Otto Schieder

Mendelian Transmission of Genes Introduced into Plants by the Ti Plasmids of Agrobacterium tumefaciens

SummaryInsertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity. rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

Intergeneric nuclear gene transfer between somatically and sexually incompatible plants through asymmetric protoplast fusion

SummaryA stable intergeneric transfer of nuclear genes from Physalis minima into the genome of Datura innoxia has been achieved through asymmetric protoplast fusion. No hybrid plants could be obtained from these species either by traditional methods of sexual breeding or by somatic hybridization via fusion of protoplasts containing complete nuclear genomes. The incompatibility barriers were bypassed by the fusion of highly X-irradiated (LD100) wild-type Physalis with nuclear albino mutant Datura protoplasts. In this intergeneric reconstruction, 1.15% of the total heterokaryons restored the chlorophyll synthesis in their regenerants. Two representative transformed lines, TRL-A and TRL-D, were further characterized, showing 0.43–0.78 pg. additional nuclear DNA (4.45–8.07% nuclear DNA of P. minima). Since chromosomes of the species of Datura and Physalis were distinguishable, the mitotic complement of the transformed regenerants showed only 3 and 1 chromosomes of the donor in tetraploid (2n=48) and octoploid (2n=96) genomes of the recipient, respectively. The introduction and expression of limited genes of Physalis in Datura have also been confirmed by the allelic expression of various isoenzymes. Such stable gene transfer via asymmetric fusion of protoplasts has been discussed in relation to its application in the genetic manipulation of plants.

Selection of somatic hybrids after fusion of protoplasts from Datura innoxia Mill. and Atropa belladonna L.

After fusion of protoplasts from a diploid (2n=24) and a tetraploid (4n=48) chlorophyll-deficient mutant of Datura innoxia Mill. with diploid (2n=72) green wild-type protoplasts of Atropa belladonna L. thirteen somatic hybrids could be selected, most of which had already started to produce leaves and shoots. Hybrid calli were recognizable by the production of hairs, typical for Datura innoxia, and the green colour, derived from Atropa belladonna. Further proof for the hybrid nature was furnished by cytological investigations. The metaphase chromosomes of both species are easily distinguishable in their size: chromosomes of Datura innoxia are about twice as large as those of Atropa belladonna. The chromosome numbers of the hybrids varied from ca. 84 to ca. 175.

Correction of nitrate reductase defect in auxotrophic plant cells through protoplast-mediated intergeneric gene transfers

SummaryNitrate reductase-deficient cells of Nicotiana tabacum cv Gatersleben (coded cnx-68) lacking active molybdenum-cofactor were corrected by introducing the genes from Physalis minima and Datura innoxia into NR- genomes. In these itergeneric reconstruction experiments, X-irradiated inactive mesophyll protoplasts of Physalis and Datura were fused separately with the cultured cell protoplasts of cnx-68 Nicotiana. A total of 45 cell colonies, 37 transformed by Physalis and 8 by Datura, were selected from about 1.7×103 heteroplasmic fusion products. The selection of transformants was made by their ability to grow on a medium containing nitrate as the sole nitrogen source. Some of these transformants were further characterized with respect to nitrate reductase, xanthine dehydrogenase and glutamate dehydrogenase activities, chlorate sensitivity, and chlorophyll synthesis. The restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana. Such stable transformations via fusion of normal and highly irradiated protoplasts may have a considerable application in higher plants for introducing desirable characters from diverse genomes.

Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species

Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.

Regeneration von haploiden und diploiden Datura innoxia Mill. Mesophyll-Protoplasten zu Pflanzen

Summary Haploid and diploid protoplasts from mesophyll cells of Datura innoxia Mill , were isolated after enzymic digestion from leaves of axenic cultures. The washed protoplasts were suspended in DPD medium ( Durand et al., 1973). Seven-tenth of a millilitre of suspension containing approximately 0.7 × 10 4 protoplasts were plated in a PETRI-dish. First divisions of haploid and diploid protoplasts could be observed after 40 h. After 6 days, up to 20% of the protoplasts showed divisions. Eight days later the cell clumps were transfered to the agar medium B5 ( Gamborg et al., 1968) with an addition of 0.5 mg/L BAP. In general, differentiation into leaves and shoots could be observed after 5 to 6 weeks on B5 medium. Although higher ploidy were encountered, 50 percent of calluses derived from haploid mesophyll protoplasts were haploid. Shoot differentiated from haploid calluses were also haploid.

Comparison of somatic and sexual incompatibility between Datura innoxia and Atropa belladonna

After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.

Somatic hybrids in tobacco mediated by electrofusion

Abstract Somatic hybridization mediated by electrofusion was carried out with protoplasts isolated from suspension cultures of two complementing nitrate reductase-deficient mutant cell lines of Nicotiana tabacum cv. Gatersleben. Protoplasts were aggregated to chains between platinum wire electrodes by mutual dielectrophoresis with a non-uniform alternating current (a-c)-field. The fusion process of protoplasts was initiated with high intensity direct current (d-c)-square wave pulses of short duration. Somatic hybrids were selected by their vigorous growth on a nitrate-containing culture medium and were found to have an active nitrate reductase system. The results demonstrate that somatic hybrids derived from electrofusion of plant protoplasts were viable, regenerated to calli and exhibited organogenetic response by shoot differentiation.

Selektion einer somatischen hybride nach fusion von protoplasten auxotropher mutanten von Sphaerocarpos donnelliiAUST

Summary Using the method of protoplast fusing, attempts were made to isolate hybrids between a normal green, nicotinic acid deficient female (nic 2 ) and a pale green, glucose deficient male (pal 2 ) of the liverwort Sphaerocarpos donnellii Aust . The fusion medium consisted of 0,2 M Ca(NO 3 ) 2 adjusted with 0.05 M glycine and 0.05 M NaOH to pH 9. Female plants possess 7 autosomes together with a larger heterochromatic X-chromosome, while male plants possess, in addition to the 7 autosomes, one smaller Y-chromosome. From numerous fused protoplasts, one hybrid developed to form an autotrophic green diploid plant. This plant has 14 autosomes and both X and Y sex chromosomes, showing it to be a somatic hybrid between the female nic 2 and the male pal 2 .

Isolation of mutants with altered pigments after irradiating haploid protoplasts from Datura innoxia Mill. with X-rays

SummaryTen different mutants with altered pigment patterns were isolated following X-irradiation of approximately 105 haploid protoplasts of Datura innoxia Mill. Seven of the selected strains gave rise to shoots and 3 to leaves only. The mutants were selected from light green or white calli, which had developed 4 weeks after transfer of developing cell clusters onto B5 agar medium containing 0.5 mg/l BAP (Gamborg et al., 1968). Of the 10 mutant strains 5 were light green, two were yellow, one was pale yellow and one was white. One additional strain does not possess anthocyanin in its stems; a feature chracteristic of the wildtype is the possession of anthocyanin. This strain is able to grow in soil and has now flowered. None of the mutants obtained is haploid. Nine are diploid and the other is tetraploid. The chlorophyll deficient strains can be propagated on B5 agar medium supplemented with higher concentration of sucrose than normally required for the growth of the wild-type.