| Poplar is the most useful model plants. Transgenic poplar genetic transformation mechanism, methods and the scope of transformation have been studied, and made a great progress, and also received many different varieties poplar transgenic plants. In fact, in the process of the experiment of poplar genetic transformation, only a few of transgenic poplar was obtained according to repeated experiments. FOX-Hunding System, namely the Full-length cDNA_Over-eXpressor gene Hunding System (full-length cDNA efficient expression of functional genes discovery System). The full length cDNA was transfer into plants efficiently. The phenotype was changed according to the gene expression and achieve the gene function. In this part the FOX hunting system technology was applied in the woody plants of poplar, and a efficient poplar genetic transformation method was established. Success of plant genetic transformation was based on a efficient plant regeneration system. The establishment of the transformation system and the regeneration system is inseparable. The Populus davidiana Dode×Populus bollena Lauche leaf as the experiment material, efficient regeneration system was established. Based on the efficient regeneration system, the efficient genetic transformation system was established. Poplar as a woody-plant model can be used in mutant library construction and FOX hunting system, improve the transformation frequency is significant. The salix cDNA Agrobacterium library has been built, and the library was transfer into populus using the efficient genetic transformation system. A great many of salix FOX populus was obtained. The resistance plants were treated by NaCl and NaHC03, and the salt-stress populus was generated. It was the foundation of screening functional genes using the FOX hunting system technology. The main research was as follows:A efficient regeneration system was established, and lay a foundation for the genetic transformation.(1) At2%NaCIO for sterilization, different time was used for screening sterilization time, the results showed that using2%NaCIO,15min for sterilization of populus can achieve a good effect.(2) Different concentration of6-BA (0.1,0.3,0.5mg/L) and NAA (0.01,0.03,0.05,0.08,0.1mg/L) were studied in the process of shoot differentiation. The results showed that the highest frequency of shoot regeneration (90%) from leaf was obtained on1/2MS medium supplemented with0.3mg/L6-BA and0.08mg/L NAA.(3) For elongation and proliferation of the differentiation shoot, the shoot explants were put on the medium containing6-BA (0.3,0.5,0.8mg/L) and NAA (0.05,0.1,0.15mg/L), the result showed that1/2MS+6-BA0.05mg/L+NAA O.lmg/L was the most suitable for elongation and proliferation.(4) Effect of NAA on rooting capacity was investigated. The medium containing NAA (0.1,0.2,0.25,0.3,0.4mg/L) was used for rooting. The result showed that NAA at0.25mg/L was most effective for root regeneration(100%), and the growth status of roots were length and polyrhizal.(5) The plants of the populus in the medium was transplanted after hardening, and the potted seedlings was obtained. The survival rate was upto90%.On the basis of the efficient regeneration system, a efficient genetic transformation system was established for obtaining a great many of cDNA transgenic plants.(1) Kan as a selection marker in genetic transformation. Screening a suitable concentration was important in the genetic transformation. Experimental result showed that30mg/L Kan was suitable for differentiation selection,40mg/L Kan was appropriate for rooting. Cef as bacteriostatic in the genetic transformation. Cef can restrain the growth of the Agrobacterium, but also affected the normal growth and development of plants. Experimental result showed that200mg/L Cef for bacteriostatic was suitable.(2) The different bacteria concentration (OD600=0.6,0.8,1.0,1.2) and infection time (10min,20min,30min,40min) were carried on the experiment. The experimental samples were observated by scanning electron microscopy (SEM). The experimental result showed that in the process of genetic transformation bacteria concentration between OD600=0.8-1.0, and the infection time20-30min were suitable for transformation.(3) The effect of AS on the transformation was studied, the result showed that in the process of genetic transformation, adding AS in the cocultivation medium was no effect on the genetic transformation efficiency.(4) Based on above (1)-(3) of the optimal conditions, the effect of different leaf age (30days,60days,90days) on transformation efficiency was studied. The experimental sample was observated by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for selecting the suitable leaf explant for poplar genetic transformation. The experimental result showed that30-day-old leaf as explant for genetic transformation improved the genetic transformation efficiency obviously. The status of Agrobacterium attachment to leaf surface was observed by scanning electron microscopy. The result showed that Agrobacterium attachment gradually reduced with increasing leaf age of explants. The30-day-old leaf surface showed substantial Agrobacterium attachment. There were fewer Agrobacterium cells attached to leaf surfaces of60-day-old and90-day-old. According to transmission electron microscopy observations, there were more Agrobacterium invasions in the30-day-old leaf explants than60-day-old and90-day-old explants. Our result suggested that younger leaves had higher transformation efficiency (-30%) than older leaves (10%).Using the green fluorescent protein (GFP) marker, the expression of pBI121-MD-GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode x P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants.In this section, Salix linearistioularis Hao as the research object. The. Agrobacterium cDNAs library of Salix was transformated into the populus and obtained the218Salix FOX Populus resistance plants. The resistance plants was treated by NaCl and NaHCO3.2resistance plants stress to NaCl and NaHCO3respectivly. It is a foundation for using FOX hunting system technology screening functional genes in woody plants... |
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