Construction Of Agrobacterium Transgene System For Mature Seeds Of Sorghum Strain 32

Sorghum(Sorghum bicolor(L.))Gramineae sorghum family sorghum is an important dry grain crop.There are also 500 million people in the world who use sorghum as the main grain.Sorghum is used for edible brewery and fodder.At present,sorghum is mostly used for feeding in China.The main goal of breeding is to pursue the quality of forage sorghum,but the problems such as incompatibility of distant hybridization and long time consuming are the problems that need to be faced and solved in sorghum breeding.Compared with other crops,sorghum is difficult to establish efficient tissue culture and transformation system,so the research work on genetic transformation is relatively slow.At present,the most commonly used methods of crop genetic transformation at home and abroad are gene gun and Agrobacterium tumefaciens mediated method.As for the genetic transformation of sorghum,the technology of microprojectile transformation is more and more,and the progress of agrobacterium-mediated method is slow.In addition,that research on the genetic transformation of sorghum at home and abroad mainly takes the embryo as an explant,but the material of the young embryo is subject to time and geographical limitation,so that the continuous supply of the explant can not be ensured,and the operation of the young embryo is time-consuming and time-consuming when the young embryo is inoculated.Therefore,the establishment of Agrobacterium-mediated transgenic system for sorghum mature seeds is of great significance for sorghum genetics and breeding.In this study,a sorghum line 32 selected from 120 sorghum lines was used to optimize sorghum tissue culture system and to construct Agrobacterium tumefaciens transgenic system.The effects of normal temperature and freezing treatment on callus induction rate of sorghum mature seeds were compared.The results showed that the rate of callus induction was significantly enhanced by freezing treatment,and the callus induction rate was 85.07%.In order to determine the effect of lipoic acid on the induction rate,the callus induction rate of the mature seed of sorghum was compared.The callus induction rate and browning rate were significantly different under the five lipoic acid concentrations,but the germination rate was not significantly different,among which 1.5 mg/L induction rate was the highest,and the browning rate was the lowest.The effects of 5 different concentrations of CuSO4 on callus induction rate were further compared.When CuSO4 concentration was 8.5 ?mol/L,the callus induction rate was significantly enhanced by 83.17%.After 15 days of subculture,the rate of callus induction was not significantly different in 3subculture media with 2,4-D concentrations.The results indicated that lipoic acid could significantly increase the rate of callus differentiation.The effect of the concentration of 4 kinds of lipoic acid on the rate of differentiation was compared.The difference of the rate of differentiation of the four kinds of lipoic acid was significant,and the rate of differentiation was the highest when the concentration of the lipoic acid was 2 mg/L.The rooting rate of 1/2MS was faster than that of MS medium and the growth potential was good.On the basis of this study,a transgenic system of Agrobacterium tumefaciens was constructed for sorghum strain 32.The effects of five Silwet-77 concentrations on the conversion were compared.The conversion efficiency was significantly higher when the concentration of Silwet-77 was 0.005%than that of the other four treatments.The effects of five acetyleugenones on the conversion were compared in co-culture.The results showed that there was a significant difference between the five concentrations,and the highest conversion rate was obtained when the concentration of acetyringone was 50 ?mol/L.The results showed that the appropriate cysteine concentration could significantly increase the conversion rate.The effects of 5 cysteine concentrations on conversion were compared.The results showed that the conversion of cysteine at the concentration of 100?mol/L was significantly higher than that of the other four concentrations.At the same time,the effects of five cysteine concentrations on differentiation were compared,and the differentiation rates were significantly different under these five concentrations.The highest differentiation rate was at the concentration of 100 ?ml/L.PCR detection and histochemistry analysis of GUS gene were carried out in T0 generation plants which were transferred into PBI121 vector.The results showed that the GUS gene on PBI121 vector was successfully transferred into regenerated plants and could be expressed normally in transgenic plants.This experiment laid a good foundation for transgenic breeding and gene function research of sorghum.