CN102239807A

CN102239807A - Establishing method of peanut regenerating system by taking deembryonated cotyledon as explant

Info

Publication number: CN102239807A
Application number: CN 201110179847
Authority: CN
Inventors: 耿丽丽; 束长龙; 张�杰; 宋福平; 黄大昉
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Zhongzhi Kechuang Biotechnology Co., Ltd.
Priority date: 2011-06-29
Filing date: 2011-06-29
Publication date: 2011-11-16
Anticipated expiration: 2031-06-29
Also published as: CN102239807B

Abstract

The invention relates to an establishing method of a peanut regenerating system by taking deembryonated cotyledon as explant, belonging to the technical field of biology. The establishing method comprises the following steps of: taking the deembryonated cotyledon as the explant of the peanut regenerating system, reducing the concentration of the 6-BA after inducing buds from a bud inducing culture medium containing 6-BA, promoting adventitious buds to elongate on an elongating culture medium containing 6-BA, then cultivating and rooting on a rooting culture medium containing NAA to obtain rooting plantlets, and finally transplanting and cultivating according to a conventional method. The highest bud induction rate of the peanut regenerating system taking the deembryonated cotyledon as the explant established by the invention is 48.3%.

Description

To remove scutellum is the method for building up of explant peanut regenerating system

Technical field

The present invention relates to biological technical field, particularly a method for building up of cultivating peanut regenerating system.

Background technology

Peanut is one of important oil crop in the world, and the peanut yield of China occupies first place in the world.Some biologies and abiotic stress all can influence output, especially soil insect, fungi and the bacteriosis of peanut.Although traditional crossbreeding can obtain some pest-resistant disease-resistant strain (Garcia et al., 2006), but low (the Kochert et al. of peanut cultivating variety genetic diversity, 1991), the crossbreeding cycle is long, common and other the unexpected linkages of characters of the disease-resistant proterties of breeding, therefore, cultivate peanut varieties by the molecular breeding means at present, the improvement peanut quality has remarkable advantages.

The efficient of regenerating system is the key of genetic transformation success or not, the plant regeneration system is exactly to utilize the totipotency of cell, by tissue culture approach or other non-tissue culture approach, under the effect of exogenous hormone, take place or the idiosome cell high-efficient, stably obtain regeneration plant through organ.The selection of explant requires that stronger regeneration capacity is arranged, to the importing and the integration of antibiotic sensitive, suitable foreign gene.The tissue culture of peanut starts from the forties in 20th century, and China then originates in the seventies, but regeneration efficiency is lower.Therefore, set up a peanut regenerating system efficiently, improvement peanut quality, raising peanut yield are had great importance.

Summary of the invention

At the defective in the above-mentioned field, the invention provides a kind of is the method for building up of explant peanut regenerating system to remove scutellum, and the bud induction rate of this method can reach about 48%, and the subculture well-grown can become the good regenerating system of genetic transformation.

To remove scutellum is the method for building up of explant peanut regenerating system, comprises the steps:

(1) turn out indefinite bud on the bud inducing culture that will go scutellum just placing to contain 6-BA, described just being changed to position contact medium away from embryo, the concentration of 6-BA is 5-20mgl ^-1

(2) will have the scutellum that goes of indefinite bud, and transfer to and promote the indefinite bud elongation on the elongation medium that contains 6-BA, the concentration of 6-BA is 5-10mgl ^-1

(3) indefinite bud of elongation is gone scutellum to forward to contain cultivate on the root media of NAA and take root, the concentration of NAA is 0.5-1mgl ^-1

(4) the seedling implanting and cultivating according to a conventional method of taking root.

Described bud inducing culture is the MS basal medium.

The concentration of 6-BA in the described bud inducing culture is 15-20mgl ^-1, step (1) incubation time was two weeks.

Described elongation medium is the MS basal medium.

The concentration of 6-BA in the described elongation medium is 5mgl ^-1, step (2) incubation time was two weeks.

The MS basal medium that described root media is.

The concentration of NAA in the described root media is 0.5mgl ^-1, step (3) incubation time is a week.

The described scutellum that goes is 1/2 cotyledon or a undivided full cotyledon separately.

Described going needs make aseptic process to peanut before the scutellum taking-up.

The method of described aseptic process is: peanut shells, and is positioned over 1min in 75% ethanol, abandons ethanol, places 4min in 0.1% mercuric chloride, and sterilization washing 3 times is positioned on the aseptic paper to drying.

The present invention adopts and removes the explant of scutellum as the peanut regenerating system, contain induce on the bud inducing culture of 6-BA sprout after, reduce the concentration of 6-BA, containing promotion indefinite bud elongation on the elongation medium of 6-BA, on the root media that contains NAA, cultivate again and take root, obtain the seedling of taking root, according to a conventional method implanting and cultivating.The regenerating system that the present invention sets up, its bud induction rate is up to about 48%.

Description of drawings

Fig. 1 explant prepares modes of emplacement,

1. just put, 2. be inverted,

It is the explant induction bud that Fig. 2 removes scutellum with 1/2,

A:1/2 goes scutellum B:1 week to produce indefinite bud C at nearly embryo place: induced bud (two weeks)

The influence (1/2 cotyledon) of Figure 36-BA concentration to removing the scutellum bud induction rate,

The influence (full cotyledon) of Figure 46-BA concentration to removing the scutellum bud induction rate,

Fig. 5 is elongation, the transplanting and solid of explant induction bud to remove scutellum.

Embodiment

The bud inducing culture is the MS basal medium, adds the 6-BA (being 6-benzyl aminopurine) of variable concentrations

Elongation medium is the MS basal medium, adds the 6-BA of variable concentrations

Root media is the MS basal medium, adds the NAA (being methyl) of variable concentrations

Peanut is commercially available arbitrary kind.The MS basal medium can be commercially available, also can prepare voluntarily.

Embodiment 1

1. be determining of explant bud inducing culture to remove scutellum

1. peanut shells, and is positioned over 1min in 75% ethanol, abandons ethanol, places 4min in 0.1% mercuric chloride, and sterilization washing 3 times is positioned on the aseptic paper to drying;

2. with aseptic scalpel, with two cotyledons of peanut separately, remove embryo after, cotyledon vertically is cut into two parts, 1/2 cotyledon is placed on contains variable concentrations 6-BA (0,5,10,15 and 20mgl ^-1) the bud inducing culture on, modes of emplacement is divided into two kinds: just put and be inverted and (just put, away from the position of embryo contact medium; Be inverted the position contact medium of nearly embryo), see Fig. 1, or the cotyledon of cutting not, just be seated in and containing 20mgl ^-1On the bud inducing culture of 6-BA, approximately place 35 explants on every kind of medium, the independent repetition 3 times;

2. add up bud induction rate after two weeks,

2 is the peanut regenerating system of explant to remove scutellum

1. peanut shells, and is positioned over 1min in 75% ethanol, abandons ethanol, places 4min in 0.1% mercuric chloride, sterilization washing 3 times, place with aseptic paper on to drying;

2. with aseptic scalpel, with two cotyledons of peanut separately, remove embryo after, cotyledon just is being seated in containing 20mgl ^-1On the bud inducing culture of 6-BA, the indefinite bud of inducing after two weeks produces;

3. will produce the explant subculture of indefinite bud, and transfer to and contain 5mgl ^-1On the elongation medium of 6-BA, about two weeks, the indefinite bud elongation;

4. the induced bud that extends is forwarded to and contain 0.5mgl ^-1On the root media of NAA, take root about a week;

The little transplantation of seedlings that to take root is transferred in the flowerpot that contains nutritious soil and vermiculite (volume ratio is 2/1), moves on to hot-house culture.The result

Removing scutellum with 1/2 is explant (Fig. 2 A), is just placing on the medium that contains 6-BA illumination cultivation.After 1 week, there is indefinite bud to produce (Fig. 2 B) at nearly embryo place.About two weeks, differentiation adventitious buds becomes obvious visible budlet (Fig. 2 C).On the 6-BA of 5 kinds of variable concentrations, except the medium that does not add 6-BA does not have the generation of evoking adventive bud, the evoking adventive bud that other 4 kinds of medium all can be in various degree, the inductivity of indefinite bud is (Fig. 3) between 4.2%～16.0%.Containing 15mgl ^-1The inductivity of indefinite bud is the highest on the medium of 6-BA, reaches 16%.Inversion can not be induced and be sprouted.

Based on top result, selecting the scutellum that goes of not cutting again for use is explant, is just placing illumination cultivation on the medium that contains 6-BA.After two weeks, at the nearly embryo end generation induced bud of explant.At the 6-BA of variable concentrations (5,10,15,20 and 30mgl ^-1) on the medium, bud induction rate from 11.6% to 44.0% (Fig. 4) is containing 20mgl ^-1Induce most effectively on the medium of 6-BA, through repeating, maximum bud induction rate can reach 48.3% (table 1).The result shows, with cutting not to remove scutellum be explant is higher than significantly that to remove scutellum with 1/2 be the outer planting height containing the efficient that produces induced bud on the medium of 6-BA.

Determining of the maximum bud induction rate of table 1

With the bud subculture of inducing, transfer to 6-BA (5mgl ^-1) on the medium that reduces of concentration, about two weeks, the bud elongation, leaf stretches, the regrowth robust growth.The seedling of elongation is transferred to and contained 0.5mgl ^-1On the root media of NAA, take root about two weeks.Healthy and strong plantlet of transplant, it is also solid to bloom normally after the suitable photoperiod.(see figure 5)

Claims

1. be the method for building up of explant peanut regenerating system to remove scutellum, comprise the steps:

2. method according to claim 1, described bud inducing culture is the MS basal medium.

3. method according to claim 2, the concentration of the 6-BA in the described bud inducing culture is 15-20mgl ^-1, step (1) incubation time was two weeks.

4. method according to claim 1, described elongation medium are the MS basal medium.

5. method according to claim 4, the concentration of the 6-BA in the described elongation medium are 5mgl ^-1, step (2) incubation time was two weeks.

6. method according to claim 1, the MS basal medium that described root media is.

7. method according to claim 6, the concentration of the NAA in the described root media are 0.5mgl ^-1, step (3) incubation time is a week.

8. method according to claim 1, the described scutellum that goes is not cutting cotyledon or 1/2 cotyledon.

9. method according to claim 1, described going needs make aseptic process to peanut before the scutellum taking-up.

10. method according to claim 9, the method for described aseptic process is: peanut shells, and is positioned over 1min in 75% ethanol, abandons ethanol, places 4min in 0.1% mercuric chloride, and sterilization washing 3 times is positioned on the aseptic paper to drying.