Yheml Transformation Into Banana To Improve Its Cold Tolerance

Banana is one of the most important tropical and sub-tropical economic crop in the world. Low temperature is an important limiting factor influencing the quality, yield and planting area of banana. However, cultivated banana is nonsexually-produced plant,the special biological properties make it difficult to use conventional breeding methods for genetic improvement. In recent years, technology of tissue culture and genetic engineering made it cometrue。5-aminolevulinic acid (ALA) is a naturally occurring in plants.It has been suggested being ability to improve plant resistant. In animal and yeast, ALA-S is a rate-limiting enzyme for ALA synthesis pathway.In Arabidopsis, GluTR is encoded by HemAl,and HemAl is regulated by light.We introduced a recombinant gene YHemlwhere the yeast ALA synthase gene(Heml) was the functional gene, which was controlled by Arabidopsis HemAl promoter, into banana thin-cell-layers. By detection of Km, GUS, PCR, RT-PCR and endogenous ALA levels, we have showed that two of those resistant buds we got from study could biosynthesize more endogenous ALA and higher capacity of chlorophyll fast fluorescence characteristics under low temperature tolerance. The study laid the foundation for breeding new varieties of cold tolerance of banana. It is the main results of the research below:1We set up a higher regeneration system of thin-cell-layer explants of’Brazil’banana, by reseach on plant growth regulators, explant selection and other aspects. The results suggested that before the5th generation, on MS medium with0.1mg/L NAA+3mg/L6-BA+2mg/L AgNO3, and after cultured in darkness for2weeks, the buds regeneration percentage reached95%. And regenerated buds could root on MS medium with0.4mg/L NAA.2On above basis of regeneration system,we studied several factors that affect banana genetic transformation, such as:secondary infection method, concentrations of Km (kanamycin) and co-culture condition.Then we established genetic transformation system of banana. The study showed that put the explants in the bacterial liquid30minutes with OD600=0.8～1.0, then incubated in MS+0.1mg/LNAA+3mg/L6-BA+100μmol/L AS+2mg/L Pro for4-6days. After co-culture, the infected explants were transfered into selection medium, which contained MS+0.1mg/L NAA+3mg/L6-BA+250mg/L Km+600 mg/LCarb for3weeks, then we got7kanamycin-resistant plant lines.3We get15buds(regeneration rate of Km resistant buds is about20%), and7rooted plantlets. After GUS, PCR, RT-PCR detection, two of them were found transgenic, who could biosynthesize endogenous ALA, and endogenous ALA levels of transgenic banana were greatly more than that of wild type plants.4Analysis to the results of SPAD, fast chlorophyll fluorescence characteristics, OJIP curves and oxidation and reduction ability of PSI reaction center of leaves of transgenic banana and wild-type at25℃and7℃low temperature tolerance for1.5h, we know that transgenic plants get higer SPAD and chlorophyll fluorescence characteristics. Low temperature decreased the OJIP phase and the rate of oxidation and reduction of PSI reaction center of wild type significantly, but not to genic lines.The result of fluorescence parameters showed that, before and after low temperature tolerance, the energy conversion (ABS/CS, TRo/CS, and ETo/CS) and the photosynthetic performance index(PIABS and PICS), oxidation and reduction ability of PSI reaction center of transgenic banana were higher than the wild type, but the energy dissipation through heat (DIo/RC) was less than the wild type.All of these suggested that, over-production of ALA propected PS II phyotosynthetic of banana leaves and increased the accumulation of photosynthetic products, also is the protection of the banana plant resistance to low temperature.