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CN-121986719-A - Method for promoting somatic embryogenesis of fir by using histone deacetylase inhibitor

CN121986719ACN 121986719 ACN121986719 ACN 121986719ACN-121986719-A

Abstract

The invention belongs to the field of seed seedling cultivation, and particularly relates to a method for promoting embryogenesis of fir by utilizing a histone deacetylase inhibitor, which comprises the steps of taking immature zygotic embryos of fir as explants, inoculating the explants to a DCR culture medium to induce callus after disinfection, culturing in the dark for about 30 days to obtain embryogenic callus, transferring the embryogenic callus to a DCR induced embryogenic embryo culture medium (the final concentration is 0.5 μm, 2 mu M, 5 mu M, and optimally 2 mu M) containing a histone deacetylase inhibitor TSA, and culturing in the dark for 25-30 days to induce differentiated somatic embryos. According to the invention, through accurately regulating and controlling the adding stage and concentration of TSA, the somatic embryo differentiation efficiency of fir is improved from below 20% to above 60% in the prior art, the embryo state maintenance time can be prolonged, the problems of low efficiency and poor stability in the prior art are solved, the operation is simple and convenient, and the method is suitable for mass propagation of fir fine variety.

Inventors

  • HAN XIAOJIAO
  • YANG YUYING
  • LI JIAYUE
  • ZHUO RENYING

Assignees

  • 中国林业科学研究院亚热带林业研究所

Dates

Publication Date
20260508
Application Date
20260331

Claims (9)

  1. 1. A method for promoting somatic embryogenesis of fir by using histone deacetylase inhibitor is characterized by comprising the following steps: Callus induction culture, namely selecting immature zygotic embryos of fir as explants to remove endosperm, placing the embryos on a DCR solid medium for callus induction, and obtaining embryogenic callus under the conditions of 23-25 ℃ and dark culture; Transferring the embryogenic callus obtained by induction into a differentiation medium added with TSA, and continuously culturing at 23-25 ℃ in dark culture to differentiate the embryogenic callus into somatic embryos; the final concentration of TSA in the medium is 0.5-5. Mu.M.
  2. 2. The method for promoting embryogenesis of fir wood using a histone deacetylase inhibitor according to claim 1, wherein the process of removing endosperm is: The explant is soaked in ethanol solution with the volume fraction of 75% for 30-60s, hgCl 2 solution with the mass fraction of 0.1% for 8-12min, and finally washed with sterile distilled water for 5-6 times, so that disinfection is completed.
  3. 3. The method for promoting embryogenesis of fir wood using a histone deacetylase inhibitor according to claim 1, wherein the DCR solid medium contains 6.0 mg/L2, 4-D, 0.5 mg/L6-BA, 0.5g/L hydrolyzed casein, 0.45g/L L-glutamine, 0.1g/L inositol, 2g/L activated carbon, 20g/L maltose and 3.5g/L gellan gum, and the pH is adjusted to 5.3 to 5.5.
  4. 4. The method for promoting somatic embryogenesis of fir utilizing a histone deacetylase inhibitor according to claim 1, wherein the pH of the DCR solid medium is adjusted to 5.3-5.5.
  5. 5. The method for promoting embryogenesis of fir wood using a histone deacetylase inhibitor according to claim 1, wherein the differentiation medium comprises 6.0mg/L ABA, 0.5mg/L GA3, 0.5g/L hydrolyzed casein, 170g/L PEG, 2g/L activated carbon, 25g/L maltose, and 3.5g/L gellan gum.
  6. 6. The method for promoting somatic embryogenesis of fir utilizing a histone deacetylase inhibitor according to claim 1, wherein the pH of the differentiation medium is adjusted to 5.8-6.0.
  7. 7. The method for promoting somatic embryogenesis of fir utilizing a histone deacetylase inhibitor according to claim 1, wherein the time for the callus induction culture is 30 days.
  8. 8. The method for promoting somatic embryogenesis of fir utilizing a histone deacetylase inhibitor according to claim 1, wherein the time for the somatic embryo-induced culture is 25-30 days.
  9. 9. The method for promoting somatic embryogenesis of fir utilizing a histone deacetylase inhibitor according to claim 1, wherein the efficiency of somatic embryo induction is 24.1-63%.

Description

Method for promoting somatic embryogenesis of fir by using histone deacetylase inhibitor Technical Field The invention belongs to the field of seed seedling cultivation, and particularly relates to a method for promoting somatic embryogenesis of fir by utilizing a histone deacetylase inhibitor. Background China fir (Cunninghamia lanceolata) is a special fast-growing high-quality wood tree species and plays an important role in forestry production, ecological protection and wood processing industries. The traditional fir propagation mainly depends on seed seedling raising and cutting propagation, but the seed propagation is easily influenced by genetic character separation, the purity of good varieties is difficult to ensure, the cutting propagation has the problems of limited cutting source, unstable rooting rate, low propagation coefficient and the like, and the large-scale and standardized seedling production requirements cannot be met. The somatic embryogenesis (somatic embryogenesis for short) technology is an important breakthrough in forest asexual propagation, has the advantages of high propagation coefficient, good genetic stability, capability of realizing industrialized seedling culture and the like, and provides an effective way for rapid propagation of fir excellent germplasm resources. However, the existing fir embryogenesis technology still faces a plurality of bottlenecks that the somatic embryo induction efficiency is generally lower than 20%, a large number of explants cannot be normally differentiated to form embryogenic callus, meanwhile, the induced embryogenic tissue always has the problems of low structuring degree and poor differentiation synchronism, so that the subsequent somatic embryo maturation rate and germination rate are seriously affected, and the industrialized application of the technology in fir seedling cultivation is restricted. Histone acetylation modification is one of the important ways of epigenetic regulation, which regulates the differentiation and development processes of plant cells by affecting chromatin structure and gene transcription activity. Histone deacetylase inhibitors (HDACi) can inhibit histone deacetylase activity, increase histone acetylation level, and activate key gene expression related to cell differentiation and embryo development. Trichostatin A (TSA) is used as a high-efficiency and specific histone deacetylase inhibitor, has potential in embryogenesis regulation of arabidopsis, rice and other mode plants and partial woods, but is applied to the process of embryogenesis of fir, and research on proper addition stage, concentration and action mechanism of trichoderma has not been reported. Therefore, development of a fir embryogenesis optimization method based on TSA regulation has important significance in breaking through the bottleneck of the prior art. Disclosure of Invention In order to overcome the defects in the prior art and solve at least one technical problem in the background art, the invention provides a method for promoting the embryogenesis of fir wood by utilizing a histone deacetylase inhibitor. By adding TSA with proper concentration in the specific culture stage of fir embryogenesis, the histone acetylation level is accurately regulated and controlled, and embryogenic related gene expression is activated, so that the somatic embryo induction efficiency and the structuring and synchronizing degree of embryogenic tissues are obviously improved, and technical support is provided for efficient propagation of fir good germplasm. The technical scheme adopted for solving the technical problems is that the method for promoting the embryogenesis of fir by utilizing the histone deacetylase inhibitor comprises the following steps: Callus induction culture, namely selecting immature zygotic embryos of fir as explants to remove endosperm, placing the embryos on a DCR solid medium for callus induction, and obtaining embryogenic callus under the conditions of 23-25 ℃ and dark culture; Transferring the embryogenic callus obtained by induction into a differentiation medium added with TSA, and continuously culturing at 23-25 ℃ in dark culture to differentiate the embryogenic callus into somatic embryos; the final concentration of TSA in the medium is 0.5-5. Mu.M. As a further technical scheme of the invention, the endosperm removing process comprises the following steps: The explant is soaked in ethanol solution with the volume fraction of 75% for 30-60s, hgCl 2 solution with the mass fraction of 0.1% for 8-12min, and finally washed with sterile distilled water for 5-6 times, so that disinfection is completed. As a further technical scheme, the DCR solid culture medium contains 6.0 mg/L2, 4-D, 0.5 mg/L6-BA, 0.5g/L hydrolyzed casein, 0.45g/L L-glutamine, 0.1g/L inositol, 2g/L activated carbon, 20g/L maltose and 3.5g/L gellan gum, and the pH is regulated to 5.3-5.5. As a further technical scheme of the invention, the pH of the DCR solid culture me