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High-efficiency regeneration method using cunninghamia lanceolata cotyledon as explant

A high-efficiency regeneration and explant technology, applied in the field of plant breeding and reproduction, can solve the problems of insufficient genetic transformation and large-scale reproduction, browning to reduce the regeneration frequency, and no transgenic plants are obtained, and achieve a good regeneration system, convenient material acquisition and cost. low effect

Active Publication Date: 2019-05-17
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, some domestic scholars have used the embryo, cotyledon and hypocotyl of Chinese fir as explants to obtain regenerated plants through direct organogenesis, but the regeneration frequency is low, which is not enough to be used as the basis for genetic transformation and large-scale reproduction. platform
However, the in vitro regeneration research on Chinese fir with stems as explants obtained regenerated plants, and the regeneration frequency also met the basic requirements of transgenic operation technology, but no transgenic plants were obtained. The main reason is that browning reduces the regeneration frequency and fixed Buds (axillary buds) causing false positives

Method used

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  • High-efficiency regeneration method using cunninghamia lanceolata cotyledon as explant
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  • High-efficiency regeneration method using cunninghamia lanceolata cotyledon as explant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A kind of high-efficiency regeneration method taking Chinese fir cotyledons as explants, it comprises the steps:

[0026] 1) Sowing: Select mature and plump seeds, soak them in tap water for 24 hours, and then sow them in a mixture of nutrient soil, vermiculite and perlite at a ratio of 2:2:1 (v / v) at room temperature (25±2°C). in the matrix;

[0027] 2) Disinfection: 20 days after the seeds germinate, select healthy seedlings and clean them with tap water. After removing the roots, soak them in 75vol% alcohol for 30 seconds, 0.1vol% mercuric chloride solution for 5 minutes, and rinse them with sterile water for 5 times;

[0028] 3) Induction culture: the cotyledons of the sterilized seedlings were taken as explants, and inoculated on the induction medium, under the light rate of 80 μmol·m -2 ·s -1 , light time 16 h / d, and culture temperature 25±2°C for 30 days to obtain adventitious buds; the formulation of the induction medium is: DCR+NAA 0.2 mg / L+TDZ 0.005 mg / L+6- ...

Embodiment 2

[0034] A kind of high-efficiency regeneration method taking Chinese fir cotyledons as explants, it comprises the steps:

[0035] 1) Sowing: Select mature and plump seeds, soak them in tap water for 24 hours, and then sow them in a mixture of nutrient soil, vermiculite and perlite at a ratio of 2:2:1 (v / v) at room temperature (25±2°C). in the matrix;

[0036] 2) Disinfection: 20 days after the seeds germinate, select healthy seedlings and clean them with tap water. After removing the roots, soak them in 75vol% alcohol for 45 seconds, soak them in 0.1vol% mercuric chloride solution for 6 minutes, and rinse them with sterile water for 5 times;

[0037] 3) Induction culture: the cotyledons of the sterilized seedlings were taken as explants, and inoculated on the induction medium, under the light rate of 80 μmol·m -2 ·s -1 , light time 16 h / d, and culture temperature 25±2°C for 30 days to obtain adventitious buds; the formulation of the induction medium is: DCR+NAA 0.2 mg / L+TDZ 0...

Embodiment 3

[0043] A kind of high-efficiency regeneration method taking Chinese fir cotyledons as explants, it comprises the steps:

[0044] 1) Sowing: Select mature and plump seeds, soak them in tap water for 24 hours, and then sow them in a mixture of nutrient soil, vermiculite and perlite at a ratio of 2:2:1 (v / v) at room temperature (25±2°C). in the matrix;

[0045] 2) Disinfection: 20 days after the seeds germinate, select healthy seedlings and clean them with tap water. After removing the roots, soak in 75vol% alcohol for 1 minute, 0.1vol% mercury chloride solution for 7 minutes, and rinse with sterile water for 5 times;

[0046] 3) Induction culture: the cotyledons of the sterilized seedlings were taken as explants, and inoculated on the induction medium, under the light rate of 80 μmol·m -2 ·s -1 , light time 16 h / d, and culture temperature 25±2°C for 30 days to obtain adventitious buds; the formulation of the induction medium is: DCR+NAA 0.2 mg / L+TDZ 0.005 mg / L+6- BA 1.0mg / L; ...

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Abstract

The invention discloses a high-efficiency regeneration method using a cunninghamia lanceolata cotyledon as an explant. The method comprises the steps of sowing, disinfecting, inducing culture, proliferating culture, rooting culture and seedling hardening, and specifically, a medium containing different hormone ratios is utilized to provide factors needed for the explants to take roots and strengthen seedlings, so that a good rooting condition is provided, the health and robustness of the seedlings are promoted, and regenerated seedlings of cunninghamia lanceolata are obtained. The cultivationmethod has the advantages of convenient material obtaining, low cost, simple operation, high expanding propagation speed and high reproduction coefficient, and can provide an efficient regeneration system for a genetic transformation system of cunninghamia lanceolata.

Description

technical field [0001] The invention belongs to the technical field of plant breeding and reproduction, and in particular relates to a high-efficiency regeneration method using fir cotyledons as explants. Background technique [0002] Chinese fir( Cunninghamia lanceolata (Lamb.) Hook.) is one of the most important evergreen conifer species in southern my country. Due to its good processing performance and strong natural corrosion resistance, fir wood is widely used in construction, furniture, papermaking and other industries, and is an important commercial timber tree species in my country. [0003] At present, some domestic scholars have used the embryo, cotyledon and hypocotyl of Chinese fir as explants to obtain regenerated plants through direct organogenesis, but the regeneration frequency is low, which is not enough to be used as the basis for genetic transformation and large-scale reproduction. platform. However, the in vitro regeneration research on Chinese fir wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 丁文沙叶善汶刘爱琴马祥庆朱强
Owner FUJIAN AGRI & FORESTRY UNIV
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