J. Appl. Hort.

, 3(2):82-84, July-December, 2001

Micropropagation studies in ‘single’ vs ‘double’

types of tuberose (Polianthes tuberosa L.)

K.B. Krishnamurthy1, J.B. Mythili2* and Meenakshi Srinivas3

1
Division of Horticulture, University of Agricultural Sciences, Bangalore-560065, 2Division of Biotechnology, 3 Division
of Ornamental Crops, Indian Institute of Horticultural Research, Hessaraghatta, Bangalore-560089.E-mail:
[email protected]. *Corresponding Author

Abstract
Micropropagation of tuberose  (Polianthes tuberosa L.) was attempted for the rapid  multiplication of single (Shringar) and double
(Suvasini) types of tuberose and to study their in vitro response. Decontaminated terminal and axillary stem scale section explants
were used  for  initiating the culture. Although  the presence  of cytokinin was enough  to induce  multiple shoots, addition of auxin
increased the regeneration response and response up to 100% was obtained with BAP 2 mg l-1  and IAA 0.1 mg I-1. However, the
length of shoots was improved with increase in the level of BAP used. Successful rooting of up to 100% with maximum number of
roots in proliferated shootlets was obtained in 1/2 strength MS medium with IAA 0.2 mg l-1 and  IBA 0.25 mg l-1. Maximum root length
was however obtained in media containing IBA 0.5 mg l-1. The rooted plantlets could be successfully hardened and transferred ex
vitro. The two hybrids differed in their in vitro response with the double type Suvasini exhibiting higher regeneration and multiplication
rate than the single type Shringar.

Key words: Polianthes tuberosa L., tuberose, single type, double type, micropropagation, regeneration, in vitro response

Introduction for varied periods of time followed by several washes in distilled

water before sterilization with 0.1% HgC12 for 10-15 min. After
Tuberose, an ornamental flowering plant is generally propagated 5-6 washings in sterile distilled water, the explants were cultured
through bulbs. Propagation of tuberose through bulbs, has the risk on Murashige and Skoog (1962) medium supplemented with
of perpetuating the disease causing pathogens which result in 3% sucrose, 0.25% phytagel (Sigma, St Louis, USA). The pH of
significant loss of productivity. Since, the genetic variability is the medium was adjusted to 5.8 before autoclaving at 121°C for
limited in tuberose, there is scope for development of new varieties 15 min.
through somaclonal variation and genetic engineering.
Standardizing the protocols for micropropagation is a pre-requisite In vitro multiplication and rooting: The shoot tips from the
for clonal propagation of plants that are produced through sprouted axillary and terminal scale sections were placed in media
biotechnological manipulations. Micropropagation is also required containing BAP 2-4 mg 1-1 alone or in combination with IAA
to augment the supply of planting material of any newly developed 0.1 mg l-1 . The regenerated shoots were placed in different
cultivar in a short period of time and will also help in germplasm rooting media comprising of half strength MS media with
conservation and exchange. There are not many reports on different auxins viz., IAA, IBA (0.25 - 1.0 mg 1-1) alone or in
micropropagation of tuberose. Muralidhar and Mehta (1982) and combination. The observations on rooting %, root number and
Bose et. al. (1987) used bulb scales as the explant and single type days taken for rooting were recorded after 20 days of culturing.
of tuberose. Bose et al. (1987) obtained adventitious shoots from Twenty shoot tips and 20 shoots were used for each treatment
callus which is not a desirable method of in vitro propagation. In for each type with 5 replications in experiments on regeneration
this paper, we report micropropagation of tuberose through axillary of multiple shoots and rooting respectively. The experiment was
shoot propagation and compare the in vitro response of single and repeated twice. The multiplication rate per explant was
double types of tuberose. determined 60 days after culture, and the data was subjected to
analysis of variance.
Materials and methods Acclimatization: The rooted plantlets were successfully
hardened using the closed sachet technique (Ravindra and
Plant Material: Two cultivars of tuberose viz., Shringar (single Thomas, 1995). The procedure involved planting of rooted
type) and Suvasini (double type) , released by the Indian Institute shootlets in polybags containing a mixture of autoclaved soil,
of Horticultural Research, Bangalore, were used in the study. sand and soilrite mixture TC (Perlite - Vermiculte - Irish peat
Decontamination treatment: Terminal or axillary stem scale moss mixture, KEL Perlite Bangalore) (1:2:1) previously
sections were used as explants and were disinfected in a solution watered to field capacity. The bags were misted, closed, kept
of fungicide and/ or bactericide viz., Bavistin (1000 ppm), Kavach under a 16 h light (30-40 mol m-2s-1) opened after 3 weeks
(1000 ppm) and Cetrimide (500 ppm) alone or in combination and after a week transferred to glasshouse.
 Krishnamurthy et al.- Micropropagation of ‘single’ and ‘double’ tuberose 83

Results and discussion Rumynin et al., 1990, Misra and Singh, 1999). Although the
presence of cytokinin was found to be enough to induce multiple
Decontamination of cultures: Decontamination of explants shoot proliferation, addition of auxin increased the regeneration
obtained from underground parts has been reported to be a very response and response up to 100% with BAP 2 mg 1-1 and IAA
difficult task by several workers (Seabrook,1990, Hol and Vander 0.1 mg 1-1. The same treatment gave rise to maximum number
Linde,1992).They partly solved the problem by very elaborate
decontamination procedures including hot water treatment of
bulbs, high concentration of decontaminants or by the use of
fungicide containing media. We also encountered fungal Fig. 1
contamination to be the major deterrent in aseptic culture
establishment as there was no problem of bacterial contamination.
However, Debergh and Maene (1981), have reported bacterial
contamination as a major limitation in the establishment of aseptic
culture. In the present study treatment with Bavistin (1000 ppm)
+ Kavach (500 ppm) + Cetrimide (500 ppm) overnight followed
by HgC12 (0.1%) for 15 minutes was found to be effective in
establishment of maximum per cent i.e., 26.6 and 30.0, 23.3
and 30.0 of axenic cultures in case of axillary and terminal stem
scale sections of cvs ‘Shringar’ and ‘Suvasini’, respectively. This
treatment helped to control fungal contamination totally. Dilta C
A B D
et al. (2000) used Bavistin, Dithane and HgC12 for sterilization
in lilium.
Multiple shoot induction : Terminal and axillary stem scale Fig. 2
sections were used for the study and both the explants could be
successfully established and proliferated. The response of both
the explants was identical and hence both the explants were used.
In general, almost all organs of bulbous plants have been shown
to be able to regenerate plantlets, but the best suited ones were
obtained from bulb scales, stems or bulbs (Vander Linde and
Aartrijk, 1986). Earlier Bose et al. (1987), have also used stem
scale sections for tuberose micropropagation.
Explants did not show any morphogenetic response in a media
devoid of hormones. Generally cytokinins stimulate development
Fig. 1a. Culture of terminal stem scale section of explant, 1b. Culture
of axillary buds whose growth is inhibited by apical dominance of shoot tip obtained from explant, 1c In vitro proliferation of shoots
(Hussey, 1976 a,b) and it was effective in successful bud from shoot-tip culture and  1d. In vitro rooting of shoots. Fig. 2. Hardened
emergence from axillary buds of gladiolus corms (Hussey, 1977, plants established ex vitro
Table 1. Regeneration response to the tuberose cv. Shringar and Suvasini, 60 Days after culturing
S. Treatment Regeneration (%) Average Shoot number Average shoot length
No (mg 1-1) Shringar Suvasini Mean Shringar Suvasini Mean Shringar Suvasini Mean
1 BAP 2.0 60.0 82.0 71.0 3.20 3.13 3.17 1.90 1.92 1.91
2 BAP 4.0 80.0 100.0 90.0 4.10 5.72 4.91 2.90 3.38 3.14
3 BAP2.0 + IAA 0.1 100.0 100.0 100.0 5.24 6.54 5.89 2.36 2.92 2.64
4 BAP4.0 + IAA 0.1 90.0 100.0 95.0 4.04 4.70 4.37 3.32 2.78 3.05
Mean(A)  82.5 95.5 4.15 5.02 2.62  2.75
CD (P=0.05) Variety (A) 3.1 0.22 NS
Treatment (B) 4.3 0.31 0.29
AxB 6.1 0.44 0.41
Table 2. Rooting response of the tuberose cv. Shringar and Suvasini, 20 days after culture
Sl. Treatment Rooting (%) Average root number Average root length(cm) Days for rooting
No (mg 1-1) Shringar Suvasini Shringar Suvasini Shringar Suvasini Shringar Suvasini
1 IBA 0.5 100 100 14.20 14.5 1.39 1.33 12 12
2 IBA 1.0 65 75 9.50 10.0 0.94 0.80 13 12
3 IAA 0.5 90 90 10.48 9.0 1.24 1.20 13 12
4 IAA 0.25 + IBA 0.25 100 100 16.70 15.8 1.30 1.30 10 10
5 MS Basal 0.0 0.0 0.0 0.0 0.0 0.0 0 0
SEM + 2.3 1.5 0.2 0.3 0.05 0.06 0.4 0.4
CD (p=0.05) 6.9 4.6 0.6 0.9 0.14 0.19 1.2 1.1
CV (%) 7.4 4.8 4.7 6.9 10.59 16.08 9.6 9.8
84 Krishnamurthy et al.- Micropropagation of ‘single’ and ‘double’ tuberose

of shoots. The concentration of BAP was also critical as the House, New Delhi.
number of shoots obtained is reduced significantly, while the Bose, T.K. and B.K. Jana, 1977. Regeneration of plantlets in
length of the shoots increased significantly with the increase in Hippeastrum in vitro. Indian J. Hort., 34:446-447.
BAP level 4 mg 1-1 used singly or in combination with IAA 0.1 Bose, T.K, B.K. Jana and J. Moulik, 1987. A note on micropropagation
mg l-1 (Table l and Fig lA-C). of tuberose from stem scale sections. Indian J. Hort., 44:100-101.
Debergh, P.C. and L.J. Maene, 1981. A scheme for commercial
Regulation of both, organ differentiation and growth in tissue propagation of ornamental plants by tissue culture. Scientia Hort.,
cultured plants by interplay of cytokinins and auxins has been 14:335-345.
reported by several workers beginning with Miller and Skoog Dilta, B.S., O.P. Sehgal, B.P. Sharma and D.R. Sharma, 2000. In vitro
(1957). The ratio of cytokinin to auxin used depends on the level multiplication of Lilium hybrids. Abstract, National Seminar on
of endogenous cytokinin present in the plant and thus varies Hi-tech Horticulture June 26-28, Bangalore. pp34-35.
with plant species used. However, there are species such as Hol, G.M. and P.C.G. Vander Linde, 1992. Reduction of contamination
Lilium which did not require either plant growth regulators in bulb explant cultures of Narcissus by a hot water treatment of
parent bulbs. Plant Cell Tissue Organ culture, 31:75-80.
(Niimi, 1985). In the present study, variations in the response of
Hussey, G. 1976a. In vitro release of axillary shoots from apical
two hybrids of tuberose viz., Shringar and Suvasini was noted. dominance in monocotyledonous plantlets. Ann Bot., 40:1323-
The double type (Suvasini) had significantly higher regeneration 1325.
potential than the single type Shringar. The double type also Hussey, G. 1976b. Plantlet regeneration from callus and parent tissue
gave rise to significantly higher number of shoots than the single in Ornithogaluym thyrsoides. J. Exp. Bot., 27:375-382.
type Shringar in all the hormonal treatments except with BAP Hussey, G. 1977. In vitro propagation of gladiolus by precocious axillary
2mg 1-1. shoot formation. Scientia Hort., 6:287-289.
The better morphogenetic response in Suvasini (double type) Hussey, G, J. Milton and P. Lumsden, 1979. In vitro propagation of
Alstroemeria. John.Inces. Institute, Seventeenth Annual Report. p
may be related to the physiological differences in growth of the 56.
two types. There are hardly any reports on this aspect in the Kim, Y.J., P.M. Hasegawa and R.A. Bressan, 1981. In vitro propagation
literature. However, differences in the morphological characters of hyacinth. Hort Sci., 16:645-647.
of single and double type have been reported by Bhattacharjee Miller, C. O. and F. Skoog, 1957. Chemical regulation of growth and
(1995). He states that the single type is characterized by more organ formation in plant tissue culture in vitro. Symp. Soc. Exptl.
number of leaves (60-80), with greater spike length (90-120cm) Biol., 11:118-130.
as compared to double type which has 35-45 leaves and smaller Misra, S. and R. Singh, 1999. In vitro propagation of gladiolus Cv.
spike length (75-95cm). Despite this, double type yields more American Beauty. J. Ornamental Horticulture, 2:67-70.
number of flowers as compared to single type which may be Mukhopadhyay, A. and H.T. Nagaraju, 1988. Changes in growth,
related to the better translocation or utilisation of photosynthate flowering and chemical composition of the tuberose “single”.
necessary for the production of flowers (Mukhopadhyay and Herbertia., 44:61-67.
Nagaraju, 1988, Pathak et al., 1980). Muralidhar, C.E. and A.R. Mehta, 1982. Clonal propagation of three
ornamental plants through tissue culture methods (pp.693-694). In
In vitro rooting: Use of auxins for root induction has been A. Fujiwara. (ed) Plant Tissue Culture, Proc. 5 th Intl. Cong. Pl.
reported in several bulbous ornamentals. In this study, among Tissue & Cell Culture, Jap. Assoc. Plant Tissue Cult., Tokyo.
different treatments tried, 100% root induction, early rooting Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth
and maximum number of roots (16.7 and 15.8) was obtained in and bioassay with tobacco tissue cultures. Physiol. Plant., 15:473-
479.
half strength MS medium with IAA 0.25 mg 1-1 and IBA 0.25
Niimi, Y. 1985. Factors affecting the regeneration and growth of bulblets
mg 1-1 in Shringar and Suvasini respectively (Table 2 and Fig in bulbscale cultures of Lilium rubellum Baker. J. Jap. Soc. Hort.
ld). Maximum root length (1.39 and 1.33) was obtained in media Sci., 54:82-86.
containing IBA (0.5 mg 1-1) in Shringar and Suvasini respectively Pathak, S., M.A. Choudhary and S.K. Chatterjee, 1980. Germination
(Table 2). Similar observations of maximum root length in media and flowring in different sized bulbs of tube rose (Polianthes
supplemented with IBA has been reported (Hussey et al., 1979) tuberosa L.). Indian J. Plant Physiol., 23:47-54.
in Alstromeria. Better response of rooting observed in media Ravindra, M.B. and P. Thomas, 1995. Sachet technique- an efficient
containing IAA and IBA could be due to the synergistic effect method for the acclimatization of micropropagated grapes (Vitis
of auxins. In Hippeastrum, it took 40 days to root on media vinifera L.). Curr. Sci., 68:546548.
containing IAA alone (Bose and Jana, 1977). Rumynin V.A., I.V. Agadzhanyan and A.G. Slyusarenko, 1990. Mass
clonal propagation of gladiolus plants. In: Glavnogo
Acclimatization: Both the cultivars could be hardened with100% Botanicheskogo Sada, USSR, Bull No. 156:6878.
survival using closed sachet technique (Fig 2). The presence of Seabrook, J.E.A. 1990. Narcissus (Daffodil). In: Ammirato, P.V, Evans,
soilrite may have enhanced the survival rate due to optimum D.A, Sharp, W.R and Bajaj, Y.P.S (eds). Handbook of Plant Cell
conditions of aeration, water holding capacity and nutrients Culture vol V: Ornamental Species, McGraw-Hill Publishing
Company, New York, pp577-597.
present in this media. The hardened plants established with 100%
success when transplanted to pots. Survival rate ranging from Vander Linde, P.C.G. and V.J. Aartrijk, 1986. Micropropagation of
flower bulb crops. In: Anderson, P.F, Dulforce, W.M (eds).
60% in gladiolus (Ziv, 1979, Misra and Singh,1999) to 90% in Micropropagation in Horticulture, pp 123-134.
hyacinth (Kim et al., 1981) has been reported. Ziv, M. 1979. Transplanting gladiolus plant propagated in vitro. Scientia
Hortic., 11:257-260.
References
Bhattacharjee, S.K. 1995. Cultural requirements of tuberose (pp757-
774). In K.L. Chadha and S.K. Bhattacharjee (eds.), Advances in
Horticulture Vol 12 (Part2) Ornamental plants, Malhotra Publishing