E. Jacobsen

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.

Three Pea Mutants with an Altered Nodulation Studied by Genetic Analysis and Grafting

Summary In this study three independently isolated nodulation mutants of pea ( Pisum sativum L./cv. Rondo) were compared, i.e.: supernodulating mutant nod3 and the nodulation resistant mutants K5 and K24. It was observed that the root- and shoot morphology of mutant nod3 were also changed in comparison with the wild type. Both the altered nodulation and morphology of mutant nod3 appeared to be due to a pleiotropic effect of one mutated gene. Mutant K5 and K24 did not differ morphologically from the wild type. All mutants were non-allelic, indicating that three different genes were involved. Crossing experiments between nod3 and both nod - mutants showed independent segregation and resulted in the isolation of two different recombinant genotypes. A difference in nodulation resistance between mutant K5 and K24 was observed. Both mutants did not form nodules when grown in liquid medium, but only mutant K24 showed nodulation resistance when grown in soil. Microscopical investigations on inoculated lateral roots of mutant K5 showed the presence of infection threads, in contrast with mutant K24, where only curling of root hairs was observed. Grafting showed that the altered nodulation of nod3 and K5 was dependent on the root, while the inhibited nodulation of mutant K24 was dependent on the root and the shoot. Grafting- and adventitious root regeneration experiments showed that the cotyledons were not involved in the mutant characters.

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.

Isolation of a Nitrate Reductase Deficient Mutant of Pisum sativum by Means of Selection for Chlorate Resistance

SummaryAfter EMS treatment of seeds of the Pisum variety ‘Rondo’ a chlorate resistant mutant was isolated which showed a decrease in the in vitro activity of the enzyme nitrate reductase of roughly 95%. The mutation is monogenic and recessive. The mutant shows a decrease in protein content, and an increase in the amount of nitrate accumulated and in the activity of the enzyme nitrite reductase. On a liquid nutrient medium containing nitrate as the sole nitrogen source and in soil, the mutant grows very poorly due to necrosis of the leaves. On liquid medium containing ammonium, either with or without nitrate, growth is as good as that of the parent variety.

Cloning and Characterization of R3b; Members of the R3 Superfamily of Late Blight Resistance Genes Show Sequence and Functional Divergence

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.

Ploidy levels in leaf callus and regenerated plants of Solanum tuberosum determined by cytophotometric measurements of protoplasts

SummaryIn potato (Solanum tuberosum) instabilities in nuclear DNA content in callus cells of a monohaploid and a dihaploid and in tuberprogenies of a regenerated, doubled dihaploid plant were assessed by cytophotometry in isolated, purified protoplasts. Leaf protoplasts of a tetraploid variety have nuclei with 3.6±0.08 pg of DNA. In callus protoplasts a dihaploid showed 3 and a monohaploid 6 ploidy levels. Progeny of a plant, doubled and regenerated from dihaploid callus, showed mixo-euploidy, indicating an instability similar to the dihaploid callus; in addition, there are mixo-aneuploid cells.These findings confirm and extend earlier observations based on chromosome counts in other genotypes of potato. The experimental approach can be applied to a variety of other purposes.

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.

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.