David M. Hunter

AtHD2D Gene Plays a Role in Plant Growth, Development, and Response to Abiotic Stresses in Arabidopsis thaliana

The histone deacetylases play important roles in the regulation of gene expression and the subsequent control of a number of important biological processes, including those involved in the response to environmental stress. A specific group of histone deacetylase genes, HD2, is present in plants. In Arabidopsis, HD2s include HD2A, HD2B, HD2C, and HD2D. Previous research showed that HD2A, HD2B, and HD2C are more related in terms of expression and function, but not HD2D. In this report, we studied different aspects of AtHD2D in Arabidopsis with respect to plant response to drought and other abiotic stresses. Bioinformatics analysis indicates that HD2D is distantly related to other HD2 genes. Transient expression in Nicotiana benthamiana and stable expression in Arabidopsis of AtHD2D fused with gfp showed that AtHD2D was expressed in the nucleus. Overexpression of AtHD2D resulted in developmental changes including fewer main roots, more lateral roots, and a higher root:shoot ratio. Seed germination and plant flowering time were delayed in transgenic plants expressing AtHD2D, but these plants exhibited higher degrees of tolerance to abiotic stresses, including drought, salt, and cold stresses. Physiological studies indicated that the malondialdehyde (MDA) content was high in wild-type plants but in plants overexpressing HD2D the MDA level increased slowly in response to stress conditions of drought, cold, and salt stress. Furthermore, electrolyte leakage in leaf cells of wild type plants increased but remained stable in transgenic plants. Our results indicate that AtHD2D is unique among HD2 genes and it plays a role in plant growth and development regulation and these changes can modulate plant stress responses.

Biological Activity of the tzs Gene of Nopaline Agrobacterium tumefaciens GV3101 in Plant Regeneration and Genetic Transformation

Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species.

Impact of Plum pox virus (PPV-D) infection on peach tree growth, productivity and bud cold hardiness

Abstract In 2000, the Dideron (D) strain of Plum pox virus (PPV) was detected in commercial peach and nectarine orchards in the Niagara region of Ontario where most of Canada’s stone fruit crops are produced. As part of a disease management research programme, peach (Prunus persica L. Batsch) trees in a commercial orchard at Niagara-on-the-Lake were assayed for PPV annually for 3 years. The orchard consisted of two blocks of the cultivars ‘Allstar’ and ‘Brighton’, of which 4 of 288 and 5 of 252 trees, respectively, were infected with PPV-D. The growth and health of these PPV-infected and non-infected trees were evaluated based on the annual growth rates, vigour (chlorophyll content) and bud winter hardiness. Comparative fruit quantity and quality index values were based on total yield and marketable yield per tree, fruit size and weight, fruit pH, titratable acidity, total soluble solids, flesh firmness, and fruit skin colour. Results from these preliminary studies showed that trees infected with this mild Ontario isolate of PPV produced slightly more fruit of smaller size that ripened earlier than non-infected trees. However, yield efficiencies based on weight of fruit relative to the trunk cross-sectional area did not differ statistically. Screenhouse studies on three graft-inoculated fresh market peach cultivars (‘Babygold’, ‘Catherina’, and ‘Garnet Beauty’) similarly did not demonstrate any differences in growth or fruit production in the second and third year post inoculation, but fruit on infected trees matured somewhat earlier.