In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment

It has been proposed that transgenic plants of cereals can be generated by inoculating florets with Agrobacterium at or near anthesis. This procedure is shown to lead to the production of embryos of wheat and barley with enhanced resistance to antibiotic selection. It has also been possible to recover plants of wheat, barley and maize that gave positive hybridization signals with probes produced from within the T-DNA of the Agrobacterium vector. However, no evidence was found for transmission of the bands detected by hybridization in the progeny of the putative transgenic plants nor could enzyme activity associated with the resistance genes be found in plant extracts. Furthermore, undigested genomic DNA from the plants that were positive when probed with the T-DNA, showed hybridization to bands smaller than the genomic DNA. It is suggested that the apparent transformation is an artifact of the procedure and does not reflect transformation of the plant nuclear genome.     

Plant regeneration of wildGlycine species from suspension culture-derived protoplasts

Protoplasts isolated from four-week old cell suspension cultures ofGlycine canescens F. J. Herm andG. clandestina Wendl. were cultured in 8P or modified 8P to a multicellular stage. Colonies of 0.5 to 1.0 mm diameter were transferred to solid media for callus growth and regeneration. Callus consisted of friable masses with compact green nodular areas. Organogenesis of both species occurred primarily from the green nodular areas. Shoot buds ofG. clandestina did not mature, but shoots ofG. canescens proliferated on MS medium, with B5 vitamins, 0.33 mgL^–1 each BA, KN, ZN, and 0.15 mgL^–1 NAA. Shoots failed to root after multiple subcultures on four different rooting media.In vitro grafting ofG. canescens scions ontoG. max root stocks allowed plants to be transferred to soil. An overall protoplast division efficiency of 48% was achieved with moderately efficient shoot regeneration inG. canescens. Division efficiencies forG. clandestina were lower (11%). Refinements of this protocol should result in high efficiencies of regeneration which would allowin vitro manipulations of these wild soybean relatives at the single cell level and would make the derivation of somatic hybrid plants possible within the genusGlycine.Abbreviations BA 6-Benzyladenine - KN kinetin - ZN Zeatin - NAA Napthaleneacetic acid - 2,4-D 2–4-dichlorophenoxyacetic acid - PIC Picloram - CH casein hydrolysate - Gln glutamine - Met methionine - MES 2[N-morpholine] ethanesulfonic acid     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

A segment of rye chromosome 1 enhances growth and embryogenesis of calli derived from immature embryos of wheat

Summary The influence of the short arm of rye chromosome 1 (1RS) from Secale cereale var. Imperial on the growth and differentiation of callus cultures from wheat Triticum aestivum var. Chinese Spring immature embryos was analysed. This chromosome arm was found to stimulate both embryogenesis and the rate of growth of calli. Recombinant lines carrying segments of 1RS were used to delineate the regions of 1RS responsible for the tissue culture effects. The enhancement of embryogenesis and the stimulation of growth were shown to be associated with two distinct genetic regions of the chromosome arm; the former is located between the centromere and the Sec 1 locus, while the latter is situated in the immediate vicinity of the Sec 1 locus.     

Insertional tagging of regulatory sequences in tritordeum; a hexaploid cereal species

As an approach to isolate novel cereal promoters, promoterless uidA constructs and particle bombardment were used to transform tritordeum. Five of eight transgenic lines containing uidA sequences showed evidence of promoter tagging. Expression of uidA was detected in four lines as: constitutive expression, expression in short cells of the epidermis of the spikelets, expression in pollen grains and in cells of the epidermis of the spikelet, and expression in anther primordia and pollen grains. In the fifth line, the uidA was shown by RT-PCR to be transcribed, but no GUS activity was detected. The different patterns of uidA expression indicate that different regulatory sequences were tagged in each of these lines. Analysis of the progeny resulting from self-fertilisation of the primary tagged plants, indicate that the transgenes integrated at one or two loci and the patterns of expression were stably inherited. To our knowledge, this is the first report of promoter tagging in cereals by direct gene transfer.     

Transgenic Plant Monitor (TPM): a relational database for the management and analysis of information in plant tissue culture and transformation laboratories

Increased public concern and strict statutory regulations relating tothe generation and exploitation of genetically modified organisms, make itimperative to track accurately individual plants through DNA transformationprogrammes. The ability to rapidly retrieve information associated withspecifictransgenic events and to provide accurate reports on demand is an increasinglyimportant feature for public research laboratories. Transgenic Plant Monitor(TPM) has been developed as a database structured to allow efficient recording,monitoring and analysis of the extensive and complex data generated in planttissue culture and transformation experiments. TPM is built upon the widelyavailable Microsoft Access database engine and can be readily adoptedand/or adapted by other users. The key features and the utility of TPM as aresearch tool are discussed in this article.