Callus induction and regeneration
The cotyledons, hypocotyls, roots, leaves, and petioles were used as explants for callus induction. Briefly, cotyledons, hypocotyls, and roots were taken from germinated 4-day-old sterile seedlings. The cotyledons were dissected into 2-3 sections, and the hypocotyls and roots were cut cross-sectionally into 2-3 mm long longitudinal segments (Supplementary Material: Fig. S1A and S1B). The petioles of 30-day-old aseptic plantlets were excised into 3-4 mm fragments, and the leaves were divided into 3-4 parts (Supplementary Material: Fig. S1C and S1D).
The cotyledon sections were taken as explants to determine the hormonal ratios for callus induction. The callus tissues were induced on the MS basal medium containing different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 1, 2, 3, 5 mg·L−1) and 0.4 mg·L−1 6-benzyl amino purine (6-BA). The composition of the media is shown in Supplementary Material: Table S1. Furthermore, the pH of the media was adjusted to 5.8 before autoclaving at 121℃. The explants were initially grown in the dark for 20 days, and initiation efficiency was counted after cultivation on various clover callus mediums (CCM1-CCM5). Calluses were then sub-cultured after every 20 days on fresh medium. Callus browning rate, callus thickness, and callus area were measured at the 25th day of sub-culturing.
To examine the callus formation effect, the cotyledons, hypocotyls, roots, leaves, and petioles were inoculated into CCM2 containing 2 mg·L−1 2,4-D and 0.4 mg·L−1 6-BA. The whole process of callus induction was accomplished in a dark environment. Each medium and explant type was subjected to three independent experiment replicates (60 tissues per set of experiments).
60-day-old fresh embryogenic callus differentiation was tested on MS media supplemented with naphthalene acetic acid (NAA; 0.1 mg·L−1) and disparate quantities of 6-BA (0.5, 1, 1.5, 2 mg·L−1). Supplementary Material: Table S2 shows the combinations of different clover differentiation mediums (CDM1-CDM4). Each concentration was verified with three replicates of about 150 calluses. Regenerated shoots and embryoids were cultivated on MS medium containing 0.1 mg/L NAA (clover rooting medium, CRM) (Ahuja et al. 1983) for root development under growth chamber at 23℃ with a 16 h /8 h light/dark photoperiod.
Agrobacterium tumefaciens strain and plasmid vectors
The binary plasmid PBI121 (Gene Bank accession: AF485783.1), carrier of the neomycin phosphotransferase II (NPTII) and the β-glucuronidase (GUS) reporter gene, was applied for transformation. PBI121 vector was introduced into white clover tissues using the Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993) containing a disarmed Ti plasmid. Single colonies of Agrobacterium EHA105 were incubated in 50 ml of YEB liquid medium with 20 mg·L−1 rifampicin (Rif) and 50 mg·L−1 kanamycin (Kan) at 28℃, and shaken at 180 rpm for 65 h.
Agrobacterium -mediated white clover transient transformation
To evaluate the efficiency of Agrobacterium-mediated transient transformation, cotyledons explants and 60-day-old calluses were infected with the Agrobacterium suspension. The treatments were as follows: (1) Agrobacterium bacteria were suspended in liquid clover infection mediums (CIM) supplemented with 10 mg·L−1 acetosyringone (AS). The optical density at 600 nm (OD600) was adjusted to 0.1, 0.2, 0.3, 0.5, and 0.7, respectively. Tissues were placed in the suspended solutions for 20 min and incubated on the co-cultivation medium (CM) for 3 days at 22℃. (2) Agrobacterium cells were resuspended to an OD600 of 0.3 and added with 0, 10, 20, or 40 mg·L−1 AS, respectively. Tissues were immersed in suspension for 20 min and cultivated on CM for 3 days. (3) Following 20 min infection in CIM (OD600=0.3) containing 20 mg·L−1 AS, tissues were dried on sterile filter paper to remove excessive Agrobacterium and co-cultured on CM in dark conditions for 1-5 days. These tissues were stained daily with 5-bromo-4-chloro-3-indolyl-glucuronide (X-Gluc) after being co-cultivated on CM.
Two protocols of Agrobacterium tumefaciens-mediated white clover transformation and plant regeneration
Protocol A: Agrobacterium-mediated transformation in white clover ‘Ladino’ after callus formation from 4-day-old roots
Root explants of 4-day-old white clover ‘Ladino’ seedlings were placed on CCM2 and cultured at 22℃ in a dark environment. After 40-50 days, the callus was formed and utilized for transformation. The Agrobacterium bacteria was centrifugally agglomerated for 20 min at 4500 rpm on the date of transformation. The supernatant was thrown out, and the cells were delicately resuspended in liquid CIM1 with 20 mg·L−1 AS and 600 mg·L−1 casein peptone at an optical density of 0.5 (600 nm). Uniform ready-to-infection calluses were collected in CIM1 and gently agitated (80 rpm) for 20-25 min at 28℃. Following infection, the callus was laid on sterile filter paper to absorb excessive suspension. After co-cultivation with adhering Agrobacterium for 4 days on CM1, the callus was washed 3-4 times with distilled water. The calluses were then sub-cultured under a 16 h photoperiod on CDM2 selection medium (CDM2-S) containing 50 mg·L−1 Kan to select transformed cells and 300 mg·L−1 Cefotaxime sodium salt (Cef) to inhibit the further growth of A. tumefaciens. After 20 days, the resistant green shoot buds and embryoids of Kan were transferred to the same CDM2-S media for an additional 20-40 days to develop shoots. Regenerated green shoots under Kan selection were then placed on CRM selection medium (CRM-S) at 23℃ on a 16 h/8 h light/dark photoperiod for whole-plant development. After roots formation within 30-40 days, the regenerated plantlets were transplanted in the soil. The composition of the media for Protocol A has been given in Supplementary Material: Table S3.
Protocol B: Agrobacterium-mediated transformation in ‘Ladino’ clover before callus initiation from cotyledons
Cotyledons explants from 4-day-old white clover ‘Ladino’ seedlings were immersed in liquid CIM2 supplemented with A. tumefaciens (OD600=0.5) and then incubated at 28℃ for 20-25 min at 80-100 rpm. Subsequently, the explants were dried on sterile filter paper and co-cultivated with Agrobacterium on CM2 at 23°C for 4 days. After washing 3-4 times with sterile water, the explants were transferred to CCM2 selection medium (CCM2-S) supplemented with 300 mg·L−1 Cef and 50 mg·L−1 Kan and incubated at 23℃. The selection was implemented in the dark for 50-60 day with media alteration after every 20 days. The differentiated structure induction and roots development procedure were the same as described in protocol A on CDM2-S and CRM-S medium, under a 16 h /8 h light/dark photoperiod at 23℃. The green-white clover plantlets were transferred into the greenhouse about 40 days after root formation. The media composition for Protocol B has been shown in Supplementary Material: Table S4.