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Methods of Proliferation and Maintenance of Embryogenic Callus of Pinus massoniana

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

Active Publication Date: 2022-03-01
GUANGXI FORESTRY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Methods of Proliferation and Maintenance of Embryogenic Callus of Pinus massoniana
  • Methods of Proliferation and Maintenance of Embryogenic Callus of Pinus massoniana

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 method for the proliferation and maintenance of embryogenic callus of pine massoniana. The embryogenic callus obtained by separating the embryogenic cell mass in a silky shape obtained after conventional somatic embryo induction for 3 to 4 weeks from the female gametophyte Put it in a culture dish with medium Ⅰ, remove the dead cells and excess tissue in the embryogenic callus after 4 to 6 weeks, transfer it to a centrifuge tube, add medium Ⅱ, mix and shake well, and then shake well to obtain Place the embryogenic culture of the culture medium III in a petri dish. After 6-8 weeks, take an appropriate amount of embryogenic callus and directly transfer it to medium IV for normal subculture. The subculture period is 10-14 days, and finally An embryogenic cell line of Pinus massoniana that grows rapidly, is vigorous and can be subcultured for a long time was obtained. The embryogenic callus of the present invention has no obvious browning and water-soaking phenomenon in long-term subculture, the embryogenic callus grows fast, the multiplication effect is stable and high, and the multiplication coefficient reaches more than 25, which has good economic and social benefits .

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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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 姚瑞玲王胤
Owner GUANGXI FORESTRY RES INST
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