Haploid Culture and Double Haploid Induction in Medicago sativa L. cv. XinJiangDaYe

Alfalfa (Medicago sativa), a perennial cross-pollinated plant, is one of the most important forage crops in semi-arid and arid areas (Rong et al., 2017; Zhang et al., 2017) with greater commercial and ecological significance (Yin et al., 2018). However, cultivated alfalfa is a self-incompatible autotetraploid (2n = 4x = 32) plant with a very complex genome that hinders efforts to genetically improve the varieties (Chen et al., 2020). Due to the large number of imported varieties and long-term artificial selection, the genetic base of alfalfa tends to be narrow. Research on generation and utilization of haploid and double haploid plants has important significance for widening the genetic base of alfalfa and also for accelerating the breeding of improved varieties. Crossing with double haploid plants could greatly improve the heterozygosity of F1 generation and could contribute to increased yield, quality and resistance (Riday et al., 2002). The whole genome of alfalfa (Medicago sativa L. ‘XinJiangDaYe’) was published and this knowledge together with the availability of double haploids could be of greater genetic significance for research on genetic transformation and to determine genetic origin.
 
Haploid plants were obtained effectively by combining high-frequency induction regeneration system with Flow cytometry (Galbraith et al., 2001). With the development of very efficient physical and chemical doubling techniques, double haploid (DH) plants of many species have been successfully developed (Sharma et al., 2019). However, cell polyploidy is a complex process, which is influenced by culture methods (solid and liquid), reagent selection for doubling, explant selection, penetrant, treatment time and culture conditions. Moreover, suitable doubling conditions do vary by species. So far, haploid and double haploid plants have been successfully obtained by tissue culture with several plants including: maize (Jiang et al., 2015), tobacco (Schedel et al., 2016), rice (Joann et al., 2017) and others, but there were only a few reports of progress from tissue culture of alfalfa.
 
In the previous research, we obtained the haploid plants of alfalfa (Medicago sativa L. ‘XinJiangDaYe’), and we propagated the haploid plants by using cuttings from tissue culture (Xu et al., 2019; Cui Nan, 2017). In the current study, we aimed to determine: 1) which of the two double culture methods (solid and liquid) is better?; 2) which of the three explants (leaves, shoot tips and root tips) has the best outcome? and 3) what is the optimal condition for generating double haploid of alfalfa (Medicago sativa L. ‘XinJiangDaYe’)?

The anther tissue culture and regeneration system was established using Medicago sativa L. ‘XinJiangDaYe’, which was planted in 2015 in the forage experimental field of Inner Mongolia Agricultural University Hohhot (37°282 N, 97°202 E) in Inner Mongolia, P.R. of China. The experiments on anther tissue culture and ploidy identification of regenerated plants were conducted at Key Laboratory of Grassland Resources, Ministry of Education P.R. of China. The experiment began in July 2017 and ended in December 2019.
 
Developing regenerated plants by anther tissue culture
 
In June 2017, the buds at the uninucleate eccentric stage (the bud length: 2~3mm) were selected according to Zagorska and Dimitrov (1995). The anothers were removed by dissection needle and placed in the callus induction medium (MS basal salts and vitamins, 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, 0.2 mg/L NAA, 3.0 mg/L kinetin, 30 g/L sucrose, 0.01 g/L activated carbon, pH 5.8). Dark culture for 60 days in the shaking bed at 25°C with a rotating speed of 110 rpm. After subculture for 20 days, all callus tissues were transferred to shoot induction medium (MS basal salts and vitamins, 1.5 mg/L 2,4-D, 1.0 mg/L 6-BA, 0.2 mg/L NAA, 20 g/L sucrose, 7 g/L agar, pH 5.8). After 30 days, the regenerated shoots were transferred to root induction medium (MS basal salts and vitamins, 0.1 mg/L NAA, 20 g/L sucrose, 7g/L agar, pH 5.8).
 
Ploidy identification of regenerated plants
 
The ploidy of regenerated plants from another culture was identified by Beckman coulter cytoFLEX at 488 nm (Loureiro et al., 2006). The external standard control materials were the known ploidy seedlings of M. ruthenica (2n=2x=16) and M.varia Martin ‘Caoyuan No.1’ (2n=4x=32). In addition, the nuclear suspensions of the regenerated plants were prepared using LB01 according to Galbraith et al., (1983).
 
Solid and liquid colchicine treatment medium for doubling
 
The colchicine treatment for doubling experiment started in January 2019. Using MS as the base medium, orthogonal tests were conducted on the three factors and four levels of colchicine concentration (Cc), osmotic Dimethyl sulfoxide (DMSO) concentration and treatment time (Tt) according to Wang et al., (2013). The gradient was set according to L₁₆ (4³). 20 g/L sucrose was added into the liquid medium (pH 5.8) and dark culture was conducted in a shaking bed at 25°C with a rotating speed of 110 rpm. The solid medium had the same composition as the liquid, but 7g/L agar was added to solidify it. Explants identified from haploid plants were excised and placed in solid and liquid colchicine doubling treatment medium.
 
Improved culture of callus and regeneration culture of explants after doubling treatment
 
After colchicine treatment for doubling, the explants were rinsed with sterile water, dried with filter paper, and placed in the callus induction medium by the positive insert, and 10 explants per bottle were placed and this process was repeated 3 times. Most of the callus induced from the explants treated by colchicine were non-embryogenic. The two-factor random combination of 2,4-D (0.5, 1.0mg/L) and 6-BA (0, 0.5mg/L) was used to improve culture conditions (Li et al., 2010). The improved callus underwent the same differentiation and rooting culture as above. Regenerated seedlings were used to identify the ploidy by using Beckman coulter cytoFLEX as described above.
   Statistical analyses
 
The callus induction rates of each orthogonal experiment were evaluated via analysis of variance (ANOVA, P < 0.05). The ploidy ratio of regenerated plants was calculated after doubling treatment.

Ploidy identification of regenerated plants from another culture of alfalfa
 
The regeneration process of anther tissue culture is shown in Fig 1 (A to D). As indicated in Fig 2, there are two types of ploidy, tetraploids and diploids, in the regenerated plants. The main peak value of diploid plants is located in the linear graph of PE Channel 7 to 9 and the main peak value of tetraploid is located in 15 to 18. Among all the 76 regenerated plants, 18 haploids (23.7%) and 56 tetraploids (76.3%) were identified by Flow cytometry.
 

 

 
Effect of colchicine treatment on callus rate
 
Compared with the CK, the callus induction rate of shoot tip, leaf and root tip after solid treatment decreased by 43.46% to 81.24% (Table 1). The callus induction rate of the three explants decreased with the increase of colchicine concentration, DMSO concentration and treatment time. These results are consistent with the results from studies on Miscanthus and Limonium bellidifolium (Mori et al., 2016; Katarzyna et al., 2010). After liquid treatment, the callus induction rate of the three explants was reduced by 51.99 to 88.90%. Moreover, the callus induction rate of three explants treated with colchicine was compared, and the highest was obtained from leaf, followed by shoot tip and root tip. The callus induction rate of shoot tip or leaf was significantly higher than that of root tip (P < 0.05). This may be due to the fact that root tip is more sensitive to colchicine toxicity than the other two explants (Katarzyna et al., 2010).
 

 
Improved culture of non-embryogenic callus
 
Callus could be induced after both solid and liquid doubling treatment, but most of them were non-embryonic callus. This may be due to the colchicine toxicity which had a negative effect on embryogenic callus formation and multiple shoots regenerated (Karen et al., 2003; Ganga and Chezhiyan, 2002).  In our study, hormone combination improved the non-embryogenic callus (Table 2). After solid culture with colchicine treatment, the browning callus became yellow-green obviously (Fig 2 E and F) when MS medium was supplemented with 2,4-D 0.5 mg/L and 6-BA 0.5 mg/L. After colchicine liquid treatment, adding only 2,4-D at 0.5 mg/L was more advantageous to the formation of embryogenic callus. The improvement rate after colchicine solid treatment was 17.46 to 36.76% and that after liquid culture was 17.65 to 47.27%. Even if the non-embryogenic callus was improved, the number of regenerated plants was not necessarily large (Karen et al., 2003; Ganga and Chezhiyan, 2002). The efficiency was found to be genotype-dependent, primarily due to differences in the ability to regenerate plants from embryogenic callus (Karen et al., 2003).
 

 
Effects of solid and liquid culture on the doubling rate of alfalfa haploid
 
The doubling rate of the two culture methods were different (Fig 3, Table 3). Under the condition of colchicine solid doubling treatment, double haploid plants only accounted for 18.75% of the regenerated plants, and 37.50% of the regenerated plants did not have ploidy change, which were still diploid. Under the condition of colchicine suspension doubling treatment, the total ratio of ploidy changed percentage was 81.82%, the ratio of diploid was 27.28% lower than that of solid doubling treatment. In addition, the ratio of mixed ploidy increased by 24.69%. Double haploid plants accounted for 27.27% of regenerated plants, which is 45.44% higher than solid treatment. Colchicine and DMSO were added to the liquid medium, which was easier to penetrate the explants, and contributed to the improvement of the doubling rate (Currah and Ockendon, 1987). The doubling effect of colchicine liquid treatment was found to be better than that of solid medium treatment.

 

 
Effects of different explants on doubling rate
 
The ratio of each ploidy from three explants treated with colchicine was calculated. In both solid and liquid culture methods, at the explant level, the polyploidy efficiency of the leaves was the highest (Karen et al., 2003). Although the number of regenerated plants was higher than the root tip, the proportion of double haploid was lower 6.25% to 18.18% than the leaf with the shoot tip as explants. When the root tip was used as explants, only 6.25% of the regenerated plants were obtained and all the plants were diploid without any change in ploidy. In general, the proportion of double haploid is low, most of which are either mixed ploidy or of original ploidy (James et al., 1987). Similar results were reported from studies on cherry rootstocks (James et al., 1987) and bananas (Ganga and Chezhiyan, 2002). Selection of suitable explants is a prerequisite for obtaining more double haploid plants, and in our study, the leaf as the explants had the best doubling effect.
 
The optimal doubling condition for alfalfa double haploid
 
In the medium treatment of double haploid plants obtained of this study, medium A7 and medium A6 were combined with low concentration for a long time, medium A15 was treated with high concentration for a short time. Considering the time and doubling rate of the test process, A15 was found to be the most appropriate treatment. Colchicine concentration and treatment time are the two most important and direct factors which have a certain correlation, and most of the results of doubling treatment study used were either low concentration for a long time or high concentration for a short time (Sharma et al., 2019). Likewise, the addition of DMSO, a penetration aid, would also be beneficial to the doubling effect, which can reduce the toxic effect of colchicine and increase the rate of generation of double haploid (Currah and Ockendon, 1987).

Thanks to the Key Laboratory of Grassland Resources, Ministry of Education P.R. of China for providing facilities for investigation. This study was supported by the Science and technology project of Inner Mongolia Agricultural University (YZGC2017008) and the Science and technology projects of Inner Mongolia Autonomous Region (2019GG244).