| Cucumis melo is in the ninth place in the world common fruits and china is the greatest countries that produce and consume melon. In china, Cucumis melon with high quality, large size, and thick skin mainly plant in the Northwest china. Due to short ripening period and long distantance between producing area and market place, keeping fresh and prolonging shelf life of the postharvest melon is a urgent problem that should be solved as early as possible in the practice. The investigation is a part of the program supported by the national natural foundation (30160055) "transformation of melon cultivated species with melon ACC oxidase antisense gene to improve fruit store and transport quality". The aim is to achieve transformation of one or two main cultivated species of melon with thick shin quality with the target gene on the basis of the successful transformation of melon with thin skin, which make the research production change to the application. To compare regeneration difference and effect on transformation rate of different genetypes ,two cultivates 'Lvbaoshi'and 'Gantian-1#',the former was a thick shin species and the later was a thin skin species , were selected to carry out the regeneration experiment with the cotyledons as the explants on the same culture condition .The results showed that the regeneration response of 'Gantian-1#'was significantly superior to that of the 'Lvbaoshi'.The main characters were explants last green for long time and did not excessively expand.the adventitious buds induced at the cut were small and dense and the adventitious buds induce rate (the number of the explants inducing adventitious buds divided by the total number of the explants treated )were high. After analysis and discussion, to overcome the difficulty of the transformation of the thick skin melon, we believe,a high rate regeneration should be established, the aim of which is to improve transformation rate. Many researches concerning muskmelon tissue cultures think that the different genotypes exists the hypostatic difference on the reborn ability, and we agree it. The regeneration ability of six melon cultivars were studied. The results showed that adventitious buds of different melon genotypes were different on the same culture medium(MS+6-BA1.0 mg·L-1). In these melon cultivars, the regeneration rate of adventitious bud of 'Lvbaoshi'is the highest, at 100%, followed by 'Xiyu-1'and 'Huanghemi', at 96.67% and 73.33%, respectively.The lower induction rates are for 'Yujinxiang'and 'Huangzuixian', and the lowest is for 'Bailangua', at 6.67%. Then, the experiment selects 'Lvbaoshi'and 'Lvbaoshi'to transform. After this work, the regenerate rate of shoots in several induction media from cotyledons of two melon cultivars 'Lvbaoshi'and 'Huanghemi'were studied. The result showed that inducing rate of adventitious buds had obvious difference in different media and cultivars. For example, the highest regenerate rate of adventitious buds in 'lvbaoshi'was obtained on the medium with 2.0 mg?L-16-BA,which was 93.75%. The result discovered the highest regenerate rate of adventitious buds in 'Huanghemi'was obtained on the medium with 1.0 mg?L-16-BA,which was 73.33%.Meanwhile, 6-BA and IAA had effects on calli. To overcome the difficulty of the transformation of the thick skin cucumis melon, we also studied the transformation procedure about Agrobacterium tumefacien mediated and the regeneration procedure about selection. We discovered that transformation needed 24 hours pre-culture, 0.30 for inoculation concentration, 30min for inoculation times,108 hours for co-cultivation , 75mg?L-1 for Kanamycin selection concentration in buds differentiation and 25mg?L-1 for Kanamycin selection concentration in buds elongation on 'Huanghemi'. But transformation needs 0.35 for inoculation concentration, 10 minutes for inocula-tion times, 62 hours co-cultiva-tion and dark culture on'Lvbaoshi', the same as 'Huanghemi'in Kanamycin selection concentration. The studies on transformation of ACC oxidase antisense gene in melon used two vectors: pBl-aACO1 and pCB-aACO1. The characteristics of the former basing on the pBI 121 is that nptⅡand CM-aACO1 catenated close, but GUS has been displaced. NptⅡ, CM-aACO1 and GUS are series-wound in pCB-aACO1. The research carries on 27 experiments, and the average number of the explants of each is 200. Through the regeneration and selection by Kanamycin, 10 plants roots in 'Lvbaoshi'and 20 plants roots in'Huanghemi'. By GUS gene detecting, some of the calli also expressed GUS positive reaction. In future studies, it is expected that the adventitious buds or embryoid have been induced from the calli, and the complete transgenic plants have been obtained.
|
PDF Full Text Request