Aug 23, 2002 - (barley, buckwheat, fingermillet, grain amaranth, foxtail, proso ... The office of the Science Advisor, USAID, Washington D.e., U.S.A..
Fagopyrum 19: 55-58 (2002)
In vitro growth of Nepalese buckwheat embryos in response to culture media and vessels Bal Krishna JOSHI* and Hari Prasad BIMB Biotechnology Unit, Agriculture Botany Division, NARC, Khumaltar, PO Box 1135 Katmandu, Nepal Received April 20, 2002; accepted in revised form August 23, 2002
Key words: Nepalese buckwheat, culture media, culture vessels, embryo culture
ABSTRACT A study was conducted considering three factors, buckwheat species, tite (Tartary) and mithe (common) buckwheat , culture media (tap water and 1/2 MS) and culture vessels (Petri dish and jam-bottle) to determine the response of Nepalese buckwheat cultivars to in vitro embryo culture. Mature seeds from the field were collected for the embryo culture . Growth patterns of the embryos were measured after 6 days of culture. Tite and mithe buckwheat responded differently to growth of the embryo in vitro for germination, length of main root, seedling height and root branches. Embryos of mithe buckwheat cultured in Petri dishes responded very well. Supplemented media was necessary for growing the embryos. Culturing mature embryos of mithe buckwheat in supplemented media in a Petri dish may be an appropriate method for conducting further research on tissue culture.
five hybrids between F. cymosum and other species: F. esculentum, F. tataricum, F. homotropicum, F. pilus. Uchinomiya (1990, cited by Adachi, 1990) succeeded in ovule culture just three days after pollination. Ujihara et a1. (1990) also reported on successful buckwheat ovule culture. Ovule culture has facilitated the production of inter-specific hybrids to overcome the post zygotic barriers (Woo and Adachi, 1997; Wang and Campbell, 1998). Woo et al. (1999) suggest that ovule culture is an easy and effective tool for obtaining wide inter-specific hybrids in the genus Fagopyrum . Effective and efficient embryo culture techniques are necessary for embryo rescue, which helps in the development of hybrids within the genus Fagopyrum. The culture of embryos in vitro, to increase the number of growth cycles per year, is one of the strategies used in breeding programs for breaking the dormancy period. The present study was conducted on tite and mithe buckwheat with the objective being to develop an efficient method for embryo culture of buckwheat in vitro.
INTRODUCTION Buckwheat is an irreplaceable crop in the mountainous agro-ecological regions of Nepal. Although research on buckwheat was initiated in 1973 in Nepal, a high yielding variety has not yet been formally released. It is a minor food and cash crop in Nepal, nevertheless, it is a staple of life in the remote food deficit areas of the country (Baniya et al., 1995). Many distinct types of buckwheat have been found in Nepal as the Himalayan region is very rich in buckwheat genetic resources. Farmers grow both tite (Tartary) and mithe (common) buckwheat. A significant variation within and among Nepalese buckwheat has been found (Ujihara, 1983; Baniya et al., 1992; Rajbhandhary and Hatley, 1993; Bimb et al., 2001 ). Some important traits of both mithe and tite buckwheat encouraged scientists to attempt to cross them to generate new genotypes. However, conventional breeding techniques have not been successful in crossing mithe and tite buckwheat. Suggestions from many scientists were made for using plant cell and tissue culture techniques as a means to overcome breeding barriers in buckwheat (Neskovic et al., 1986; Lachmann, 1991; Bohanec, 1995; Joshi and Bimb, 2001). A number of studies in relation to the potential of inter-specific hybrids to overcome cross-incompatibility have used embryos or ovule culture system. As mentioned by Campbell (1995), successful hybridization of Tartary buckwheat with common buckwheat at the diploid level has been accomplished using conventional breeding techniques by crossing the two species and then growing the resulting ovules on media. Woo et a1. (1999) used embryo rescue to develop
MATERIALS AND METHODS Three factors, species (tite and mithe buckwehat), culture media (tap water and 1/2 MS) and culture vessels (Petri dish and jam-bottle) were evaluated in this study. Seeds of tite and mithe buckwheat were collected from the Hill Crop Unit of Agriculture Botany Division, Khumaltar. These seeds originally came from a landrace from Dolpa, Nepal for tite and from a landrace from Mustang, Nepal for mithe buckwheat. They were grown in a field at Khumaltar, Nepal during the growing season of 2001.
*Corresponding author
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Fertilizer and irrigation were not applied. The weeds were manually controlled. Mature seeds (after the seed coat had changed colour) from the field were collected for embryo culture. The seeds were surface sterilized with 70% ethyl alcohol for 5 minutes and 0.1 % sodium hypocloride for 15 minutes and then rinsed 3 times with sterile distilled water. Half strength MS media (Murashige and Skoog, 1962) and tap water were sterilized in an autoclave at 121 DC for 15 minutes prior to use. In the medium treatment, 20 ml and 40 ml of medium was applied per petri dish and per jam-bottle, respectively. In the tap water treatment, filter paper sterilized under ultraviolet rays was used. Embryos dissected from the surface sterilized seeds were placed in the various treatments according to the experimental design. The experiment was designed as a 32 factorial with a completely randomized design with 3 replications at the Biotechnology Unit, Khumaltar, Nepal. All together there were 24 treatments. Six embryos were cultured in each Petri dish and 3 embryos in each jam-bottle. The average of measurements on these embryos was treated as an entry. In case of jam-bottle, two jam bottles were used per entry to adjust the number of embryos per entry. All the indoor activities were conducted in a laminar flow hood. The embryos were cultured under constant light at 21°C temperature. The growth patterns of the embryos were recorded after 6 days of culture. The number of germinated embryos, root and seedling height and number of root branches were recorded. The data were analyzed using MSTAT software and DMRT were applied to separate the means.
RESULTS AND DISCUSSION The number of germinating embryos and seedling height are given in Table 1. The largest number of germinating embryos was obtained in mithe buckwheat cultured in jam-bottles supplemented with 1/2 MS media. The least number of Tite buckwheat embryos germinated in a Petri dish containing tap water. The number of germinated embryos were higher in mithe than in tite buckwheat in all treatments. Most of the responses of mithe buckwheat were significantly different from tite buckwhet. A similar type of growth response was observed in seedling height. The lowest seedling height was in tite buckwheat cultured in a Petri dish containing tap water. The seedling height of mithe buckwheat was higher than tite buckwheat in all cases. Early growth characters, such as the number of germinated embryos, was better in mithe buckwheat cultured in a jam-bottle with 1/2 MS media. But later the growth response was better in mithe buckwheat cultured in a Petri dish containing 1/2 MS media. The species gave a significant difference in both number of germinated embryos and seedling height, but
the media used produced a significant difference only in seedling height. All the interactions, including culture vessels, were non significant for these two characters (Table 1). The effect of these treatments on root length and number of root branches are given in Table 2. The response pattern of these traits was similar to that for seedling height. Species, media and the interaction among them were significant for the number of root branches. The longest root and the highest number of root branches were found in mithe buckwheat cultured in a Petri dish containing 1/2 MS media. Rooting was not observed in tite buckwheat cultured in a Petri dish containing tap water. Wang and Campbell (1999) found that after embryos were transferred from beakers into Petri dishes, the seedling were found to grow better in 1/2 MS medium than in tap water. Mithe buckwheat responded well to this in vitro technique, possibly this is due to its long history of cultivation or domestication . It also suggests that mithe buckwheat can be artificially manipulated easier. For just germinating embryos, media was not necessary, but water must be supplemented for further growth of the buckwheat plant. It was observed that media affected buckwheat growth more with an increase in the growth stage. The possibility for successful embryo rescues from crosses between mithe and tite may possibly be expected if mithe buckwheat is used as the female parent. More of the cellular contents that come from mithe buckwheat in the embryo, may improve the chances of better embryo response when cultured in vitro. Successful hybrids between F. esculentum and F. tartaricum were developed using mithe buckwheat as female parent with the help of embryo rescue (Wagatsuma and Un-no, 1995; Rumyantsava et al., 1989). The possibility for successful development of hybrids using mithe buckwheat, was supported by Woo et al. (1995) who reported that F. esculentum has relatively high capacity to allow foreign pollen penetration. By simply using the technique of embryo rescue the dormancy period of buckwheat seeds can be broken, resulting in an increase in the number of growth cycles per year. Tite and mithe buckwheat responded differently to the treatments for growth of the embryo in vitro for germination number, length of main root, seedling height and number of root branches. Significant differences between 1/2 MS media and tap water were found for length of main root, seedling height and the number of root branches. A significant interaction between species and vessels, and media and vessels was found only for the number of root branches. Embryos of mithe buckwheat cultured in a Petri dish with 1/2 MS media had the longest root, highest seedling height and largest number of root branches. A significant difference between culture
Nepalese buckwheat embryo culture
Table I.
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Number of germinated embryos and seedling height of buckwheat embryos cultured in vitro
Culture media (M)
Tap water
1/2MS
Culture vessel (V)
No. of germinated
Seedling height,
embryos'
em'
Tite
Mithe
Tife
Mithe
0.67 d
4.33 ab
0.17 c
1.57 be
Jam-bottle
1.00 cd
4.67 ab
0.55c
1.14 c
Petri dish
2.00bcd
3.67 abc
1.59 bc
3.47 a
Jam-bottle
1.00 cd
5.33 a
1.68 be
Petri dish
2.92 ab
F, Species (S)
**
F (M)
ns
** **
F (SxM)
ns
ns
F (V)
ns
ns
F (SxV)
ns
ns
F(MxV)
ns
ns
F (SxMxV)
ns
ns
** Significant at 1% level. t Means followed by the same letterls are not significantly different at 5 % level. Tab le 2.
Root length and number of root branches of buckwheat embryos cultured in vitro
Culture media (M)
Tap water
1/2MS
Length of main root,
Culture vessel (V)
em'
No. of root branches"
Mithe
Tite
Petri dish
Tite 0.00 c
Mithe
1.63 be
O.OOc
2.31 ab
Jam-bottle
0.97 be
1.35 be
2.33 ab
3.20 a
Petri dish
1.60 be
3.63 a
0.33c
3.42 a
Jam-bottle
2.00 ab
2.36 ab
0.00 c
1.23 be
F, Species (S)
**
F (M)
ns
** *
F (SxM)
ns
ns
F (V)
ns
ns
F (SxV)
ns
F (MxV)
ns
** **
F (SxMxV)
ns
ns
*, ** Significant at 5% and 1% level respectively. Means followed by the same letterl s are not significantly t
different at 5% leve l.
vesse ls was not observed, however Petri dishes may be considered a suitable culture vessel, as the embryos cultured in Petri dishes responded very well as compared to those in a jam-bottle. A supplemented media is neces sary for growing embryo . Tap water could not supply the nutrients needed by the growing embryos . Wang and Campbell (\ 999) found a similar type of result. Culturing mature embryo s in a supplemented media in a Petri dish may be considered an efficient technique for further research in tissue culture. These techniques may be equally applicable to immature embryos for in vitro culture.
ACKNOWLEDGEMENTS NARC provided the financial support. B. Dongol and J. Shrestha assisted in laboratory works.
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Bimb , H.P., S. Fukuoka, K. Ebana and T. Nagamine, 200!. Genetic diversity in Nepalese populations of Fagopyrum tataricum revealed by RAPD assays . In: Bimb , H.P. and B.K. Joshi (eds.), Research and Development on buckwheat: An important yet a neglected crop in Nepal. Proc . National Work shop, 13-14 Sept 2001, Nepal, pp.108-116. Bohanec, B., 1995. Progress of buckwheat in vitro culture techniques with special aspect on induction of haploid plants . Proc . 6th IntI. Symp . Buckwheat at Ina: 205-209. Campbell, G.C., 1995. Inter-specific hybridization in genus Fagopyrum. Proc . 6,hIntl , Symp. Buckwheat at Ina: 225-263. Joshi, B.K. and H.P. Bimb , 2001. Prospects and possibilities of buckwheat development through biotechnology. In: Bimb , H.P. and B.K. Joshi (eds .), Research and Development on buckwheat: An important yet a neglected crop in Nepal. Proc. National Workshop, 13-14 Sept 2001, Nepal, pp. 83-94. Lachmann, S., 1991. Plant cell and tissue culture in buckwheat: an approach towards genetic improvements by means of unconventional breeding techniques. Proc . ICOBB in Miyazaki 145-154. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures . Physio . Plant. 15: 473497. Neskovic, M., V. Srejovic, R. Vujicic , 1986. Buckwheat iFagopyrum esculentum Moench) . In: Y.P.S . Bajaj (ed.), Biotechnology in agriculture and forestry 2, Crops I pp. 579-593. Rajbhandary, B.P. and E. Hatley , 1993. Exploitation of genetic yield potential of common buckwheat iFagopyrum esculentumt ecotypes in different regions of Nepal. Annual Report-3. The office of the Science Advisor, USAID, Washington D.e. , U.S.A. Rumyantseva, N.!., N.V . Sergeeva, L.B. Khakinova, V.V. Salnikov , E.A .
Gumeroa and V.V. Lozovaya, 1989. Organenesis and somatic embryogeneiss in culture of two buckwheat species . Sov. Plant Physi o. 36: 152-158. (Engl translation) Ujihara, A., 1983. Studies on the ecological features and the potential as breeding material s of Asian common buckwheat varieties (Fagopyrum esculentum M.). Ph. D. Thesis . Lab. Crop Sc. and Plant Breeding, Faculty of Agriculture, Shinshu Uni versity, Japan. Ujihara , A., Y. Nakamura and M. Minami , 1990. Interspecific hybridi zation in genus Fagopyrum-properties of hybrids (F. esculentum Moench. x F. cymosum Meissn .) through ovule culture. Gamma Field Symp. Inst Radiation Breeding NIAR, MAFF, Jap an 29: 4551. Wagatsuma, T. and Y. Un-no, 1995. In vitro culture of interspecific ovule between buckwheat (F. esculentumi and tartary buckwheat (F. tataricums. Breed . Sci. 45 (Suppl. 2): 312. Wang, Y. and e. Cambell, 1999. Culture of buckwheat embryos in petri dishes to speed up the breeding cycle . Fagopyrum 16: 37-41. Wang, YJ. and C. Campblell, 1998. Interspecific hybridization in buckwheat among Fagopyrum esculentum, F. homotropicum and F. tataricum . Proc. 6 th Intl, Symp . Buckwheat at Ina: 1-12. Woo, S.H . and T. Adachi , 1997. Production of interspecific hybrids between Fagopyrum esculentum and F. homotropicum through embryo rescue . SABRAO J. 29: 89-95. Woo, S.H., Q.S. Tsai and T. Adachi, 1995. Possibility of interspecific hybridization by embryo rescue in the genus Fagopyrum. Proc. 6,h IntI. Symp. Buckwheat at Ina: 225-237. Woo, S.H., YJ. Wang and e.G. Campbell , 1999. Interspecific hybrids with Fagopyrum cymosum in the genus Fagopyrum. Fagopyrum 16: 13-18.
