Insert copy number, chromosome number, pollen stainability, and crossability ofAgrobacterium-transformed diploid potato

Abstract

Two 2n-pollen producing cultivated diploid, 2n = 24, potato genotypes DM56.4 and US-W5337.3 were inoculated with binaryAgrobacterium vectors. The T-DNA contained a neomycin phosphotransferase II coding region fused to a nopaline synthase promoter providing gene expression in plants and resistance to kanamycin. A total of 28 clones regenerated as adventitious shoots from callus tissue, growing on medium with kanamycin, was analyzed for chromosome number, pollen stainability, and crossability. Several clones were tested and found to retain crossability with diploid and tetraploid clones. Nine out of 10 of the regenerants from DM56.4 had been doubled; seven of these were tetraploids with 2n = 48, one clone had 2n = 49, and another 2n = 46. Five of 14 regenerants of US-W5337.3 had been doubled, and three of these had fewer than 2n = 48. Pollen stainability revealed that the tetraploid regenerants from DM56.4 were male sterile, whereas tetraploids from US-W5337.3 had stainable pollen. All of the diploid regenerants produced 2n pollen. Crossability of several diploid and tetraploid regenerants was high. The number of T-DNA inserts revealed by Southern blot analysis was also determined. Six, 10, and 9 regenerants had 1, 2, and 3 inserts of T-DNA, respectively. There were single regenerants each with 5, 6, or 7 inserts. Selection for higher insert number among regenerants should be feasible based on the range of insert number found. Identical restriction patterns within three groups of clones indicated that the 28 clones analyzed emerged from only 21 cells derived from independent transformation events. The variability in chromosome number and pollen stainability suggests that variation in other traits would be obtainable.