Biotechnology and its uses in improvement of canker resistance in citrus trees.

摘要

'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) is one of the leading commercial cultivars in Florida because of its high yield potential and early maturity. 'Hamlin' also has a high regeneration capacity from protoplasts and is often used in transformation experiments. Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem. The Xa21 gene is a member of the Xa21 gene family of rice that provides broad spectrum Xanthomonas resistance in rice. Since the citrus canker pathogen is in the same genus, this gene may have the potential to function against canker in citrus. The wild-type Xa21 gene contains an intron, and there is some question as to whether dicot plants can process genes containing monocot introns (the cDNA is intron free). The development of canker resistant citrus has become an important research objective.; Genetic transformation has become a widespread tool in both basic research and commercial plant breeding programs for disease resistance. Plasmid DNA (pARS108) encoding the non-destructive selectable marker Green Fluorescent Protein (GFP) gene, and the plasmid cDNA of the Xa21 gene (pCR506-mtaq) were co-transformed into 'Hamlin' orange protoplasts using polyethylene glycol. Also, plasmid DNA (pAO3), encoding the GFP gene and the cDNA of the Xa21 gene, was transformed into 'Hamlin' orange protoplasts. Following protoplast culture in liquid medium and transfer to solid medium, transformed colonies were microscopically selected via expression of GFP, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryoids were recovered. Over a thousand transgenic plantlets were regenerated from about 80 independent transformation events. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in some of the transgenic plantlets. The recovery of multiple transgenic plants was expedited by in vitro grafting. The transgenic plants have shown normal growth and stable GFP expression for over a year in the greenhouse. Transgenic greenhouse plants include 400 growing on different rootstocks and over 200 plants on their own roots. This is the first time to report a large population of transgenic 'Hamlin' sweet orange plants using protoplast/GFP transformation system. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in the transgenic greenhouse plants. Some of the plants have only GFP. Southern analysis shows integration of the cDNA into different sites ranging from 1-5 sites per plant. Real-Time PCR shows integration of the cDNA into different sites in citrus genome ranging from 1-4 copies per plant. Western analysis shows the expression of the cDNA of the Xa21 gene in the transgenic citrus plants. This is the first time that a gene from rice has been stably integrated and expressed in citrus plants.