Allan Zipf

SOMATIC EMBRYO INITIATION AND GERMINATION IN DIPLOID COTTON (GOSSYPIUM ARBOREUM L.)

SummaryThe diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A2-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M2 [0.04M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)] On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M1 (0.2M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M2 medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.

Ecological impact of Bt cotton.

Abstract An overview of the ecological impact of transgenic crops, especially Bt cotton, is given in this paper. Crops expressing insecticidal crystal protein (ICP) genes from Bacillus thuringiensis (Bt) were among the first transgenic products approved for commercial use in the USA and several other countries. The cotton farming community in the USA embraced the transgenic technology because of potential benefits such as reducing (1) costs of operation and (2) damage to the environment and ground water supply, due to the repeated use of pesticides. However, concerns about genetically modified crops and foods remain in the USA and elsewhere. The transfer of Bt genes to wild relatives and neighboring crops, impact of Bt on non-target organisms, effects on crop yield and the possibility of insect pest populations developing resistance have all been items of concern. Key aspects of these benefits and concerns are summarized here.

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were ‘Corbit’ (first subculture); ‘GAF/Park’ and ‘88Ab3073’ (second subculture); and ‘GAF/Park’ and ‘87Ab5932’ (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. ‘GAF/Park’, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes ‘GAF/Park’, ‘88Ab3073’, and ‘Tibor’. The smaller clusters only regenerated plants for ‘GAF/Park’ and ‘88Ab3073’. From one gram of callus used to initiate suspensions of ‘GAF/Park’ and ‘88Ab3073’, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of ‘GAF/Park’, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.

Putrescine-enhanced somatic embryos and plant numbers from elite oat (Avena spp. L.) and reciprocal crosses

SummaryMature embryos from hulled, regenerable GP-1 (A. sativa L.), hull-less, recalcitrant Tibor (A. nuda L.) and reciprocal crosses were cultured in vitro on a putrescine- (Put) containing medium. Hormone-free Murashige and Skoog medium (MS-0) or shoot proliferation medium (SPM) [2.0 mgl−1 (9.0 μM) 2,4-dichlorophenoxyacetic acid (2,4-D)], with and without 0.5 mM Put or 1 mM Put, were tested for effects on somatic embryogenesis and plant regeneration. Put/SPM (0.5mM) was the best medium for both somatic embryos (SEs) and plant numbers per gram of callus, regardless of genotype. This effect was most evident in Tibor, which produced no somatic embryos or plants on SPM, a previously published regeneration medium, and in Tibor ×GP-1, which produced reduced numbers of SE and plants on the remaining media. The number of SEs per gram of callus for GP-1 and GP-1× Tibor showed little significant differences between the different media. Put treatments produced plants from the four genotypes but the regeneration efficiency on Put-containing medium was similar or even better than on SPM for explants containing maternal GP-1 germplasm. This suggests that Put-containing MS-0 medium can be used for testing regeneration of other oat lines. In addition, SPM containing 0.5 mM Put can be used to induce significant regeneration of plants from normally recalcitrant genotypes. This improvement greatly increases the number of potential germplasms for further transformation efforts.

Genetic Analysis of Plant Regenerability in Oats (Avena spp. L.)

Abstract The objective of this study was to estimate genetic effects for transfer of regenerability in oats. Corbit, an agronomically important oat cultivar, was crossed with the highly regenerable, but agronomically undesirable line, GP-1. Callus was induced from mature seeds of each parent (P1 and P2); F1, F2 and their reciprocals; and backcross (BC1 and BC2) generations. The number of somatic embryos was recorded before transfer to regeneration medium and the number of plants regenerated was recorded. Gene effects, using generation mean analysis, were computed when GP-1 was the maternal parent (Set 1) and when Corbit was the maternal parent (Set 2). From this study we conclude that selection for callus weight and plant number would be expected to produce only small gains per cycle because of the substantial negative d X d and dominance effects and these two traits might not be improved simultaneously when selection is practiced for one of them. However, two important characters-callus fresh weight and plant number-were positively correlated when GP-1 was chosen as the maternal parent. Therefore, back-cross strategies for improvement would need to take into consideration the direction of the cross as the highly regenerable plant characteristics observed were considerably influenced by maternal inheritance.

Evaluation of promoters and visual markers for transformation of eastern white pine

This report serves to evaluate possible promoters for use in theproduction of transgenic eastern white pine (Pinus strobus L.).Embryogenic cultures of eastern white pine were bombarded with goldparticles coated separately with a variety of gene constructs containingthe UidA β-glucuronidase (GUS) or green fluorescentprotein (GFP) reporter gene. Transient expression of the UidAgene, driven by a novel algal virus adenine methyl transferase genepromoter, as well as five other promoters used in angiospermtransformation, were evaluated. The maize alcohol dehydrogenase promoterwas not effective in eastern white pine cultures. The construct with thedoubled Cauliflower Mosaic Virus 35S promoter plus Alfalfa Mosaic Virusenhancer showed the highest levels of expression. GUS expression wasdetected within 24 hours, but decreased after 5 days and was notdetectable 15 days after bombardment. Expression of GUS activity wasrecorded mainly in somatic embryonal heads of various stages ofdevelopment and occasionally in suspensor cells. Similar to GUSexpression, modified green fluorescent protein (GFP) was detected in theembryonal head cells 24 hours after bombardment. GFP-expressingsuspensor cells were both more infrequent and difficult to detect, astheir highly vacuolate nature rendered the GFP presence less visibleagainst the yellow background autofluorescence.