Multi-bud Regeneration System And Transgenic Method Of Cotton Cotyledonary Node

Upland cotton(Gossypium hirsutum L.)is one of the important economic crops.The genetic improvement of cotton has always been the focus of cotton breeders.At present,cotton tissue culture and transgenic systems with hypocotyls as explants have been widely used in cotton transgenic research,but the experimental process of this system requires repeated transfer culture,which is easy to contaminate and takes a long time.In order to explore a transgenic system with short time-consuming and simplified experimental procedures,this study explored the use of cotyledonary nodes as explants to induce the production of multiple shoots in adventitious embryos.At the same time,the establishment of genetic transformation system was explored by Agrobacterium-mediated method.Four new land cotton products(species)of Xinluzao No.15,Xinluzao No.42,Qianmian No.3 and W0 were selected as experimental materials,and cotyledonary leaves were used as explants,and different hormone or hormone ratios were analyzed.And AgNO3 and the like have an effect of inducing multiple shoots through the organogenesis pathway,and explored the effect of introducing ABD2-mCherry fluorescent marker gene into cotton plants by Agrobacterium infecting cotyledonary nodes.The main results of this study are as follows:(1)Using upland cotton varieties as test materials,using cotyledonary nodes as explants,adding 6-BA,KT,6-BA+KT to induce multiple shoots in MSB medium,and adding a single cytokinin 6 When BA or KT was used,the multi-bud induction rate was low,and the multi-bud induction rate was significantly increased when used in combination.The multi-bud induction rate of different varieties was different,and the multi-bud induction rate was affected by genotype.The average rate of multi-bud induction was the highest in the 1.5mg/L 6-BA and 1.5mg/L KT,and the average yield could be 3 buds.,can produce up to 6-7 buds,the induction rate can reach 66.7%;W0 has the highest induction rate at 2mg/L 6-BA and 2mg/L KT,with an average of 3.3 buds,and can produce up to 4 buds.The bud induction rate can reach 66.7%;Xinluzao 15 has the highest induction rate at 2mg/L 6-BA and 2mg/L KT,and can produce 3 buds on average,and can produce up to 7 buds.The multi-bud induction rate can reach 63.6%.(2)Using AgNO3 instead of hormone for multi-bud induction,it was found that the multi-bud induction efficiency was higher,up to 88.9%,the average number of multiple buds could reach 6.6,and up to 8 buds could be produced.In addition,AgNO3 can also significantly reduce the browning of cotton tissue.(3)The addition of GA3 or ZR in MSB medium had no obvious effect on the elongation induction of multiple buds,while in MSB medium without hormone added,the bud elongation was the highest,and the average bud length was 2.3 cm.(4)The rooting rate of multiple shoots was low,and the average rooting rate was 5%.The rooting rate of the roots was the highest in Xinluzao 42 using 2mg/L AgNO3,which could reach 42.9%.(5)The RFP gene ABD2-mcherry was introduced into Xinluzao No.15,Yumian No.3 and W0 by Agrobacterium-mediated method.By comparison,it was found that when the concentration of bacteriophage OD6000.6-0.8,infestation for 10min,dark culture for 2d,the multi-bud induction rate was the highest.(6)The transformed multiple shoots were screened for Kan resistance,and the Kan sensitive concentration of the resistant plants was selected to be 500 mg/L.The target gene was detected by PCR,and the PCR amplified fragment was recovered and sequenced.It was proved that the target gene sequence was consistent.The positive plant could detect red fluorescence under fluorescence microscope,indicating that ABD2-mCherry had introduced multiple buds and was correctly expressed.In this paper,the cotton leaf cotyledonary node was used as explant,the establishment of cotton organ regeneration system was completed,and the target gene was successfully introduced into cotton.The whole test cycle only took 3 months,and only 3 transfer cultures were needed.Transgenic multiple shoots are available.It has the advantages of short time period,saving labor and labor,and is expected to be applied to rapid,high-throughput transgenic research of cotton.