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Somatic embryogenesis method for cunninghamia lanceolata

A technology of embryogenesis and somatic cells, applied in horticultural methods, botany equipment and methods, plant regeneration, etc., can solve the problems of loose EMS structure, limited proliferation efficiency, browning, etc., to solve the problem of low frequency of somatic embryogenesis, improve Browning phenomenon, cost saving effect

Active Publication Date: 2014-06-25
NANJING FORESTRY UNIV
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Problems solved by technology

[0003] At present, Chen Jinhui et al. (ZL 201010275858.0) have established a successful method of indirect somatic embryogenesis by using immature zygotic embryos of Chinese fir tree to induce ESM, proliferating ESM through liquid, and inducing somatic embryogenesis to achieve plant regeneration. Affected by natural attributes, Chinese fir is rich in secondary metabolites, and its EMS structure is relatively loose. After being subjected to liquid shear force, the embryo stalk structure is easily damaged, and the browning is serious when the EMS is multiplied by liquid, so the multiplication efficiency is very limited. , when inducing somatic embryogenesis, the formula is not perfect enough, it is greatly affected by the state of the material, the induction frequency is unstable, and there is a big gap from industrial production

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  • Somatic embryogenesis method for cunninghamia lanceolata
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  • Somatic embryogenesis method for cunninghamia lanceolata

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Induction of embryogenic stalk (EMS).

[0039] The test materials were the immature cones of the excellent Chinese fir trees collected from the seed orchards of the Chinese fir clones grafted in Shunchang and Weimin, Fujian, respectively, every week from June to July. The seed development stages corresponding to each seed collection time point were the developmental stages from immature embryo to mature embryo. After the cones are collected, put them in a pre-frozen ice box on site and wrap them with moist gauze to prevent water loss; then quickly put them in a 4°C refrigerator for storage for later use. Before inoculation, take the spare cones out of the refrigerator, use a scalpel and tweezers to take out the well-developed immature seeds with seed wings in the cones, and use Diaopai detergent rich in high-efficiency compound active agents to make a concentration of 1:50 Soak in the lotion for 10 minutes, then rinse with running water for 30 minutes. In a ...

Embodiment 2

[0041] Example 2 Maintenance and proliferation culture of EMS.

[0042] The maintenance and proliferation medium for EMS has the same composition as the basic medium for inducing EMS, but the concentration of auxin is reduced. The maintenance and proliferation culture of Chinese fir ESM uses DCR as the basic medium, supplemented with 2,4-D 2~6 mg / L, 6-BA 0.5~1.0mg / L, KT 0.5mg / L, Vc 10 mg / L, hydrolyzed casein Protein 0.5 g / L, maltose 15 g / L, Ac 2.5 g / L, crystal agaric 2.3 g / L solid medium, add PSK (0.01~1 mg / L), subculture cycle 25 days, 23 ℃, dark to cultivate.

[0043] It can be observed that on the proliferation medium added with PSK, the EMS stalk cells develop better and the materials disperse better.

Embodiment 3

[0044] Example 3 Development and maturation of somatic embryos.

[0045] (1) Indirect somatic embryogenesis

[0046] A. Liquid suspension culture

[0047] The liquid proliferation medium is the same as DCR as the basic medium, supplemented with 2,4-D 0.5~2 mg / L, 6-BA 0.2mg / L, glutamine 0.45 g / L, hydrolyzed casein 0.5 g / L, maltose 30g / L. The initial density of the cultured cells was 1.20% (V / V), the rotation speed of the shaker was 85~90r.p.m, and the culture was carried out at 23°C in the dark, and the time of proliferation culture was controlled to be about 7 days. And add different concentrations of PSK (0.01~1mg / L) in the liquid proliferation medium to carry out the culture test.

[0048] Liquid pretreatment medium (DCR, 5-10mg / L ABA, 0.1-1.0mg / L GA, 10mg / L vitamin C, 0.5g / L hydrolyzed casein, 30g / L maltose) to remove all additional hormones, add ABA (Abscisic acid) 5~10mg / L, stop the ESM to continue to split, and turn to the mature development process of somatic embryo...

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Abstract

The invention discloses a somatic embryogenesis method for cunninghamia lanceolata, which comprises the steps of initial induction of an embryogenic suspensor cell mass, maintenance and multiplication of an ESM (embryogenic suspensor mass), and development and maturity of a somatic embryo, wherein the development and maturity of the somatic embryo can adopt an indirect somatic embryogenesis way or a direct somatic embryogenesis way. With the adoption of the somatic embryogenesis method for the cunninghamia lanceolata, browning in an establishment process of the conventional liquid suspension system can be effectively improved, cell division under a low density condition is started, and the stability of suspensor cells is maintained, so that a stable multiplication liquid suspension cell line is established. The somatic embryogenesis frequency of the cunninghamia lanceolata can be increased by about 20 times at most through the direct and indirect somatic embryogenesis ways, the problem of low somatic embryogenesis frequency caused by an incomplete somatic embryogenesis system of the cunninghamia lanceolata can be well solved, the cost is saved, conditions are prepared for factory production of the cunninghamia lanceolata, and a pace of industrial production of the cunninghamia lanceolata is accelerated.

Description

technical field [0001] The invention belongs to the technical field of forestry biology, and specifically relates to a method for somatic embryogenesis of Chinese fir, which optimizes the somatic embryogenesis system of Chinese fir and increases the frequency of somatic embryogenesis of Chinese fir. Background technique [0002] Chinese fir( Cunninghamia lanceolata (Lamb.) Hook) belongs to the genus Cunninghamia of Taxodiaceae, and is one of the endemic evergreen conifer species in my country, widely distributed in the mountainous areas of southern China. Chinese fir grows fast, has a straight and round stem shape, is corrosion-resistant, and has a very beautiful wood texture. It is an important commercial timber and afforestation tree species. The history of Chinese fir cultivation can be traced back to more than 3,000 years ago. After the systematic research of the "Sixth Five-Year" to "Ninth Five-Year" Chinese fir research plan, a large number of Chinese fir good proven...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 施季森周小红陈金慧赵芳芳
Owner NANJING FORESTRY UNIV
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