W.J. Feenstra

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

SummaryAn amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.

Rapid estimation of the amylose/amylopectin ratio in small amounts of tuber and leaf tissue of the potato

SummarySmall amounts of potato tuber and leaf tissues are extracted in perchloric acid. After staining with I2-KI solution absorbancies at 618 and 550 nm are measured. The amylose/amylopectin ratio can be estimated from the ratio of the absorbancies by using a formula or a graph in which the specific absorptions of the two compounds are introduced. Weighing of samples is not required. The method can be used for the estimation of the starch composition of minitubers and is especially suitable for the detection of mutants with an altered starch composition.

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.

Molecular cloning and partial characterization of the gene for granule-bound starch synthase from a wildtype and an amylose-free potato (Solanum tuberosum L.)

Abstract The gene encoding granule-bound starch synthase (GBSS), which determines the presence of amylose in reserve starches, has been isolated from a wild-type and from an amylose-free potato by using a potato GBSS cDNA. From the analysis of five genomic GBSS clones, isolated from the wild-type potato, it is shown that GBSS is a single copy gene in potato. GBSS messenger RNA was shown to be present in a number of different tissues, but was most abundantly found in stolons and tubers. Southern blot analysis of the GBSS genes from both potato genotypes revealed that the amylose-free mutant, which lacks GBSS activity and protein, does not contain a large structural lesion in the GBSS gene. The GBSS messenger RNA was even found to be present far more abundant in the mutant than in the wild-type potato.

EFFICIENT TRANSFORMATION OF POTATO (SOLANUM-TUBEROSUM-L) USING A BINARY VECTOR IN AGROBACTERIUM-RHIZOGENES

SummaryWe transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 1∶1 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and β-glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing β-glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the “mixed” transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed β-glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the β-glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.

CHROMOSOMES IN SOMATIC HYBRIDS BETWEEN NICOTIANA-PLUMBAGINIFOLIA AND A MONOPLOID POTATO

SummaryLeaf mesophyll protoplasts of the monohaploid potato (Solanum tuberosum L.) clone H7322 were fused with callus protoplasts of nitrate reductase deficient (NR−) mutants Cnx 20 and NA 36 of Nicotiana plumbaginifolia. Somatic hybrid lines were selected for nitrate reductase proficiency. All callus lines tested appeared to be stable for the retention of the potato chromosome carrying the compensating NR gene when grown for over 1.5 years in the absence of nitrate. Shoots were regenerated from six different fusion lines of Cnx 20 + H7322 24 months after fusion. Chromosomal analysis in callus cultures revealed that in both fusion combinations 40–120 N. plumbaginifolia chromosomes were present, as were 9–20 potato chromosomes. Cells with 17 potato chromosomes in combination with a relatively small number (31) of N. plumbaginifolia chromosomes were found in one line. Preferential loss of species-specific chromosomes was not observed. Analysis of regenerating tissue from three lines of Cnx 20 + H7322 revealed that after 24 months of culture intra- and intergeneric translocations, fragments and deletions were present. Elimination of the potato and N. plumbaginifolia chromosomes had taken place before and after genome doubling.

Complementation of the amylose-free starch mutant of potato (Solanum tuberosum.) by the gene encoding granule-bound starch synthase

SummaryAgrobacterium rhizogenes-mediated introduction of the wild-type allele of the gene encoding granulebound starch synthase (GBSS) into the amylose-free starch mutantamf of potato leads to restoration of GBSS activity and amylose synthesis, which demonstrates thatAmf is the structural gene for GBSS. Amylose was found in columella cells of root tips, in stomatal guard cells, tubers, and pollen, while in the control experiments using only vector DNA, these tissues remained amylose free. This confirms the fact that, in potato, GBSS is the only enzyme responsible for the presence of amylose, accumulating in all starch-containing tissues. Amylose-containing transformants showed no positive correlation between GBSS activity and amylose content, which confirms that the former is not the sole regulating factor in amylose metabolism.

Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato.

SummaryGranule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Analysis of antisense RNA mediated inhibition of GBSS gene expression in large numbers of tubers from in vitro grown, greenhouse grown and field grown transgenic potato plants revealed stable and total inhibition of GBSS gene expression in one clone. In three other transgenic genotypes partial and unstable inhibition was found. In these genotypes both GBSS activity and amylose content were remarkably reduced compared with the non-transformed control genotype. No relationship was found between the level of inhibition of GBSS gene expression and yield and dry matter content.

Expression and inheritance of inserted markers in binary vector carrying Agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.)

SummaryTransgenic shoots were regenerated from eight diploid potato hairy root clones obtained by transformation with Agrobacterium rhizogenes harboring next to its wild-type Ri-plasmid a binary vector containing the neomycin phosphotransferase and the β-glucuronidase genes. The plants exhibited the typical hairy root phenotype. Of the plants isolated, 58% were tetraploid and 38% were diploid. Flowering and tuberization was much better in the diploid than in the tetraploid plants. Transgenic plants formed a significantly larger root system when grown on kanamycin-containing medium as compared to growth on kanamycin-free medium. Direct evidence for genetic transformation was obtained by opine, neomycin phosphotransferase and β-glucuronidase assays, and by molecular hybridization. Fourteen flowering diploid plants were reciprocally crossed with untransformed S. tuberosum plants, but only six were successful. Seedlings obtained from four crosses showed that all traits were transmitted to the offspring. Molecular analysis confirmed the presence of multiple integrations (copies) of both vector T-DNA and Ri-T-DNA. The genetic data, furthermore, suggest that the traits derived from Ri-T-DNA and binary vector T-DNA are linked, as no recombination between the different traits was observed.

In situ hybridization to somatic metaphase chromosomes of potato

SummaryAn in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a “ladderpattern” in genomic Southerns of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.