Research Of Chloroplast Transformation Technologies In Wheat (Triticum Aestivum L.) And Cloning Of Monolignol Glucosyltransferase Genes From Poplar (Populus Tomentosa Carr.)

The first part of this thesis is about the chloroplast transformation technologies in wheat. Compared with nuclear transformation, chloroplast transformation offers a number of unique advantages, such as high-level of transgene expression, transgene containment, multi-gene transformation, lack of gene silencing and position effects. Chloroplast transformation was looked as the "Environment friendly" biotechnology. This technology has been accomplished in a number of dicotyledonous plants including tobacco, tomato, potato, Arabidopsis, Lesquerella, oilseed rape, petunia and lettuce. Recently, plastid transformation was reported in rice-a monocotyledonous plant specie, and this transplastomic plant could transmit transgenes to the next generation. However, the chloroplast transformation of wheat, one of the major staple food crops, has not been developed, thus could not be used in the genetic improvement of wheat.Major obstacles to the extension of chloroplast transformation technology to monocotyledonous cereal crop which regenerate via somatic embryogenesis including: The proplastids rather than chloroplasts existed in the wheat callus, a nearly exclusively explant for the wheat regeneration in tissue culture; the smaller size of proplastid than chloroplast, causing more difficult for targeting in transformation; the difference of gene expression and gene regulation between proplastid and chloroplast; and the inability to obtain homoplastomic plants via subsequent rounds of regeneration using leaves as explants.In order to overcome the barriers of wheat chloroplast transformation existed in the tissue culture, we studied the tissue culture technologies related to chloroplast transformation and homoplastomy for the first time. This study lays the foundation for the development of wheat chloroplast transformation. The paper has got the results below:1. The establishment of induction and subculture technologies of recipient material for wheat chloroplast transformation. We determined the optimum conditions for inducing immature embryos to form embryogenic callus from two wheats (JN177 and CS respectively), and found that the size and state of this immature embryos were very important for the induction and subculture of embryonic callus. Yellowish and compact embryogenic callus should be chosen for subculture, and callus that subcultured within 1-6 months should be chosen as recipient material for wheat chloroplast transformation due to the higher regeneration frequency.2. The establishment of the repeated regeneration system of wheat plantlet, which lays an important technical foundation for getting the homoplastomy in wheat chloroplast transformation. This was the first time to achieve the second round regeneration from regenerated leaves of wheat plantlet. This technology had applied for national patent (application No.200910230815.8). The results showed that the optimal materials for callus induction were 1-2mm leaf segments from the basal parts of 4cm high regenerated plantlets. The optimal callus induction medium was MS basal medium supplemented with 2mg/L 2,4-D and lmg/L NAA, while the optimal differentiation medium was MS basal medium supplemented with lOmg/L AgNO3 and lmg/L TIBA.3. The initial establishment of callus greening technology of wheat. This was the first time to obtain green callus of wheat which has characteristics of embryonic cells and developed chloroplasts. This green callus would be the favorable recipient material for wheat chloroplast transformation.4. The study of screening technology of transformants. We determined the suitable type and concentration of selection agents and established the related technologies of transformant screening. We found that the optimal screening condition for antibiotic resistant callus and plantlets of wheat were:200mg/L streptomycin for the selection of resistant callus, while 100mg/L streptomycin for the selection of resistant plantlets. 5. We conducted chloroplast transformation of callus tissue in wheat by particle bombardment and obtained some resistant plantlets through streptomycin resistance selection.The second part of this thesis is about cloning of monolignol glucosyltransferase genes from poplar. Lignin is aromatic polymers that are present mainly in secondarily thickened plant cell walls. Researches have elucidated the main biosynthetic routes toward the monolignols and demonstrated that lignin amounts and the phydroxyphenyl /guaiacyl/ syringyl (H/G/S) composition can be engineered by changing the activities of enzymes in the lignin biosynthetic route.Monolignol synthesis is in the cytoplasm, but polymerization of monolignols is outside the cell membrane. How these monolignols transport across membrane are still lack of study. Glycosylation of small molecule compounds is believed to have a function in altering molecule transport capacity. Recently, a small cluster of three closely related genes was determined to glucosylate monolignols in Arabidopsis, thus proposing a hypothesis for transmembrane transport of monolignols. Poplar lignocellulose is the important raw material for papermaking and biomass energy industry. Lignins have attracted significant research attention because they represent a major obstacle in chemical pulping and processing of plant biomass to biofuels. These industries would benefit from processing biomass with either less lignin or a lignin that is easier to degrade.In order to study the mechanism of poplar lignin synthesis and regulation, we cloned the candidate genes of monolignol glucosyltransferases from poplar(Populus tomentosa Carr). This work would lay the foundation for the further study on mechanism of poplar lignin synthesis and regulation. The paper has got the results below:1. Four candidate genes for monolignol glycosyltransferases of poplar were predicted by sequence alignment.2. Full cDNA of these candidate genes were cloned through RT-PCR from Populus tomentosa Carr.3. Prokaryotic expression vectors and plant expression vectors of these candidate genes were constructed respectively.