Rule Of Floral Buds Differentiation And Molecular Regulation Of Flowering Development In Populus Tomentosa Carr.

Populus tomentosa Carr (Chinese white poplar) is a native species in section Leuce with fast-growing and high wood quality in the north of China. However, a long juvenile period and pollen & catkin pollution hinders its research and extensive application. With the development of a transformation system in poplars, it provides us an effective approach to incorporate alien species genes into this valuable tree species. It is a good approach to construct sense & antisense and RNAi vectors to transform into male and female clones for revealing the molecular mechanism of flowering in P. tomentosa, and plays as an important base for shortening the breeding cycle and suppressing contamination of pollen and catkins.A serial studies were adopted, ranging from developmental rule of male & female floral buds observed, male & female floral buds cDNA libraries respectively constructed; regenerative and transgenic condition and antibiotic concentration screened, effective regeneration system and transgenic system established to transform into tobacco and LM50 and 5082 clones, molecular tests and field test results acquired too.Seasonal changes (2-yr period) in the development of reproductive structures are documented for only one male and one female tree of P. tomentosa. Serial paraffin sections were prepared to observe the morphological changes and the structure in microscope level of female and male reproductive systems, The organogenesis of male and female floral buds and the mechanism of male sterility was discussed in P. tomentosa.Male and female buds (containing all stages in reproductive development process) cDNA library were constructed separately. The results showed primary libraries had a titer of 8×10⁵ pfu/mL and 7.2× 10⁵ pfu/mL, amplified libraries had a titer of 2.6×10⁸ pfu/mL and 2.56×10⁸ pfu/mL in female and male clones respectively. The combination ratio reached 90% and the size of inserts was 400-2500 bp. The results indicated that cDNA libraries have been successfully constructed.PtAP3, an AP3 homologous gene from P. tomentosa was isolated by PCR using genomic DNA of male clone of P tomentosa (LM50) as template. The result indicated that the sequence was 1 813 bp (BamH I and Sac I were introduced at the 5'and 3'end) including 7 exons and 6 introns, coding 238 amino acids. The result of Southern blot analysis indicated that there were double copies of PtAP3 or two members which had a high homology to each other in P. tomentosa (L50 , male) genomic DNA, and there was single copy PtAP3 in P. tomentosa (5082 , female) genomic DNA.Regenerative efficiency of different stage, state, place of poplar leaves were compared. The optimum concentration of Kan was respectively 20 mg/L and 25 mg/L at least at the stage of screening, propagation and rooting. As to cef and Cb, the optimum concentration was 200-300 mg/L at the stage of screening, propagation, while 200-600 mg/L at the stage of rooting. The inhibitive effects of Cef and Cb upon Agrobacterium tumefaciens varied with the strains of Agrobacterium. Cef is better choice to inhibit Agrobacterium LBA4404 and GV3101. And the optimum regeneration system of tobacco and Chinese white poplar was founded as follows: Regeneration medium for leaf-explant in tobacco was MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+sugar 30 g/L+agar 5.5 g/L, and for poplars' leaves, the regenerate medium was MS+6-BA 2.0 mg/L+NAA 0.1 mg/L+sugar 30 g/L+agar 5.5 g/L; Rooting media both was MS+NAA 0.3 mg/L+sugar 20 g/L+agar 4.5 g/L.Establishment of genetic transformation system: The test demonstrated the best leaves for adventitious buds regeneration stem from rooted plants. Sense and anti-sense expression vectors of PtAP3 and PtLFY-IR were constructed by PCR and restriction enzymes digestion identification, and were fused to a CaMV 35S promoter of pBI121 to transform into LBA4404 and GV3101. The optimized condition for transformation was infected with A .tumefaciens concentration of OD600=0.3-0.5 about 5-10 min and co-cultured with an engineered A. tumefaciens strain carrying PtAP3, Ptap3 and PtLFY-IR genes for 2-3 days. The integration of foreign genes into tobacco was confirmed by PCR-Southern hybridization and PCR analysis.It had been developed, 15 clones with PtAP3 gene, 10 clones with antisense Ptap3 and 10 clones with PtLFY-IR. Great phenotypic differences in transgenic tobacco plants have been observed. Almost all transgenic tobaccos T₀ of sense PtAP3 showed a higher growth rate than wild type and antisense transformants and a few developed pregnant earlier than wild type seedlings under the same conditions. Furthermore T₀ tobacco seeds of PtAP3 were harvested and phenotypic characteristic of T₁ transgenic PtAP3 tobacco showed flowering earlier and growing higher than wild type either. Thus it indicates that the sense PtAP3 has been transmitted to the next generation in these lines.The protocols of a routine in vitro regeneration and transformation for P. tomentosa to propose to develop genetic transformation of PtAP3, Ptap3 and PtLFY-IR genes into clones L50 and 5082 of P. tomentosa. Now a few transformants (LM50) have been acquired with sense & antisense PtAP3 and PtLFY-IR, and initiate their molecular analysis, PCR detection indicates that the constructs have been transformed into male clone LM50. Further research is carrying on to regulate flowering development in P. tomentosa.