Establishment of homozygous transgenic rice lines after elimination of unlinked albino traits from transgenic events through segregation

In an analysis of 339 independent T ₀ transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T ₁ generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T ₀ plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T ₁ generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino plants in the T ₁ generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait from the transgenic trait in the lines O54 and O36 was confirmed in T ₂ and T ₃ generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T ₁ generation itself. Thus, we demonstrate that when an albino trait appears in the T ₁ generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous transgenic lines free from albinos can be established.     

Constitutive over-expression of two wheat pathogenesis-related genes enhances resistance of tobacco plants to Phytophthora nicotianae

The potential role in plant defence of the two wheat pathogenesis-related proteins of class 4 Wheatwin1 and Wheatwin2, possessing high in vitro antimicrobial activity against several pathogens, was investigated through over-expression of their encoding genes wPR4a and wPR4b in transgenic tobacco plants. Several independent transformants were obtained, expressing high levels of either transgene when analysed by northern and western blotting. Accumulation of the wPR4b-encoded protein Wheatwin2 in the apoplast of transgenic plants was also demonstrated. When homozygous transgenic lines in the T4 generation were tested for increased tolerance to Phytophthora nicotianae, they were found to be significantly more resistant than both the wild type and their isogenic, non-wPR4 transgenic lines. These results suggest that both Wheatwins might have in vivo antimicrobial activity, confirming earlier indications from in vitro assays.     

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T₀ and T₁generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.     

Investigations of copy number of transgene,fertility and expression level of an introduced GUS gene in transgenic NERICA produced by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated methods

In the use of genetic transformation in breeding, there are several possible problems including multiple copy insertion of transgene, sterility caused by somaclonal variation and gene silencing. In this study, we characterized transgenic New Rice for Africa (NERICA) produced by Agrobacterium-mediated methods with respect to copy number of transgene, fertility, and expression level of an introduced GUS gene. Southern blot analysis of primary transformants demonstrated that about half of the events carried a single copy of the transgene regardless of the cell density of Agrobacerium for inoculation. We examined ten procedures, consisting of different time periods and times of subculture for callus formation and the starting times of hygromycin-based selection of transformed cells, for transformation of NERICA cultivars to produce transformants within a short culture period at high frequency. A new culture method developed in this study required only about 1.5 mo from the beginning of tissue culture to transformants, whereas a standard protocol we developed previously needed about 2 mo of culture; however, it did not significantly reduce percentages of sterile plants. Fertile T₀ plants produced fertile T₁ plants at higher frequency. However, fertility was not inherited in a simple fashion: both fertile and partially sterile T₀ plants produced fertile, partially sterile and sterile T₁ plants. Expression assay of an introduced GUS gene revealed position effects in seven independent homozygous transformed lines carrying one copy of the transgene. Points to pay attention to in the use of genetic transformation in breeding are discussed.     

Decreased peroxidase activity in transgenic tobacco and its effect on lignification

An antisense gene construct of a peroxidase gene (Shpx6a) from a tropical pasture legume Stylosanthes humilis was transferred into tobacco cells via Agrobacterium tumefaciens to test whether peroxidase activity could be decreased and what effect this would have on lignification. A large number of tobacco cell lines were regenerated on selective media and stable integration of the transgene was confirmed in randomly selected putative transformants. Analyses of the primary transgenic plants and their progeny (T ₁) demonstrated that the total peroxidase activity was significantly decreased (up to 36%) as compared to that measured in untransformed control plants. Importantly, reduction in peroxidase activity is accompanied by decreases (up to 23%) in lignin content in several transgenic lines.     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

Heat-tolerant basmati rice engineered by over-expression of hsp101

Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp101) in imparting thermotolerance to cells, we introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T₁ lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T₂ transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance.     

Anthocyanin expression in marker free transgenic wheat and triticale embryos

Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T₀ lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale. Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented embryos were only observed visually in mature T₁ seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic lines.     

Aspects of comparative thermal ecology of sympatric hinge-back tortoises (Kinixys homeana and Kinixys erosa) in the Niger Delta, southern Nigeria

Aspects of the thermal ecology of two sympatric hinge‐back tortoises, Kinixys erosa and Kinixys homeana, were studied in the rainforest zone of south‐eastern Nigeria (West Africa). There were no significant differences in mean body temperatures (T_b) among K. homeana males, K. homeana females, K. erosa males and K. erosa females. In both species, there was a significant correlation between T_b and air temperature (T_a), and the regression line of T_a plotted against T_b differed significantly from the line of ideal poikilothermy. These regressions did not differ significantly between sexes in either species. There were no significant differences in terms of mean T_b in K. homeana during the various day‐hour intervals, whereas the mean T_b of K. erosa varied significantly among the various daytime intervals, attaining the higher values at 11.01–15.00 and 15.01–19.00 hours. In both species, T_bs were nearly always below T_as, and the negative difference ‘T_b ‐ T_a’ increased with increasing T_a. The hourly‐time course of the difference between T_b and T_a showed significant differences in K. homeana (with the higher negative differences at 11.01–15.00 and 15.01–19.00 hours), whereas no such difference was observed in K. erosa. In general, tortoises of both species showed behavioural thermoregulation in their avoidance of overheating in tropical environments, taking advantage of shady forest environments.     

The Tomato spotted wilt virus cell‐to‐cell movement protein (NSM) triggers a hypersensitive response in Sw‐5‐containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw‐5b resistance gene copy

Although the Sw‐5 gene cluster has been cloned, and Sw‐5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum ‘SR1‘ plants transformed with a copy of the Sw‐5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw‐5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non‐structural TSWV proteins tested, the TSWV cell‐to‐cell movement protein (NS_M) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non‐replicative pEAQ‐HT expression vector system. HR was induced in Sw‐5b‐transgenic N. benthamiana as well as in resistant near‐isogenic tomato lines after agroinfiltration with a functional cell‐to‐cell movement protein (NS_M) from a resistance‐inducing (RI) TSWV strain (BR‐01), but not with NS_M from a Sw‐5 resistance‐breaking (RB) strain (GRAU). This is the first biological demonstration that Sw‐5‐mediated resistance is triggered by the TSWV NS_M cell‐to‐cell movement protein.     

Diurnal body temperature patterns in free‐ranging populations of two southern African arid‐zone nightjars

Endotherms allocate large amounts of energy and water to the regulation of a precise body temperature (T_b), but can potentially reduce thermoregulatory costs by allowing T_b to deviate from normothermic levels. Many data on heterothermy at low air temperatures (T_a) exist for caprimulgids, whereas data on thermoregulation at high T_a are largely absent, despite members of this taxon frequently roosting and nesting in sites exposed to high operative temperatures. We investigated thermoregulation in free‐ranging rufous‐cheeked nightjars Caprimulgus rufigena and freckled nightjars Caprimulgus tristigma in the southern African arid zone. Individuals of both species showed labile T_b fluctuating around a single modal T_b (T_b‐mod). Average T_b‐mod was 39.7°C for rufous‐cheeked nightjars and 39.0°C for freckled nightjars. In both species, diurnal T_b increased with increasing T_a. At T_a ≥ 38°C, rufous‐cheeked nightjar mean T_b increased to 42°C, equivalent to 2.3°C above T_b‐mod. Under similar conditions, freckled nightjar T_b was on average only 1.1°C above T_b‐mod, with a mean T_b of 40.0°C. Freckled nightjars are one of the most heterothermic caprimulgids investigated to date, but our data suggest that during hot conditions this species maintains T_b within a narrow range above T_b‐mod, possibly reflecting an evolutionary tradeoff between decreased thermal sensitivity to lower T_b but increased sensitivity to high T_b. These findings reveal how general thermoregulatory patterns at similar T_a can vary even among closely related species.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Production of insect-resistant transgenic rice plants for use in practical agriculture

Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T₀ transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T₀ transgenic lines were generated and underwent extensive analysis. We selected four T₃ lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.     

The TaDREB3 transgene transferred by conventional crossings to different genetic backgrounds of bread wheat improves drought tolerance

Drought tolerance of the wheat cultivar Bobwhite was previously enhanced by transformation with a construct containing the wheat DREB3 gene driven by the stress‐inducible maize Rab17 promoter. Progeny of a single T₂ transgenic line were used as pollinators in crosses with four elite bread wheat cultivars from Western Australia: Bonnie Rock, IGW‐2971, Magenta and Wyalkatchem, with the aim of evaluating transgene performance in different genetic backgrounds. The selected pollinator line, BW8‐9‐10‐3, contained multiple transgene copies, had significantly improved drought tolerance compared with wild‐type plants and showed no growth and development penalties or abnormalities. A single hybrid plant was selected from each cross‐combination for three rounds of backcrossing with the corresponding maternal wheat cultivar. The transgene was detected in all four F₁BC₃ combinations, but stress‐inducible transgene expression was found in only three of the four combinations. Under well‐watered conditions, the phenotypes and grain yield components of the F₂BC₃ transgene‐expressing lines were similar to those of corresponding recurrent parents and null‐segregants. Under severe drought conditions, the backcross lines demonstrated 12–18% higher survival rates than the corresponding control plants. Two from four F₃BC₃ transgenic lines showed significantly higher yield (18.9% and 21.5%) than control plants under limited water conditions. There was no induction of transgene expression under cold stress, and therefore, no improvement of frost tolerance observed in the progenies of drought‐tolerant F₃BC₃ lines.     

Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics

Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T₀ generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T₄ generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance.