Study On Plant Regeration From Cotyledon Node And Genetic Transformation In Luffa Acutangula

Ridge gourd[Luffa acutangula（L.） Roxb.]is one of two cultivated species of the genus Luffa grown extensively in southern China.Due to its narrow distribution area,there is a limited genetic variation which can be explored in breeding program.The transgenic technology has been widely and successfully employed for broadening the genetic base.However,there are few reports in Luffa species.In this experiment,the plant regeneration and the genetic transformation system was studied using a ridge gourd cultivar‘Sweet-crisp’as plant material.The main results obtained are as follows:1.A system for directly regenerating adventitious buds from cotyledon nodes has been established.In order to get rid of the contamination,the seeds were sterilized twice.Firstly,the seeds were treated with 0.1%Hg Cl₂for 7 mins.The seed coats were softened by soaking the seeds in sterilized water for 24 h and were removed under sterile condition.The testa-removed seeds were sterilized again with 0.1%Hg Cl₂for 10 mins.The best explant type was determined as cotyledon node after comparing the six explants（abaxile cotyledon,proximal cotyledon,cotyledon node,rough leaf,epicotyl and hypocotyls）,which could directly produce adventitious buds without callus stage.The appropriate seedling age for cutting off cotyledon nodes was 5-7 d after sowing when cotyledon remaining green and un-flattened.The optimal induction medium was MS+1mg/L 6-BA+0.5 mg/L IBA with adventitious bud induction rate 91.92%and the average number of buds per explant 5.29.The best medium for bud elongation was MS+1 mg/L6-BA,on which the buds could extend to 2-3 cm in length within 10 days.The optimal rooting medium was MS+0.2 mg/L NAA,on which all buds could produce strong root system.2.The GFP gene was transformed into the ridge gourd‘Sweet-crisp’through the Agrobacterium-mediated method using the above established regeneration system.The dark pre-infection period of 2 d and the dark co-cultivation period of 2 d favored the infection.Cefotaxime of 500 mg/L could effectively inhibit Agrobacterium multiplication and 20 mg/L hygromycin（Hyg）as selective agent could inhibit nearly half of the cluster buds.After three successive subcultures on selective medium of MS+1 mg/L 6-BA+0.5mg/L IBA+20 mg/L Hyg+500 mg/L Cef culture,a batch of resistant buds were obtained.The transformation frequency reached 85.1%by fluorescence microscopy observation and the PCR detection of GFP gene.In summary,this experiment has established an efficient system for the direct induction of adventitious buds and genetic transformation of cotyledon nodes of Luffa acutangula,which laid a solid foundation for the further molecular breeding of this crop.