In vitro direct organogenesis and regeneration of Medicago sativa

Abstract

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm -3 thidiazuron (TDZ) and 3 mg dm -3 AgNO 3 . When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm -3 α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.Additional key words: alfalfa, cotyledonary node, mature embryo, silver nitrate, thidiazuron, vitrification ⎯⎯⎯⎯ Increased quality, disease resistance and productivity have been the main goals for the improvement of alfalfa (Medicago sativa L.), but most of the achievements in the past were obtained by traditional breeding methods rather than genetic engineering techniques (Volenec et al. 2002). This is because of the lack of successful regeneration protocol for a wide range of alfalfa genotypes. Some progress has been reported via indirect organogenesis or somatic embryogenesis on a variety of explants of alfalfa (Zagorska et al. 1997, Gilmnur et al. 1987, Barbulova et al. 2002, Tian et al. 2002, Liang et al. 2003), but mostly based on a very small number of highly tissue culture-adapted germplasms did not overcome the limitation of genotype-dependence. Furthermore, compared with the direct regeneration protocols, plants regenerated from tissue or organ with a stage of callus formation could increase the possibility of somaclonal variation (Piccioni et al. 1997, Loureiro et al. 2007). Therefore, the objective of this study was to establish a rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes via direct organogenesis.Seeds of Medicago sativa L. cv. Eureka were surface sterilized in 75 % ethanol for 2 min and in 0.14 % HgCl 2 , for 15 min, followed by several rinses with sterile water and dipped in a shallow layer of water. After the overnight imbibition at 4 ºС, seed coats, cotyledons, and radicles were removed (Fig. 1A). The cotyledonary nodes with 1 -2 mm segments of hypocotyl were cultured in modified Murashige and Skoog (1962; MS) media (mMS) consisted of MS salts, B5 vitamins (Gamborg et al. 1968), 30 g dm -3 sucrose, 8 g dm -3 agar and 0 -1.0 mg dm -3 thidiazuron (TDZ). After culture for 10 d at temperature of 25 ºС and 14-h photoperiod (fluorescent tubes providing irradiance of 40 µmol m -2 s -1 ), the main shoot developing from cotyledonary node was excised and explants were subcultured onto mMS media with different TDZ concentrations (0.01 -0.1 mg dm -3 ). Six days later, the newly developed ax...

In vitro direct plant regeneration and Agrobacterium‐mediated transformation of lucerne (Medicago sativa L.)

“…Li et al . () also used TDZ (along with AgNO 3 ) and reported a maximum of six shoots per explant in M. sativa . TDZ has also been used in different plant species to induce high‐frequency shoot regeneration (Khalafalla and Hattori, ; Caramori et al ., ; Seedhabadee et al ., ; Kumar and Chandra, ; Kumar and Bhat, ).…”

“…Recently, Li et al . () reported direct organogenesis from cotyledonary nodes of M. sativa using thidiazuron (TDZ) and AgNO 3 . Plant regeneration via direct organogenesis has become a preferred path because it maintains genetic fidelity, as there is no intermediate callus phase involved.…”

High frequency plant regeneration from the mature seeds of Garcinia indica

“…The smaller shoots were again cultured on elongation medium. Kalia et al (2007) and Li et al (2009) reported that removal of the elongated shoots at regular intervals was necessary to allow the elongation of smaller shoot buds.…”

High frequency in vitro propagation of Trichopus zeylanicus subsp. travancoricus using branch–petiole explants

“…Sequential rooting strategies, induction on auxin supplemented medium and expression on growth regulatorfree medium, have been successfully applied in many medicinal plants. Induction of roots by auxin pulse treatment and subsequent culture on basal media has also been reported in Sequoiadendron giganteum (Berthon et al 1987), Robinia pseudoacacia (Kanwar et al 2008) and Medicago sativa (Li et al 2009). The emergence of roots on half-strength basal media with NAA may be due to the inhibition by the continuous exposure to NAA.…”