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.     

Agrobacterium rhizogenes rol genes induce productivity-related phenotypical modifications in “creeping-rooted” alfalfa types

Summary Agrobacterium rhizogenes rol genes were transferred individually or in combination into the forage legume Medicago sativa L. (alfalfa). Kanamycin resistant, neomycin phosphotransferase II positive plants showed the presence of the rol inserts in their genome. Phenotypical evaluation of transgenic populations indicated significant morphological alterations of the root system, stem number per plant and plant structure. A possible utilization of these transgenics in breeding programs of the so-called creeping-rooted alfalfa strains is discussed.Abbreviations NPTII neomycin phosphotransferase II - ORF open reading frame - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - CaMV cauliflowermosaic virus     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

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.     

Ri-plasmid as a helper for introducing vector DNA into alfalfa plants

Genetic engineering of legumes and other important dicotyledonous plants is limited because of the difficulty of regenerating plants via cell culture. Since a considerable number of crop plants can be regenerated only from root culture, the introduction of foreign genes into Agrobacterium rhizogenes-induced hairy roots may expand the list of crop plants that could be genetically engineered. Here we report genetic transformation of alfalfa (Medicago sativa L.), a valuable forage legume, using a virulent strain of Agrobacterium rhizogenes containing, in addition to its Ri-plasmid, a binary vector containing a nopaline synthase gene. Plant cells transformed by this vector can be easily identified by their ability to produce nopaline. Transformed alfalfa plants were recovered from A. rhizogenes-induced hairy roots. These transgenic plants were characterized by normal leaf morphology and stem growth but a root system that was shallow and more extensive than normal. These plants were also fertile, set seeds upon self-pollination and outcrossing. Nopaline was detected in R1 progeny. Southern blot analysis confirmed the presence of multiple copies of T-DNAs from the Riplasmid in the plant genome in addition to the vector T-DNA.     

Effect of <Emphasis Type="Italic">Medicago sativa</Emphasis> ferritin gene on stress tolerance in transgenic grapevine

The effect of Medicago sativa (alfalfa) ferritin gene (MsFer) on abiotic stress tolerance was tested using transgenic Vitis berlandieri × Vitis rupestris cv. ‘Richter 110’ grapevine rootstock lines. Leaf discs from transgenic plants maintained higher photosynthetic activity after NaCl, tert-butyl-hydroperoxide (t-BHP) or paraquat treatment than control ones. These results indicate that the increased production of ferritin significantly improved abiotic stress tolerance in transgenic grapevine plants.     

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.     

MicroRNA156 as a promising tool for alfalfa improvement

A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down‐regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.     

Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.     

Nucleotide polymorphism of the Srlk gene that determines salt stress tolerance in alfalfa (Medicago sativa L.)

Based on legume genome syntheny, the nucleotide sequence of Srlk gene, key role of which in response to salt stress was demonstrated for the model species Medicago truncatula, was identified in the major forage and siderate crop alfalfa (Medicago sativa). In twelve alfalfa samples originating from regions with contrasting growing conditions, 19 SNPs were revealed in the Srlk gene. For two nonsynonymous SNPs, molecular markers were designed that could be further used to analyze the association between Srlk gene nucleotide polymorphism and the variability in salt stress tolerance among alfalfa cultivars.     

Delay of leaf senescence in <Emphasis Type="Italic">Medicago sativa</Emphasis> transformed with the <Emphasis Type="Italic">ipt</Emphasis> gene controlled by the senescence-specific promoter SAG12

We report the successfull delay of leaf senescence in Medicago sativa. A highly regenerable clone of alfalfa was transformed with the construct SAG12-IPT, an approach that has already proved efficient in other crops. Several independent transformants were obtained as determined by Southern analysis and all the transformants expressed the transgene as measured by RT-PCR. In vitro and in vivo analyses showed that SAG12-IPT plants exhibited a stay-green phenotype that has the potential to greatly improve the quantity and quality of alfalfa forage.     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn 1 mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn ₁ was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn ₁ mutation.     

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa,lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the recipient parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.