| Pumpkin(Cucurbita spp.)germplasm resources are very rich,wide-range in application and widely grown in the world.With the pumpkin industrialization development,new varieties with strong resistance has become the main breeding objective.But the existing pumpkin resources is not enough to meet the needs of breeding goals.Because traditional breeding method has long cycle,great difficult and instability of genetic trait genetic engineering is applied to improve the stress tolerance of pumpkin.To It is an important prerequisite for the development of pumpkin genetic engineering that establishment a highly efficient and stable regeneration system of pumpkin in vitro,and also lay the foundation for further research on gene function,genetic transformation and other relevant breeding process.In order to provide the basis for genetic transformation of pumpkin,the inbred line C.maxima ‘Beiguan' was applied to explore the inducement of callus different explants,the conditions of callus in subculture,and the regeneration system by callus tissues,and to find out the influence factors of regeneration,the optimize the regeneration system in vitro using cotyedonary node of C.maxima ‘Beiguan',and to investigate the main affecting factors of genetic transformation and optimize the genetic transformation system in pumpkin,it also to confirm the optimum conditions of regeneration and Agrobacterium mediated transformation of StNHX1 gene into pumpkin.The main work and results are as follows:(1)Study on callus induction and establishment of regeneration system of pumpkin: The results showed that the callus could be induced from the hypocotyls,cotyledons,true leaves and stem segments of C.maxima ‘Beiguan' inbred seedlings,and cotyledons and stem as explants was easier to induce callus than hypocotyls and true leaves.MS + 1.0 mg/L 6-BA(or 2.0 mg/L KT)+0.2 mg/L NAA medium was best in callus subculture to obtain high quality callus.In the induction of callus regeneration culture,none of the adventitious buds were inducted from all callus explants with different phytohormone combinations.(2)Optimization of regeneration system in vitro of pumpkin cotyledon node: The shelled seed were sterilized with 75% alcohol for 30 s,0.1% mercuric chloride for 9min,washed4-5 times with distilled and sterile water,soaked with sterile water for 5h,and then inoculated inMS medium on the super-clean bench.That was cultured under conditions of darkness with 28 degree celsius,and cultured under the light when the radicle length was about 0.5cm,this can get high quality aseptic seedlings.The best regeneration system in vitro of pumpkin cotyledon node was based on that the cotyledon(1/4 of cotyledon + 2mm hypocotyl)from 5 days seedling stage were used as explants,with MS + 1.5 mg/L 6-BA + 4 mg/L AgNO3 as cotyledon induction medium,with MS + 0.5 mg/L 6-BA + 0.05 mg/L GA3 as adventitious bud elongation medium,with MS + 0.1 mg/L NAA as rooting medium.(3)Optimization of genetic transformation conditions of pumpkin cotyledon node: with Agrobacterium(Agrobacterium tumefaciens EHA105)mediated transformation adding carbenicillin with 500mg/L in culture medium can effectively inhibit the excessive proliferation of Agrobacterium,and will not inhibit the differentiation of explants.The optimal concentration of bialaphos antibiotics was 5mg/L.When Agrobacterium mediated transformation of pumpkin cotyledon node ultrasound,under the condition of ultrasonic 60 W power for 3min,the transformation rate could be improved to a great extent,and the damage was the least in explants. |
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