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Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T₀ transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

L-ascorbic acid (vitamin C) is an abundant metabolite in plant cells and tissues. Ascorbate functions as an antioxidant, as an enzyme cofactor, and plays essential roles in multiple physiological processes including photosynthesis, photoprotection, control of cell cycle and cell elongation, and modulation of flowering time, gene regulation, and senescence. The importance of this key molecule in regulating whole plant morphology, cell structure, and plant development has been clearly established via characterization of low vitamin C mutants of Arabidopsis, potato, tobacco, tomato, and rice. However, the consequences of elevating ascorbate content in plant growth and development are poorly understood. Here, we demonstrate that Arabidopsis lines overexpressing a myo-inositol oxygenase or an L-gulono-1,4-lactone oxidase, containing elevated ascorbate, display enhanced growth and biomass accumulation of both aerial and root tissues. To our knowledge, this is the first study demonstrating such a marked positive effect in plant growth in lines engineered to contain elevated vitamin C content. In addition, we present evidence showing that these lines are tolerant to a wide range of abiotic stresses including salt, cold, and heat. Total ascorbate content of the transgenic lines remained higher than those of controls under the abiotic stresses tested. Interestingly, exposure to pyrene, a polycyclic aromatic hydrocarbon and known inducer of oxidative stress in plants, leads to stunted growth of the aerial tissue, reduction in the number of root hairs, and inhibition of leaf expansion in wild type plants, while these symptoms are less severe in the overexpressers. Our results indicate the potential of this metabolic engineering strategy to develop crops with enhanced biomass, abiotic stress tolerance, and phytoremediation capabilities.

A program for producing new seedless table and raisin grape cultivars was initiated using embryo culture methods. F₁ descendants of the cross Delight × Ruby Seedless (DRs) were used as the female parent and crossed with various seedless cultivars. The berries of DRs have excellent table traits but they retain small seed traces at maturity. Blush Seedless and DRs were used as female parents, and 354 ovules from five crosses were tested. The percentage of embryos that developed using DRs as the female parent was higher (26.7–35.8%) than when a seedless cultivar was the female parent (5.9–6.7%). The best ovule excision time [days after flowering (DAF)] for ovule inoculation was tested in another set of crosses and found to be DAF 48, 50, and 50 for DR1 × Monukka, DR6 × Thompson Seedless, and DR7 × Zhengguo Seedless, respectively. A total of 569 hybrid progeny were obtained from 13 hybrid combinations; 311 of these survived and were established in soil after hardening. We used three molecular markers (SCC8, SCF27, and GSLP1) to analyze 15 parents used in the hybrid combinations. SCC8 had a 1018-bp band in the seedless parents and in some of the DR parents, while SCF27 had a 2000-bp band in all seedless and DR parents. GSLP1 had a 569-bp band in all of the seedless parents tested, whereas the seeded and DR parents had no bands of this size. Therefore, GSLP1 was used to screen the progeny for possible seedlessness. Eight strains had the 569-bp band; these were preliminarily identified as being seedless since they are in juvenile phase, but they have not yet been evaluated for fruiting characteristics. If the strains identified by using GSLP1 are confirmed to be seedless, this marker will be a valuable tool in breeding for seedlessness in grape.

We have obtained transgenic plants of Nicotiana sylvestris expressing an arylalkylamine N-acetyltransferase (AANAT) gene and a hydroxyindole-O-methyltransferase (HIOMT) gene using Agrobacterium tumefaciens-mediated transformation. Both AANAT and HIOMT are key enzymes in melatonin synthesis. Transgenic plants of N. sylvestris were characterized by polymerase chain reaction (PCR) and reverse transcription PCR analyses. The content of melatonin was significantly higher in transgenic plants than in nontransgenic plants. The highest melatonin content of transgenic plant leaves reached 50.4 μg/g dry weight, while almost no melatonin could be detected in the nontransformed plants. To investigate effects of the expression of the AANAT and HIOMT genes on melatonin function in plants, isolated protoplasts of N. sylvestris were exposed to ultraviolet (UV)-B radiation for different durations. DNA damage was evaluated by single-cell gel electrophoresis, which showed that the TailDNA percentage value in transgenic protoplasts was lower than that in the nontransformed protoplasts in the range of 0–30 s of UV-B radiation. DNA damage caused by UV-B was therefore reduced in transgenic N. sylvestris plants.

Cisgenic engineering involves isolation and modification of genetic elements from the host genome, which are reinserted to develop plant varieties with improved characteristics. As a first step toward production of fungal-disease resistant cisgenic grapevines, the Vitis vinifera thaumatin-like protein (vvtl-1) gene was isolated from “Chardonnay” and reengineered for constitutive expression. Embryogenic cultures of “Thompson Seedless” were initiated from leaves and transformed with Agrobacterium to regenerate cisgenic VVTL-1 plants. Cisgene presence and copy number were confirmed by PCR and quantitative real-time PCR. Protein expression was measured using ELISA. Among the plant lines tested, two exhibited a 7–10 day delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests. Berries exhibited a 42.5% reduction in sour-bunch rot disease incidence compared to non-transformed controls after 3 wk of storage at room temperature. Although plants recovered in this study contain viral promoters and reporter/marker genes, this is the first report of a cisgenic approach to obtain broad-spectrum fungal-disease resistance in genetically engineered grapevine.

Ma bamboo (Dendrocalamus latiflorus Munro) is a widespread culm and shoot-producing species in southern China. However, low temperatures reduce Ma bamboo shoot production and delay its development. In an attempt to enhance its cold-tolerance, a bacterial CodA gene encoding choline oxidase was introduced into Ma bamboo by Agrobacterium-mediated transformation, an approach that had not been previously utilized in bamboo. PCR and Southern blot analyses confirmed that CodA had integrated into the Ma bamboo genome. RT-PCR results showed that expression of CodA driven by the Arabidopsis Rd29A promoter was induced by cold stress in the transgenic bamboo lines. Following treatment at 4°C for 24 h, the content of glycine betaine (GB) increased to 83% and 140% in control plants (wild type (WT)) and CodA transgenic Ma bamboo plants, respectively. Superoxide dismutase, peroxidase, and catalase activities increased in both transgenic and WT plants. However, increases in these enzymes activities were much greater in the transgenic lines than in the WT plants under cold stress. The accumulation of malondialdehyde and electrolyte leakage (REL) in CodA transgenic Ma bamboo plants was less than that in control plants. Collectively, these results suggest that increased cold-tolerance induced by accumulation of GB in vivo was associated with the enhancement of antioxidant enzyme activities, which led to reduced accumulation of reactive oxygen species and stabilization of membrane integrity against extreme temperatures in transgenic plants.

Wheat streak mosaic virus (WSMV) is one of the major wheat viruses found in the Great Plains of the USA. Cultural practices are the primary methods of disease management, though not fully effective. Although genetic resistance is available, it is temperature sensitive and is sometimes closely linked with traits having negative agronomic effects. Alternative approaches to viral resistance are clearly needed. RNA interference (RNAi) has been shown to play a role in viral defense response and has been successfully used as a biotechnological tool to preprogram viral resistance in transgenic plants. In this work, a portion of the coat protein of WSMV was used as a hairpin construct and was co-transformed with pAHC20-bar to elicit viral resistance. Eleven WSMV RNAi independent transgenic events were obtained. Thirteen T₁lines were resistant as evident by the lack of viral RNA within the tissue. Beginning in the T₂generation, single-plant lineages were selected, selfed, and evaluated for resistance and presence of the transgene until the T₅generation. Families were then evaluated for the presence of the transgene, presence of the selectable marker, and WSMV resistance. Each of the lines in the T₅generation were resistant to the virus. Generational selection has maintained expression of the transgene and resistance to WSMV.

The aim of this work was to improve existing transformation protocols and to transform specific genotypes of Paspalum notatum (bahiagrass) for functional analyses of candidate genes involved in reproduction. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos, and shoot meristems. Plant regeneration was achieved with all explant types, but mature seeds produced the optimal rate (78.0%) and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed, which could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the optimal selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: (1) the reporter egfp gene cloned under the rice act1 promoter and (2) the selector bar gene driven by the ubiquitin promoter. A total of 27 (64.2%) transgenic plants out of 42 resistant plants analyzed were obtained. The presence of the transgenes in regenerated plants was confirmed by polymerase chain reaction and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.

Osmotin and osmotin-like proteins belong to the PR-5 pathogenesis-related group of proteins and are induced in response to various types of biotic and abiotic stresses in several plant species. Carrot was transformed with a tobacco osmotin gene that encodes a protein lacking the vacuolar-sorting motif that is composed of a 20-amino-acid sequence at the C-terminal end, under the control of the cauliflower mosaic virus 35S promoter, using Agrobacterium-mediated transformation. Transgene integration and expression were confirmed by Southern and western blot analyses, and three selected transgenic lines were evaluated for their ability to tolerate drought stress. Under drought stress conditions, all transformants exhibited slower rates of wilting compared with the wild-type plants and recovered faster when the drought stress was alleviated. Transformants showed lower levels of hydrogen peroxide accumulation, reduced lipid peroxidation and electrolyte leakage, and higher leaf water content under drought stress. Our results provide additional evidence for the protective ability of the osmotin protein against drought stress conditions and suggest a possible means to achieve tolerance against this abiotic stress in plants.

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25 ± 2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.