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Method for in-vitro rapid propagation of clivia miniata by using mature embryos as explants

A technology for explants and Clivia, applied in the field of plant reproduction, can solve the problems of low dedifferentiation rate and regeneration rate, limit the application of Clivia tissue culture technology, etc., achieves good growth state, low cost, and overcomes the effects of transgenic silence.

Inactive Publication Date: 2011-10-19
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Referring to the relevant international and domestic literature, we know that although the previous Clivia tissue culture experiments have been initially successful, the dedifferentiation rate and regeneration rate are low, and an average of one explant can regenerate a few tenths to two tenths of seedlings depending on the variety.
This limits the application of Clivia tissue culture technology in actual production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] 1. Explant detoxification, inoculation and callus induction: get the seed embryo in the mature fruit, soak it in 75% (volume / volume) alcohol for 30 seconds in a sterile ultra-clean bench, and use 0.1% (mass / volume) liter of Soak in mercury solution for 2 minutes, rinse with sterile water for 3 times, cut off the dense yellow end of about 2 mm in length on the naked embryo, and inoculate it in a medium containing 2,4-D 2 mg / L, 3% sucrose (mass / volume), 0.45 agar % (mass / volume) MS medium, 20 ℃, 40W fluorescent lamp every day for 8 hours. Subculture once every 40 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.

[0013] 2. Induction of adventitious buds: transfer the yellow callus or green swollen tissue in a good growth state to the induction differentiation medium: MS+KT0.25mg / L+2, 4-D0.75+3% (mass / volume) Sucrose + 0.45% (mass / volume) agar, subculture once every 40 days, and light for 10 hours a day at 20°C...

Embodiment 2

[0017] 1. Explant detoxification, inoculation and callus induction: get the embryo in the mature fruit in a sterile ultra-clean bench, soak it for 60 seconds with 75% (volume / volume) alcohol, and use 0.2% (mass / volume) liter of Soak in mercury solution for 5 minutes, rinse with sterile water 5 times, cut off the dense yellow end of the naked embryo with a length of about 2 mm, and inoculate it on a medium containing 2,4-D 4 mg / L, 4% sucrose (mass / volume), 0.7 agar % (mass / volume) MS medium, irradiated with 40W fluorescent lamp for 9 hours every day at 30°C. Subculture once every 50 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.

[0018] 2. Induction of adventitious buds: the yellow callus with good growth state or the green expanded tissue are transferred to the induction differentiation medium: MS+KT0.5mg / L+2, 4-D1.5mg / L+4% (quality / volume) sucrose+0.7% (mass / volume) agar, subculture once every 50 days, and lig...

Embodiment 3

[0022] 1. Explant detoxification, inoculation and callus induction: get the seed embryo of ripe fruit and soak in 75% (volume / volume) alcohol for 40 seconds in a sterile ultra-clean bench, and use 0.15% (mass / volume) mercuric chloride Soak in the solution for 4 minutes, rinse with sterile water 4 times, cut off the end of the dense yellow about 2 mm in length on the naked embryo, and inoculate it in a medium containing 2,4-D 3 mg / L, 3.5% sucrose (mass / volume), 0.6% agar (mass / volume) MS culture medium, 25 ℃ 40W daylight lamp irradiation 8.5 hours every day. Subculture once every 48 days, and induce differentiation when the callus (yellow) or green swelling grows to a diameter of about 5 mm.

[0023] 2. Induction of adventitious buds: transfer the yellow callus or green swollen tissue in good growth state to the induction differentiation medium: MS+KT (0.4mg / L)+2,4-D (1.0mg / L)+3.5 % (mass / volume) sucrose+0.5% (mass / volume) agar, subculture once every 45 days, and light for 8 h...

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Abstract

The invention belongs to a plant propagation method, and particularly relates to a method for the asexual rapid propagation of flowers. The method utilizes the technique such as detoxification, inoculation and callus induction of explants, induction of adventitious shoots, root induction and transplant of regeneration plants for the asexual rapid propagation of clivia miniata. When the method is used, the selection of raw materials is not limited by seasons, the method is suitable for agrobacterium-mediated transgenes, and the silence of the transgenes is overcome to some extent; the mature embryos from ten plants of mature clivia miniata are used as the explants, and the dedifferentiation rate and the differentiation rate both reach 70 to 100 percent; the regeneration coefficient is high, and an original callus tissues can grow into hundreds of cluster buds by repeated sub-culture; the rooting rate of the cluster buds is 100 percent, and the transplanting survival rate is 100 percent. When the method is used, the clivia miniata in different varieties can mass propagate the plants which have the same genetic background and smooth appearances in a shorter time; and after the plantsare transplanted, the growing state is good, the cost for the average individual-plant regeneration seedling is low, and the method is suitable for industrialized production.

Description

Technical field [0001] The invention belongs to a plant propagation method, and specifically relates to a vegetative and rapid propagation method of flowers. Background technique [0002] Plant tissue culture is a sterile plant culture technology. It is based on the totipotency of plant cells and uses isolated plant organs (such as roots, stems, leaves, stem tips, flowers, fruits, etc.) and tissues (such as cambium, epidermis, cortex, endosperm, etc.) or cells (such as megaspores, microspores, somatic cells, etc.) and protoplasts, under sterile and suitable artificial culture media and artificial conditions such as light and temperature, callus, adventitious buds, Techniques of adventitious roots that form complete plants or produce other products of economic value. Regenerated plants produced by plant tissue culture can basically maintain the excellent traits of the parents (explant-derived plants) except for extremely low somatic clonal variation. Plant tissue culture te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 王丽王钦美邹凡雨
Owner NORTHEAST NORMAL UNIVERSITY
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