Genetic Transformation Of Cotton With Endo-1,4-β-glucanase Gene And Regeneration Of Transgenic Plants

Cotton fiber is the most important natural textile material, and plays animportant role in Chinese national economy. So, it is of great significancetheoretically and practically to elucidate fiber development process by molecularbiological methods.Previously, we had isolated a full-length cDNA clone(designated GhCEL,GenBank accession number: AY574906) which is highly homologous to KOR geneof Arabidopsis thaliana, a gene necessary for plant cellulase biosynthesis.In this study, a over-expression and a anti-expression vector of GhCEL genewere constructed and transformed successfully into cotton plant viaAgrobacterium-mediated genetic transformation respectively to investigate theeffects of GhCEL on cotton fiber development. The main results are as follows: 1.Improvement of cotton genetic transformation system using hygromycin as aselectable marker.The results of research indicated that callus induction medium with double foldKNO₃ was advantageous for initiation and proliferation in the early stage oftransformation; 5mg/L of original concentration of hygromycin was optimal in theearly stages of cotton genetic transformation system using hypocotyl as recipient.Hygromycin concentration could be adjusted up to 10mg/L in the calli growth andproliferation stage.After subculturing this medium for two or three times, calli couldbe transferred onto medium without hygromycin to make them more easily grow anddifferentiate.2. Transformation of interest gene and obtaining of embryogenic callus linesIn this study, two expression vectors were transformed successfully intorecipient W₀. Resistant calli were peeled off explants when their diameter are 0.5cmor so, then transferred onto medium supplemented with 10mg/L hygromycin and nottransferred onto embryogenic callus induction medium until the diameter increasedto 1.0cm or so. Finally, 62 different resistant calli lines were obtained, among which42 calli lines were positive detected by PCR and GUS, average positive rate up to 67.7%.3. Molecular detection of transgenic cotton plantsAbout 300 T0 plants were obtained, 239 plants were selected to perform PCRand 190 of them were positive, counting for 79.5%. 110 sense tansgenic plants weredetected by PCR with Hpt gene specific primers and promoter-gene specific primers52.7% positive transgenic plants were confirmed by promoter-gene specific primers,while 79.1% were positive by Hpt gene specific primers. Only transgenic plantsconfirmed by both two kinds of primers wero thought to be positive plants. 7 T₀plants which were detecteded as positive primarily by PCR reaction were furtheranalyzed with Southern blotting using Hpt gene as the probe, and the results showedthat the Hpt gene had been inserted to the cotton genome.4. Grafting and transplantation of transgenic plantsTotle 50 tansgenic plants were grafted in the greenhouse, among which 35plants survived, with a survival rate of 70%. While only 4 plants survived among 20directly transplanted plants, with a survival rate of 20%. Compared with regenerativeplants of W₀, only individual plant structure in T₀ of RNAi became more compactand the others had no change; however, the fibers got shorter than that of control.These reasons of altering plant structure and fiber quality need further study andanalysis.