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Effect of genotype on adventitious bud formation from leaves of rapeseed (Brassica napus L.)

Yoko Akasaka-Kennedy1,2, Hidefumi Yoshida2, Masao Watanabe2 and Yoshihito Takahata2

1 Genesis Research Institute Inc., Nagoya, Aichi 451-0051 Japan, www.konpon.com/jpn/kaian.html Email yokok@iwate-u.ac.jp
2
Iwate University, Morioka, Iwate 020-8550 Japan, www.iwate-u.ac.jp/index.html Email ytakahata@iwate-u.ac.jp

Abstract

We examined the frequency of bud formation in twenty genotypes of rapeseed (Brassica napus L.) from leaves. Two of the twenty genotypes reviewed are commercial cropping types, one a spring and the other a winter type. Excised leaves were cultured on B5 basal medium containing various PGRs. About three weeks after the initiation of culture, adventitious buds were formed at the cut surface of explants. The frequency of bud formation was strongly influenced by genotype, ranging from 0% to 91%. However, no relationship was found between spring and winter types.

Media summary

The frequency of bud formation from rapeseed leaves was strongly related to genotypes, however, there was no dependence on cropping type.

Key Words

Rapeseed; regeneration; plant genotype

Introduction

Various kinds of tissues had been used as explants for plant regeneration and genetic engineering in rapeseed, such as hypocotyls, flower stalks, microspores, cotyledon and thin cell layer. However few studies have reported plant regeneration from rapeseed leaves. The response of regeneration in tissue culture is strongly influenced by genotype (Ono et al. 1994). In this study, we investigate the genotypic variation in regeneration ability from leaves of rapeseed.

Materials and methods

Twenty genotypes of rapeseed were used. The seeds were surface-sterilized in sodium hypochlorite solution (1% active chlorine) for ten minutes, and then rinsed three times in sterile distilled water. The seeds were placed on 0.6% agar-solidified B5 basal medium (Gamborg et al. 1968). Leaves were excised from 1.5-month-old seedlings and cultured onto basal medium containing various plant growth regulators (PGRs) for bud induction according to the reported method (United States Patent No. 6515206) with minor modifications. Regenerated shoots were transferred to basal medium without PGRs for rooting. Plantlets, which had been acclimatised, were transferred to pots filled with vermiculite, and grown in a greenhouse. All cultures were incubated at 25C under a 16-h photoperiod (50 μmol photons/m2/s).

Results

After two days of culture, whole leaf explants had expanded and turned dark green on basal medium containing 1.0 mg/l BA. Adventitious buds were observed at the cut surface of the explants after three weeks of culture in basal medium containing 3 mg/l BA and 1 mg/l Zeatin (Figure 1). The frequency of bud formation ranged from 2.5% in ‘IWT-44’ to 91% in ‘IWT-3’, however ‘IWT-48’ did not exhibit any buds from leaves (Figure 2). There was no relationship between spring and winter type on bud formation. The number of buds per explant also ranged from 1.0 in ‘IWT-44’ to 7.5 in ‘IWT-9’. The number of induced buds per explant strongly related to the frequency of bud formation. Most adventitious buds developed only leaves without shoot meristem, however adventitious shoots were differentiated occasionally from some of the buds one month after being transferred onto basal medium contained no PGRs. The normal shoots produced roots two weeks after transference onto basal medium without PGRs. Acclimatized regenerated plants were grown in pots under greenhouse conditions, the plants flowered and set seeds.

Figure 1. Adventitious bud formation on basal medium containing 3mg/l BA and 1mg/l Zeatin after three weeks of culture.

Conclusion

Rapeseed is considered amenable to tissue culture and to transformation, however there was marked variation in organogenesis and embryogenesis among genotypes. In this study, we found great variation in shoot regeneration from rapeseed leaves. In rapeseed, microspore embryogenesis was established in a limited number of cultivars, however these cultivars are still recalcitrant to organogenesis and transformation. ‘IWT-10’ which was used for microspore embryogenesis showed a high efficiency of shoot regeneration, and we obtained regenerated plants successfully from this cultivar.

References

Gamborg O, Miller R and Ojima K (1968) Nutrient requirements of suspension cultures if soybean root cells. Experimental Cell Research 50, 151-158.

OnoY, Takahata Y and Kaizuma N (1994) Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed (Brassica napus L.). Plant Cell Reports14, 13-17

United States Patent No. 6515206 Plastid transformation of Brassica

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