CN103238519A

CN103238519A - Rapid seedling raising method of switchgrass tissue culture

Info

Publication number: CN103238519A
Application number: CN2013101780078A
Authority: CN
Inventors: 杨冉; 黄萍; 杜道林; 杨淞惠; 祁珊珊
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2013-05-14
Filing date: 2013-05-14
Publication date: 2013-08-14
Anticipated expiration: 2033-05-14
Also published as: CN103238519B

Abstract

The invention relates to a rapid seedling raising method of switchgrass tissue culture and particularly relates to a tissue culture technology of a biological energy plant switchgrass. The method disclosed by the invention is carried out according to following process steps of: treating seeds, inducing calluses and buds, and carrying out rooting culture. According to the rapid seedling raising method disclosed by the invention, a switchgrass seed tissue culture method is adopted, so that good varieties can be reproduced, excellent seedlings can be selected from seed seedlings to be used for a genetic transformation experiment, and a lot of money and energy are prevented from spending. The method disclosed by the invention is feasible, is simple to operate, and provides an effective way for carrying out crossbreeding and genetic transformation experiments by people.

Description

A kind of switchgrass tissue culturing fast seedling-cultivating method

Technical field

The present invention relates to a kind of field of plant tissue culture technique, particularly relate to the tissue culture technique of bioenergy plant switchgrass.

Background technology

Switchgrass (English name Switchgrass, latin name Panicum virgatumlinn), claim again " energy grass ", because of its culture technique simple, wide adaptability, the ability of checking winds and fixing drifting sand is strong, impoverishment tolerant, the biological yield height is defined as producing the first-selected bioenergy plant of dyestuff ethanol in the world, and has dropped into great amount of manpower and material resources and researched and developed.

Utilizing transgenic technology that energy-source plant is carried out genetic improvement is the new breakthrough that the modern genetic engineering technology is used at agriculture field.Switchgrass has become one of main target of genetic transformation as a kind of important new bio energy-source plant.Wherein, adopt the resistance of transgenic technology enhancing switchgrass, improve light and efficient, increase biological yield, reducing aspects such as content of lignin and raising alcohol yied has become present research focus and has shown good prospects for application.

Yet similar to the tissue cultivation of most of gramineous plantses, switchgrass is also existing a lot of difficulties aspect artificial induction's callus.Therefore, the research of the external propagation method of passing through callus plantlet of strengthening switchgrass is just necessary, will play very big facilitation to the biotechnology research of switchgrass.

Summary of the invention

Defective and deficiency at the external propagation technique existence of above-mentioned existing switchgrass, the objective of the invention is to, the external propagation method of a kind of switchgrass is provided, the switchgrass tissue is cultivated and the genotype scope of genetic transformation to widen, utilize transgenic technology that energy-source plant is carried out genetic improvement with realization, and then realize the new breakthrough that the modern genetic engineering technology is used at agriculture field.

The object of the present invention is achieved like this: a kind of employing switchgrass seed tissue is cultivated seedling-cultivating method, and its special type is that it comprises following processing step:

Choose 100 full seeds (fresh weight is 0.225 g, and the seed length of choosing is at 0.2 ~ 0.5 cm, and width is 0.1 ~ 0.2 cm) and use material as experiment,

(1), seed treatment

A is that 0.225 g switchgrass seed is put into 5 ~ 10 ml 6% (v/v) NaClO with soaking 2 h among switchgrass seed 6% (v/v) NaClO by every fresh weight, during do not stop to stir;

After b cleans 3 times with distilled water, in the dark place standing over night;

C soaks 20 min by every fresh weight 0.225 g switchgrass seed with 5 ~ 10 ml 6% (v/v) NaClO;

After d cleans seed, under aseptic condition, it is seeded in carries out callus of induce on the callus inducing medium and cultivate.

(2), callus and bud induces

With the inoculation media transfer to incubator, illumination every day 14 h, intensity of illumination 1400-1500 lux, 23 ℃ ± 1 ℃ of temperature, cultivate 3 ~ 5 d under the aseptic condition after, seed begins to sprout, begin to have callus to form after 1 week, and being attended by the formation of sprouting or young leaves, it is the MS basal medium that callus induction is selected medium for use, replenishes 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH are 5.8.

(3), culture of rootage

After 4 weeks, the callus that will newly grow up under aseptic condition is transferred on the root media.Root media is selected the MS basal medium for use, replenishes 2,4-D, 5 mg/L, 6-BA 1.2 mg/L, and NAA 1.5 mg/L, pH are 5.8.23 ℃ ± 1 ℃ of temperature, illumination 14 h, light intensity 1500 ~ 2000 lux begin to take root behind 7 ~ 10 d, and speed is very fast, after 2 weeks, the switchgrass seedling of taking root can be carried out transiting cultivation.

Wherein, callus inducing medium is to add 2,4-D, 5 ~ 6 mg/L on the MS basal medium among step (1) d, 6-BA 1.2 ~ 1.5 mg/L, and NAA 1.0 ~ 1.2 mg/L, pH are 5.8.

Research method of the present invention is described by concrete experiment, has following advantage and result with respect to prior art:

The present invention adopts switchgrass seed tissue breeding method, both can breed improved seeds, can choose excellent young plant again as the genetic transformation experiment from seedling from seed, avoids spending mint of money and energy.The inventive method is feasible, and is simple to operate, can provide a kind of valid approach for people's cross-breeding and genetic transformation experiment.

Shortened the cultivation cycle of switchgrass, utilized orthogonal experiment, found out the suitableeest hormone combination that to induce the switchgrass callus to form, the formation that can promote callus largely and sprout, take root, the cultivation cycle of shortening switchgrass.

Description of drawings

Fig. 1 is inoculated into the seed photo of sprouting behind 3 d on the callus of induce medium;

Fig. 2 is the photo that seed forms callus and plumule behind inoculation 10 d;

Fig. 3 is the photo that forms a large amount of differentiated tissues behind inoculation 14 d at medium;

Fig. 4 is the photo of the switchgrass that takes root that forms at root media;

Fig. 5 is the photo of hardening.

Above accompanying drawing is the experiment effect figure among the embodiment one.

Embodiment:

Describe in detail below in conjunction with example and accompanying drawing

The amino fast cry of certain animals (6-BenzylaminoPurine) of 6-BA 6-b benzyl

NAA a-methyl (1-NaPhthylaceticacid)

2,4-D 2,4-dichlorphenoxyacetic acid (2,4-Dichlorophenoxyacetic acid)

Embodiment one:

It is explant that the present invention selects the switchgrass seed, adds different hormone combinations at the MS medium, evoked callus, differentiation and root induction.Concrete production process is:

Choose explant

Choose 100 in the switchgrass seed (0.225 g) of mature and plump.

Callus of induce and bud differentiation

Seed is placed culture dish, adopts 10 ml 6% (v/v) NaClO to carry out surface sterilization 2 h, during place and do not stop on the shaking table to stir;

Discard clorox, clean up, spend the night with distilled water immersion;

With the NaClO of 10 ml 6% (v/v), 20 min that sterilize, put into medium under the aseptic condition.Culture medium prescription is

Add 2,4-D, 5 mg/L on the MS basal medium, 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH are 5.8.

24 ℃, in illumination box, cultivate 10 ~ 15 d and induce the acquisition callus, when continuing to cultivate 30 ~ 40 d, can from callus, bear a large amount of buds of growing thickly again, concrete experiment effect is seen Fig. 1, Fig. 2 and Fig. 3.

Through statistics, 100 switchgrass mature seeds (0.225 g) induced bud differentiation rate can reach more than 90%.

The blastogenesis root of growing thickly is cultivated

The callus that shifts differentiation and bud formation carries out the root induction cultivation to root media, and 24 ℃, cultivate 20 ~ 30 d under the illumination condition, root induction obtains regeneration plant (Fig. 4).Wherein, the culture of rootage based formulas is to add 2,4-D, 5 mg/L on the MS basal medium, 6-BA 1.2 mg/L, and NAA 1.5 mg/L, pH are 5.8.

Transplanting is to the greenhouse

Open blake bottle and seal, with regeneration plant hardening 4 ~ 6 d, transplanting gets final product (Fig. 5) to the greenhouse.

Embodiment two:

(1) chooses explant

Choose 100 in the switchgrass seed (0.225 g) of mature and plump.

(2) callus of induce and bud differentiation

Seed is placed culture dish, adopts NaClO surface sterilization 2 h of 5 ml 6% (v/v), during place and do not stop on the shaking table to stir;

Discard clorox, clean up, spend the night with distilled water immersion;

With the NaClO sodium of 5 ml 6% (v/v), 20 min that sterilize, put into medium under the aseptic condition.Culture medium prescription is

Add 2,4-D, 6 mg/L on the MS basal medium, 6-BA 1.5 mg/L, NAA 1.2 mg/L, pH are 5.8.

24 ℃, in illumination box, to cultivate 15 ~ 20 d and induce the acquisition callus, differentiation is cultivated, and during 30 ~ 40 d, can bear a large amount of buds of growing thickly again from callus.

Through statistics, 100 switchgrass mature seeds (0.225 g) induced bud differentiation rate can only reach about 40%.

(3) the blastogenesis root of growing thickly is cultivated

The callus that shifts differentiation and bud formation carries out the root induction cultivation to root media, and 24 ℃, cultivate 20 ~ 30 d under the illumination condition, root induction obtains regeneration plant.Wherein, the culture of rootage based formulas is to add 2,4-D, 5 mg/L on the MS basal medium, 6-BA 1.2 mg/L, and NAA 1.5 mg/L, pH are 5.8.

(4) transplant to the greenhouse

Open blake bottle and seal, with regeneration plant hardening 4 ~ 6 d, transplanting gets final product to the greenhouse.

Claims

1. switchgrass tissue culturing fast seedling-cultivating method, carry out according to following step:

(1) seed treatment

A soaks 2 h with the switchgrass seed in 6% v/v NaClO, be that 0.225 g switchgrass seed is put into 5-10 ml 6% v/v NaClO by every fresh weight, during do not stop to stir;

C is that 0.225 g switchgrass seed soaks 20 min with 5-10 ml 6% v/v NaClO by every fresh weight;

After d cleans seed, under aseptic condition, it is seeded in carries out callus of induce on the callus inducing medium and cultivate;

(2) callus and bud induces

With the inoculation media transfer to incubator, illumination every day 14 h, intensity of illumination 1400-1500 lux, 23 ℃ ± 1 ℃ of temperature, behind the cultivation 3-5 d, seed begins to sprout under the aseptic condition, begin to have callus to form after 1 week, and being attended by the formation of sprouting or young leaves, it is the MS basal medium that callus induction is selected medium for use, replenishes 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH are 5.8;

(3) culture of rootage

After 4 weeks, the callus that will newly grow up under aseptic condition is transferred on the root media, 23 ℃ ± 1 ℃ of temperature, illumination 14 h, light intensity 1500 ~ 2000 lux begin to take root behind the 7-10 d, and speed is very fast, after 2 weeks, the switchgrass seedling of taking root can be carried out transiting cultivation.

2. switchgrass tissue culturing fast seedling-cultivating method according to claim 1, it is characterized in that callus inducing medium is to add 2,4-D 5-6 mg/L on the MS basal medium, 6-BA 1.2-1.5 mg/L among step (1) d, NAA 1.0 ~ 1.2 mg/L, pH are 5.8.

3. switchgrass tissue culturing fast seedling-cultivating method according to claim 1 is characterized in that root media is selected the MS basal medium for use described in the step (3), replenishes 2,4-D, 5 mg/L, 6-BA 1.2 mg/L, and NAA 1.5 mg/L, pH are 5.8.

4. callus inducing medium that is used for the described switchgrass tissue culturing fast seedling-cultivating of claim 1, its composition is to add 2,4-D 5-6 mg/L on the MS basal medium, 6-BA 1.2-1.5 mg/L, NAA 1.0-1.2 mg/L, pH are 5.8.

5. root media that is used for the described switchgrass tissue culturing fast seedling-cultivating of claim 1, its composition is that the MS basal medium replenishes 2,4-D, 5 mg/L, 6-BA 1.2 mg/L, NAA 1.5 mg/L, pH are 5.8.