K. Sree Ramulu

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. ‘Bintje’)

SummaryA wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar ‘Bintje’. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.

Flow cytometric and karyological analysis of polysomaty and polyploidization during callus formation from leaf segments of various potato genotypes

SummaryFlow cytometry and karyological analysis were used to study polysomaty and polyploidization during the first 15 days of callus formation in leaf segments from shoot cultures and greenhouse-grown plants of various lines and genotypes of Solanum tuberosum and S. phureja. The greenhouse-grown plants showed a higher degree of polysomaty (77% and 49% of polyploidized nuclei) than the shoot cultures (< 3%). During the in vitro culture period, polyploidization occurred through endoreduplication. Segments of the five shoot cultures showed up to 87%, 53%, 59%, 45% and 56% polyploidization, respectively; the DNA content of corresponding interphase nuclei amounted to 8C, 16C, 16C, 16C and 8C, and the chromosome numbers to 96. Segments from the two greenhouse-grown genotypes showed up to 87% and 84% polyploidization; the DNA content amounted to 32C and 16C, and the chromosome numbers to 192 and 96. The number of reduplication cycles was species-dependent; the degree of polyploidization was dependent on the initial ploidy level of the genotypes. Cell proliferation did not take place at a constant rate. The maximum frequencies of metaphases (52–171 per segment) occurred after 1 week of culture and were correlated with the ploidy level of the genotypes. Cells were triggered to mitosis rather than to endoreduplication. Cell cycles with normal monochromosomes could be shorter than 1 day, and those with diplochromosomes lasted at least 1 day. Polysomaty, degree of polyploidization and abnormal nuclear processes are discussed in relation to the origin of genetic instability early in culture.

Regeneration and characterization of plants from potato root lines transformed by Agrobacterium rhizogenes

SummaryTransformed root lines were obtained after infection of leaf segments and tuber discs of tetraploid potato cvs Bintje and Desirée with Agrobacteriumrhizogenes. In response to shoot induction, about 10% of the root lines produced shoots through callus formation. The tests for opine suggest that all 26 shoot lines of cv Bintje (Ri-Bintje) and 13 of Desirée (Ri-Desirée) were transformed. All shoot lines were tetraploid except for one octoploid subshoot line of cv Desirée; no aneuploids were observed. With the exception of two shoot lines derived from the same root line, all other Ri-Bintje plants showed a pattern of phenotypic variation, generally observed among transformed plants. In contrast, the phenotype of Ri-Desirée plants was uniform and normal; variation was observed in tuber form and size. Phenotypic variation observed among Ri-plants appeared to be mainly root line-dependent, particularly for height of plants and tuber size and form. Variation was also observed within root and shoot lines and was more pronounced among the Ri-Bintje plants. Segregation of phenotypic characteristics was observed among transformed plants, resulting in the occurrence of phenotypes resembling the control. Chromosomal stability and the frequent reversion to normal phenotype of Ri-plants make A. rhizogenes particularly suitable as a virulence vector in the binary transformation system for the transfer of desirable genes.

Mitotic dynamics of micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer in plants

SummaryMitotic dynamics and the kinetics of mass induction of micronuclei after treatment of Nicotiana plumbaginifolia cell suspensions with the spindle toxin amiprophos-methyl (APM) are reported. The addition of APM to suspension cells resulted in the accumulation of a large number of metaphases. The course of mitosis was strikingly different from normal. Metaphase chromosomes showed neither centromere division nor separation of chromatids. Single chromosomes and groups of 2 or more chromosomes were scattered over the cytoplasm. After 5–6 h of APM treatment, chromosomes decondensed and formed micronuclei. When treatment duration was increased, the frequency of cells with micronuclei as well as those showing lobed micronuclei increased. Similarly, with an increase in APM concentration the frequency of cells with micronuclei increased. After removal of APM, chromosome grouping disappeared, cells showing lobed micronuclei further increased and mitoses with doubled chromosome numbers appeared in the next cell division. Cytological observations and DNA measurements revealed that several sub-diploid micronuclei containing 1 or a few chromosomes can be obtained, and that flow cytometry can detect and sort out these micronuclei. The applications of micronuclei for genetic manipulation of specific chromosomes and gene mapping are indicated.

Fate of introduced genetic markers in transformed root clones and regenerated plants of monohaploid and diploid potato genotypes

SummaryAgrobacterium transformation of stem internodes of four monohaploid (839-79, 849-7, 851-23, 855-1) and two diploid (M9 and HH260) potato genotypes using hairy root-inducing single (LBA 1020, LBA 9365, LBA 9402) and binary (LBA 1060KG) vectors is reported. Various media and successive culture steps were tested for plant regeneration from different transformed root clones. The fate of introduced genetic markers in root clones and regenerated plants (hairy root phenotype, hormone autotrophy, opine production, kanamycin resistance, β-glucuronidase activity), the ploidy stability and protoplast yield were analysed. The transformation efficiency of stem internodes (hairy root production) and the regeneration capacity of the transformed root clones greatly differed within and between the various potato genotypes. The regenerated plants obtained after transformation with both types of vectors often showed the absence of one or more genetic markers. However, transformation with the binary Agrobacterium vector generally resulted in the stable presence of the opines in all transformed root clones and most regenerated plants. In HH260, transformation efficiency, plant regeneration of transformed root clones, protoplast yield and ploidy stability were the highest as compared to the other genotypes. The application of these transformed plants as marker lines in gene mapping and gene expression studies is indicated.

Somatic hybridization between potato and Nicotiana plumbaginifolia

SummaryElectrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, β-glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.

Isolation and characterization of microprotoplasts from APM-treated suspension cells ofNicotiana plumbaginifolia

SummarySubprotoplasts with a DNA content of less than the G1 level (microprotoplasts) were isolated from micronucleated cells of transformedNicotiana plumbaginifolia (‘Doba’ line resistant to kanamycin) and characterized cytologically as well as by flow cytometry and Feulgen microdensitometry. Micronuclei were induced upon treatment of the suspension cells with the anti-microtubule drug amiprophos-methyl (APM). Protoplasts were fractionated on a continuous iso-osmotic gradient of Percoll; this resulted in several visible bands. Flow cytometric analysis of fluorescein and nuclear DNA contents after staining with fluorescein and DAPI respectively showed that the main band contained mostly evacuolated, intact (sub)protoplasts. Microprotoplasts contained one or a few micronuclei surrounded by a thin rim of cytoplasm and an intact plasma membrane. A maximum of 40% of the microprotoplasts in the fraction just below the main band had a DNA content less than the G1 level, in other fractions this maximum was 20%. Some of these contained an amount equivalent to that of one or a few chromosomes. The application of microprotoplasts for chromosome-mediated gene transfer in plants is indicated.

Mentor pollen effects on gametophytic incompatibility in Nicotiana, Oenothera and Lycopersicum

SummaryAttempts were made, through mentor pollen techniques, to overcome self-incompatibility in species belonging to the genera Nicotiana and Oenothera and in a hybrid of Lycopersicum, which are characterized by a gametophytic system of incompatibility. While radiation-killed incompatible pollen did not generate mentor effects in any of the material tested, radiation-killed compatible pollen was found to promote a high level of illegitimate fertilizations by incompatible pollen in N. alata. No evidence was obtained that radiation-killed compatible pollen could induce mentor effects in strictly self-incompatible clones of O. organensis and of the interspecific hybrid L. esculentum x L. peruvianum.

Somatic hybridization between potato and Nicotiana plumbaginifolia : 2. Karyotypic modification and segregation of genetic markers in hybrid suspension cultures and sublines.

SummarySeveral hybrid callus lines were produced through somatic hybridization between the diploid transformed Solanum tuberosum plant clone 413 (2n = 2x = 24) and a diploid wild-type plant clone of Nicotiana plumbaginifolia (2n = 2x = 20). The hybrid callus lines with subdiploid numbers of potato chromosomes were studied for karyotypic evolution as well as for segregation of the transformation marker characters (i.e. hormone autotrophy, opine synthesis, kanamycin resistance and β-glucuronidase activity). Initially, these hybrids (cultured in kanamycin-containing medium) expressed all of the transformation characters. Six callus lines were selected for the establishment of cell suspension cultures; two of these were also used to initiate sublines, one from single cells of a suspension culture, and the other from callus-derived protoplasts. The cell suspension cultures and the sublines were cultured in kanamycin-free medium. After prolonged culture, karyotypic analysis of the various cell suspension lines revealed independent evolution of both parental genomes. Out of the six suspension lines, four showed a considerably reduced number of potato chromosomes as compared to the original hybrid callus lines, whereas the karyotypes of the individual sublines generally reflected the karyotypic diversity of the original cultures. The fate of the marker characters in various suspension cultures and sublines revealed independent segregation of the markers of TL-DNA (hormone autotrophy) and vector T-DNA (kanamycin resistance and β-glucuronidase activity). Loss of the TR-DNA marker (opine synthesis) was observed only in combination with the simultaneous loss of the TL-DNA marker and the vector T-DNA markers. The results on segregation patterns of marker characters are discussed in the light of specific chromosome loss in the hybrid lines and gene linkage relationships.

Failure of EMS to induce S-locus mutations in Nicotiana alata (Link and Otto)

Abstract Attempts were made by EMS treatment of pollen mother cells to induce S-locus mutations in a self-incompatible clone of Nicotiana alata . While the EMS treatments induced high frequencies of M 2 chlorophyll mutations, no S-locus mutations were induced. The mechanism of generation of S-locus mutations and the action of EMS are discussed.