Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’

Background: Medicago ruthenica L. ‘Zhilixing’ is a new variety with superior forage and seed yield compared to the wild type. The cold, drought and salt tolerance of Zhlixing are better than those of alfalfa, suggesting that this variety can serve as a high-quality genetic resource for improving the stress resistance of alfalfa. However, because of the lack of tissue culture regeneration system, it is difficult to perform genetic transformation studies on stress resistance genes. This study aimed to establish an efficient tissue culture regeneration system for Zhilixing variety. Methods: Three types of explants were selected and tested on four types of basal media supplemented with different combinations of auxin and cytokinin for callus induction and differentiation, based on orthogonal tests to select the combinations of auxin and cytokinin suitable for callus induction and differentiation. Two-factor combination method was used to formulate a suitable rooting medium. Result: The hypocotyledonary axis was found to be an excellent explant for callus induction on MS medium. The optimum callus induction medium contained thidiazuron (TDZ, 0.5 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/L) and naphthaleneacetic acid (NAA, 0.5 mg/L) where the callus induction rate was 93.33%. The differentiation medium was supplemented with TDZ (0.75 mg/L), 2,4-D (0.25 mg/L) and 6-benzyladenine (6-BA, 1.5 mg/L) where the differentiation rate was 63.33%. Thidiazuron played the key role in both processes of callus induction and differentiation. Half-strength MS containing 0.1 mg/L of NAA was the most efficient rooting medium.

Utilization of Transcriptome, Small RNA, and Degradome Sequencing to Provide Insights Into Drought Stress and Rewatering Treatment in Medicago ruthenica

Drought is a major limiting factor in foraging grass yield and quality. Medicago ruthenica (M. ruthenica) is a high-quality forage legume with drought resistance, cold tolerance, and strong adaptability. In this study, we integrated transcriptome, small RNA, and degradome sequencing in identifying drought response genes, microRNAs (miRNAs), and key miRNA-target pairs in M. ruthenica under drought and rewatering treatment conditions. A total of 3,905 genes and 50 miRNAs (45 conserved and 5 novel miRNAs) were significantly differentially expressed in three test conditions (CK: control, DS: plants under drought stress, and RW: plants rewatering after drought stress). The degradome sequencing (AllenScore < 4) analysis revealed that 104 miRNAs (11 novel and 93 conserved miRNAs) were identified with 263 target transcripts, forming 296 miRNA-target pairs in three libraries. There were 38 differentially expressed targets from 16 miRNAs in DS vs. CK, 31 from 11 miRNAs in DS vs. RW, and 6 from 3 miRNAs in RW vs. CK; 21, 18, and 3 miRNA-target gene pairs showed reverse expression patterns in DS vs. CK, DS vs. RW, and RW vs. CK comparison groups, respectively. These findings provide valuable information for further functional characterization of genes and miRNAs in response to abiotic stress, in general, and drought stress in M. ruthenica, and potentially contribute to drought resistance breeding of forage in the future.

Environment-Driven Adaptations of Leaf Cuticular Waxes Are Inheritable for Medicago ruthenica

Cuticular waxes covering the plant surface play pivotal roles in helping plants adapt to changing environments. However, it is still not clear whether the responses of plant cuticular waxes to their growing environments are inheritable. We collected seeds of Medicago ruthenica (a perennial legume) populations from 30 growing sites in northern China and examined the variations of leaf cuticular waxes in a common garden experiment. Four wax genes, MrFAR3-1, MrFAR3-2, MrCER1, and MrKCS1, involved in biosynthesis of predominant wax classes (primary alcohol and alkane) and wax precursors, were isolated to test the contributions of genetic variations of the coding sequences (CDS) and the promoter sequences and epigenetic modifications. The plasticity responses of the cuticular waxes were further validated by two stress-modeling experiments (drought and enhancing ultraviolet B). Great variations in total wax coverage and abundance of wax classes or wax compounds were observed among M. ruthenica populations in a common garden experiment. Stress-modeling experiments further validated that M. ruthenica would alter leaf wax depositions under changed growing conditions. The transcriptional levels of the wax genes were positively or negatively correlated with amounts of cuticular waxes. However, the analysis of promoter methylation showed that the methylation level of the promoter region was not associated with their expressions. Although both promoter sequences and CDS showed a number of polymorphic sites, the promoters were not naturally selected and insignificant difference could be observed in the numbers and types of acting elements of the four wax genes among populations. In contrast, the CDS of the wax genes were naturally selected, with a number of missense mutations resulting in alterations of the amino acid as well as their isoelectric points and polarities, which could impact on enzyme function/activity. We conclude that long-term adaptation under certain environments would induce genetic mutation of wax biosynthesis genes, resulting in inheritable alterations of cuticular wax depositions.

Leaf Transcriptome Analysis of Medicago Ruthenica, Revealing Its Response and Adaptive Strategy to Drought Stress and Rehydration

Background: Recently, drought stress has brought tremendous loss on the production of agriculture and animal husbandry. In realistic production, plants are often in cyclic wet-dry environment. Therefore, the factors that affect the final yield of plants in adversity including the resistance and tolerance to drought and the ability of plants to resume from the previous damage after rehydration. So it’s necessary for us to study the response and adaptive strategies of plants to drought and rehydration. Generally, the yield of herbage with strong resistance is relatively low. However, Medicago ruthenica(L.)cv.Zhilixing has the advantages of strong resistance and high yield concurrently. This made it can be used for raising livestock, natural grassland improvement, as a good parent for breeding and a new and high quality resource of stress resistance genes. Now, there are still many problems need to be solved when compared with other important legume forages. Therefore, we analyzed the changes of Medicago ruthenica(L.)cv.Zhilixing on transcription level under drought stress and rehydration, explored its phased response strategies.Results: We obtained 191 DEGs in drought stress, and the three treatments has 43 DEGs in common. Galactose metabolism, Starch and sucrose metabolism, Arginine and proline metabolism, TCA cycle, Photosynthesis-antenna proteins, were involved in the adaptation of Medicago ruthenica to 9 days of drought stress. The regulation of Arginine and proline metabolism, Cysteine and methionine metabolism, Photosynthesis-antenna proteins, Ascorbate and aldarate metabolism were conducive to the resistance of Medicago ruthenica to severe drought stress. The regulation of Starch and sucrose metabolism, Flavonoid biosynthesis, Valine, leucine and isoleucine degradation, Circadian rhythm-plant was beneficial to the post drought recovery of Medicago ruthenica.Conclusions: We preliminarily analyzed the adaptation mechanism of the plant under different drought and rehydration conditions. Medicago ruthenica(L.)cv.Zhilixing adopts different strategies to adapt to different degrees of drought stress and rehydration. The research discovered the genes that can be used as candidate genes to improve stress resistance and drought adaptability of plants. Our transcriptome data dramatically enriches the resources of stress resistance genes. It can provide theoretical support for further adaptation mechanism research of the plant under different drought and rehydration conditions.