CN1883258A - Method for building regenerative system of kostelezkya virginica - Google Patents

Abstract

The invention relates to a method for buildsing-up a regeneration system for beach mallow, using beach mallow mature embryos as explants, comprising following steps: (1)explants surface sterilization, (2)tissue culture, with MS basal culture medium containing 0.5-2.0mg/L of 2,4-dichlorophe noxyacetic acid,(3)tissue subculture, with MS basal culture medium containing 0.3mg/L of heteroauxin and 0.3mg/L of zeatin, (4)adventitious bud culture, with MS or 1/2 MS basal culture medium containing 0.1-0.2mg/L of heteroauxin and 0.3-0.5mg/L of zeatin, (5) rooting culture, with MS basal culture medium free of hormones, (6)obtusifolia seedlings transplanting and hardening. The method is provided with a tissue culture rate, a adventitious bud culture rate, a rooting rate up to 86.8%,64.2% and 95.2% respectively, and the survival rate of regenerating seedlings up to 80%.

Description

A kind of method of setting up regenerative system of kostelezkya virginica

Technical field

The present invention relates to plant regeneration, relate in particular to and utilize the kostelezkya virginica mature embryo to set up the method for regenerating system by tissue culture technique.

Background technology

Kostelezkya virginica (Kostelezkya virginica L.Presl.) be in recent years China from one of the salt-tolerant plant of external introduction (Yin Zengfang etc., plant resources and environment journal, 2006,15 (1): 14-17), originate in coastal saliferous marshland, eastern united states, its growth substrate salt content can be the herbaceos perennial of suitable beach area, beach growth up to 2.5%.The kostelezkya virginica seed is rich in fat and protein, and seed oil can be used as power fuel and uses, and being important biodiesel, (Yin Jin comes etc., Jiangsu agricultural science, 2000 (6): 29-31 with one of plant resources; Ruan Chengjiang, Dalian Nationality College's journal, 2006 (1): 13-16); The kostelezkya virginica update cycle generally is 8～10 months, and this its plantation of characteristics decision does not need too many work input, therefore is very beneficial for carrying out establishing in large scale in the many less beach areas of people; In addition, kostelezkya virginica is an indefinite inflorescence, and the florescence is long, and flower shape is the toy trumpet shape, rediance, and establishing in large scale can beautify the natural landscape of beach, and also quite helpful (Yin Jin comes etc., Jiangsu agricultural science, 2000 (6): 29-31) to developing coastal tourist industry.Therefore, the introducing of kostelezkya virginica has been opened up bright prospects with cultivation for the coastal beach of improvement China, development solonchak agricultural and exploitation " biodiesel " new forms of energy.

Yet the kostelezkya virginica percentage of seedgermination is low, and the comparatively serious empty seedling or the phenomenon of being short of seedling can appear in direct sowing and seedling, causes in reality cultivation the demand to seed big, and waste is serious.This has seriously limited the commerial growing of kostelezkya virginica.

For popularizing planting kostelezkya virginica in large area at short notice, the rapid propagation system of setting up stabilization characteristics of genetics is very important.Quick propagating technology based on plant tissue culture technique can effectively address the above problem.Carry out plant regeneration by plant tissue culture technique, can preserve parent's good genetic character effectively, become the fast and homogeneous of seedling.In addition, tissue culture is the indispensable part of modern biotechnology, is that to set up regenerative system of kostelezkya virginica efficiently be the important prerequisite that further applying transgene technique carries out the genetic character improvement on the basis with the tissue culture technique.

Deborah etc. attempted to utilize kostelezkya virginica stem section and mature embryo to set up tissue culture and regenerating system in 1989, but had only obtained tissue cultivating seedling in the callus that the stem section is induced, for the tissue culture and the never success of regeneration of mature embryo.(Deborah A Cook etc., plant cell, tissue and organ culture (Plant CellTissue and Organ Culture), 1989,17:111-119).This research group tests according to the method that Deborah etc. provides, but does not obtain tissue cultivating seedling according to results reported from the callus that the stem section is induced.

Summary of the invention

The present invention is that explant carries out cultured in vitro with the kostelezkya virginica mature embryo, induces explant to form callus, and callus is sprouted and takes root, and with high frequency, stably realize plant regeneration, and provides good acceptor material for genetic transformation.

The objective of the invention is to realize by the following technical solutions:

With the kostelezkya virginica mature embryo is explant, comprises the cultured in vitro of callus induction, adventitious bud inducing and culture of rootage, and then sets up the method for regenerative system of kostelezkya virginica, and this method comprises following concrete steps:

(1) acquisition of explant and surface sterilization;

(2) carry out callus induction containing on the MS basal medium of hormone, described hormone be concentration be 0.5～2.0mg/L the 2,4 dichloro benzene ethoxyacetic acid (2,4-dichlorophenoxyacetic acid, 2,4-D);

(3) containing the successive transfer culture that carries out callus on the 1/2MS basal medium of hormone, described hormone is that concentration is heteroauxin (the indole-3-acetic acid of 0.3mg/L, IAA) be zeatin (zeatin, combination ZT) of 0.3mg/L with concentration;

(4) indefinite bud induces, employed medium comprises basal medium and hormone, described basal medium is MS basal medium or 1/2MS basal medium, and described hormone is that concentration is that 0.1～0.2mg/L heteroauxin and concentration are the combination of the zeatin of 0.3～0.5mg/L;

(5) culture of rootage is used the MS basal medium that does not contain hormone;

(6) transplanting of aseptic seedling and hardening.

Also all containing sucrose and the concentration that concentration is 30g/L in above-mentioned steps (2), (3), (4), (5) in the employed medium is the agar of 8g/L, and the pH value of medium is 5.8.

In the method for setting up regenerative system of kostelezkya virginica of the present invention, step (2), promptly the preferred concentration of employed hormone 2,4 dichloro benzene ethoxyacetic acid is 0.5mg/L in the inducing of callus.

Step (4), promptly in the inducing of indefinite bud, preferred basal medium is the 1/2MS basal medium; Preferred hormone combinations is that concentration is that heteroauxin and the concentration of 0.1mg/L is the zeatin of 0.5mg/L.

In the method for setting up regenerative system of kostelezkya virginica of the present invention, the inducing of callus, promptly above-mentioned steps (2) comprises two stages:

A) dark induction period, this stage continues 72 hours from the inoculation back;

B) half light cultivation stage, periodicity of illumination is 14h/day, and intensity of illumination is 2500lx, and this stage is right after above-mentioned dark induction period, continues for 2 weeks;

Described step (2), promptly the condition of culture of the induction period of callus also comprises: 26 ± 2 ℃ of temperature, relative moisture 70%～80%.

The present invention is that explant carries out cultured in vitro with the kostelezkya virginica mature embryo, success makes up the regenerating system of kostelezkya virginica, callus induction rate, adventitious bud induction frequency, rooting rate best result do not reach 86.8%, 64.2% and 95.2%, the regrowth survival rate reaches 80%, for the commerial growing of China's kostelezkya virginica provides the technical foundation of growing seedlings.

Embodiment

The present invention is described further below in conjunction with embodiment, but this does not limit the scope of the invention.

Embodiment 1:

With the kostelezkya virginica mature embryo is that explant carries out cultured in vitro, set up the method for regenerative system of kostelezkya virginica, mainly comprise the inducing and the transplanting and the hardening of aseptic seedling of the inducing of acquisition, callus induction, callus successive transfer culture, indefinite bud, root of explant, concrete steps are:

1. as the acquisition and the surface sterilization of the kostelezkya virginica mature embryo of explant

(1) gets some in ripe kostelezkya virginica seed,, make kind of a skin deliquescing with concentrated sulfuric acid immersion treatment 1h;

(2), carefully peel off covering of a seed with tweezers and scalpel and complete embryo occurs more than 5 times with the kostelezkya virginica seed after the distilled water flushing concentrated sulfuric acid immersion treatment;

(3) excision cotyledon and radicle stay the plumular axis part as explant;

(4) surface sterilization of explant: above-mentioned plumular axis as explant is immersed in 75% ethanol 10s, rinses well with distilled water then.

2. callus induces

Above-mentioned explant after surface sterilization is inoculated on the callus inducing medium, and this medium is for containing 0.5mg/L 2,4-D, and the MS basal medium of 30g/L sucrose and 8g/L agar, this medium pH value is 5.8; Change culturing room after the inoculation over to and cultivate, culturing room's temperature is 26 ± 2 ℃, relative moisture 70%～80%; The phase I of inducing cultivates for dark, and this stage finishes from the explant inoculation, continues 72 hours; The second stage of inducing is that half light is cultivated, and this stage periodicity of illumination is 14h/day, and intensity of illumination is 2500lx, continues for 2 weeks; The inductivity of callus is 86.8%.

3. the successive transfer culture of callus

Change the callus that induces in the step 2 over to subculture medium, this medium is the 1/2MS basal medium that contains 0.3mg/LIAA, 0.3mg/L ZT, 30g/L sucrose and 8g/L agar, this medium pH value is 5.8, and callus successive transfer culture condition is identical with callus induction second stage condition of culture.Callus is through behind the successive transfer culture, and the callus piece obviously increases.

4. indefinite bud induces

To change the adventitious bud induction culture base through the callus behind the successive transfer culture over to, this medium is the 1/2MS basal medium that contains 0.1mg/L IAA, 0.5mg/L ZT, 30g/L sucrose and 8g/L agar, this medium pH value is 5.8, the adventitious bud induction culture condition is identical with the successive transfer culture condition, after inducing for 2 weeks, bud ratio is 64.2%.

5. culture of rootage

Change the callus that has produced indefinite bud in the above-mentioned steps 4 over to root media, this medium is the MS basal medium that contains 30g/L sucrose and 8g/L agar, and this medium pH value is 5.8, and the culture of rootage condition is identical with the successive transfer culture condition, after 2 weeks, rooting rate is 95.2%.

6. transplanting, the hardening of the aseptic seedling that will produce through above-mentioned steps, last planting percent is 80%.

Embodiment 2:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed callus inducing medium is to contain 1.0mg/L 2 in the step 2, the MS basal medium of 4-D, 30g/L sucrose and 8g/L agar, the inductivity of callus are 75%.

Embodiment 3:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed callus inducing medium is to contain 1.5mg/L 2 in the step 2, the MS basal medium of 4-D, 30g/L sucrose and 8g/L agar, the inductivity of callus are 86.8%.

Embodiment 4:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed callus inducing medium is to contain 2.0mg/L 2 in the step 2, the MS basal medium of 4-D, 30g/L sucrose and 8g/L agar, the inductivity of callus are 81.8%.

Embodiment 5:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed adventitious bud induction culture base is the MS basal medium that contains 0.1mg/L IAA, 0.3mg/L ZT, 30g/L sucrose and 8g/L agar in the step 4, and the bud ratio of indefinite bud is 45.9%.

Embodiment 6:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed adventitious bud induction culture base is the MS basal medium that contains 0.1mg/L IAA, 0.5mg/L ZT, 30g/L sucrose and 8g/L agar in the step 4, and the bud ratio of indefinite bud is 62.1%.

Embodiment 7:

Present embodiment is described to be thereby that explant carries out the method that cultured in vitro is set up regenerative system of kostelezkya virginica with the kostelezkya virginica mature embryo, the concrete operations step is identical with embodiment 1, but employed adventitious bud induction culture base is the MS basal medium that contains 0.2mg/L IAA, 0.5mg/L ZT, 30g/L sucrose and 8g/L agar in the step 4, and the bud ratio of indefinite bud is 53.8%.

Claims (5)

1. a method of setting up regenerative system of kostelezkya virginica is characterized in that with the kostelezkya virginica mature embryo being that explant carries out cultured in vitro, and this method comprises following concrete steps:

(1) acquisition of explant and surface sterilization;

(2) carry out callus induction containing on the MS basal medium of hormone, described hormone is that concentration is the 2,4 dichloro benzene ethoxyacetic acid of 0.5～2.0mg/L;

(3) containing the successive transfer culture that carries out callus on the 1/2MS basal medium of hormone, described hormone is that concentration is that heteroauxin and the concentration of 0.3mg/L is the combination of the zeatin of 0.3mg/L;

(4) indefinite bud induces, employed medium comprises basal medium and hormone, described basal medium is MS basal medium or 1/2MS basal medium, and described hormone is that concentration is that 0.1～0.2mg/L heteroauxin and concentration are the combination of the zeatin of 0.3～0.5mg/L;

(5) culture of rootage is used the MS basal medium that does not contain hormone;

(6) transplanting of aseptic seedling and hardening,

Also all containing sucrose and the concentration that concentration is 30g/L in above-mentioned steps (2), (3), (4), (5) in the employed medium is the agar of 8g/L, and the pH value of medium is 5.8.

2. method according to claim 1 is characterized in that employed hormone is that concentration is the 2,4 dichloro benzene ethoxyacetic acid of 0.5mg/L in the described step (2).

3. method according to claim 1 is characterized in that employed basal medium is the 1/2MS basal medium in the described step (4).

4. method according to claim 1 is characterized in that employed hormone in the described step (4) is that concentration is that heteroauxin and the concentration of 0.1mg/L is the combination of the zeatin of 0.5mg/L.

5. method according to claim 1 is characterized in that the callus induction of described step (2) comprises 2 stages:

A) dark induction period, this stage continues 72 hours from the inoculation back;

B) half light cultivation stage, periodicity of illumination is 14h/day, and intensity of illumination is 2500lx, and this stage is right after above-mentioned dark induction period, continues for 2 weeks;

The condition of culture of described step (2) also comprises: 26 ± 2 ℃ of temperature, relative moisture 70%～80%.