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Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in melon (Cucumis melo L.)
哈密瓜的有效植株再生和體胚誘導農桿菌轉型
We established an efficient plant regeneration and transgenic system using somatic embryogenesis in two melon cultivars: Vedrantais
(Cucumis melo L., var. cantalupensis) and Earl’s Favourite Fuyu A (C. melo L., var. reticulatus). These model cultivars are used for genetic
and development studies of melon. As explants, 1- to 4-mm2 sections of mature seeds were used, including the cotyledon and hypocotyl. To
induce embryo formation, the explants were cultured in embryo-induction (EI) medium, which consisted of liquid MS medium containing
2 mg/l 2,4-d and 0.1 mg/l BA. After 4 weeks of culture, an average of 116.7 and 148.6 embryos per seed were produced for Vedrantais and
Earl’s Favourite Fuyu A, respectively. The frequency of germination 2 weeks after transfer into MS liquid medium was 80% in Vedrantais
and 35% in Earl’s Favourite Fuyu A. More than 60% of the regenerated plants were diploid, and exhibited normal phenotypes when grown
in the field. Using this protocol, Agrobacterium-mediated transformation in Vedrantais was developed. Calli and embryos were obtained in
EI selection medium containing 25 mg/l kanamycin and 375 mg/l Augmentin 9 weeks after bacterial inoculation, and over 80% of these
were transgenic. Putative transgenic plants were regenerated from these embryos in 1.0% agar-solidified MS medium containing 50 mg/l
kanamycin, and were grown in a greenhouse. Transformation was confirmed with a histochemical GUS assay, PCR analysis, and Southern
blot analysis of the transgenic plants. We obtained transgenic plants successfully and the transgenic rate exceeded 2.3%, which is sufficient
for practical use.
我們利用體細胞胚胎在Vedrantais (Cucumis melo L., var. cantalupensis)和Earl’s Favourite Fuyu A (C. melo L., var. reticulatus)，建立有效植株再生和基因轉殖系統，並且使用在甜瓜的基因發展研究上，培植體，是使用１～４平方公釐的成熟種子切片，並且包含子葉和胚軸。接著誘導體胚形成。培植體在EI培養基中培養，此為MS液態培養基再加入2 mg/l 2,4-d和0.1 mg/l BA.。經過四個禮拜的培養之後，Vedrantais和Earl’s Favourite Fuyu，每個種子的平均胚胎數分別為116.7和148.6，換到液態培養基兩個禮拜後，Vedrantais的發芽率是８０％而Earl’s
Favourite Fuyu的發芽率是30%。此成長過程中，超過60%的再生殖株是二倍體跟正常表現型。在實驗顯示，農桿菌轉殖在Vedrantais的生長情形較好。最後經過九個禮拜的細菌培養，產生的癒合組織和胚胎基因改變超過80%，推測這些轉基因植物從胚胎再生，是在1.0% ＭＳ固態培養基加入50 mg/l kanamycin並且在溫世長大，使用ＧＵＳ組織化學染色法、ＰＣＲ分析法和南方墨點法分析做基因分析。我們成功的得到了轉基因植物，可以作為實用的更超出了2.3%
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