Determination of the most suitable combination of explant and corresponding culture medium for alfalfa regeneration system
In the 1st Section of study, the three different explants of cotyledons, cotyledonary nodes, and hypocotyls from the four alfalfa cultivars, including alfalfa ‘Qingshui’, ‘Sandeli’, ‘Xinjiangdaye’, and ‘Gannong1’ showed the different percent callus induction when cultured on the callus induction medium containing 4.47 g L− 1 MS, 30 g L− 1 sucrose, 0.2 mg L− 1 6-BA, 2 mg L− 1 2,4-D, and 8 g L− 1 agar (Fig. 2). Among the four tested alfalfa cultivars, each explant of cotyledons, cotyledonary nodes, or hypocotyls from the ‘Qingshui’ had a greater percent callus induction compared to those of for other three alfalfa cultivars. For instance, the mean percent callus induction of cotyledons from ‘Qingshui’ was 73.2%, which was significantly greater than that of 27.2% for ‘Sandeli’, 30.1% for ‘Xinjiangdaye’, and 17.2% for ‘Gannong1’. The greatest mean percent callus induction value for cotyledonary nodes from ‘Qingshui’ was also observed among those cultivars tested. Regarding the percent callus induction for hypocotyl, while no significant difference among the three cultivars of ‘Qingshui’, ‘Sandeli’, and ‘Gannong1’was revealed by the Fisher’s LSD test, the percent callus induction value of ‘Qingshui’ (98.5%) was greater than those of 90.0 and 95.2% for the two cultivars, respectively. When averaging across the percent callus induction of the three explants for each cultivar, the mean value of ‘Qingshui’ was greatest (80.6%), followed by ‘Gannong1’ (55.5%), ‘Sandeli’ (52.3%), and ‘Xinjiangdaye’ (46.9%). Therefore, alfalfa ‘Qingshui’ was tested for optimizing the callus induction medium.
As shown in Fig. 3A, the percent callus induction of the three explants from ‘Qingshui’ varied greatly when cultured on various callus induction medium containing different hormones (6-BA and 2,4-D) concentrations (Table 2). Overall, all those three explants cultured on medium C24 (79.3, 92.7, and 100% for cotyledon, cotyledonary node, and hypocotyl, respectively) had the greater percent callus induction compared to those of values for other mediums. Additionally, compared to cotyledon and cotyledonary node explants, the hypocotyl always showed a greater percent callus induction (92.5–100%) except when cultured on mediums C13 (81.3%) and C32-C35 (79.6–87.0%). Therefore, C24 medium (0.4 mg L− 1 6-BA, 4 mg L− 1 2,4-D, 4.47 g L− 1 MS, 30 g L− 1 sucrose, and 8 g L− 1 agar) was determined the best callus induction medium for inducing callus.
As the hypocotyl explant showed a consistent greater percent callus induction, the callus developed from this explant was further used to optimize the bud differentiation medium, which contained various hormones (6-BA and KT) combinations (Table 2). All the callus induced from hypocotyl explant (totally 150 callus, 30 callus in each medium with 5 replicates) cultured on each of medium B11 and B12 developed bud achieving 100% bud differentiation rate, which was significantly greater than that of 28.6–82.5% for callus cultured on other mediums (Fig. 3B). As both the B11 and B12 mediums showed the greater percent bud differentiation, finally B11 medium (each of 0.25 mg L− 1 6-BA and KT, 4.47 g L− 1 MS, 30 g L− 1 sucrose, and 8 g L− 1 agar) was determined the suitable bud differentiation medium used in alfalfa genetic transformation system. The regenerated seedlings rooted on MS basal medium, followed by transplanting and cultivating in pot when grown up to 10 cm in height.
Based on the results presented above, an optimized alfalfa regeneration system with using ‘Qingshui’ hypocotyl as poexplant for callus induction and bud differentiation (cultured on C24 and B11 medium, respectively) was established.
Agrobacterium -mediated transformation of alfalfa using eGFP as a visual marker
To improve the transformation efficiency, three different agrobacterium infection approaches were tested using the hypocotyl callus of ‘Qingshui’ and compared for percent bud differentiation and infection (Figs. 4 and 5). Ultrasonic-assisted agrobacterium infection for 10 mins showed the greatest percent bud differentiation (76.5%) and infection (92.1%) compared to that of direct infection for 15 mins (35.2 and 85.7%, respectively) and vacuum infection for 10 mins (46.9 and 85.1%, respectively) approaches (Fig. 4). The Fig. 5 showed the eGFP signal detection in the hypocotyl callus at co-cultivation stage after agrobacterium infection (Fig. 5D), as well as the fluorescent signal in developed leaf (Fig. 5E) and root tissues (Fig. 5F) after transplanted into the pot (Fig. 5H). Considering the percent callus induction, bud differentiation, and agrobacterium infection, the final percent transformation of alfalfa ‘Qingshui’ was approximately 72%.
The genetically transformed alfalfa ‘Qingshui’ plant could normally grow and flower under natural conditions with producing eGFP-tagged pollen grains (Fig. I-K). The PCR analysis further evidenced the introgression of eGFP into the alfalfa plants and pollens from the flowers of those transformed plants (Fig. 5L). The Fig. 6 illustrated the detailed procedure of an agrobacterium-mediated transformation of alfalfa using eGFP as a visual marker.
Application of the established transformation protocol to other Medicago species
The established transformation system was also applied to the annual M. polymorpha (‘Jinhuacai’) and perennial M. falcata for evaluating its feasibility in other Medicago species. Following Fig. 6, it took about 85–120 days for regenerating the complete M. polymorpha and M. falcata plants. The subsequent fluorescence detection on leaf and root tissues and PCR amplification using plant genomic DNA demonstrated that those regenerated ones are eGFP-positive plants (data not shown). The percent transformation was 64% for M. polymorpha (‘Jinhuacai’) and 68% for Medicago falcata (accession No. YD-CX-NO. 1), respectively.