Optimization Of Cotton Transformation System And The Transformation Of Resistant Gene To Verticillium Wilt

Verticillium wilt,caused by Verticillium dahliae(V.dahliae),is one of fungal diseasedecreasing not only fiber yield by 20-60% but also fiber quality,and it exist widely inworldwide cotton planting area.Now,it has been one of major limiting factors in cottonproduction.Results from previous researchers showed that cotton varieties could be improvedin resistance of disease,pest and herbicide and fiber quality etc by using gene engineering.Inrecent years,the gene transformation via Agrobacterium-mediated system was used widelyand its mechanism of transformation is very clear.However,many problems still exist incotton gene transformation.The main reasons are a higher degree of genotype dependence inplant regeneration of cotton tissue culture,lower efficiency in gene transformation of cottonand foreign gene silence in transgenic plant.In this paper,an optimized system wasestablished for cotton tissue culture and gene transformation.An Alfalfa antifungal peptide(AlfAFP)gene,cloned from seeds ofMedicago sative,was introduced into cotton genome andtransgenic plants of enhanced resistance to V.dahliae were obtained.Development of cotton fiber was regulated by many genes.However,because of longduration and a lot of work in cotton gene transformation,large-scale analysis of gene functionin fiber development is very difficult by using cotton stable transformation system.Therefore,it is not clear that which and how genes regulate fiber development.It is very necessary toestablish a gene transient expression system to evaluate gene function in fiber cell by asimpler and quicker test method.In this paper,two transient gene expression systems in fibercell were established by particle bombarding ovules and injecting ovaries through pollen tubepathway.The main results are listed below:1.Optimization of effect factors in cotton tissue cultureCotton hypocotyl pieces(3-5 mm)from 5 to 7 days-old sterile seedlings were culturedon 15 kinds of callus induction media,which included different kinds of hormones in LS orMSB basic media.Results indicated that calli growth on LS media were better than these onMSB media when hormones and hormone concentrations were same.Most of calli on LSmedia were yellow-green and had capacity to differentiate into embryonic calli.In all hormone treats,calli growth on LS medium with 0.1 mg/L KT and 0.2 mg/L 2,4-D were thebest,and the calli were yellow-green and loose.Calli on the other media were white and hardor brown and very soft,which were not impossible to differentiate into embryonic calli.Yellow-green calli were placed on 4 proliferation media and showed a better proliferation onMSB medium than on LS medium.Proliferation efficiencies were decreased when ZT oractive carbon were added in MSB medium.When calli on MSB medium were transferred on6 kinds of media with different hormones,the MSB medium with 0.3mg/L KT and 0.5 mg/LIBA was the best and could produced the highest number of embryo and cotyledon embryo inall treats.If somatic embryos were developed into plantlets on 1/2MSB medium with 0.5mg/LNAA,differentiation efficiency of embryogenic calli and plantlet regeneration was increasedwhen 5μmol/L Zn²⁺and Ni²⁺were added in medium.It was suggested that plantlets withroots should be transplanted into greenhouse when plants heights were about 5-8 cm andSeedlings without roots were grafted on rootstock in greenhouse.2.Optimization of effect factors in cotton gene transformationEffect factors of transformation processes were examined in efforts to improve theproduction efficiency of transgenic cotton.The result showed that strain LBA4404 wassignificantly better than EHα101 and EHα105.The efficiency of transformation could beimproved when bacterium density was 0.5-0.7 at OD₆₀₀.15 minutes is the most appropriatetime for cotton hypocotyl pieces to be dipped into Agrobacterium suspension.Relative lowco-cultivation temperature(21℃)and long co-cultivation duration(60h)were optimal fordeveloping a highly efficient method of cotton transformation.Concentration ofacetosyringone at 200μmol/L during co-cultivation significantly increased transformationeffiency.3.Construction of plant expression vector and development of transgenie plantsAn alfalfa antifungal peptide gene(alfAFP),which was cloned from seeds of Medicagosative,was ligated in pCAMBIA1301 to form a recombinant pCAMBIA1301-alf.The alfAFPgene expression was under the CaMV35S promoter control in recombinant binary vector.Gene alfAFP was delivered into an upland cotton line 5983 via Agrobacterium-mediatedhypocotyl system.It took about 8-10 months that 15 plants were regenerated by cultivatingthe transformed cotton tissues.Transgenic plants were transplanted in greenhouse when their roots were about 5cm in length.50mg/L hygromycin solution.was spread onto the yongleaves oftransgenic plants.7-10 days later,results showed that there were 3 negetive plants in15 transgenic plants.PCR products confirmed the integration of the alfAFP gene in thegenome of 12 transformants.Southern blot analysis showed that 6 transformants were onecopy and another 6 transformants were two copies of the alfAFP gene.RNA from roots oftransformants was used for Northern blot and gene transcripts were present in all 12transgenic plants.4.Resistance to V.dahliae,changes of related enzymes and agronomy characters oftransformantsIn vitro assays showed that crude leaf protein extracts from transforrnants was able tosignificantly(p＜0.01)inhibit the growth and proliferation of V.dahliae by 46.2-86.8%compared to extracts from control plants.In vivo assays with fungal pathogen showed thattransformants displayed a significant reduction in disease symptom compared to controlplants.At harvest stage,evaluation of disease degree demonstrated that 5 plants of 12transformants were immune to verticillium wilt,3 plants each showed the disease grade of 1,another 3 plants were scored 2,respectively.1 plant showed severe disease symptom with thedisease grade of 3.Results from assays in vitro and in vivo suggested that the alfAFP genewas able to express in the upland cotton and its product of gene expression was active to thegrowth inhibition of V.dahliae.Activities of four related enzymes in control and transgenic cottons were measured afterinoculated by V.dahliae suspension.Results showed that activity trends of four relatedenzymes were similar.After inoculated,enzyme activities of control plants dramaticallyincreased,and then decreased rapidly.There were peaks at 3 or 5 d after inoculation.Intransgenic plants,changes of enzyme activity of transgenic plants with part verticillium wiltsymptom were similar to that of control plants,but had lower peaks.However,the enzymeactivity of immune-verticillium wilt cotton was maintained at low level and did not show anyobvious peak.Agronomy characters of transgenic cotton plants were influenced by the alfAFPintroduction.Results showed that transgenic plants were reduced in height,fruiting branchesand nodes,bolls and boll weight,but was not affected in fiber lint percentage.In characters of fiber quality,transgenic plants showed lower fiber strength and higher micronaire value,buthad significantly unstable change in fiber length,some became longer and others becameshorter than control plants.5.Establishment of transient expression system of foreign gene in cotton fiber cell1DPA ovules were cultivated for 1 d on BT medium,and then they were used for thebolistic transformation.The optimal parameters were as follows:Helium pressure at 1100psi,target distance at 9 cm,the vaccum pressure at 28 inches Hg,vector concentration at 2μg perbombardment.Foreign gene expression could be continually detected in fiber cells onculturing ovules from 2 to 20 d after bombardment.After 20 d,gene expression disappeared.It is indicated that function analysis of genes lelated with the fiber developmet may beavailable by the bolistic transformation system when ovules were cultured in the period from3 to 22d in fiber development.Vector concentration of 0.4-0.7μg/μl was the optimum for gene transformation for 1DPA ovules by the method of ovary-injection in vivo.GUS gene in fiber cells on ovules couldcontinually express within 9 days after transformation,and its expression efficiency was themost high at 3 d after injection.Therefore,the duration from 2 to 10 d may be the best timefor function evaluation of genes related with fiber development by the ovary-injection system.