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Pinus massoniana embryogenic callus proliferation and maintenance culture method

An embryogenic callus, proliferation culture technology, applied in the field of forest tree reproduction, can solve the problems of difficulty in embryogenic callus induction, hindering somatic embryo seedling raising technology, and decreasing callus vigor, etc. Operational and instructive, proliferative effect

Active Publication Date: 2019-10-08
GUANGXI FORESTRY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there have been related studies on somatic embryogenesis of Pinus massoniana, but due to the difficulty in the induction of embryogenic callus, and when the subculture time exceeds half a year, the embryogenic callus in subculture will appear obvious browning and water staining , the callus viability decreased significantly, hindering the development of somatic embryonic seedling technology

Method used

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  • Pinus massoniana embryogenic callus proliferation and maintenance culture method
  • Pinus massoniana embryogenic callus proliferation and maintenance culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The immature coniferous female gametophytes of Pinus massoniana zygotic embryos in the cleaved polyembryonic stage were used as explants. After conventional somatic embryo induction for 3 to 4 weeks, when the diameter of the embryogenic callus was ≥ 1 cm, the colorless, The transparent, water-free silken embryogenic cell mass and the female gametophyte were separated by conventional methods and used as embryogenic callus for proliferation and maintenance culture.

[0031] The isolated embryogenic callus was placed in a 90 mm diameter petri dish filled with medium I, and cultured in the dark for 4 weeks at a culture temperature of 20±0.5°C. The medium I mentioned is: modified DCR medium + 2,4-D 0.8 mg·L -1 + NAA2.0mg·L -1 + Meta-Topolin (MT) 1.0 mg·L -1 + sodium thiosulfate 135 mg·L -1 + Glutathione 100mg·L -1 + Inositol 100 mg·L -1 + Glutamine 500 mg·L -1 + Acid Hydrolyzed Casein 1000 mg·L -1 + sucrose 20000 mg·L -1 + Lactose 10000 mg·L -1 + Agar 4500 mg·L -1...

Embodiment 2

[0036] The immature coniferous female gametophytes of Pinus massoniana zygotic embryos in the cleaved polyembryonic stage were used as explants. After conventional somatic embryo induction for 3 to 4 weeks, when the diameter of the embryogenic callus was ≥ 1 cm, the colorless, The transparent, water-free silken embryogenic cell mass and the female gametophyte were separated by conventional methods and used as embryogenic callus for proliferation and maintenance culture.

[0037] The isolated embryogenic callus was placed in a 90 mm diameter petri dish filled with medium I, and cultured in the dark for 6 weeks at a culture temperature of 20±0.5°C. The medium I mentioned is: modified DCR medium + 2,4-D 0.8 mg·L -1 + NAA2.0mg·L -1 + Meta-Topolin (MT) 2.0 mg·L -1 + sodium thiosulfate 135 mg·L -1 + Glutathione 100mg·L -1 + Inositol 100 mg·L -1 + Glutamine 500 mg·L -1 + Acid Hydrolyzed Casein 1000 mg·L -1 + sucrose 20000 mg·L -1 + Lactose 10000 mg·L -1 + Agar 4500 mg·L -1...

Embodiment 3

[0042] The immature coniferous female gametophytes of Pinus massoniana zygotic embryos in the cleaved polyembryonic stage were used as explants. After conventional somatic embryo induction for 3 to 4 weeks, when the diameter of the embryogenic callus was ≥ 1 cm, the colorless, The transparent, water-free silken embryogenic cell mass and the female gametophyte were separated by conventional methods and used as embryogenic callus for proliferation and maintenance culture.

[0043] The isolated embryogenic callus was placed in a 90 mm diameter petri dish filled with medium I, and cultured in the dark for 5 weeks at a culture temperature of 20±0.5°C. The medium I mentioned is: modified DCR medium + 2,4-D 0.8 mg·L -1 + NAA4.0mg·L -1 + Meta-Topolin (MT) 2.0 mg·L -1 + sodium thiosulfate 135 mg·L -1 + Glutathione 100mg·L -1 + Inositol 100 mg·L -1 + Glutamine 500 mg·L -1 + Acid Hydrolyzed Casein 1000 mg·L -1 + sucrose 20000 mg·L -1 + Lactose 10000 mg·L -1 + Agar 4500 mg·L -1...

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Abstract

The invention discloses a pinus massoniana embryogenic callus proliferation and maintenance culture method. The method includes: placing a filamentous embryogenic callus cell cluster obtained by induction of 3-4 weeks of a conventional somatic embryo and an embryogenic callus obtained by separation of a female gamete into a petri dish with a culture medium I; removing dead cells and unnecessary tissues in embryogenic calli in 4-6 weeks; transferring into a centrifugal tube, adding a culture medium II, mixing and well shaking; adding an embryogenic culture obtained after well shaking into a petri dish with a culture medium III; after 6-8 weeks, directly transferring an appropriate size of the embryogenic calli into a culture medium IV for normal subculture in a subculture period of 10-14d to finally obtain a pinus massoniana embryogenic cell line which is fast in growth, vigorous and available for long-term subculture. Evident browning and water soaking of the embryogenic calli in long-term subculture are avoided, the embryogenic calli grow fast, proliferation effects are stably high, the proliferation coefficient reaches 25 or above, and high economic benefits and social benefits are achieved.

Description

technical field [0001] The invention belongs to the technical field of tree propagation, and relates to a tissue culture embryogenic callus subculture proliferation culture technology, in particular to a method for the proliferation and maintenance of masson pine embryogenic callus. Background technique [0002] Masson pine ( Pinus massoniana Lamb.) is the main tree species used for ecological construction and afforestation in southern my country. As a tree species with high comprehensive utilization value and broad promotion prospects, masson pine can not only be used to produce three-board, papermaking and chemical fiber industrial manufacturing, but also can be used to produce industrial raw materials such as rosin and turpentine. The long breeding cycle of Pinus massoniana, coupled with the aging of the mother tree in the seed orchard in recent years, the decline in seed setting, and the lack of improved varieties have led to large differences in genetic differentiatio...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 姚瑞玲王胤
Owner GUANGXI FORESTRY RES INST
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