CN-120323326-B - Method for promoting somatic embryogenesis and maturation of cryptomeria fortunei

CN120323326BCN 120323326 BCN120323326 BCN 120323326BCN-120323326-B

Abstract

The application discloses a culture method for promoting somatic embryogenesis and maturation of cryptomeria fortunei, belonging to the technical field of somatic embryogenesis. The application discloses a method for somatic embryogenesis and maturation of cryptomeria fortunei, which comprises the steps of taking immature seed embryo as an explant, inducing to obtain embryogenic callus, transferring the embryogenic callus after multiplication culture into a somatic embryo induction culture medium for culture to obtain somatic embryo, transferring the somatic embryo into a somatic embryo maturation culture medium for dark culture to obtain mature cotyledon embryo, transferring the mature cotyledon embryo into a somatic embryo germination basic culture medium for illumination culture to obtain somatic embryo seedlings, and after hardening and transplanting the somatic embryo seedlings, obtaining regenerated plants. The result of the application shows that embryogenic callus can be stably obtained from different families, the somatic embryo maturation capability and maturation rate are high, a large number of high-quality seedlings with stable genetic characters can be cultivated in a short time, the character separation problem caused by gene recombination in the seed propagation process is avoided, and the offspring plants can inherit the excellent characteristics of parents.

Inventors

XU JIN
Jin Mengshuang
LUO YI

Assignees

南京林业大学

Dates

Publication Date: 20260505
Application Date: 20250523

Claims (3)

1. A method of promoting embryogenesis and maturation of a cedar body comprising: 1) Peeling immature seed embryo from cone of good family of Cryptomeria fortunei as explant, wherein the good family of Cryptomeria fortunei is selected from 8#, 32# and 3#; 2) Inoculating the explant to an induction solid culture medium under the aseptic condition, and inducing to obtain embryogenic callus, wherein the formula of the induction solid culture medium is DCR basic culture medium, and 2, 4-D2.0 mg/L, 6-BA1.0mg/L, KT 0.50.50 mg/L, L-glutamine 1.0mg/L, acid hydrolyzed casein 0.5mg/L, sucrose 30.0g/L, active carbon 2.0g/L and crystal watercress 2.4g/L are added; 3) Transferring embryogenic callus to a proliferation culture solid culture medium for proliferation culture, wherein the formula of the proliferation culture solid culture medium is DCR basic culture medium, 2, 4-D1.0 mg/L, 6-BA 0.5mg/L, L-glutamine 1.5g/L, sucrose 25.0-30.0g/L, activated carbon 2.0g/L and crystal seaweed 2.4g/L are added, and 15 days later are taken as one round; 4) Transferring the embryogenic callus after multiplication culture into a somatic embryo induction culture medium for culture to obtain somatic embryos, wherein when the excellent family of the Japanese cedar is 8# of the excellent family of the Japanese cedar, the formula of the somatic embryo induction culture medium is 1/2DCR basic culture medium, 10mg/L, PEG8000 170g/L of ABA, 40.0g/L, L-glutamine 0.5g/L, 0.5g/L of acid hydrolyzed casein, 0.5g/L, L-aspartic acid 0.2g/L, 3.0g/L of crystal seaweed and 2.0g/L of activated carbon are added, and when the excellent family of the Japanese cedar is 32# or 3# of the excellent family of the Japanese cedar, the formula of the somatic embryo induction culture medium is 1/2DCR basic culture medium, 10.0mg/L, PEG8000 150g/L of ABA, 30.0g/L, L-glutamine 0.5g/L, 0.5g/L of acid hydrolyzed casein, 0.5g/L of inositol, 0.5g/L, L-2 g/L of crystal seaweed and 2g/L of activated carbon are added; 5) Transferring somatic embryo into somatic embryo maturation culture medium for dark culture to obtain mature cotyledon embryo, wherein the formula of the somatic embryo maturation culture medium is DCR basic culture medium, and GA 3 3.0.0 mg/L, L-glutamine 0.5g/L, active carbon 2.0g/L, sucrose 30.0g/L, and herba Solani Nigri 2.4g/L are added 6) Transferring the mature cotyledon embryo to a somatic embryo germination basic culture medium for illumination culture to obtain a somatic embryo, hardening off and transplanting the somatic embryo to obtain a regenerated plant.
2. The method of claim 1, wherein the somatic embryo germination minimal medium is formulated as MS minimal medium, 0.25g/L glutamine, 0.1g/L acid hydrolyzed casein, 30g/L sucrose, 2.4g/L crystal psyllium.
3. The method according to claim 1, characterized in that the specific steps comprise: 1) Peeling immature seed embryo from cone of excellent family 8#, 32# or 3# of Cryptomeria fortunei as explant; 2) Inoculating the explant to an induction solid culture medium under aseptic condition, culturing at 23 ℃ in a dark way for 20-25 days, and inducing to obtain embryogenic callus; 3) Transferring the embryogenic callus to a proliferation culture solid medium, and carrying out proliferation culture at 23 ℃ in a dark culture way for 15 days; 4) Transferring the embryogenic callus after proliferation culture into a somatic embryo induction culture medium for dark culture at 23 ℃ for 5-6 weeks to obtain somatic embryos; 5) Transferring the somatic embryo into a somatic embryo maturation medium for dark culture at 23 ℃ for 15 days to obtain mature cotyledon embryo; 6) Transferring the mature cotyledon embryo to a somatic embryo germination basic culture medium for 16 hours, lighting for 8 hours and culturing at 23 ℃ to obtain somatic embryo seedlings, hardening and transplanting the somatic embryo seedlings to obtain regenerated plants.

Description

Method for promoting somatic embryogenesis and maturation of cryptomeria fortunei Technical Field The invention belongs to the technical field of somatic embryogenesis, and particularly relates to a method for promoting somatic embryogenesis and maturation of cryptomeria fortunei. Background In the field of forestry biotechnology, somatic Embryogenesis (SE) technology is of paramount importance. It can make plant somatic cell grow into complete plant in the development mode similar to that of zygotic embryo without cell fusion under specific condition. The technology is widely applied to plant, especially forest research, including basic biological research, improved variety in vitro rapid propagation, germplasm resource protection and utilization, excellent variety breeding popularization, construction of transgenic and gene editing breeding technology and the like. Many conifers have successfully built stereo embryogenic systems, but different tree species, even different genotypes of the same tree species, differ greatly in embryogenic capacity, and this genotype dependence severely limits the application of the technology in forest genetic improvement, germplasm innovation and elite breeding. The cedar (Cryptomeria fortunei Hooibrenk ex Otto et Dietr) belongs to the genus cedar of the family Cunninghamiae, and is an important species of the rural tree in China. The wood material is light and soft, has straight texture, fine structure and strong corrosion resistance, is easy to process, and is a high-quality material for industrial buildings. The young cedar is slightly shade-tolerant and can grow rapidly in mountainous areas with warm and moist, acid and rich soil and good drainage. However, current cedar propagation mainly depends on traditional methods such as seed propagation and cutting propagation. The seed propagation is easily influenced by seed quality and environmental factors, so that offspring have high genetic diversity and are difficult to maintain good characters, and the cutting propagation has the problems of low propagation coefficient, difficult rooting and the like. The establishment of a high-efficiency cedar embryogenesis system has great significance for genetic improvement and improved variety breeding of the cedar. By the system, the large-scale propagation of excellent clone can be realized, and a large number of high-quality seedlings with consistent genetic quality can be provided for forestry production. Meanwhile, the somatic embryogenesis system is an ideal platform for developing molecular biological researches such as gene transformation, functional gene verification and the like, and is helpful for deeply understanding the growth and development and stress resistance mechanism of the cedar. However, the somatic embryogenesis system of the cedar is not perfect so far, and the somatic embryogenesis efficiency is low, the maturation rate is low, and the proportion of malformed embryos is high, so that the somatic embryogenesis system becomes a key difficult problem for limiting the application of the somatic embryogenesis system. Thus, there is a need to explore effective methods for promoting embryogenic maturation of cryptomeria fortunei. Disclosure of Invention Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide a method for promoting the embryogenesis and maturation of the cedar body, which is used for promoting the breeding of the improved variety of the cedar. In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method of promoting embryogenesis and maturation of a cedar body comprising: 1) Peeling immature seed embryo from cone of excellent family of Cryptomeria fortunei as explant; 2) Inoculating the explant to an induction solid culture medium under the aseptic condition, and inducing to obtain embryogenic callus; 3) Transferring the embryogenic callus to a proliferation culture solid medium for proliferation culture; 4) Transferring the embryogenic callus after proliferation culture into a somatic embryo induction culture medium for culture to obtain somatic embryos; 5) Transferring the somatic embryo into a somatic embryo maturation medium for dark culture to obtain mature cotyledon embryo; 6) Transferring the mature cotyledon embryo to a somatic embryo germination basic culture medium for illumination culture to obtain a somatic embryo, hardening off and transplanting the somatic embryo to obtain a regenerated plant. The good family of the cedar is selected from 8#, 32# and 3# good families of the cedar. The formula of the induction solid culture medium is DCR basic culture medium, and 2, 4-D2.0 mg/L, 6-BA 1.0mg/L, KT 0.50.50 mg/L, L-glutamine 1.0mg/L, acid hydrolyzed casein 0.5mg/L, sucrose 30.0g/L, active carbon 2.0g/L and crystal watercress 2.4g/L are added. The solid culture medium for proliferation culture has a formula of DCR basic culture medium, and