CN103238519B

CN103238519B - Rapid seedling raising method of switchgrass tissue culture

Info

Publication number: CN103238519B
Application number: CN201310178007.8A
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: 2015-03-04
Anticipated expiration: 2033-05-14
Also published as: CN103238519A

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), also known as " energy grass ", because its cultivation technique is simple, wide adaptability, ability of checking winds and fixing drifting sand is strong, impoverishment tolerant, biological yield is high, is defined as producing the first-selected bioenergy plant of dyestuff ethanol in the world, and has dropped into a large amount of human and material resources and research and develop.

Utilizing transgenic technology to carry out genetic improvement to energy-source plant is the new breakthrough that modern genetic engineering technology is used at agriculture field.Switchgrass as a kind of important new bio energy-source plant, one of major objective becoming gene transformation.Wherein, adopt transgenic technology to strengthen the resistance of switchgrass, improve light and efficiency, increase biological yield, reducing content of lignin and improving the aspects such as alcohol yied has become current study hotspot and has shown good application prospect.

But similar to the tissue culture of most of grass, switchgrass also also exists a lot of difficulty in artificial induction's callus.Therefore, the research strengthening the vitro propagation method by callus plantlet of switchgrass is just necessary, will play very large promoter action to the biotechnology research of switchgrass.

Summary of the invention

The defect existed for above-mentioned existing switchgrass vitro propagation technology and deficiency, the object of the invention is to, a kind of vitro propagation method of switchgrass is provided, to widen the genotype scope of switchgrass tissue culture and genetic transformation, to realize utilizing transgenic technology to carry out genetic improvement to energy-source plant, and then realize the new breakthrough that modern genetic engineering technology applies at agriculture field.

The object of the present invention is achieved like this: a kind of switchgrass seed tissue that adopts cultivates method for culturing seedlings, 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 chosen is at 0.2 ~ 0.5 cm, and width is 0.1 ~ 0.2 cm) and use material as experiment,

(1), seed treatment

A soaks 2 h by switchgrass seed 6% (v/v) NaClO, is that 0.225 g switchgrass seed puts into 5 ~ 10 ml 6% (v/v) NaClO by every fresh weight, and period does not stop to stir;

After b distilled water cleans 3 times, in dark place hold over night;

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

D, by after wash seeds, aseptically, is seeded on callus inducing medium and is carried out callus of induce cultivation.

(2), the induction of callus and bud

By the media transfer of inoculation to incubator, illumination every day 14 h, intensity of illumination 1400-1500 lux, temperature 23 DEG C ± 1 DEG C, after cultivating 3 ~ 5 d under aseptic condition, seed starts to sprout, start have callus to be formed after 1 week, and being attended by the formation of sprouting or young leaves, callus induction selects substratum to be MS basic medium, supplements 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH is 5.8.

(3), root culture

After 4 weeks, aseptically the callus newly grown up to is transferred on root media.Root media selects MS basic medium, and supplement 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.5 mg/L, pH are 5.8.Temperature 23 DEG C ± 1 DEG C, illumination 14 h, light intensity 1500 ~ 2000 lux, starts to take root after 7 ~ 10 d, and speed, after 2 weeks, the switchgrass seedling of taking root can be carried out transiting cultivation.

Wherein, in step (1) d, callus inducing medium is that MS basic medium adds 2,4-D 5 ~ 6 mg/L, and 6-BA 1.2 ~ 1.5 mg/L, NAA 1.0 ~ 1.2 mg/L, pH is 5.8.

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

The present invention adopts switchgrass seed tissue method of cultivation, both can breed improved seeds, can choose again excellent young plant and be used as genetic transformation experiment, avoid cost mint of money and energy from seedling from seed.The inventive method is feasible, simple to operate, can be cross-breeding and genetic transformation experiment, provide a kind of effective approach for people.

Shorten the culture cycle of switchgrass, utilize orthogonal experiment, find out the suitableeest hormone combination inducing switchgrass Callus formation, the formation that can promote callus largely and sprout, take root, shorten the culture cycle of switchgrass.

Accompanying drawing explanation

Fig. 1 is inoculated into the seed photo that calli induction media is sprouted after 3 d;

Fig. 2 is the photo of Seed Development callus and plumule after inoculation 10 d;

Fig. 3 is the photo forming a large amount of differentiated tissue after inoculation 14 d on substratum;

Fig. 4 is the photo of the switchgrass taken root formed on root media;

Fig. 5 is the photo of hardening.

Above accompanying drawing is the experiment effect figure in 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-naphthylacetic acid (1-NaPhthylaceticacid)

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

Embodiment one:

The present invention selects switchgrass seed to be explant, MS substratum adds different hormone combinations, evoked callus, differentiation and root induction.Concrete production process is:

Choose explant

(0.225 g) to choose 100, the switchgrass seed of mature and plump.

Callus of induce and Bud polarization

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

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

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

MS basic medium adds 2,4-D 5 mg/L, and 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH is 5.8.

24 DEG C, in illumination box, cultivate 10 ~ 15 d inductions obtain callus, when continuing cultivation 30 ~ 40 d, can bear a large amount of Multiple Buds again from callus, specific experiment effect is shown in 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%.

Multiple Buds root culture

The callus of transfer differentiation and bud formation carries out root induction cultivation, 24 DEG C to root media, and cultivate 20 ~ 30 d under illumination condition, root induction, obtains regeneration plant (Fig. 4).Wherein, prescription of rooting medium is that MS basic medium adds 2,4-D 5 mg/L, and 6-BA 1.2 mg/L, NAA 1.5 mg/L, pH is 5.8.

Transplant to greenhouse

Open culturing bottle sealing, by regeneration plant hardening 4 ~ 6 d, transplant and get final product (Fig. 5) to greenhouse.

Embodiment two:

(1) explant is chosen

(0.225 g) to choose 100, the switchgrass seed of mature and plump.

(2) callus of induce and Bud polarization

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

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

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

MS basic medium adds 2,4-D 6 mg/L, and 6-BA 1.5 mg/L, NAA 1.2 mg/L, pH is 5.8.

24 DEG C, in illumination box, cultivate 15 ~ 20 d inductions obtain callus, differentiation culture, during 30 ~ 40 d, can bear a large amount of Multiple Buds again from callus.

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

(3) Multiple Buds root culture

The callus of transfer differentiation and bud formation carries out root induction cultivation, 24 DEG C to root media, and cultivate 20 ~ 30 d under illumination condition, root induction, obtains regeneration plant.Wherein, prescription of rooting medium is that MS basic medium adds 2,4-D 5 mg/L, and 6-BA 1.2 mg/L, NAA 1.5 mg/L, pH is 5.8.

(4) transplant to greenhouse

Open culturing bottle sealing, by regeneration plant hardening 4 ~ 6 d, transplant to greenhouse.

Claims

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

(1) seed treatment

Switchgrass seed is soaked 2 h by a in 6% v/v NaClO, is that 0.225 g switchgrass seed puts into 5-10 ml 6% v/v NaClO by every fresh weight, and period does not stop to stir;

After b distilled water cleans 3 times, in dark place hold over night;

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

D, by after wash seeds, aseptically, is seeded on callus inducing medium and is carried out callus of induce cultivation;

(2) induction of callus and bud

By the media transfer of inoculation to incubator, illumination every day 14 h, intensity of illumination 1400-1500 lux, temperature 23 ± 1 DEG C, after cultivating 3-5 d under aseptic condition, seed starts to sprout, start have callus to be formed after 1 week, and being attended by the formation of sprouting or young leaves, callus inducing medium selects MS basic medium, supplements 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.0 mg/L, pH is 5.8;

(3) root culture

After 4 weeks, aseptically the callus of differentiation and bud formation is transferred on root media, temperature 23 ± 1 DEG C, illumination 14 h, light intensity 1500 ~ 2000 lux, starts to take root after 7-10 d, and speed, after 2 weeks, the switchgrass seedling of taking root is carried out transiting cultivation;

Described in step (3), root media selects MS basic medium, and supplement 2,4-D 5 mg/L, 6-BA 1.2 mg/L, NAA 1.5 mg/L, pH are 5.8.