Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

Drought-induced responses of organic osmolytes and proline metabolism during pre-flowering stage in leaves of peanut (Arachis hypogaea L.)

Peanut （Arachis hypogaea L.）, an improtant oil crop, usually encounters drought stress in the process of growth and development, especially at pre-flowering stage. In order to gain insight into the drought tolerance potentials based on osmolyte accumulation and metabolism of proline aspects of peanut, pot experiments were conducted with a split-plot design in Tai＇an, Shangdong Province, China in 2013 and 2014. Pre-flowering drought （PFD） stress and optinum irrigation （control, CK） were served as the main plots and the two peanut cultivars Shanhua 11 and Hua 17 served as sub-plots. Shanhua 11 was drought-tolerant cultivar and Hua 17 was drought-sensitive. The content of soluble sugars, soluble protein, free proline and other free amino acids, the activities of enzymes involved in proline metabolism, and malondialdehyde （MDA） content and ion leakage were all investigated in the two cultivars at pre-flowering stage. Results showed that PFD stress significantly increased the levels of soluble protein, free proline and free amino acid, and increased Al-pyrroline-5-car- boxylate synthetase （P-5-CS, EC 2.7.2.11） activity in the leaves of drought-tolerant and drought-sensitive cultivars. The activity of proline dehydrogenase （proDH） （EC 1.5.99.8） decreased under PFD stress in both cultivars. The leaves of the tolerant cultivar maintained higher increments of osmolyte levels, lower increments of MDA content and ion leakage, as well as a higher increased proportion of P-5-CS activity and higher inhibited proportion of proDH activity under water stress compared with the drought-sensitive cultivar. The study suggests that proline accumulation in peanut leaves under PFD can be explained by the higher enhanced activities of P-5-CS and higher inhibition of proDH. The results will provide useful information for genetic improvement of peanut under drought tolerance.

Arbuscular mycorrhizal fungi and endophytic fungi differentially modulate polyamines or proline of peach in response to soil flooding

Symbiotic fungi are involved in plant flooding tolerance,while the underlying mechanism is not yet known.Since polyamines(PAs)and proline are also associated with stress tolerance,it is hypothesized that the enhancement of stress resistance by symbiotic fungi is associated with changes in PAs and/or proline.The aim of this study was to analyze the effect of inoculation with Funneliformis mosseae and Serendipita indica on plant growth,PAs,and proline and the metabolisms in peach(Prunus persica)under flooding.Two-week flooding did not affect root colonization frequence of F.mosseae,while it promoted root colonization frequence of S.indica.Under flooding,plants inoculated with F.mosseae and S.indica maintained relatively higher growth rates than uninoculated plants.Funneliformis mosseae promoted root ornithine(Orn)contentration and arginine(Arg)and Orn decarboxylase activities under flooding,which promoted putrescine(Put),cadaverine(Cad),and spermidine(Spd)contentrations.Conversely,S.indica decreased contentrations of Arg,Orn,and agmatine and Arg decarboxylase activities,thus decreasing PA contentrations under flooding.Polyamines were negatively correlated with the expression of PA uptake transporter genes,PpPUT1 and PpPUT2,in peach.Polyamine transporter genes of F.mosseae(FmTPO)and S.indica(SiTPO)were regulated by flooding,of which FmTPO1 was positively correlated with Put,Cad,and Spd,along with positive correlations of Spd with SiTPO1,SiTPO2,and SiTPO4.Under flooding,F.mosseae decreased proline concentration,while S.indica increased proline concentration and correlated with expression of a△^(1)-pyrroline-5-carboxylate synthetase gene,PpP5CS2.It was thus concluded that F.mosseae modulated polyamine accumulation,while S.indica induced proline accumulation to tolerate flooding.