Generation and properties of ascorbic acid‐deficient transgenic tobacco cells expressing antisense RNA for L‐galactono‐1,4‐lactone dehydrogenase

Summary In higher plants, the terminal step of l‐ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L‐galactono‐1,4‐lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA‐deficient transgenic tobacco BY‐2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY‐2 cells using PCR. Two transgenic cell‐lines, AS1–1 and AS2–2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild‐type BY‐2 cells. In synchronous cultures, division of AS1–1 and AS2–2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild‐type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild‐type cells. The calli of AS1–1 and AS2–2 appeared to be sticky and soft. Back extrusion method also showed that AsA‐deficient BY‐2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1–1 and AS2–2 cells were abnormally slender, suggesting a potential for a significant and a uni‐axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.