I.INTRODUCTION
Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world (Kechang et al., 2009). It has the highest yield potential and feeding values and plays an important role in livestock industry (Hill et al., 1991). It is also a high quality forage crop and an important source of biological nitrogen fixation. Deak et al. (1986) first described that transgenic alfalfa plants were generated using Agrobacterium-mediated gene transfer technique and there have been a few reports of stably transformed conifers using this technique (Ding et al., 2003; Chabaud, 2003; Rosellini et al., 2007). Plant transformation using Agrobacterium tumefaciens is advantageous due to single copy DNA insertion compared to particle bombardment transformation (Vain and Thole, 2009). Herbicide-resistant transgenic alfalfa is cultivated in more than 200,000 hectares in USA (James, 2011) and also there are several reports concerning the introduction of new traits to improve abiotic stresses such as salinity (Yan et al., 2012; Zhang et al., 2012), drought (Jiang et al., 2009) and heavy metal contamination (Zhang and Liu, 2011). To improve its genetic traits, it is necessary to optimize regeneration protocol in order to increase regeneration frequency and then to improve transformation efficiency of alfalfa for further study of molecular biological functions. Plant regeneration under in vitro conditions are affected by several different parameters such as selection of the genotype, concentration of phytohormones, bacterial strain, laboratory conditions, and the growth response of the explant to the regeneration medium. In addition, explant types could influence transformation efficiency (Bregitzer, 1992). The objective of this research was to develop a regeneration system for the selected cultivars and to optimize the conditions for transformation that could be effectively used for genetic improvement of alfalfa.
II.MATERIALS AND METHODS
1.Explant source of alfalfa cultivars
Seeds of five alfalfa cultivars (Xinjiang Daye, ABT405, Vernal, Wintergreen and Alfagraze) were collected from National Institute of Animal Science, Rural Development Administration (RDA), Republic of Korea. The seeds were first soaked in 70% (v/v) ethanol for 1 min and then treated with 30% (w/v) sodium hypochlorite (NaOCl) for 30 min, followed by five time rinses with sterile water for surface sterilization. The sterilized seeds were germinated on hormone-free half-strength MS medium (Murashige and Skoog, 1962) containing 2% (w/v) sucrose and 0.3% (w/v) Gelrite (Table 1). Then cultured in a growth chamber at 24 ± 2°C in a 12 h light/12 h dark cycle. In vitro grown seedlings were used as the source of hypocotyl segments and cotyledonary explants.
2.Transformation vector and bacterial strain
Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 was used for alfalfa transformation experiments. The binary vector consisted of the GUS gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and the hygromycin phosphotransferase (HPT) under the control of the CaMV 35S promoter promoter (Fig. 1).
The Agrobacterium tumefaciens culture was grown overnight in 50 mL YEP medium (10 g/L yeast extract, 10 g/L bacto peptone, 5 g/L NaCl, pH 7.0) containing 50 mg/L kanamycin at 28°C on a rotary shaker at 200 rpm. To prepare Agrobacterium tumefaciens inoculum, an overnight culture of bacteria was centrifuged at 4,000 rpm for 10 min at 4°C and the cells were resuspended in 100 mL of liquid AI medium (Table 1) to the optical density at 600 nm reaches 0.6-0.8.
3.Plant transformation and regeneration
For transformation of selected cultivars, fully developed hypocotyl segments and cotyledonary explants from 7 days old seedlings were used as explants. Hypocotyls 1~3 mm explants were carefully excised from the seedlings without including any of the meristematic axillary buds and the cotyledonary were excised. The hypocotyl and cotyledonary segments were gently shaken in the bacterial suspension for about 30 min and blotted dry on a sterile filter paper. The hypocotyl and cotyledonary segments were transferred to the solid CCM medium (Table 1). Following 5 days of cocultivation, hypocotyl and cotyledonary segments transferred onto shoot induction SIM medium containing 250 mg/L carbenicillin and 15 mg/L hygromycin, and then, subcultured into a fresh SIM medium every 3 weeks. The green healthy shoots from the embryos were subjected to 2~3 more passages of selection by repeated excision of buds and their exposure to selective shoot elongation medium (SEM). Green healthy shoots were excised and transferred to shoot elongation SEM medium and developed shoots were transferred to root induction RM medium (Table 1). Rooted plants transferred to the soil 3~4 weeks later. Callus induction and regeneration ratios of different genotypes and explants were calculated.
4.Polymerase chain reaction (PCR) analysis of putative transgenics
Total genomic DNA was isolated from young leaves of hygromycin-resistant transgenic lines and a non-transformed control plant using the cetyl trimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). PCR analysis was performed with the extracted genomic DNA to verify for the presence of transgenes in putative transformants using primer pairs that amplify each GUS and HPT transgenes. Optimized PCR conditions were performed according to Lee et al. (2009; 2010). PCR products were separated by gel electrophoresis on 1.2% agarose gels, stained with ethidium bromide and visualized with a UV transilluminator.
5.Histochemical staining of β-glucuronidase (GUS) activity
Histochemical staining of the shoots was performed according to the method of Jefferson et al. (1987). Hygromycin-resistant plants were incubated in 5-bromo-4- chloro-3-indolyl glucuronide (X-Gluc) at 37°C for overnight. Non-transgenic regenerated shoots were used as negative controls. After staining, the samples were washed with 70% ethanol for 2~3 days to remove plant pigments. The ethanol bleach mixture was replaced three or four times then washed with double distilled H2O.
III.RESULTS AND DISCUSSION
1.Selection of an explant type for the regeneration of alfalfa cultivars
In the preliminary experiments, the in vitro regeneration potential of selected cultivars was tested in two types of plant tissue such as hypocotyl and cotyledonary segments. Developing an efficient regeneration system of alfalfa by Agrobacterium-mediated transformation were evaluated by the percent of regenerated callus from hypocotyl and cotyledonary explants. Explants from hypocotyl resulted in high regeneration into calli compared to those from the cotyledonary explants (Table 2). Regeneration efficiency of cotyledons ranged from 0% (cv. Alfalgraze) to 9.3% (cv. Vernal) and hypocotyls from 2.2% (cv. Alfalgraze) to 18.5 % (cv. Xinjiang Daye). Also For all genotypes, callus inductions from hypocotyls were faster than cotyledons. Results are summarized in Table 2. It is well known that regeneration efficiency is an essential requirement for transformation efficiency.3
2.Efficiency of Agrobacterium-mediated transformation
In this study, we evaluated transformation potential of the five alfalfa cultivars and optimized conditions for obtaining transgenic alfalfa plants using Agrobacterium-mediated transformation into hypocotyl explants. Following the transformation of the hypocotyl explants, putative transformants were selected based on regeneration under hygromycincontaining B5 plates. In this study, carbenicillin was selected as an antibiotic used for suppressing the growth of Agrobacterium (Chabaud et al., 2007). Transformation rate was calculated as the proportion of healthy explants among all the tested genotypes, with an average transformation rate of 2.16%. Xinjiang Daye varieties grew well and had 7.9 % transformation efficiency. Among the two genotypes Alfalgraze and Wintergreen showed 0% transformation rates.
To our knowledge, this is the report of the successful transformation of cultivars ABT405, Vernal and Xinjiang Daye. High transformation efficiencies were observed in alfalfa cultivars Xinjiang Daye. Genetic modification of an economically important alfalfa cultivar, Xinjiang Daye, gives us the possibilities to adapt them as a good candidate to improve genetic traits against current climatic changes, such as cold, heat, salinity and drought stresses.
3.Analysis of transgenic alflalfa plants
There were no morphological differences between control and putative transgenic plants. The genomic DNA from the leaves of 2-month-old putative transgenic plants were extracted and amplified with specific primers for the GUS and HPT genes. For PCR analysis, DNA was extracted from hygromycin resistant shoots expressing GUS signal. The amplified fragments by PCR were expected size 0.4 kb for GUS gene and 0.8 kb for HPT gene, while the control plants (wild type) did not show any bands in PCR amplification (Fig. 2). Histochemical GUS assay was performed by incubation of putatively transgenic alfalfa leaves with X-gluc overnight and blue color formation occurred in the expression sites where X-gluc was catabolized by GUS gene product. Histochemical GUS staining analysis confirmed that the stable integration and expression of the GUS gene in the genome of the regenerated shoots of transgenic alfalfa plants and wild type (WT) plants did not show any blue staining (Fig. 3A).
IV.CONCLUSION
Agrobacterium tumefaciens-mediated transformation is generally used for genetic transformation for higher plants. Efficiency of Agrobacterium-mediated transformation in five alfalfa cultivars (Xinjiang Daye, ABT405, Vernal, Wintergreen and Alfagraze) was examined. As mentioned above, successful regeneration and genetic transformation systems for alfalfa are genotype dependent. The protocol designed could achieve a stable transformation frequency of Xinjiang Daye varieties having 7.9% efficiency for hypocotyl explants. Results on PCR analysis and histochemical GUS staining revealed the successful integration of transgene into alfalfa genome via Agrobacterium-mediated transformation. Based on our results, a case study is required to adapt the transformation techniques to a wide range of alfalfa cultivars. The low frequency of transformation may be attributed to many factors such as genotype, explant, bacterial strain, agro infection, laboratory conditions etc. Our procedure would be valuable in further research on genetic transformation with agronomically important genes involved in tolerance and/or avoidance against environmental stress such as drought, temperature, salinity and heavy metal contaminations.
