Drought Response Of Callus And Molecular Characterization Of A Dwarf Mutant In Zoysia Matrella

Zoysia matrella[L.]Merr.,also known as Manila grass,is widely used in subtropical and tropical areas as a warm-season turf grass because of its fine-texture,dense lawn formation,fast spread,wearing and shade tolerance,and low nutrient requirements.However,Z.matrella requires sufficient water and regular mowing for maintenance,which is labor and cost intensive.To reduce the water supplyment,frequency of mowing and cost of lawn management,somaclonal variation were used to obtain plants with drought tolerance and dwarf mutant.In this study,the effects of the brassinosteroid?BR?phytohormone on the somatic embryogenesis of Z.matrella were investigated by exogenous application of epibrassinolide?EBL?to optimize and improve long-term maintenance of in vitro cultures and the regeneration capacity of this species.PEG-6000 was used to simulate drought stress and the regeneration of drought tolerant Z.matrella plants was assessed during the development of somatic embryos.The exogenous application of EBL was used to explore the role of BR in conferring drought tolerance in Z.matrella.Additionally,a Z.matrella dwarf mutant was isolated from ⁶⁰Co-?-irradiated calli and used to study the potential mechanism of phytohormone in regulating plant growth.We conducted morphological,physiological,biochemical,and transcriptional analyses to reveal the dwarfing mechanism in the mutant.The candidate genes involved in Z.matrella dwarfism were identified using RNA-sequencing,which provided a molecular basis for genetic engineering methods that can improve lawn quality.1.The phytohormone BR promoted somatic embryogenesis of Z.matrella.Exogenous application of EBL significantly promoted callus growth and regeneration.As EBL concentration increased,the growth status of the callus and plant regeneration both initially increased and then decreased.High concentrations of EBL inhibited the growth and regeneration of the callus.A comparative analysis of the morphology and physiology of Z.matrella indicated that 0.02?mol·L^-1 EBL was the optimal concentration of exogenous treatment because it greatly increased the proliferation and regeneration rates of calli,as well as antioxidant enzyme activity.2.The differentiation and regeneration abilities of Z.matrella calli were inhibited by drought stress.Different concentrations of PEG were used to select for somatic variation in Z.matrella,and plants with drought tolerance were isolated from 5%PEG and 10%PEG treatments.3.The phytohormone BR can significantly improve drought resistance in Z.matrella grown under drought stress.Exogenous EBL treatment significantly alleviated the damage from drought stress and Z.matrella calli were able to regenerate.These results suggested that 0.05,0.1 and 0.5?mol·L^-1 EBL promoted the regeneration of calli grown in simulated drought conditions.Moreover,BR promoted the drought stress response by increasing catalase?CAT?,guaiacol peroxidase?G-POD?,and superoxide dismutase?SOD?activity,and reducing the malondialdehyde?MDA?content in Z.matrella.4.The plant hormones indole acetic acid?IAA?and abscisic acid?ABA?may regulate the dwarf mutant identified from 60Co-?-irradiated Z.matrella calli through changes in their synthetic,transportation,and metabolism pathways.The phenotypic and physiological indexes of the dwarf mutant maintained stability for five years of asexual propagation in the greenhouse.Phenotypically,the dwarf mutant showed shorter stems,wider leaves,a lower canopy,and a darker green color than the wild-type?WT?control.We found that the phenotypic changes in the dwarf mutant were associated with the physiological responses of CAT,POD,SOD,MDA,lignin,and chlorophyll.Of the four endogenous hormones measured in leaves,both IAA and ABA levels were decreased in the mutant compared to those in the wild type.A transcriptomic comparison between the dwarf mutant and WT leaves revealed 360differentially expressed genes?DEGs?,including 62 up-regulated and 298down-regulated unigenes.The major DEGs related to auxin transportation?PIN1?,ABA degradation?Abscisic acid 8'-hydroxylase 2-like?,and cell wall development?CESA1?,as well as homologous expansin genes were all down-regulated,indicating their potential contribution to the phenotypic changes observed in the dwarf mutant.Indeed,IAA biosynthesis,IAA transportation,ABA degradation,and expansin function all likely contribute to the dwarfism observed in this Z.matrella mutant.Overall,0.02?mol·L^-1 EBL was found to be the optimal concentration of exogenous treatment,which can significantly promote the proliferation and regeneration of callus.Plants with drought tolerance were isolated from 5%PEG and10%PEG treatments.EBL can significantly improve drought resistance in Z.matrella regeneration under drought stress by enhancing the activity of antioxidant enzymes.IAA synthesis and transport,ABA degradation,and expansin function may be the main causes of the dwarf mutant in Z.matrella.