| In recent years,the soybean import is increasing in China.Most of the imported soybean varieties are genetically modified,which have advantages in terms of yield and price compared with domestic soybeans.Therefore,it is urgent for us to develop soybean genetic transformation methods in order to increase soybean yield,improve quality,obtain resistance to pests,diseases,herbicides,and increase the competitiveness of domestic soybean production to ease the contradiction between supply and demand in the soybean market.An efficient transformation method is the prerequisite for the study of gene function and molecular breeding.Agrobacterium-mediated transformation method is the preferred method for many plant spcies.However,compared with other crops such as rice and wheat,soybean transformation efficiency is still low.Therefore,improving the transformation efficiency of soybean is an urgent problem to be solved.In this study,we try to optimize the factors affecting the Agrobacterium infection efficiency and soybean regeneration efficiency to improve the genetic transformation efficiency of soybean.And we also explored the effects of overexpressing two plant hormone-related genes,ZmKN1(Zea Mays Knotted 1)and GmGID1s(Glycine max GA Insensitive Dwarf 1),on the regeneration and transformation efficiency,as well as morphological traits of soybean,by constructing expression vectors containing ZmKNl and GmGID1s,respectively.The main results are as follows:1.Optimization of Agrobacterium infection efficiency and soybean explants regeneration efficiency to improve the transformation efficiency of soybean.The vector we used for this experiment was pTF102 and the Agrobacterium strain was EHA101.The results showed that infection efficiency of Agrobacterium can be reached above 96%(the soybean variety was Jack Purple and Tianlong 1)when we collected Agrobacterium at the concentration of OD650=0.6,and 154.2 mg/L DTT was added to the resuspension medium to infect the explants(which was obtained by 1-d imbibition),then co-cultivated for five days.On the other hand,we found that using 1.0 mg/L GA3(Gibberellin)and 0.1 mg/L IAA(Indole-3-acetic acid)in the shoot elongation medium can obtain the highest elongation efficiencies(34%and 26%for Jack Purple and Tianlong 1,respectively),which is higher than that of five other combinations of GA3 and IAA concentrations.This concentration combination of GA3 and IAA increased the elongation efficiency by 18%and 11%for Jack Purple and Tianlong 1,respectively,compared to that of the original concentration combination of GA3 and IAA(0.5 mg/L GA3 and 0.1 mg/L IAA).At this optimized hormone concentration,the transformation efficiencies of Jack Purple and Tianlong 1 were 7%and 10%respectively,which was 2%and 6%higher than that of the original concentration combination of GA3 and IAA,respectively.2.Cytokinin is one of the most important plant hormones,which can promote the division and proliferation of plant cells,induce bud differentiation,and relieve the apical dominance.KN1 gene was found to be able to increase the cytokinin content in plant cells,induce adventitious shoot formation,and increase transformation efficiency in plants such as Arabidopsis,tobacco,and rice.Therefore in this study,we would like to investigate the effect of KN1 gene on the regeneration and transformation efficiency of soybean.First we constructed the pCAMBIA3301-35S-ZmKN1-NOS vector.Then,we transformed the vector into Agrobacterium EHA105 for soybean transformation.We found that ZmKN1 gene may be able to promote the elongation of soybean adventitious shoots during transformation.But the elongation of false positive shoots(non-transgenic)was also found to be promoted by ZmKN1 gene.The phenotypes of ZmKN1 overexpressing soybean plants were observed.Comparing with the control plants,the leaves of ZmKNl overexpressing plants were curled and the petioles were twisted,leaf veins were protruded irregularly,the angle between the petiole and the main stem was almost parallel,flowered earlier,plants were smaller,and the average number of seeds per pod was less than two,which was significantly different from control plants(more than two seeds).These results indicated that overexpression of ZmKN1 gene could promote the elongation rate of soybean explants,but there were some problems such as high false positive rate and negative effects on plant growth and development.It is necessary to construct a vector with inducible promoter to regulate the expression of ZmKN1 gene in future study.3.Our previous studies had shown that the concentration of exogenous GA in the tissue culture medium affects the elongation rate of explants and transformation efficiency of soybean.Therefore,we next studied the effect of GA-related genes,GID1s(encoding GA receptors),on the explant elongation and transformation efficiency in soybean.The GA receptor binds to GA directly and then binds to DELLA-related protein that inhibits plant growth.It is recognized by SCF and ubiquitinated by E3,which degrades DELLA and releases the inhibition of plant growth,and therefore promoting plant growth.Through BLAST search,we found that there are five genes in soybean that are homologous to AtGID1 from Arabidopsis thaliana,and their proteins contain the conserved domains of HGG and GDSSGG that bind to active GA to sense GA signals.These five genes were denominated as GmGID1-1～GmGID1-5 based on their chromosome positions.The length of GmGID1s ORF are 1029～1041 bp,encoding proteins with the molecular weight of 38.67～39.39 KDa and the number of amino acids are 342～346.The expressions of GmGID1s genes were induced by exogenous spraying of 100 mg/L GA on soybean leaves.The expression of GmGID1s in soybean leaves were significantly higher than the 0 h control except GmGID1-1 gene,and the expression of GmGID1s in soybean stems were significantly higher than the 0 h control except GmGID1-3 gene(P<0.05).4.Five vectors containing GmGID1-1～GmGID1-5 respectivily(pCAMBIA3301-35S-GID1s-NOS)were transferred into Agrobacterium EHA105 and transformed into soybean.Comparing the five GmGID1s genes,the elongation efficiencies of soybean explants containing GmGID1-1 and GmGID1-2 genes were 36.13%and 30.84%,respectively,which were higher than the other three genes,and the transformation efficiency obtained by transforming the GmGID1-2 gene was the highest,reaching 6.68%,followed by GmGID1-3,which was 3.77%.Through the comparison of five GmGID1s genes,it was found that GmGID1-2 gene has a certain role in promoting the elongation of soybean adventitious shoots,but the experiment still needs to be repeated several times to verify the conclusion.In addition,the average height of GmGID1-2 overexpressing plants was 109.6 cm,which was significantly(P<0.05,t-test)higher than the average plant height of 20.5 cm for control plants.The effects of GmGID1-2 on other agronomic traits of soybean need to be observed in the future. |
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