Somatic hybridisation using a double mutant of Nicotiana tabacum

Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs

Interrelationship of cultivated rices Oryza sativa and O. glaberrima with wild O. perennis complex

Phylogenetic relationship of the cultivated rices Oryza sativa and O. glaberrima with the O. perennis complex, distributed on the three continents of Asia, Africa and America, and O. australiensis has been studied using Fraction 1 protein and two repeated DNA sequences as markers. Fraction 1 protein isolated from the leaf tissue of accessions of different species was subjected to isoelectric focusing. All the species studied have similar nuclear-encoded small subunit polypeptides and chloroplast-encoded large subunit polypeptides, except two of the O. perennis accessions from South America and O. australiensis, which have a different pattern for the chloroplast subunit. Two DNA sequences were isolated from Eco R1 restriction endonuclease digests of total DNA from O. sativa. One of the sequences has been characterized as highly repeated satellite DNA, and the other one as a moderately repeated DNA sequence. These sequences were used as probes in DNA/DNA hybridization with restriction endonuclease digested DNA from some accessions of the different species. Those accessions that are divergent for large subunit polypeptides of Fraction 1 protein (O. australiensis and two of the four South American O. perennis accessions) also lack the satellite DNA and have a different hybridization pattern with the moderately repeated sequence. All other accessions, irrespective of their geographical origin, are similar. We propose that various accessions of O. perennis from Africa and Asia are closely related to O. sativa and O. glaberrima, and that the dispersal of cultivated and O. perennis rices to different continents may be quite recent. The American O. perennis is a heterogeneous group. Some of the accessions ascribed to this group are closely related to the Asian and African O. perennis, while others have diverged

Repeated DNA sequences and ribulose bisphosphate carboxylase/oxygenase as tools for the study of rice evolution

The phylogenetic relationships of the cultivated rices, O. sativa and O. glaberrima, to the complex of species grouped under the heading of O. perennis is far from clear. We have analyzed a series of accessions from each of these species for the isoelectric focusing patterns of ribulose bisphosphate carboxylase/oxygenase (rubisco), and for the presence and organization of a number of coding and noncoding repeated sequence families. The results of these studies indicate a very close relationship between O. sativa, O. glaberrima, and O. perennis accessions from Africa, Asia, and South America. Two South American accessions of O. perennis differed from the others and from the cultivated rices, indicating that O. sativa and O. glaberrima must have diverged from the O. perennis complex more recently than some species that have been hitherto considered to be members of the complex. The results are certainly not consistent with the view that the cultivated rices have been isolated from one another and from their wild progenitors since the time of continental drift

A selection method for the synthesis of triploid hybrids by fusion of microspore protoplasts (n) with somatic cell protoplasts (2n)

Microspore protoplasts (n) isolated at the tetrad stage from plants of Nicotiana tabacum Km+ (2n=4x=48) were fused with somatic cell protoplasts (2n) of WT N. rustica (2n=4x=48) to produce triploid plants. A total of 21.2×106 microspore protoplasts were fused with 11.2×106 somatic cell protoplasts using the high pH/Ca+ + method. Microspore protoplasts did not divide and WT N. rustica protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing kanamycin. A total of 104 actively growing green colonies were recovered on the selection medium. Ninety-six of these colonies were tested for their hybrid nature by PAGE of peroxidases and were found to contain bands characteristic of both parents. Hybrid nature of the plants regenerated from some of the selected colonies was confirmed by IEF of leaf esterases, by NPT II activity assay and by hybridizing total DNAs restricted with EcoR I to a cloned 18s rDNA fragment. Root tip squashes of six of the hybrid plants revealed chromosome numbers ranging from 58-72. From chromosomal and biochemical analysis, it can be concluded that the procedure of fusing microspore protoplasts (n) of species A carrying a dominant selection marker with WT somatic cell protoplasts (2n) of species B can be a convenient selection method for the synthesis of triploid plants. The significance of triploids lies in their subsequent use for transferring alien chromosomes and genes of species A to species B

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km<SUP>+</SUP>Sr<SUP>+</SUP> with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts

AFLP-based genetic diversity assessment amongst agronomically important natural and some newly synthesized lines of Brassica juncea

AFLP markers were employed to assess the genetic diversity amongst 21 established natural and nine synthetic varietes and lines of Brassica juncea originating from Asia, Australia, Canada, Eastern Europe and Russia. Six of the synthetics used for diversity studies have been developed recently. Twenty one EcoRI/MseI-based AFLP primer pairs generated a total of 1251 scorable fragments among the 30 genotypes studied, of which 778 bands were polymorphic with an average of 37 polymorphic bands per primer pair. On the basis of the similarity coefficients (F value), cluster analysis was performed using the UPGMA method. The 30 B. juncea lines could be grouped into three distinct clusters. All the Indian, Chinese and previously developed synthetics formed one cluster (cluster A), the recently developed synthetics formed a separate cluster (cluster B) and the lines from Australia, Canada, Eastern Europe and Russia formed the third cluster (cluster C). A majority of the lines were uniquely identified by one or more primer pairs due to the presence or absence of variety specific band(s). Four primer pairs were found to be most informative, since these uniquely identified all the genotypes assayed. These four primer pairs, could therefore be used as fingerprinting primers for varietal identification

Phytogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous

Chloroplast DNA (cpDNA) variability of 60 taxa of the genus Brassica and allied genera comprising 50 species was studied. RFLPs for seven enzymes were generated and F values were estimated from five frequently cutting enzymes. Phenetic clusterings indicated a clear division of Brassica coenospecies into two distinct lineages referred to as the Brassica and Sinapis lineages. Two unexplored genera, Diplotaxis and Erucastrum, also exhibited two lineages in addition to the genera Brassica and Sinapis. This finding is inconsistent with the existing taxonomic classification based on morphology. Mitochondrial DNA (mtDNA) variability studied from EcoRI RFLP patterns, by hybridizing total DNA with four cosmid clones containing non-overlapping mtDNA fragments, did not show any congruence with cpDNA variation patterns. However, at the cytodeme level, the patterns of genetic divergence suggested by the cpDNA data could be correlated with mtDNA variation. In the Brassica lineage, Diplotaxis viminea was identified as the female parent of the allotetraploid D. muralis. The chloroplast DNAs of Erucastrum strigosum and Er. abyssinicum were found to be very closely related. In the Sinapis lineage, Brassica maurorum was found to be the diploid progenitor of autotetraploid B. cossoneana. B. amplexicaulis showed a very different cpDNA pattern from other members of the subtribe. Brassica adpressa was closest to Erucastrum laevigatum and could be the diploid progenitor of autotetraploid Er. laevigatum. Based on the close similarity of the cpDNA pattern of Diplotaxis siifolia with that of D. assurgens, we have proposed the retention of this species in the genus Diplotaxis. The taxonomic positions of some other species have also been discussed