Effect Of Abscisic Acid On Callus And Embryogenic Callus Induction In Cotton(Gossypium Hisutum L.)

As a natural fiber and oil crop,cotton is one of the most important cash crops in the world.With the rapid development of gene engineering and cell engineering technology,transgenic genetic breeding has become an important technical means to improve cotton varieties.Somatic embryogenesis of cotton is one of the main ways to achieve specific transgenic cotton breeding.As important plant growth regulators,abscisic acid play a regulatory role in somatic embryogenesis.In this experiment,cotton hypocotyls were treated with exogenous abscisic acid with gradient concentration using MSB medium,callus induction medium and embryo callus induction medium as the basic medium.The effects of abscisic acid on somatic embryogenic callus induction and embryogenic callus induction of cotton were studied by morphological observation,paraffin section observation,data statistics and qRT-PCR.The experiment determined the abscisic acid concentration with the optimal comprehensive induction effect and analyzed the relationship between abscisic acid regulation and somatic embryogenesis Marker gene spatiotemporal induction expression.The main research results are as follows:1.Abscisic acid promotes callus initiation in cotton.When cotton hypocotyl was cultured in MSB medium for 5days,it dedifferentiated to produce callus,and the number of callus cells increased with the increase of ABA concentration.The qRT-PCR was used to detect the expression level of LBD gene in callus at 5 days.Compared with no ABA treatment,the relative expression levels of GhLBD16 and GhLBD29 in callus treated by ABA were significantly up-regulated,so it was speculated that ABA could promote callus initiation by influencing the expressions of GhLBD16 and GhLBD29.2.Abscisic acid significantly increased callus dedifferentiation rate(CDR)and promoted cotton callus dedifferentiation.When cultured in MSB as the basal medium for 20 days,CDR was 15%in the control group and 71.7%-84.2%in the ABA group.When the callus induction medium was used as the basic medium,the CDR of the control group was 90%and that of the ABA-treated group was 90.8%-95.8%.The results showed that ABA treatment with 0.02 ?M-0.08?M increased CDR significantly.When treated with auxin,ABA concentration of 0.2 ?M-0.8?M increased CDR.3.Abscisic acid significantly increased callus proliferation rate(CPR)and promoted cotton callus proliferation.When cultured in MSB medium for 20 days,CPR was 3.8 in the control group and 6.2-6.9 in the ABA-treated group.At 30 days,CPR in the control group was 10.2,and CPR in the ABA-treated group was 13.1-16.1.With callus induction medium as the basic medium,CPR in the control group was 3.5 at 10 days,and CPR in the ABA-treated group was 4.3-6.2.At 20 days,CPR in the control group was 11.4,and CPR in the ABA-treated group was 15.7-20.5.At 30 days,CPR in the control group was 24.7,and CPR in the ABA-treated group was 30.3-37.1,respectively.The results showed that exogenous abscisic acid significantly promoted callus proliferation in a large concentration range of 0 ?M-0.8?M,and was independent of auxin.4.Low concentration abscisic acid promotes embryogenic callus differentiation,while high concentration abscisic acid reduces embryogenic callus differentiation.Statistics were made on embryogenic callus dedifferentiation rate(ECDR)and embryogenic callus proliferation rate(ECPR).When cultured on MSB for 35 days,ECDR was 7%in the control group and 3%to 19%in the ABA-treated group.At 45 days,the ECPR of the control group was 23.0,and the ECPR of ABA-treated group was 32.2-36.5.At 45 days,ECDR was 89.6%in the control group and 24.0%-59.4%in the ABA-treated group.At 55 days,ECDR was 89.6%in the control group and 24.6%to 65.3%in the ABA-treated group.Under low concentration(0.02?M)ABA treatment,embryonic callus showed a light yellow,loose and dispersed state,while under high concentration(0.04?M-0.8 ?M)ABA treatment,embryonic callus gradually showed a weak,soft and black state with increased ABA concentration.5.The expression of Marker genes related to somatic embryogenesis regulated by abscisic acid controls callus proliferation and differentiation.At the early stage of callus proliferation(15 days),ABA promoted BBM gene up-regulation,had no effect on LEC1 gene expression,and inhibited AGL15 gene expression.ABA induced the expression of BBM,LEC1 and AGL15 genes at metaphase of callus proliferation(20 days).ABA significantly induced the expression of FUS3,LEA and ABI3 genes at the late callus proliferation stage(30 days).Above all,exogenous ABA treatment induction of somatic embryogenesis related Marker gene specificity of time and space,callus proliferation in the early and mid mainly regulate the expression of BBM,LEC1 and AGL15,late main regulation FUS3 callus proliferation,LEA,ABI3 gene expression,the body embryogenesis related gene expression level increased is the foundation of ABA regulating the proliferation and differentiation of callus.The results showed that abscisic acid could significantly promote the callus initiation,increase the rate of callus proliferation and dedifferentiation rate,low concentration abscisic acid(0.02 ?M)increase the rate of embryonic callus differentiation,high concentration of abscisic acid inhibition of embryonic callus differentiation,and the readjustment of abscisic acid and somatic embryogenesis Marker gene induced expression of space-time are closely related.The experiment provided a reference for the application of abscisic acid in the improvement of the system of somatic embryogenesis of cotton and a basis for the in-depth analysis of the mechanism of abscisic acid in somatic embryogenesis.