The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-Waxy gene

Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. “261S” plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F₂ hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F₂ hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F₂ hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

Expression of <Emphasis Type="Italic">Bruguiera gymnorhiza</Emphasis><Emphasis Type="Italic">BgARP1</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

We have previously reported that expression of salt-responsive genes, including Bruguiera gymnorhiza ankyrin repeat protein 1 (BgARP1), enhances salt tolerance in both Agrobacterium tumefaciens and Arabidopsis. In this report, we further characterized BgARP1-expressing Arabidopsis to elucidate the role of BgARP1 in salt tolerance. BgARP1-expressing plants exhibited more vigorous growth than wild-type plants on MS plates containing 125–175 mM NaCl. Real-time PCR analysis showed enhanced induction of osmotin34 in the 2-week-old transformants under 125 mM NaCl. It was also showed that induction of typical salt-responsive genes, including RD29A, RD29B, and RD22, was blunted and delayed in the 4-week-old transformants during 24 h after 200 mM NaCl treatment. Ion content analysis showed that transgenic plants contained more K⁺, Ca²⁺, and NO₃ ⁻, and less NH₄ ⁺, than wild-type plants grown in 200 mM NaCl. Our results suggest that BgARP1-expressing plants may reduce salt stress by up-regulating osmotin34 gene expression and maintaining K⁺ homeostasis and regulating Ca²⁺ content. These results indicate that BgARP1 is functional on a heterogeneous background.     

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Effects of Agrobacterium tumefaciens-mediated transformation and tissue culture on storage lipids in Brassica napus

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T₂, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T₂ generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T₃ generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transgenic ‘Mailing 26’ apple expressing the attacin E gene has increased resistance toErwinia amylovora

Summary Apple (Malus domestica) transgenic T1 was obtained byAgrobacterium tumefaciens-mediated transformation of Malling 26 rootstock using the plasmid binary vector pLDB 15. pLDB 15 contains within its T -DNA a gene encoding the lytic protein attacin E. The integration of the attacin E gene into the apple genome was confirmed by Southern analysis. Northern analysis indicated the presence of an attacin E mRNA in plants inoculated withErwinia amylovora. After inoculation ofin vitro grown plants of T1, Malling 26, and Malling 7 (resistant control) withE. amylovora, the loglo of the inoculum concentration lethal to 50% of the plants was 5.4, 4.4, and 5.6, respectively. In greenhouse trials for resistance to fire blight, T1 was significantly more resistant than ‘Mailing 26’.     

Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T₀ plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T₁ transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T₁ generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T₃ and T₄ lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.     

叶绿体型转昆虫抗冻蛋白基因烟草的耐寒性

根据已构建的大豆叶绿体表达载体pJY01,设计特异性引物,将昆虫抗冻蛋白基因MpAFP149插入此载体中构成叶绿体表达载体pJY01-MpAFP149,利用基因枪轰击法转化烟草,经壮观霉素筛选获得4株叶绿体型转抗冻蛋白基因烟草株系。PCR和PCR-Southern结果显示外源基因已整合至烟草叶绿体基因组中但同质化水平不高,RT-PCR结果也表明昆虫抗冻蛋白基因已发生了转录。将野生型烟草、叶绿体型转抗冻蛋白基因烟草及核转化T₁代转抗冻蛋白基因烟草(pCAMBIA1302- MpAFP149)于–1℃低温处理3 d,观察耐寒表型及测定相对电导率。结果表明, 叶绿体型转基因烟草的耐寒表型优于野生型烟草,但与核转化的T₁代转抗冻蛋白基因烟草无显著差异。处理3 d时,叶绿体型转基因烟草和T₁代转抗冻蛋白基因烟草的电导率分别为39.2%和38.2%,而野生型烟草已达73.7%。本实验获得的异质化转叶绿体抗冻蛋白基因烟草与转核基因烟草的耐寒力无差异。     

根癌农杆菌介导获得稗草Ecppc转基因小麦的研究

采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1），对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C₄植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型，并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株，其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示，转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。     

Effect of temperature on gametophytic selection in a <Emphasis Type="Italic">Phalaenopsis</Emphasis> F<Subscript>1</Subscript> population

Gametophytic selection has potential to increase the efficiency of breeding for temperature tolerance. Here, we describe orchid seedlings after application of low and high temperatures during gametophytic development. In addition to phenotypic traits, amplified fragment length polymorphism (AFLP) markers were used to determine the genetic variability in seedlings. Two hybrid Phalaenopsis were cross-pollinated and exposed to 30°C day/25°C night for 3 days for a warm pollination or 15°C day/10°C night for 7 days as a cold pollination treatment. The plants were returned to the greenhouse after pollination and green capsules were collected after 150 days. Protocorms obtained from these treatments were evaluated 72 days after initial plating for germination and size on a thermogradient table ranging from 10 to 30°C. Seedlings were then evaluated 1 year after initial plating. The mean number of roots per seedling (4.2) was greater for plantlets that derived from the cold pollination treatment compared to those from warm pollination (3.6). Weight of the seedlings, number of roots and the average root length were significantly affected by the interaction between pollination treatment and germination temperature. The weight, number of leaves, and average root length were significantly affected by the interaction between pollination treatment and incubator/growth chamber. The results indicated that seedlings derived from warm pollination were more vigorous under warm growing conditions and those derived from cold pollination were more vigorous under cold growing conditions. Genetic variation among 16 F₁ seedlings randomly selected from various temperature treatments was analyzed. A dendrogram based on 651 loci resulted in three major groups and one subgroup. The groups and subgroup revealed common selection pressure during the gametophytic stage. The AFLP data support genetic differentiation of Phalaenopsis hybrids pollinated under different temperatures.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.     

基于比较转录组的水稻苗期耐冷相关基因BGIOSGA032296 TALEN基因组编辑技术构建

为了挖掘水稻苗期耐冷基因,基于比较转录组分析,参考公共数据库中的基因序列信息设计靶位点,对冷胁迫条件下东乡野生稻苗期下调表达耐冷相关基因BGIOSGA032296进行TALEN基因组编辑技术构建,克隆酶切鉴定和序列比对结果表明,BGIOSGA032296 TALEN敲除植物表达载体1301TALEN-032296构建成功,利用农杆菌介导法将TALEN敲除植物表达载体1301TALEN-032296转入水稻受体品种TP309,获得了17株转基因水稻植株,经潮霉素抗性标记基因和Fok I基因特异引物PCR检测,表明获得的转基因植株皆为携带1301TALEN-032296的阳性植株。该研究为水稻BGIOSGA032296基因后续耐冷表型鉴定奠定了前期基础。     

Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase

Summary Transgenic alfalfa plants expressinBacillus licheniformis alpha-amylase and mangaese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium were produced using the Agrobacterium tumefaciens transformation system. In each case, there was a range of expression of the introduced gene among independent transgenic plants. Plants producing alpha-amylase showed no alteration of phenotype. Production of Mn-P in alfalfa, howeven, in most cases adversely affected plant growth and development. Affected plants were stunted with yellowing foliage, but survived and produced seed. Results from field trials showed that Mn-P production in transgenic alfalfa reduced dry matter yield and plant height. The extent of these symptoms and yield reduction was, for the most part, related to the level of foreign protein production as estimated by Western analysis. Field data from transgenic plants expressing alpha-amylase showed that there was no effect of foreign protein production on plant performance. Expression of Mn-P was shown to segregate in sexual progeny derived from transgenic plants.Abbreviations Mn-P manganese-dependent lignin peroxidase     

The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis

Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R₁ and R₂ generations were raised and analysed for GUS as well as 2S albumin gene expression.Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R₂ plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.Abbreviations 35S cauliflower mosaic virus 35S protein gene - GUS -glucuronidase - NPTII neomycin phosphotransferase II - LeB4 Vicia faba legumin B4 gene - 2S albumin Brazil nut (Bertholletia excelsa) 2S albumin - ER endoplasmic reticulum - rER rough endoplasmic reticulum - HPLC high pressure liquid chromatography     

Development of a large population of activation-tagged mutants in an elite indica rice variety

In this study, we have generated more than 12,000 activation-tagged mutants in a high-yielding indica rice variety, 'BPT 5204', employing maize Ac/Ds system. Different transgenic plants obtained were analysed based on expression patterns of green fluorescence protein (GFP), red fluorescence protein (RFP), herbicide (Basta) tolerance and molecular analyses. T₁ seeds of pSQ5 and pSQ5-bar transgenics, when germinated separately on hygromycin (50 mg/L) and phosphinothricin (5 mg/L) containing medium, revealed a segregation of 3 tolerant : 1 susceptible plants. The germinal transposition frequency of Ds element in different T₂ progeny of rice plants was found to be about 18.0%. Different stable tagged mutants exhibited marked increases in plant height, number of tillers, leaf size, panicle size, seed size and number of grains per plant. The overall results indicate that the genes associated with these traits are upregulated by the enhancer element in activation-tagged mutants. As such, the various tagged mutant lines appear promising and serve as a valuable genetic resource for identification of key genes determining different agronomic traits of rice.     

Transformation of modified cowpea trypsin inhibitor gene and anti-bacterial peptide gene in Brassica pekinensis protoplasts mediated by Agrobacterium tumefaciens

Summary A transformation system for Chinese cabbage protoplasts was developed using Agrobacterium tumefaciens strain LBA4404 (harbouring the plasmid pBinΩSCK and the plasmid pMOG 411 respectively). The plasmid pBinΩSCK contains a 415 bp insert derived from the Cowpea trypsin proteinase inhibitor gene and The plasmid pMOG 411 contains a 870 bp fragment which codes an anti-bacterial peptide gene. Freshly isolated protoplasts of Chinese cabbage (Brassica campestris L.ssp.pekinensis) lines were pre-treated at 4 ^∘C for 1 h, then incubated at 25 ^∘C for 2–3 days in the dark. 3 drops of A. tumefaciens solution in log-phase were added to 10 ml protoplasts and cocultivated for 48 h at 25 ^∘C. Some kanamycin-resistant plants and a number of kanamycin-resistant calli were obtained. Southern blot hybridization analysis demonstrated the presence of the CpTI gene and the anti-bacterial peptide gene in the Chinese cabbage genome. Northern blot analysis of the kanamycin-resistant plantlets and calli confirmed the accumulation of the CpTI and the anti-bacterial peptide mRNAs.     

Stable resistance against the leaf miner <Emphasis Type="Italic">Leucoptera coffeella</Emphasis> expressed by genetically transformed <Emphasis Type="Italic">Coffea canephora</Emphasis> in a pluriannual field experiment in French Guiana

Summary A pluriannual field trial of transgenic clones of Coffea canephora (the Robusta coffee tree) transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella was installed in French Guiana. Fifty-eight transformed clones produced by transformation of the C. canephora clone 126 were planted. They were harbouring the pEF1α constitutive promoter of Arabidopsis thaliana controlling either the Bacillus thuringiensis native gene for the cry1Ac insecticidal protein (eight clones) or a synthetic cry1Ac gene (53 clones). The vectors for the transformation were a strain of the bacterium Agrobacterium tumefaciens and one of Agrobacterium rhizogenes. The transformed clones were generally independent, presenting different integration patterns of the genetic construct. Four randomly distributed groups of five plants per transformed clone were planted along with 60 untransformed control trees. Over a 4-year period after plantation six releases of L. coffeella were performed. Mines on the leaves are the marks of larvae development and were counted on plants. A majority of the independent transformed clones harbouring the synthetic gene and transformed by the strain of A. tumefaciens displayed constantly much less mines than the control, therefore expressing a stable resistance. The need for complementary research is presented.     

Over-expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">FT</Emphasis> gene reduces juvenile phase and induces early flowering in ornamental gentian plants

Ornamental gentian plants are perennial and have a juvenile period of over 1 year before flowering. We transformed gentian plants with a construct comprising the Arabidopsis FLOWERING LOCUS T (FT) gene (encoding a major component protein of the flowering hormone ‘florigen’) under the control of the rolC promoter from Agrobacterium rhizogenes, which is known to induce vascular-specific expression. The resultant rolCpro-FT transgenic gentian plants showed early flowering in vitro and the earliest line formed floral buds within 4 months after transformation. Regeneration experiments from leaf explants of these rolCpro-FT transgenic plants also confirmed the early flowering phenotype. After acclimatization, these transgenic plants showed normal floral development in a closed greenhouse. There is no effective method to induce early flowering by cultivation management in gentian, therefore these lines might be very useful as annual early season cultivars.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.