Wheat protoplast culture and wheat transformation.

摘要

Regenerable embryogenic calli were obtained from anther cultures of three wheat cultivars. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in protoplast culture media without going through cell suspension culture. After 8 to 11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3-4 x 10{dollar}sp5{dollar} mL{dollar}sp{lcub}-1{rcub}.{dollar} Frequency of protocolony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, but no protocallus was produced from protoplasts of Karl 92. Green plants have been regenerated from protoplast culture of Jinghua #1 and Pavon 76.; A rice (Oryza sativa L.) chitinase gene and an insect chitinase cDNA (Manduca sexta) driven by the CaMV 35S promoter have been introduced into wheat plants through microprojectile bombardment. A total of 2311 immature embryos from three wheat cultivars were bombarded with a plasmid DNA containing a selectable marker bar gene plus a rice or a Manduca sexta chitinase gene, fifty one plantlets were obtained.; In western blot analysis for the rice chitinase, from 11 R{dollar}sb0{dollar} plants of Pavon 76, the expected 35 KDa band was detected in five plants. Out of 8 R{dollar}sb0{dollar} plants of Bobwhite, 3 plants showed the expected 35 KDa band. For expression of the Manduca sexta chitinase gene, out of 20 R{dollar}sb0{dollar} plants, only one presented an immunoreactive polypeptide with a molecular weight about 48 KDa.; Expression of the rice or the Manduca sexta chitinase genes was also found in some R{dollar}sb1{dollar} plants of Pavon 76 and Bobwhite, which suggested the segregation of rice or Manduca sexta chitinase genes in the R{dollar}sb1{dollar} generation. When genomic DNA from R{dollar}sb0{dollar} transformed plants of Pavon 76 was digested with Hind III and probed with a rice chitinase gene fragment, the 1.53 kb band was detected in three R{dollar}sb0{dollar} plants which had expression of the gene. Out of 3 R{dollar}sb1{dollar} plants from a R{dollar}sb0{dollar} plant (#15) probed with the Manduca sexta chitinase gene fragment, the 2.23 kb band was found in genomic DNA of two plants which also showed a positive reaction in western blot analysis.