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Pinus thunbergii cell embryogenesis and plant regeneration method

An embryogenesis and somatic cell technology, applied in plant regeneration, botanical equipment and methods, horticultural methods, etc., can solve problems such as shortage of afforestation seeds and seedlings, and achieve good economic and social effects, good practicability, and high reproduction rate. Effect

Inactive Publication Date: 2012-07-18
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for somatic embryogenesis and plant regeneration of black pine, to provide an efficient way for large-scale vegetative propagation of black pine, and to solve the problem of current afforestation seedlings. shortage problem

Method used

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  • Pinus thunbergii cell embryogenesis and plant regeneration method
  • Pinus thunbergii cell embryogenesis and plant regeneration method
  • Pinus thunbergii cell embryogenesis and plant regeneration method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Materials: The immature zygotic embryos of black pine, the immature cones of black pine were collected once a week in Yantai, Shandong Province from the beginning of July to the end of August, and stored in a refrigerator at 4°C for later use.

[0034] Formula of DCR medium: 400mg / L NH 4 NO 3 , 340mg / L KNO 3 , 170mg / L KH 2 PO 4 , 556mg / L Ca(NO 3 ) 2 4H 2 O, 85mg / L CaCl 2 2H 2 O, 370mg / L MgSO 4 ·7H 2 O, 0.83mg / LKI, 22.3mg / L MnSO 4 ·H 2 O, 8.6mg / L ZnSO 4 ·7H 2 O, 0.25mg / L Na 2 MoO 4 2H 2 O, 0.25mg / LCuSO 4 ·5H 2 O, 6.2mg / LH3 BO 3 , 27.8mg / LFeSO 4 ·7H 2 O, 37.3mg / LNa 2 -EDTA·H 2 O, 0.5mg / L niacin, 0.5mg / L pyridoxine hydrochloride, 1.0mg / L thiamine hydrochloride, 2.0mg / L glycine.

[0035] Formula of WPM medium: 400mg / L NH 4 NO 3 , 990mg / L K 2 SO 4 , 170mg / L KH 2 PO 4 , 556mg / L Ca(NO 3 ) 2 4H 2 O, 96mg / L CaCl 2 2H 2 O, 370mg / L MgSO 4 ·7H 2 O, 22.3mg / LMnSO 4 ·H 2 O, 8.6mg / L ZnSO 4 ·7H 2 O, 0.25mg / L Na 2 MoO 4 2H 2 O, 0.25mg / LCuSO 4 ...

Embodiment 2

[0044] The method of somatic embryogenesis and plant regeneration of Pinus nigra is the same as in Example 1.

[0045] Among them, in the induction stage of embryogenic callus, the concentration of 2,4-D in the medium was tested with three gradients of 1mg / L, 2mg / L and 4mg / L, and the concentration of 6-BA was tested with 0.5mg / L, 1mg / L / L and 2mg / L 3 gradients were tested. Among the 9 treatments in this test, from the results of induction, only 4mg / L 2,4-D+2mg / L 6-BA was induced on the DCR medium. There were 2 pieces of embryogenic callus, and the induction rate of embryogenic callus was 3.33%. Therefore, the best formula for inducing embryogenic callus of black pine at this stage is: DCR medium + 4mg / L2, 4-D + 2mg / L6-BA + 30g / L maltose.

[0046] Among them, in the mature stage of somatic embryos, when the mature culture of somatic embryos is carried out, the concentration of ABA in the medium is tested with 3 gradients of 10mg / L, 25mg / L and 30mg / L, and PEG6000 is tested with...

Embodiment 3

[0048] The method for somatic embryogenesis and plant regeneration of black pine of the present invention has high reproduction rate, and the embryogenic callus is subcultured once every 2 to 3 weeks in the proliferation stage, and the embryogenic callus proliferates very quickly, and each piece of embryogenic callus proliferates once After that, it will become 2 to 3 pieces (1 piece of embryogenic callus is about 1g), take 1 piece of pretreated embryogenic callus and put it in 20mL of hormone-free DCR suspension, and use a pipette to draw 2mL of suspension to disperse On the sterile filter paper with suction paper, after the water is blotted dry, the filter paper is placed on the mature medium, and 2 to 3 pieces of embryogenic callus are formed on the mature medium of each petri dish. The number of somatic embryos formed by the callus reached 72, so that the number of mature somatic embryos per culture dish reached 144-216. Therefore, one piece of embryogenic callus that was ...

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Abstract

The invention discloses a pinus thunbergii cell embryogenesis and plant regeneration method. Embryogenic calluses are induced from immature zygotic embryos; the embryogenic calluses are maintained, proliferated and matured to obtain somatic embryos; the somatic embryos are germinated to obtain regenerated plants; and finally the regenerated plants are transplanted. Due to the adoption of the pinus thunbergii cell embryogenesis and plant regeneration method, high reproduction rate of the pinus thunbergii plant is achived. After the embryogenic calluses are cultured for 70 days in a maturing culturing medium, 144-216 mature somatic embryos can be obtained in each culture utensil on average; the somatic embryo germination rate is 63.8%; and the plant transformation rate is 43.5%. Therefore, a large number of somatic embryo seedlings can be obtained every year, thus a foundation is laid for the pinus thunbergii cell embryogenesis and the regenerated plant bath production, and the current problem of afforestation seedling shortage is solved. The pinus thunbergii cell embryogenesis and plant regeneration method has very good practicality.

Description

technical field [0001] The invention belongs to the technical field of tree tissue regeneration, and in particular relates to a method for somatic embryogenesis and plant regeneration of black pine. Background technique [0002] Black pine (Pinus thunbergii) belongs to the gymnosperm phylum, coniferae, coniferaceae, pinaceae, pinus, evergreen tree, up to 30m high. Light-loving, resistant to drought and barrenness, not resistant to waterlogging, not cold-resistant, suitable for growing in warm and humid marine climate areas, most suitable for growth in sandy soil with deep, loose soil and humus. Because of its resistance to sea fog and sea wind, it can also grow in saline soil on the beach, with fast growth and long life. Excellent material for construction and furniture. Beaches and river beaches can be used as afforestation tree species, as well as landscaping tree species, and can also be used as bonsai tree species. Black pine is native to Japan and the eastern coastal...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCY02A40/22Y02P60/40
Inventor 叶建仁李清清朱丽华吴小芹
Owner NANJING FORESTRY UNIV
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