 36 19 IR 36/INCA LP 10
areas [6].
8 INCA LP 10/Perla de Cuba 20 Perla de Cuba/INCA LP 10
In this setting, submerged rice demands 16 000 m3/ha
9 IR1529-430/Amistad’82 21 Amistad’82/IR1529-430
on each cultivation cycle [1]. Current environmental
10 Moroberekan/Amistad’82 22 Amistad’82/Moroberekan
changes indicate the possibility of a future increase of
11 INCA LP 10/Amistad’82 23 Amistad’82/INCA LP 10
dryness. Therefore, it is necessary to seek for feasible
12 IR 1529-430/INCA LP 9 24 INCA LP 9/IR 1529-430
alternatives for a more efficient use of cultivation wa-
ter in order to reduce the irrigation needed due to its
limited availability [5-7]. emerged and shaken over the emasculated female pa-
In Cuba, a significant alternative to try to circum- nicles for pollination. This procedure was done for five
vent such limitations comes from obtaining rice cul- days, and then female panicles were completely cove-
tivars of low water consumption by genetic improve- red with semi-translucent paper and properly identified 2. Polón R, Castro R, Ruiz M, Maqueira LA.

ment procedures. In fact, improved yields have been with the cross breeding information and date. Prácticas de diferentes alturas de corte a
cultivo de rebrote y su influencia en el
reached almost exclusively by using traditional crop Afterwards, mature seeds resulting from cross- rendimiento del arroz (Oryza sativa L.) en

improvement procedures. breeding were harvested. They were disinfected with una variedad de ciclo medio. Cult Trop.
2012;33(4):59-62.
Noteworthy, biotechnological methods provide 1.5 % hypochlorite for 1 min, and washed several
new tools for improving the existing cultivars more times with distilled water. Subsequently, they were 3. ONEI. Sector agropecuario indicado-
res seleccionados. La Habana: Oficina
suitable and adapted to stressing culture conditions placed in Petri dishes containing distilled water and nacional de Estadística e información,
and providing potentially higher production yields [8]. incubated at 30 ºC. Approximately 10 days after ger- Cuba; 2016.

For instance, cell and haploid tissue culture guarantee mination they were transplanted to pots containing 4. Alfonso R, Ruiz M, González MC. Re-
achieving homozygosis in a single generation, thereby a mix (1:3) of Gley Nodular Ferruginous petroferric sultados del mejoramiento de arroz para

reducing considerably the improvement cycle, some- soil [14] and curated bagasse, respectively. bajos insumos de agua y fertilizantes en
Cuba 2010-2011. In: Encuentro Interna-
thing feasible through anther culture [8-11]. Two new After thirty days, seedlings were transplanted un- cional del Arroz del Instituto de Investig-
cultivars were recently obtained with this method and der field conditions into groves separated 40 cm, with ación de Granos. Memorias (4, 2011 jun.
2-6, La Habana). La Habana: Instituto de
included in the Registry of Agricultural Varieties of 30 cm between plants, and phytotechnical procedures Investigación de Granos; 2011. p. 105-7.
the Cuban Ministry of Agriculture. They show high were applied according to the agricultural cultivation
5. Cristo EV, González CM, Pérez N.
production potential, good cooking properties, resis- standard procedures [1]. Once completed their biolo- Evaluación de nuevos cultivares de ar-
tance to drought and to the incidence of piriculariosis, gical cycle, F1 plants seeds were harvested for each roz (Oryza sativa L.) en condiciones de

despite their limited extension to the cooperative agri- plant for subsequent application. bajos suministros de agua y fertilizante
en la Provincia de Pinar del Río. Cult Trop.
cultural sector and not to the industrial farming. More- In vitro anther culture of F2 plants resulting
2016;37(2)127-33.
over, the use of anther culture allows the generation of from cross-breeding between selected proge- 6. García A, Dorado M, Pérez I, Mon-
rice cultivars in just 5 out of the 10-year period when nitor cultivars
tilla E. Efecto del déficit hídrico en la
distribución de fotoasimilados en plantas
using traditional breeding methods [8, 12]. Therefore, de arroz (Oryza sativa L.). Interciencia.
this work was aimed to obtain double-haploid rice F2 plant seeds were seeded into the planting beds and 30 2010;35(1):46-54.
variants through in vitro anther culture and evaluate days later transplanted to field conditions into furrows
them for drought resistance in F2 generation plants. separated 40 cm, 30 cm distance between plants, and 7. Planos GEO. Síntesis informática sobre
impactos del cambio climático y medidas
applying phytotechnical procedures as recommended. de adaptación en Cuba. La Habana:
Materials and methods The most vigorous and phytosanitary-adequate state F2 PNUD; 2014.
plants resulting from cross-breeding, grown for 60-70 8. Pérez N. Obtención de cultivares
Hybridizations of selected cultivars resistant days, were selected and two or three panicles collected de arroz (Oryza sativa L.) resistentes a
to drought and showing good agronomic attending to having a 4-8 cm separation between the Pyricularia grisea Sacc. con buen com-
portamiento agronómico. Tesis de grado.
performance auricles of the last two leaves. The knots and pods of Mayabeque: Instituto Nacional de Cien-
Twenty-four hybridizations were made with selected the leaves were preserved to protect the leaves from the cias Agrícolas; 2012.

rice progenitor cultivars [13]. For this, a Topcross sta- contamination of the field pathogens. 9. Lassaga S, Bretón A, Gieco L, Milische
tistic design was followed, and each cultivar show- Then, collected vegetal material was processed at H, Dittrich A. Cultivo in vitro de anteras
de lino (Linum usitatissimum L.) in vitro
ing good agronomic performance was used either as the tissue culture laboratory. Panicles were disinfec- flax. Anthers cultura. Rev Cienc Docencia
female or male progenitor cultivar for crossing with ted in the outer side with 70 % ethanol solution and Tecnol. 2010;(40):215-233.
drought resistant cultivars (Table 1). Cultivars were further preserved with the lower part immersed into 10. Sahu M, Minj A, Chopkar R, Jha Z,
staggered planted under semi-controlled conditions water, and they were protected with a dark plastic bag Verulkar SB. Production of double haploid
considering their cycle and after 30 days they were at 8-10 ºC for 7-8 days. Afterwards, panicles were di- population in two indica rice (Orysa sativa
L.) cross safri-17xir-64 and mtu1010 vari-
transplanted to field conditions. Phytotechnical man- sinfected for 3 min with 1.5 % sodium hypochlorite ety. J Cell Tissue Res. 2015;5(3)5347-50.
agement was done as recommended by the agricul- and 0.5 % Tween 80 as dispersing agent, and washed
11. Carloni, EJ. Cultivo in vitro de anteras
tural cultivation standard procedures [1]. four or five times with distilled water. They were fur- como estrategia para el mejoramiento
Female progenitor panicles were collected when ther seeded in vitro as recommended [15]. genético de buffelgrass (Cenchrus ciliaris
L). Tesis de grado. Córdoba: Universidad
they emerged 50-60 % and anthers were extracted with Then, three liquid culture media were tested for Nacional de Córdoba. Facultad de Cien-
a needle. Male progenitor panicles were collected fully callus induction: N6-1 [16], N6-m and NL [15], three cias Agropecuarias; 2016.

4202 Biotecnología Aplicada 2018; Vol.35, No.4

Cristo-Valdés E, et al. Drought-resistant rice double haploids

replicates each, and 100 anthers were seeded for each Table 2. Direct and reciprocal crossings among rice cultivars resistant to drought and
replicate. Once seeded, flasks were kept in the dark for cultivars showing good agronomic performance which were able to form grains
30-60 days until microcallus formation. No.
Resistant male parental
No.
Resistant female parental
The number of callus-forming anthers per flask (female/male) (female/male)
were evaluated. Calluses 1-2 cm wide were transfe- 1 Amistad’82/C4 153 13 NS
rred to flasks containing solid minimal saline medium 2 Amistad’82/IR 20 14 NS
supplemented with 1 mg/L naphtaleneacetic acid 3 Amistad’82/IR 36 15 NS
(NAA), 2 mg/L indoleacetic acid (IAA), 4 mg/L 6-fur- 4 Amistad’82/Perla de Cuba 16 Perla de Cuba/Amistad’82
furilaminopurin (kinetin) and 30 g/L sucrose and 7 g/L 5 INCA LP 10/C4 153 17 NS
agar. The flasks were incubated at 26 ± 2 ºC and under 6 NS 18 IR1529-430/Amistad’82
7 NS 19 IR 1529-430/INCA LP 9
a 16 h light/8 h dark photoperiod.
8 NS 20 Moroberekan/Amistad’82
Green and albino plants regeneration was evaluated
after 30 to 40 days. NS: Reciprocal crossbreeding that did not generate seeds.

Selection and evaluation of double haploids female progenitor. Despite, a number of combinations
combining good agronomic characters and were obtained which could provide variability enough
drought tolerance as to generate recombinants favorable for the selec-
Water stress conditions were simulated by irrigation tion of new cultivars.
during germination, subsequently limited to water
passes every seven days just to guarantee soil mois- In vitro anther culture of F2 plants
ture equivalent to field capacity. Such conditions A differential response was detected among the diffe-
were maintained until the grains were pasty. The rent hybrid combinations attending to callus forma-
other agricultural practices were implemented as rec- tion per 100 anthers seeded (Table 3, Figure 1A and
ommended [1]. 1B). Not all the hybrids were able to form calluses,
Upon harvesting, plant height (cm) was assessed and variation was seen among those successful. The
to 10 plants randomly selected; their central spike- frequency of callus-forming anthers oscillated in the
lets were evaluated for the number of filled grains range 0-56.93 %, probably due to differences in geno- 12. Cristo E, González MC, Pérez N, Ven-
per spikelet. Additionally, 1000 grains weight (g) and type and culture media. Similar results were reported tura E, Cárdena RM, Blanco G, González
M. EDUAR LP-21. Nuevo cultivar de arroz
1-m lined-up grains weight were determined, spike- by different research groups while studying the in- (Oryza sativa L.) obtenida por cultivo
let number, and spikelets were weighed to determine fluence of different culture media in calluses forma- in vitro de antera, tolerante a los bajos
suministros de agua. Cult Trop. 2015;
yields (kg). Maturation cycle was established in days, tion [8-11, 15], which could be attributed to hetero- 36(Especial):134.
and industrial yield was estimated as the percentage zygosity in the loci controlling callogenesis.
of filled grains after every milling of a 100-g sample, The highest values were achieved in descending 13. Cristo E. Empleo del cultivo de anteras
para la obtención de genotipos de arroz
resistance to lodging, shattering and pest incidence. order by the INCA LP-10/C4 153 hybrid line in the (Oryza sativa L.) de buen comportamiento
NL medium (56.93 calluses per 100 anthers seeded), en condiciones de bajos suministros de
Statistical analysis followed by this hybrid but in the N6-1 medium
agua. Tesis de Maestría. Mayabeque:
INCA; 2004.
A statistical completely randomized design was (34.80 %) and the hybrid Amistad´82/Perla de Cuba
14. Hernández A, Pérez J, Bosch D, Castro
applied, with three replicates for callus formation and in the NL medium (45.06 %). A similar performance N. Clasificación de los suelos de Cuba
nine for regenerated plants. Data were transformed by for the other two culture media, but at lower values. 2015. Mayabeque: Ediciones INCA; 2015.
the squared root plus three eighths and statistically All these crossings included the Amistad´82 cultivar 15. Lentini Z, Martínez C, Roca WM. Cultivo
processed with the aid of Statgraphics Plus version as female progenitor. By the contrary, other crossings de anteras de arroz en el desarrollo de
5. Results were analyzed by a simple classification including this cultivar either as male or female pro- germoplasma; 2006 [cited 2016 Mach 20].
Available from: http://www.ciat.cgiar.org/
variance analysis, and means were compared by the genitor showed very low values (0-0.6 %), including ourprograms/Documents/cultivo_anteras
Tukey’s test for a 5 % error. Quantitative data were Amistad´82/IR-20, Amistad´82/C4 153, Perla de
16. Meifang L. Anther culture breeding of
subjected to Principal component analysis and multi- Cuba/Amistad´82, IR 1520-430/Amistad´82, INCA rice at the CAAS. China: Chinese Academy
ple correlation with Statgraphics Plus version 5. LP-10/Amistad´82 and Moroberekan/Amistad´82. of agricultural Science (CAAS); 1992.

Results and discussion Table 3. Callus formation per 100 anthers planted in three culture media, for F2 hybrid
plants resulting from crossings between cultivars resistant to drought and cultivars of
Hybridization between cultivars selected for good agronomic performance*
drought tolerance and good agronomic per- Hybrid combination Calluses Plants
formance (female/male) NL N6m N6m Green Albino
INCA LP 10/C4 153 56.93 a 8.23 h 34.80 d 25.36 a 0.00 h
Among the crossing which were able to produce
Perla de Cuba/Amistad’82 39.10 c 5.12 jk 20.10 f 20.16 b 10.56 b
grains (Table 2), only 42 % of all the 24 combinations
Amistad’82/Perla de Cuba 45.06 b 6.50 i 28.23 e 14.40 c 7.33 c
tested formed seeds. This made impossible to com-
Amistad’82/IR 20 0.00 p 2.30 l 0.26 p 0.00 h 0.90 fgh
plete the genetic design proposed.
Amistad’82/C4153 3.50 m 2.16 l 0.90 o 0.46 gh 3.26 d
Cultivar Amistad´82 seemed to have better combi- Moroberekan/Amistad’82 4.70 l 0.00 p 5.30 j 8.16 d 1.56 ef
natorial capacity than INCA LP-10 to form grains in INCA LP-10/Amistad’82 8.46 h 2.23 l 4.76 kl 5.35 e 1.16 fgh
artificial crossings. This coincides with previous re- IR 1529/INCA LP-9 1.20 o 0.00 p 6.08 i 3.26 ef 0.46 fgh
ports on using in vitro anther culture to generate lines Amistad-82/IR-36 0.00 p 0.00 p 0.06 p 1.70 fgh 2.63 de
resistant to piriculariosis, using cultivar Amistad´82 as IR1529/Amistad’82 16.20 g 5.30 j 0.20 p 0.33 gh 16.96 a
progenitor [8]. In our work, crossings were effective M 17.51 3.18 10.06 7.91 8.96
only when this cultivar was used as female progenitor. EEM 0.08 0.08 0.08 0.44 0.22
The cultivar Perla de Cuba was the only drought * Means sharing the same letter per column did not differ statistically, according to Dun-
resistant progenitor generating grains, when used as can’s test for 5 % error.

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Cristo-Valdés E, et al. Drought-resistant rice double haploids

Noteworthy, the INCA LP-10 cultivar generating the A B

hybrid INCA LP-10/C4 153 is a somaclone of cul-
tivar Amistad´82. Previous research on other crops
like tobacco, tomato, potato, barley, soybean, wheat,
flax and others, have found that genotype and cultu-
re media influence on diploid generation by in vitro
anther culture [9, 10, 15]. In this sense, callus for-
mation varied among the three culture media tested,
with the highest values shown in the NL. The three
media differ in salt concentration (macro and micro-
nutrients), vitamins and growth regulators, thereby
making difficult to correlate the observed effect of
each culture medium to a single component. Never-
theless, the NL medium is the only one containing
maltose instead of sucrose and containing silver ni-
trate. Other studies have shown that callus induction
from recalcitrant genotypes and the production of
green plants are enhanced by substituting sucrose
by maltose. In fact, this change has been recommen- C D
ded for callus induction, particularly from the indica
subspecies [8, 16].
On the other hand, the presence of silver nitrate di-
minishes anther senescence in recalcitrant indica rice.
This result points towards the possible inhibition of
callus formation by ethylene accumulated in the flask,
an effect probably reverted by the application of the
compound [16, 17].
Additionally, differences were found in the number
of green and albino regenerated plants (Figure 1 C and
D). The line INCA LP-10/C4 153 only formed green
plants (25.36 plants per 100 anthers seeded), followed
by the crossings Amistad´82/Perla de Cuba and Perla
de Cuba/Amistad´82. Nevertheless, the IR 1529-430/
Amistad´82 crossing showed the highest values in the
formation of albino plants. The close genetic relation-
ship between INCA LP-10 and Amistad´82 could ex-
plain the response seen. In this sense, variety C4 153 Figure 1. In vitro cultures of hybrid rice cultivars resistant to drought with cutivars of good
belongs to the japonica subspecies, which displays agronomic performance. A) Anthers seedings in culture media. B) Callus formation. C)
good performance in in vitro culture, specifically for Green plant formation. D) Albino plant formation.
anther culture [12, 16].
Regarding the formation of albino plants (Table In general, it could be mentioned that the best re-
3 and Figure 1 D), the INCA LP-10/C4 153 was the sults of callus formation from anthers of F2 plants
only crossing not forming albino plants. IR 1529-430/ were obtained with the NL culture medium and the
Amistad´82, followed by Perla de Cuba/Amistad´82, hybrid combinations: INCA LP-10/C4 153, Amis-
showed the higher numbers, coinciding with the Amis- tad´82/Perla de Cuba and Perla de Cuba/Amis-
tad´82 as parental cultivar. Previous reports indicated tad’82. Coincidently, these were the three hybrids
that albino plants (i.e., chlorophyll deficient plants) also showing the highest frequency of green plants
are frequent among cereals obtained by anther culture in descending order. Otherwise, the IR1529/Amis-
techniques. In rice, the frequency of albino plants va- tad’82 hybrid showed the highest frequency of al-
ries with the genotype, and a high rate of regenerants bino plants.
can be seen in certain cases, but the frequency can
range 10-100 % [17]. Similarly, this phenomenon is Selection and evaluation of double haploids
17. Marcías Suárez, JC. Obtención de
particularly predominant among plants resulting from integrating drought resistance and good agro- líneas dihaploides de arroz (Oryza L.) a
immature pollen of inter- or intra-specific hybrids bet- nomic performance través del cultivo in vitro de anteras de
10 poblaciones F1. Tesis de Licenciatura.
ween japonica and indica subspecies [15, 16]. Highly Seeds of plantlets obtained from regenerants of the Guayaquil: Facultad de Ciencias Agrarias,
variable results have been reported, for instance, 57 to different surviving hybrid combinations were harves- Universidad de Guayaquil; 2015.
75 green plants per 100 calluses and only 1-8 calluses ted and planted under field conditions, and their mor- 18. Sharmin S, Bari MA, Siddique NA, Rah-
per 100 anthers [18]; green plants were regenerated phological traits characterized and visually related to man MH. Effects of genotype on induction
15-30 days after culture with 33 % as the highest va- ploidy levels (Table 4). Haploid plants (n = x) are ge- of callus and plant regeneration potential
in vitro anther culture of rice (Oryza sativa
lue obtained, with only 3-9 calluses per 100 anther and nerally small, weak, stumpy and sterile. On the con- L.). Pakistan J Biol Sci. 2004;7(2):235-7.
1-6 plants per 100 calluses [19]. Differences not only trary, diploid plants exhibit favorable traits, they grow
19. Pantel JK, Subhassh N, Fougat RS.
among species attending to anthers but also among vigorous, fertile, with good development, and the po- In vitro callus induction and plantlet
cultivars of the same species have been described [15, lyploid plants further grow with exuberant flowering regeneration studies through anther
culture in two indica rice (Oryza sativa L.)
18-20], with emphasis in the significant effect of the structures, a large number of grains with long spike- varieties. Curr Trends Biotechnol Pharm.
genotype on the regeneration of green plants. lets and partial sterile grains [8, 15, 19]. 2014;8(2):152-9.

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Cristo-Valdés E, et al. Drought-resistant rice double haploids

Considering the Principal components analysis run Table 4. Regenerants evaluated per crossing, display-
with the lines displaying phenotypic traits characte- ing diploid-like morphology
ristic of diploid plants (Table 5), it was found that Resistant male parental Evaluated
No.
the two principal components C1 and C2 accounted (female/male) lines
for 90.89 % of the total variation, with C1 providing 1 INCA LP 10/C4 153 15
10 % of the total variance explained (Table 5). The 2 Perla de Cuba/Amistad’82 10
variable contributing the most to the C1 component 3 Amistad’82/Perla de Cuba 6
was agricultural yield, and in C2 contributed with up 4 Amistad’82/ C4 153 2
to 90 % of the total variation. The variable ‘filled gra- 5 Moroberekan/Amistad’82 4
ins per panicle’ was the major one in a negative sense, 6 INCA LP-10/Amistad’82 2
and ‘number of empty grains per panicle’ in a positi- 7 IR 1529-430/INCA LP-9 1
ve sense. This could indicate the usefulness of these 8 Amistad’82/IR-36 1
variable for differentiating among regenerants, due to 9 IR 1529-430/Amistad’82 1
their higher correlation values with the main axes. Total regenerated plants 42
Once represented the principal components C1 and
C2 for genotype distribution, which were mainly in- Table 5. Eigen values, contribution percentage and
fluenced by the main evaluated variables (Figure 2), total of components 1 and 2 and their correlations
there was evidenced the existence of a high genetic with principal components
variability among individual plants. This could derive Parameter
Principal components
from the diversity of the genetic material of the proge- C1 C2
nitors used for the crossing, since they have been obtai- Eigen value 80.873 80.873
ned by different genetic improvement methodologies, Contribution (%) 10.018 90.871
each contributing a differential source of genetic varia- Cumulative (%) – 90.891
tion. For instance, conventional methods, biotechnolo- Height 0.0056 0.0447
gical techniques or their combination contribute diffe- Panicles/m3 0.1529 0.0267
rent ranges of genetic variability. Moreover, parental Panicle length 0.0035 0.0022
cultivars resulted from genetic improvement programs 1,000 grain weight –0.0009 0.0048
for obtaining rice cultivar showing high yield potential Yield 0.9866 0.0602
and tolerant to different stress conditions, such as: sali- Filled grains/panicle 0.0450 –0.7259
nity, drought and water-deficiency [12, 13]. Empty grains –0.0454 0.6830
Regarding the PCA analysis, the taller plant geno- Grain length 0.0017 0.0063
types exhibiting the highest grain width and length Grain width 0.0022 –0.0015
and the longest panicles located in the C1 axis positive
quadrant (Figure 2). Similarly, for the C2 axis, the best
30
genotypes showing the best tolerance to water-stress,
highest yields and higher number of filled grains per
20
panicle were located in the positive quadrant, the most INCA LP 10/C4 153
relevant example for the INCA LP 10/C4153 hybrid.
10
Other studies demonstrated that prolonged drought
during the reproductive phase, which demands in-
0
Component 2

creased transpiration intensity, could lead to yield

losses up to 85% despite high regenerant yields. Im-
portantly, yield is a key character to choose which va- –10
rieties will be transferred to field production. Hence,
yield is established as depending on the components: –20
panicle number, spikelet number per panicle, spikelet
filling percentage and 1,000-grain weight. Neverthe- –30
less, there are situations in which the spikelet filling
percentage becomes more restrictive for yields than –40
spikelet number per panicle under certain climatic
conditions. Therefore, the specific causes for yield –50
variations need to be examined, as well as their com-
ponents [1, 2, 12, 20]. –60
Moreover, yield inheritance has been demonstrated 0 40 80 120 160 200 240
low and a far complex parameter, since it is influenced Component 1
not only by the genes involved in defining the yield
components, but also other characters indirectly re- Figure 2. Distribution of regenerants according to the two principal components in PCA
lated to yield. Among these last are plant height, the analysis for the variables studied.
cultivation cycle and the grain-associated characters.
Noteworthy, the environmental influence on character Moreover, the cultivars’ combinations INCA LP-
expression should be taken into account [20]. 10/ C4 153, Amistad-82/Perla de Cuba and Perla
de Cuba/ Amistad-82 showed the best responses of
Conclusions callus formation and regeneration of green plants.
20. Longping Y, Xiaojin W, Fuming L, Guo-
The new genotype established was determinant for The NL culture medium provided the best among the hui M, Qiusheng X. Hybrid Rice Technology.
the successful in vitro anthers culture in F2 plants. three media tested for callus formation from F2 plants Beijing: China Agriculture; 2003.

4205 Biotecnología Aplicada 2018; Vol.35, No.4

Cristo-Valdés E, et al. Drought-resistant rice double haploids

anthers. Advantageously, the method introduced shor- high yields and the highest panicle number. Signifi-
tened the lines’ improvement cycle as compared to cantly, a high genetic variability was found among all
traditional cross-breeding techniques, with the expec- the regenerants tested.
ted variability and character fixation through in vitro
F2 plants anther culture. It was also possible to detect Conflicts of interest statement
the regenerant INCA LP-10/C4 153 in the third gene- The authors declare that there are no conflicts of
ration under water stress conditions, which provided interest.

Received in October, 2018.

Accepted in December, 2018.

4206 Biotecnología Aplicada 2018; Vol.35, No.4