Genetic Transformation Of FT Gene And Precocious Flowering Induction On Hybrid Aspens

Poplar(Genus Populus)is a preferred tree species in forestry and engery agriculture and has planted worldwide, is a major timber wood species for short peroiod industry in China, also is a hot interesting sopts for tree breeders to select and cultivate new species . Poplar has a long juvenile period , whereas many traits only express when adult , which resulting in limitation of progress in genetics and breeding and associated researches around this species. Therefore, stimulate precocious flowering and shorten breeding period will play a positive role in poplar genetic improvement. In recent years, successful of cloning and transfermation of many genes associated with flower development, laying foundation for plant flowering stage modification through transgenic technology. FT(Flowering Locus T)is a floral stimulating gene isolated from Arabidopsis thaliana, the expression product of FT gene is the"florigen"that researchers seeked over a long period. Researchs about FT transgenic plants mostly showed early flowering ability. In this paper , based on sucessfully cloning of Arabodipsis FT gene homologs FT1 andFT2 from Populus trichocarpa , and construct of PHSP::FT driven by a heat shock promoter(HSP)from Soybean, we created constructs of PHSP::FT1 and PHSP::FT2 of FT1 and FT2 genes driven by the same heat shock promoter, developed effencient transfation system on hybird aspens either from Section Leuce including 717-1B4( female ,Populus. tremula x P. alba, following Abbreviated as 717)or 353-53(male ,P. tremula x P. tremuloides,following Abbreviated as 353). 191 PCR positive clones of FT transgenes were obtained via leaf disc incubated in agrobacterium strain AGL1, and optimization of transform conditions for the 3 FT genes on 2 poplar clones. Early flowering were successfully induced on FT transgenes after heat induction system , best flowering FT genes were selected for 2 poplar clones, all factors affect FT gene expression and transgenes flowering capacity were discussed , flower development process under heat induction treatment were set forth, and finally graft-transmissible of FT induced flowering signal was preliminary explored. Results obtained from our experiments as following:1.Created constructs of PHSP::FT1 and PHSP::FT2 driven by a heat shock promoter of FT1 and FT2 genes in poplar.2.Factors e.g. auxin NAA, cytokinin 6-BA and TDZ, cutting styles of leaf explants, inoculation style, and culture situation etc, affect on shoots regeneration on hybrid aspen clones 717 and 353 were studied through systematic tests. Also threshold sensitive concentration of canamycin was tested in shoot regeneration and rooting in 2 poplar clones. Results showed appropriate culture medium for shoots regeneration on leaf explants is MS containing 0.10 mg/L NAA, 0.5 mg/L 6-BA and 0.05 mg/L TDZ for both poplar clones 717 and 353. The likely treatments for shoot regeneration were 4mm leaf discs punched form leaf explants, leaf adaxial touch medium, and 16 hr lights/d after initial 2 days in darkness in the above medium composition. Under the above regeneration system, shoots regeneration frequency on leaf explants were 85.6% and 77.5%, with average number of shoots per explant were 12.4 and 8.6 on poplar clones 717 and 353, respectively. The threshold sensitive concentration of canamycin was 100 mg/L in shoot regeneration on leaf explants, and 25 mg/L in shoots rooting for both poplar clones 717 and 353.3.Factors affect PHSP::FT transformation on clone 717 was tested and optimized, successfully transformation of PHSP::FT1 and PHSP::FT2 on 717, and three FT constructs transformation on clone 353 via optimized system were achived, 191 PCR positive clones obtained from PHSP::FT , PHSP::FT1 and PHSP::FT2 transgenes on clones 717 and 353. Results indicated short time pre-culture enhanced transfornmation ratio on clone 717, high concentration of AGL1, long time of transformation in AGL1 solution, relative short time incubatation were essential for PHSP::FT transformation on clone 717. AS (Acetosyringone) has less effect on transformation frequency of PHSP::FT construct on clone 717. Optimized transformation condition for FT gene on clone 717 via AGL1 is, pre-culture 3 days, agrobacterium activation of OD600=0.5-0.6, transformation 60 minutes, incubation 2 days in darkness, no AS attendance, would reach a transformation frequency of 31.7%. In additional, the optimized system also appropriate for other FT constructs on clone717 and the 3 FT constructs on clone 353.4.FT gene that could express smoothly and induced transgenes flowering after heat induction on both poplar clones 717 and 353 was selected, Factors that affect FT gene expression following heat induction were discussed, appropriate heat induction situations in smooth induction express of FT gene were found, and flower development process on FT transgenic poplar were observed in detail.Heat induction results showed, no leaky expression of the FT genes under HSP promoter was observed, great diversity among early flowering induction in various FT gene origins, FT gene has better flowering ability than genes FT1 and FT2 from P. Trichocarpa, and each FT gene prefer flowering in clone 353 to clone 717. Factors affect FT gene expression following heat induction including, receptor genotype, clones within the same genotype, ages and height of transgenes when heat induction started, heat induction treatments etc. Interaction of FT gene and receptor genotype was obvious, the 3 best flowering combinations of FT genes and receptor genotype are 353/PHSP:: FT, 717 /PHSP::FT and 353/ PHSP:: FT1;FT gene expression following heat induction was varied within clones and ramets inside clone, and such variation changes associated with ages and height of transgenes when heat induction began. .Flowering frequency is positive related to plant height, larger plants appeared to flower more readily than did smaller plants, variable height were required based on different gene/clone combination, plants must higher than 20cm in 353/PHSP:: FT(or FT1), or than 30 cm in 717 /PHSP::FT were considerably more likely to flower. Ages of transgenes mainly affect flowering initiation following heat induction, whereas less affect final ramets flowering frequency, but influence clones flowering frequency within same transgene.Heat induction hours per day has less effect on transgenes flowering frequency, duration of heat induction treatment greatly affected normal development of catkin , increased duration time of heat induction could prevent catkins reverted to vegetative growth, whereas increased normal catkin products. Increased heat induction temperature (40℃)would advance FT gene expression and initial flowering on transgenes, more production of catkins, and less catkin reverted to vegetative growth.Lower room temperature in GH is favorable for FT gene expression following heat induction. Floral buds initiated from leaf axils in newly sprouted shoots which elongated in original shoots apex after heat induction started, floral organ differentiated from floral buds varied widely, kinds of floral organs differentiation depends on location of floral shoots initiated on shoots or initiation time. Only 20-40% floral buds induced following heat induction formed into catkins, 5% catkins and 30% individual flowers on catkins dispersed pollen grain. Bisexual flowers on male catkin were more normal than those on single flowers.5.Cleft grafted transgenes failed to induce non-transgenic scions to flower after heat induction, but transgenic rootstocks had less flowering frequency than did controls, indicating grafting might resulted in transmission of part of FT product in grafted transgenic rootstocks.