Studies On Agrobacterium-mediated Transformation System And Preliminary Functional Analysis Of AGAMOUS Gene In Liquidambar Formosana Hance.

Formosan sweetgum distributes in the majority of temperate and subtropical regions in China.Due to its many desirable traits,such as fast growth,wide adaptability,high resistance and red leaves in autumn,formosan sweetgum is becoming popularity in forestry and is used as a street and courtyard tree in China.But the spiny fruits of the tree disintegrate very slowly and cause a nuisance on lawns and walks.So breeding for sterility or fruitless would greatly improve the usefulness of formosan sweetgum.For the aim of fruitless formosan sweetgum,an efficient in vitro propagation system and a regeneration system via organogenesis were developed firstly.Factors affecting shoot regeneration from leaf explants,including different plant growth regulators, incubation conditions,and position of explants,were investigated in detail.Subsequently, conditions suitable for Agrobacterium tumefaciens-mediated transformation of the species were preliminarily studied.Meanwhile,we isolated AG homologue gene(LfAG) from formosan sweetgum flower buds and preliminarily analyzed its functions.The exact functions of LfAG should be studied for further researches.The major results are as follows:1.A protocol for in vitro micropropagation of formosan sweetgum was established. Effects of different PGRs on sprouting of axillary buds and shoot multiplication, elongation and rooting and survival of transplanted plantlets were investigated.The results showed that the best sprouting medium WPM+0.1mg/L NAA+0.75 mg/L BA promoted sprouting in all cultured axillary buds.Young shoots were cultured on medium for shoot multiplication,and average 5.25 and 4.17 shoots,2.57cm and 2.90cm in length were obtained,respectively.The adventitious root induced by NAA was greatly different from that by IBA.The numerous roots formed on NAA were short with big callus tissue at bottom and without secondary root.Whereas the roots formed on IBA were longer with abundant secondary roots.All the rooted plantlets were transplanted to soil and grew normally outdoors.2.Plant regeneration system from leaves of formosan sweetgum was established and optimized.The morphogenic potential of leaf explants from five genotypes was tested on the 16 media containing different concentrations of TDZ and NAA.The results indicated that among five genotypes,four genotypes(P2,P6,P9,P11) showed higher regeneration rates(53.2%-90%in average),whereas genotype J13 showed low capability of shoot regeneration on all media tested(32.6%).In general,preferable media for inducing adventitious shoots was WPM supplemented with 0.25mg/L TDZ and 0.05mg/L NAA,on which higher regeneration rate and more adventitious shoot clumps were obtained in all five tested genotypes.The organogenesis was greatly influenced by light and the optimum condition was leaves were cultured in dark for 7d and then transferred into 50-100 lx.The upper two leaves on the stem showed higher regeneration capability(100%) than the lower two leaves,which were still over 70%in regeneration capability.Transferred to WPM basal medium containing NAA,BA and GA₃,regenerated buds elongated into shoots.3.Effects of selection markers kanamycin(Kan) on shoot differentiation of leaf explants and rooting of in vitro shoots were investigated.The results indicated that formosan sweetgum was comparatively sensitive to Kan.Selection pressures were determined as:20 mg/L Kan for shoot regeneration of leaves,and 15 mg/L Kan for rooting of shoots.300 mg/L cefotaxime(Cef) were no great influences in organogenesis of adventitious shoot and root.4.Conditions suitable for Agrobacterium-mediated transformation of formosan sweetgum were studied.The results indicated that after co-cultivation with Agrobacterium,regeneration capability of the leaves decreased dramatically when cultured in selection medium.According to the transient GFP expression,three days of pre-culture with the abaxial side of leaves touched the medium promoted adhesion and invasion of Agrobacterium evidently.For formosan sweetgum,acetosyringone(AS) in the co-cultivation medium had no promoting effect on Agrobacterium mediated transformation.Based on the transient GFP expression,10-min infection in AB liquid medium and co-culture for 4d was favorable for T-DNA transfer to plant cell of formosan sweetgum.5.We have isolated the putative AG gene from formosan sweetgum,LfAG,whose deduced protein product shares 96.0%identical amino acid residues with LAG gene product from L.styraciflua,a count-part sweetgum grown in America.LfAG included full length MADS domain and K domain,and there were two consensus AG motifs in the C termination. 6.Sense,repeated anti-sense and RNAi plant expression vectors of LfAG were construted.59 repeated anti-sense and 60 RNAi transgenic tobacco plants were obtained by the leaf discs method of Agrobacterium-mediate transforomation.Analysis of transgenic tobacco plants showed that LfAG gene was involved in the stamen and carpel of flower development.Repeated anti-sense of LfAG gene expression produced a variety of mutant phonotype with either anther transformating into petal,or stigma on anther,or secondary pistil.The reason of mutants needs further researches.