Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Genetic transformation of potato with nptII-gus marker genes enhances foliage consumption by Colorado potato beetle larvae

Little is known about the effect of transgenic plants containing commonly used marker genes, such as aph(3)II (nptII encoding neomycinphosphotransferase) and uidA (gus encoding -glucuronidase) on insect feeding behaviour. We report here, for the first time, that transgenic potato plants containing only nptII and gus marker genes enhance foliage consumption by the Colorado potato beetle (CPB, Leptinotarsa decemlineata S.). Transformation of potato cultivar Désirée was performed with Agrobacterium tumefaciens. Internode explants were inoculated with different strains of bacteria, carrying either nptII-gus or nptII alone. A total of 180 transgenic and untransformed control plants were grown in the greenhouse for the analysis of food consumption by CPB. For each transformed and untransformed line tested, four bioassays were conducted each consisting of 10 second-instar larvae feeding independently on a 2 cm diameter leaf disc for 20 h. Our data show up to 50% increase of mean foliage consumption on plants transformed with the nptII-gus construct, indicating that transgenic plants containing these marker genes can affect the feeding behaviour of the insects. These results were obtained from the primary regenerants (R₀ lines) as well as from tuber-derived plants (R₁ lines). Further tests with transgenic plants containing the nptII marker gene only, showed no significant difference in feeding when compared to untransformed control plants, allowing us to rule out a direct effect of this marker gene on foliage consumption by the insect larvae. It is suggested that gus protein is involved in the increase of foliage consumption by CPB.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.     

The feeding of the larvae of Simulium austeni Edwards and Simulium (Wilhelmia) spp.

The possibility that the feeding rates of larvae of S. austeni and S. lineatum are influenced by the nature of the food material is considered in relation to natural river conditions and laboratory feeding experiments. Diatoms appeared to be the most satisfactory food source and bacteria (Pseudomonas) the poorest. Simuliid larvae attain high assimilation efficiencies when feeding on diatoms but the proportion of river suspended solids which they assimilate is probably very low.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R₀ plants, and a single copy in one of the R₀ plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R₀ plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 g/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.Abbreviations AHAS acetohydroxyacid synthase - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - FW fresh weight - GUS -glucuronidase - IC₅₀ herbicide concentration that produces a 50% reduction in the fresh weight growth of cells - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962)     

Systemic induction of a potato pin2 promoter by wounding,methyl iasmonate,and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial -glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.     

An improved rice transformation system using the biolistic method

Immature embryos and embryogenic calli of rice, both japonica and indica subspecies, were bombarded with tungsten particles coated with plasmid DNA that contained a gene encoding hygromycin phosphotransferase (HPH, conferring hygromycin resistance) driven by the CaMV 35S promoter or Agrobactenum tumefaciens NOS promoter. Putatively transformed cell clusters were identified from the bombarded tissues 2 weeks after selection on hygromycin B. By separating these cell clusters from each other, and by stringent selection not only at the callus growth stage but also during regeneration and plantlet growth, the overall transformation and selection efficiencies were substantially improved over those previously reported. From the most responsive cultivar used in these studies, an average of one transgenic plant was produced from 1.3 immature embryos or from 5 pieces of embryogenic calli bombarded. Integration of the introduced gene into the plant genome, and inheritance to the offspring were demonstrated. By using this procedure, we have produced several hundred transgenic plants. The procedure described here provides a simple method for improving transformation and selection efficiencies in rice and may be applicable to other monocots.Abbreviations bp base pairs - CaMV cauliflower mosaic virus - GUS -glucuronidase - HPH hygromycin phosphotransferase - hyg B hygromycin B - hyg^r hygromycin resistance - NOS Agrobactenum tumefaciens nopaline synthase - PCR polymerase chain reaction - X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

Genetic organization of the E. coli chromosome around the structural gene for initiation factor IF3 (infC).

Summary A set of transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from p2 which is known to cary, besides infC, the structural genes for the subunit of phenylalanyl-tRNA synthetase (pheS), the subunit of phenylalanyl-tRNA synthetase (phetT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is pheT-pheS-P12-(infC, thrS) where infC is probably between P12 and thrS. P12 is the structural gene of a 12,000 molecular weight unidentified protein.Abbreviations PRS (EC 6.1.1.20) phenylalanyl-tRNA synthetase - TRS (EC 6.1.1.3) threonyl-tRNA synthetase - IF3 Initiation factor IF3 - SDS Sodium dodecyl sulfate - PP^R pyrophosphate resistant - PP^S pyrophosphate sensitive     

The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

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.     

Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase

High temperature inhibits photosynthesis by several mechanisms including deactivation of Rubisco. The inhibition of photosynthesis by high temperature and its relationship to Rubisco deactivation was studied using tobacco (Nicotiana tabaccum L. cv W38) transformed with a Rubisco activase gene inserted in the antisense orientation and untransformed controls. High temperature (42 °C) reduced photosynthesis in both lines of plants. However, photosynthesis recovered nearly completely in wild-type plants and very little in plants lacking Rubisco activase. The F₀ level of chlorophyll fluorescence decreased and q_N increased in the control plants during heating. In the antisense plants, q_N was always high and F₀ increased slightly during heat stress. NADP-malate dehydrogenase activation was unaffected by heat stress in control plants but was increased in the transgenic plants, consistent with a high redox status in the chloroplast. In wild-type plants, the inhibition of photosynthesis could be explained by a reversible decarbamylation of Rubisco and an acceptor-side limitation imposed on photosynthetic electron transport. However, in the anti-activase plants, carbamylation was low and constant and could not explain how photosynthesis was reduced at high temperature. Because ribulose bisphosphate was saturating at high temperature, the reduction in photosynthesis must have been caused by some impairment of Rubisco function not reflected in measurements of activation state or carbamylation status. This in vivo Rubisco impairment was not relieved upon return to lower temperature. We speculate that the reversible decarbamylation of Rubisco at moderately high temperature may be a protective mechanism by which the plant avoids more serious effects on Rubisco and the rest of the photosynthetic apparatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS -subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially -methyltryptophan (MT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on MT, indicating that it might be possible to use ASA2 as a selectable marker gene and MT as the selection agent.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency

An efficient Agrobacterium-mediated method for transformation of popular Bangladeshi Indica rice genotypes has been developed. Mature embryo-derived calluses as well as immature embryos were used as the target material. Transgenic plant production frequency was higher using the immature embryos than mature embryo-derived calluses. However, 3-week-old mature embryo-derived calluses served as an excellent starting material. The super-binary vector (pTOK233) was generally more effective than the binary vector (pC1301-Xa21mSS) particularly with recalcitrant Bangladeshi genotypes such as BR22. However, transformation of the Japonica cultivar Taipei-309 was equally effective with either plasmid. Inclusion of acetosyringone (200M) in co-cultivation media proved essential for successful transformation and the optimum co-cultivation period found was to be 3days. A large number of morphologically normal, fertile transgenic plants were obtained which expressed gus as determined by histochemical staining. Integration of the hpt gene into the genome of transgenic plants was confirmed by molecular analysis. Mendelian inheritance of transgenes (hpt and gus gene) was observed in T1 progeny.