Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa

Abstract

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis

A genetic transformation method via secondary somatic embryogenesis was developed for alfalfa (Medicago sativa L.). Mature somatic embryos of alfalfa were infected by Agrobacterium strain GV3101 containing the binary vector pCAMBIA2301. pCAMBIA2301 harbors the uidA Gus reporter gene and npt II acts as the selectable marker gene. Infected primary embryos were placed on SH2K medium containing plant growth regulators to induce cell dedifferentiation and embryogenesis under 75 mg/L kanamycin selection. The induced calli were transferred to plant medium free of plant growth regulators for embryo formation while maintaining selection. Somatic embryos germinated normally upon transfer to a germination medium. Plants were recovered and grown in a tissue culture room before transfer to a greenhouse. Histochemical analysis showed high levels of GUS activity in secondary somatic embryos and in different organs of plants recovered from secondary somatic embryos. The presence and stable integration of transgenes in recovered plants were confirmed by polymerase chain reaction using transgene-specific primers and Southern blot hybridization using the npt II gene probe. The average transformation efficiency achieved via secondary somatic embryogenesis was 15.2%. The selection for transformation throughout the cell dedifferentiation and embryogenic callus induction phases was very effective, and no regenerated plants escaped the selection procedure. Alfalfa transformation is usually achieved through somatic embryogenesis using different organs of developed plants. Use of somatic embryos as explants for transformation can avoid the plant development phase, providing a faster procedure for introduction of new traits and facilitates further engineering of previously transformed lines.     

Introduction and expression of an insect proteinase inhibitor in alfalfa Medicago sativa L.

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 g/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.Abbreviations Km kanamycin - PI protease inhibitor - SDS Sodium dodecyl sulfate - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - SH Shenk and Hildebrandt (1972) medium     

The role of cuscutain-propeptide inhibitor in haustoria parasitism and enhanced resistance to dodder in transgenic alfalfa expressing this propeptide

Cuscutain is a cysteine protease produced by dodder (the most important weeds of alfalfa) that is essential for the development and penetration of the haustoria in host. The propeptide subunit of cuscutain has a specific inhibitory function and inhibits the enzymatic activity of the cuscutain. In this study, we introduced the gene encoding the propeptide segment of the cuscutain (signal peptide-less inhibitor) into alfalfa and investigated its roles in parasitism and the alfalfa resistance to C. reflexa. Results demonstrated that cuscutain is mainly expressed in haustoria and the expression of propeptide in transgenic alfalfa plants effectively inhibited cuscutain enzyme activity and consequently interrupted haustoria development at the pathogenic stage. Digitate cells of haustoria could not differentiate into the xylem and phloem hyphae in dodder grown on transgenic alfalfa. Dodder development on transgenic alfalfa lines showed an overall reduction in fecundity and vigor due to imperfect attachment of haustoria. Morphology, nodule development and biomass of transgenic plants indicate that the inhibitory transgene exhibits exquisite specificity for cuscutain enzyme and by expression of the inhibitor in transgenic plants, there was no obvious adverse effect on them. The increased development and growth of dodder-challenged alfalfa transgenic plants compared to controls, showed the efficacy of propeptide in dodder control.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens

Production of transgenic alfalfa plants by Agrobacterium-mediated transformation requires Agrobacterium infection and regeneration from tissue culture. Variation in alfalfa (Medicago sativa L.) germplasm for resistance to oncogenic and disarmed strains of A. tumefaciens (Smith & Townsend) Conn was tested in plant populations representing the nine distinct sources of alfalfa germplasm introduced into North America and used to develop modern varieties. For each of the virulent strains there was a positive correlation (p=0.05) of resistance to tumorigenesis with the trait for fall dormancy. There was also a significant correlation between plants selected for ineffective nodulation and resistance to tumorigenesis suggesting that the genetic loci required for successful symbiosis are also involved in tumorigenesis. Tissue explants of seedlings from the nine diversity groups were tested for transformation by three disarmed strains containing a plasmid with the scorable marker -glucuronidase. The strong correlation between dormancy and resistance to oncogenic strains was not observed with disarmed strains. However, there was a strong germplasm-strain interaction or transformation and embryogenesis in a highly embryogenic genotype. Thus, transformation at the whole plant level is germplasm dependent while in tissue culture the bacterial strain used is the critical variable for successful transformation.Abbreviations pTi tumor-inducing plasmid - GUS -glucuronidase     

Physiological responses to salt stress of T2 alfalfa progenies carrying a transgene for betaine aldehyde dehydrogenase

Glycinebetaine is an important quaternary ammonium compound generated in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by a Betaine Aldehyde Dehydrogenase (BADH) gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. In this study, a BADH gene was over expressed in transgenic alfalfa (Medicago sativa L) plants using Agrobacterium-mediated transformation. Transgenic alfalfa plants grown under 9‰ NaCl grew well; while non-transgenic control plants turned yellowish in color, wilted, and eventually died. Polymerase chain reaction (PCR) and Northern blot hybridization analyses demonstrated that the BADH gene was transferred into the T2 generation and segregated in a Mendelian fashion. Transgenic alfalfa plants expressing BADH showed significantly higher BADH enzyme activity and betaine contents when grown under 6‰ NaCl. Moreover, proline content in T2 lines were higher while electrolyte leakage and malonaldehyde content were lower in T2 lines compared with non-transgenic plants. These findings indicated that transgenic plants expressing BADH transgene exhibited higher salt tolerance than non-transgenic plants.     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

Summary We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH₃. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.     

农杆菌介导的苜蓿次级体细胞胚的遗传转化

采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。     

Winter survival of transgenic alfalfa overexpressing superoxide dismutase

To test the hypothesis that enhanced tolerance of oxidative stress would improve winter survival, two clones of alfalfa (Medicago sativa) were transformed with a Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the chloroplast. Although Mn-SOD activity increased in most primary transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD had lower activity in the chloroplast SOD transgenic plants than in the nontransgenic plants. In a field trial at Elora, Ontario, Canada, the survival and yield of 33 primary transgenic and control plants were compared. After one winter most transgenic plants had higher survival rates than control plants, with some at 100%. Similarly, some independent transgenic plants had twice the herbage yield of the control plants. Prescreening the transgenic plants for SOD activity, vigor, or freezing tolerance in the greenhouse was not effective in identifying individual transgenic plants with improved field performance. Freezing injury to leaf blades and fibrous roots, measured by electrolyte leakage from greenhouse-grown acclimated plants, indicated that the most tolerant were only 1°C more freezing-tolerant than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any freezing temperature. Therefore, although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing injury, and therefore, the trait is not associated with a change in the primary site of freezing injury.     

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

Transgenic silver birch (Betula pendula) expressing sugarbeet chitinase 4 shows enhanced resistance to Pyrenopeziza betulicola

A sugar beet chitinase gene driven by the (42) CaMV 35S promoter was introduced into silver birch (Betula pendula) through Agrobacterium-mediated transformation. Transgenic shoots were regenerated and grown on WPM medium supplemented with 150 mg/ml kanamycin. From a total of 220 explants, 52 transgenics were obtained and 13 transgenic lines were randomly taken for molecular analysis to confirm the presence of the introduced sugar beet chitinase 4 cDNA by polymerase chain reaction and Southern hybridisation. All 13 transgenic lines were confirmed to contain the gene and further characterised. Northern blot analysis of total RNA indicated that the transgenic lines differed with respect to the steady-state levels of chitinase mRNA. Transgenic lines with high levels of mRNA of chitinase 4 cDNA consistently showed higher levels of resistance to Pyrenopeziza betulicola than transgenics with intermediate or low mRNA levels or a non-transgenic control plant. This report demonstrates that the constitutive expression of this gene in transgenic birch lines increased the resistance of birch against the leaf spot fungus P. betulicola.     

Improvement of in vitro embryo maturation,plantlet regeneration and transformation efficiency from alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) somatic embryos using <Emphasis Type="Italic">Cuscuta campestris</Emphasis> extract

Developmental deficiency of somatic embryos and regeneration to plantlets, especially in the case of transformation, are major problems of somatic embryo regeneration in alfalfa. One of the ways to overcome these problems is the use of natural plant regulators and nutrients in the culture medium of somatic embryos. For investigating the influence of Cuscuta campestris extract on the efficiency of plant regeneration and transformation, chimeric tissue type plasminogen activator was transferred to explants using Agrobacterium tumefaciens, and transgenic plants were recovered using medium supplemented with different concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR. Somatic embryos of Medicago sativa L. developed into plantlets at high frequency level (52 %) in the maturation medium supplemented with 50 mg 1^?1 C. campestris extract as compared to the medium without extract (26 %). Transformation efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and without the extract, respectively. HPLC and GC/MS analysis of the extract indicated high level of ABA and some compounds such as Phytol, which can affect the somatic embryo maturation. The antibacterial assay showed that the extract was effective against some strains of A. tumefaciens. These results have provided a scientific basis for using of C. campestris extract as a good natural source of antimicrobial agents and plant growth regulator as well, that can be used in tissue culture of transgenic plants.     

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolyticaA1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.     

Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73–1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.