Culture method for promoting development and maturation of Chinese pine somatic embryo

An embryo development and culture method technology, applied in horticultural methods, botanical equipment and methods, horticulture and other directions, can solve the problem of restricting the development of somatic embryo technology of Pinus tabulaeformis, affecting the efficiency of somatic embryo maturation, and the limited number of embryogenic cell lines of Pinus tabulaeformis And other issues

Active Publication Date: 2020-03-24
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing research on somatic embryos of Pinus tabulaeformis mainly has the following technical defects: (1) the number of embryogenic cell lines of Pinus tabulaeformis is limited, and there are few genotypes that can produce a large number of somatic embryos; Embryo Maturation Efficiency
These problems limit the development of somatic embryo technology of Pinus tabulaeformis and its application in large-scale multiplication

Method used

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  • Culture method for promoting development and maturation of Chinese pine somatic embryo
  • Culture method for promoting development and maturation of Chinese pine somatic embryo
  • Culture method for promoting development and maturation of Chinese pine somatic embryo

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Experimental program
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Effect test

Embodiment 1

[0079] 1. Test materials

[0080] 1. Immature cones of Chinese pine

[0081] In 2017, the present invention took Shanxi Luliang State-owned Forestry Administration National Chinese Pine Fine Variety Base as the source of materials, selected Chinese pine with healthy growth, no pests and diseases, high cone seed setting rate, free pollination, and 100 cones 51 days after the loose powder was collected. , Store the collected cones in a refrigerated environment at 4°C for later use.

[0082] 2. Plant growth regulator

[0083] The plant growth regulating substance used in the present invention adopts domestic 6-benzylaminoadenine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole butyric acid (IBA), Naphthaleneacetic acid (NAA), abscisic acid (ABA), MGBG (methylglyoxalbis-guanylhydrazone; mitoguanidine hydrazone; methylglyoxalbis-guanylhydrazone).

[0084] 3. Medium

[0085] (1) mLV medium

[0086] Table 1 mLV medium formula

[0087]

[0088] The mLV minimal medium (K...

Embodiment 2

[0096] 1. Sterilization of explants

[0097] The explant sterilization method is the same as the method disclosed in Patent No. ZL201410573714.1.

[0098] The pine cones stored at 4°C on the 51st day after the loose powder were first washed with detergent to remove the oil on the surface of the cones, then washed with tap water, then cut open the cones, and took out the seeds; then put them on the ultra-clean workbench Be that 75% alcohol is soaked pine cone seed 30-60s with volume percentage concentration, then scoop up seed and rinse several times (3-5 times) with sterile water, then put into the HgCl that mass fraction is 0.1% 2 Soak in the solution for 5-10min, take out the seeds and rinse them with sterile water several times (5-6 times), put the rinsed seeds on the filter paper sterilized by high temperature and high pressure to absorb water, and open the seeds with tweezers and scissors. Skin, to obtain the female gametophyte (including immature zygote embryos), set as...

Embodiment 3

[0111] The pre-treatment culture of embodiment 3 embryogenic tissue

[0112] Inoculate 2g (usually 2-5g) of translucent, granule or filamentous proliferation-cultured embryogenic tissue after liquid proliferation culture into a 100mL Erlenmeyer flask, and add 30-40mL of Embryogenic tissue pretreatment medium, under dark conditions, suspension culture in Erlenmeyer flask, that is, the pretreatment culture of Pinus tabulaeformis embryogenic tissue, wherein the culture temperature is (25±2) ℃, embryogenic tissue pretreatment medium The amount of MGBG used in the test is shown in Table 2, ABA 30μM, hydrolyzed casein 500mg / L, glutamine 500mg / L, sucrose 10g / L, maltose 20g / L, put the Erlenmeyer flask for 100r min –1 , dark treatment on a constant temperature shaker at 25°C for 1 week, each experiment was repeated 3 times, and a blank control was set, and the growth status and morphology of embryogenic tissues were observed and recorded.

[0113] Proliferation rate (%) = fresh weight...

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Abstract

The invention discloses a method for promoting development and maturation of a Chinese pine somatic embryo (somatic embryo). The method comprises the step of carrying out liquid pretreatment culture on a Chinese pine embryonic tissue, wherein a polyamine synthesis inhibitor regulator is contained in an embryonic tissue pretreatment culture medium. According to the method, the polyamine synthesis inhibitor regulator is added into the liquid pretreatment culture medium and an embryo maturation culture medium, so that purposes of inhibiting excessive proliferation of the embryonic tissue in the embryo maturation process, promoting development and maturation of the somatic embryo and improving the quality and the quantity of the mature somatic embryo are achieved. By using the method, more mature somatic embryos and somatic embryo seedlings can be obtained from a genotype material with weak Chinese pine embryonic nature and excessive proliferation, and diversification of Chinese pine somatic embryo seedling genotypes is facilitated, so that the limitation of clone forestry genotypes can be overcome, and multi-genotype Chinese pine somatic embryo seedlings can be produced in a large-scale and industrialized manner.

Description

technical field [0001] The invention relates to a plant tissue culture method, in particular to a culture method for promoting the development and maturation of somatic cell embryos of Pinus tabulaeformis, and belongs to the technical field of cell engineering seedling breeding in the forestry industry. Background technique [0002] Chinese pine (Pinustabulaeformis C.), also known as red bark pine, short-leaved pine, etc., is a coniferous evergreen tree belonging to the genus Pinus. Often chapped and scaly, the needles are usually a bundle of 2 needles, with fine serrations on the edge, 9-14cm in length, light green when young, then dark green later, hard texture, thicker branchlets, yellowish brown, with trace The resin, the branches usually spread downward or flat, the crown of the adult tree is relatively flat, the leaf sheath is light brown at first, and gradually turns dark brown, the cones of Pinus tabulaeformis are oval or ovoid, 3-8cm long, and short Stems and male ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/005A01H4/008
Inventor 孔立生张金凤赵健李亦轩崔莹范英明
Owner BEIJING FORESTRY UNIVERSITY
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