CN105766639B

CN105766639B - A kind of method of cultivating sweet sorghum tissue culture fast seedling growing

Info

Publication number: CN105766639B
Application number: CN201610148176.0A
Authority: CN
Inventors: 杜浩; 黄萍; 杜道林; 庄义庆; 祁珊珊
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2016-03-15
Filing date: 2016-03-15
Publication date: 2017-11-17
Anticipated expiration: 2036-03-15
Also published as: CN105766639A

Abstract

The invention discloses a kind of method of cultivating sweet sorghum tissue culture fast seedling growing, it is related to field of plant tissue culture technique.Using sweet sorghum seed tissue cultivating method, improved seeds can be both bred, excellent young plant can be chosen from seedling from seed again and tested as genetic transformation, avoid spending mint of money and energy.The inventive method is feasible, simple to operate, can be that people are cross-breeding and genetic transformation is tested, there is provided a kind of effective approach.The cultivation cycle of sugar grass is shortened, using orthogonal experiment, the most suitable hormone combination that can induce sugar grass Callus formation is found out, can largely promote callus and the formation sprouted, taken root, shorten the cultivation cycle of sugar grass.

Description

A kind of method of cultivating sweet sorghum tissue culture fast seedling growing

Technical field

The present invention relates to a kind of field of plant tissue culture technique, more particularly to the tissue cultures of energy-source plant sugar grass Technology.

Background technology

Sugar grass (English name Sweet Sorghum, latin name Sorghum bicolor L.Moench) is initiated by non- The annual C4 herbaceous plant of high bar in continent torrid zone continent, because its growth is rapid, strong stress resistance, sucrose accumulation are fast, biological yield The features such as height, ethanol production are high, ethanol conversion is high, turns into a kind of excellent forage crop, sugar crop and reproducible energy Source crop, there is the good reputation of " camel in desert ", it is considered to be most one of energy-source plant of potentiality to be exploited.In petroleum resources Under the severe situation for facing exhaustion, sugar grass has been paid attention to extensively by global as energy crop, and input was a large amount of in recent years Man power and material researched and developed.

Modern genetic engineering technology especially transgenic technology achieves new breakthrough in application agriculturally.Sugar grass conduct The fifth-largest crops in the world, while be the stronger energy crop of adaptability again, it is increasingly becoming the mesh of modern agriculture genetic transformation Mark one of energy-source plant.Wherein, nutrition improvement, Resistant, low lignin is carried out to sugar grass kind using transgenic technology to contain Amount and high ethano yield etc., these researchs as current study hotspot and show good application prospect.

However, different from the tissue cultures of most of grasses, sugar grass is deposited in terms of artificial induction's callus Many difficult, it is considered to be most difficult to carry out one of kind of tissue cultures.Thus, strengthen to sugar grass callus induction And the research of the vitro propagation of regeneration plant, with regard to necessary, this makees very big promotion is played to the cultivation of sugar grass new varieties With.

The content of the invention

It is an object of the present invention to the defects of existing for above-mentioned existing sugar grass vitro propagation technology and deficiency, there is provided A kind of vitro propagation method of sugar grass, to widen the genotype scope of cultivating sweet sorghum tissue culture and genetic transformation, to realize profit Genetic improvement is carried out to energy-source plant with transgenic technology, and then it is new to realize that modern genetic engineering technology is applied in agriculture field Break through.

One kind uses sweet sorghum seed tissue culturing fast seedling-cultivating method, carries out as steps described below：

(1) seed treatment

A, sweet sorghum seed is soaked into 2h in 10% (v/v) NaClO, does not during which stop to stir；

B, after cleaning 3 times with distilled water, stood overnight in dark place；

C, 20min is soaked with 10% (v/v) NaClO；

D, in superclean bench, with sterile water wash 3~5 times, it is enterprising to be seeded in callus inducing medium Row callus Fiber differentiation.

(2) callus induction

By the media transfer of inoculation to incubator, daily illumination 16h, intensity of illumination 1400-1500lux (lux), 25 DEG C ± 1 DEG C of temperature, after cultivating 3~5d under aseptic condition, seed starts to sprout, and beginning with callus after 1 week forms, and is accompanied by Sprouting or the formation of young leaves, callus induction are MS basal mediums from culture medium, 2,4-D of supplement 1~3mg/L, 6-BA 1 ~3mg/L, sucrose 30g/L, proline 0.7~1g/L, KH₂PO₄1~2g/L, CuSO₄1~3 μM, pH 5.8.

(3) sprout culture of rootage

After 4 weeks, aseptically the callus newly grown up to is transferred on root media, 25 DEG C ± 1 DEG C of temperature, Start to take root after illumination 16h, light intensity 1500~2000lux, 7~10d, and speed, after 2 weeks, the sugar grass that will can take root Seedling carries out transiting cultivation.

Wherein, step (3) root media selects MS basal mediums, supplements 1~3mg/L of IAA, sucrose 30g/L, dried meat Propylhomoserin 0.7~1g/L, KH₂PO₄1~2g/L, CuSO₄1~3 μM, pH 5.8.

The research method of the present invention is described by specifically testing, there is as follows excellent compared with prior art Point and result：

The present invention uses sweet sorghum seed tissue cultivating method, can both breed improved seeds, again can be from seedling from seed Choose excellent young plant to test as genetic transformation, avoid spending mint of money and energy.The inventive method is feasible, operation letter It is single, can be that people are cross-breeding and genetic transformation is tested, there is provided a kind of effective approach.

The cultivation cycle of sugar grass is shortened, using orthogonal experiment, sugar grass Callus formation can be induced by finding out It most suitable hormone combination, can largely promote callus and the formation sprouted, taken root, shorten the cultivation cycle of sugar grass.

Brief description of the drawings

Accompanying drawing 1-7 is the experiment effect figure in embodiment one.

Seed sprouting after Fig. 1 cultures 3d；

Callus after Fig. 2 cultures 2 weeks；

After Fig. 3 callus stripping and slicing 2 weeks；

After Fig. 4 callus is bred 3 weeks；

The generation of Fig. 5 Multiple Buds；

The generation of Fig. 6 roots；

Fig. 7 seedlings；Wherein A is transferred in Nutrition Soil for aseptic seedling and cultivated 1 week, and B is to be cultivated 2 weeks in big flowerpot.

Embodiment：

Described in detail with reference to example and accompanying drawing

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

NAA α-naphthylacetic acids (1-NaPhthylaceticacid)

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

MS basal medium formulations are as follows:

Embodiment one：

Present invention selection sweet sorghum seed is explant, and different hormone combinations, callus induction are added on MS culture mediums Tissue, differentiation and rooting induction.Specific operation process is：

Choose explant

Choose the sweet sorghum seed 100 of mature and plump.

Callus induces and bud differentiation

Seed is placed in culture dish, surface sterilization 2h is carried out using 10ml 10%, is during which placed on shaking table and does not stop to stir Mix；

Sodium hypochlorite is discarded, is cleaned up, is stayed overnight with distilled water immersion；

20min is sterilized with 10ml 10% (v/v) NaClO, is put under aseptic condition in calli induction media.Culture Based formulas is

2,4-D 1mg/L, 6-BA 2mg/L, sucrose 30g/L, 0.7~1g/L of proline are added on MS basal mediums, KH₂PO₄1~2g/L, CuSO₄1~3 μM, pH 5.8

24 DEG C, 10~15d inductions are cultivated in illumination box and obtain callus, can be from when continuing 30~40d of culture Substantial amounts of Multiple Buds are regenerated in callus, specific experiment effect is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5.

By statistics, 100 sugar grass mature seed induction inductivities can reach more than 90%.

Multiple Buds culture of rootage

Shift and rooting induction culture is carried out on callus to the root media of differentiation and bud formation, 25 DEG C, under illumination condition 15~20d is cultivated, root induction, obtains regeneration plant (Fig. 6).Wherein, prescription of rooting medium is to add on MS basal mediums Add IAA1.5mg/L, sucrose 30g/L, proline 0.7~1g/L, KH₂PO₄1~2g/L, CuSO₄0.15~0.3mg/L, pH are 5.8。

Transplant to greenhouse

Culture bottle closure is opened, by regeneration plant 4~6d of hardening, transplanting can (Fig. 7) to greenhouse.

Embodiment two：

Present invention selection sweet sorghum seed is explant, and different hormone combination and nutrients are added on MS culture mediums Matter, evoked callus, differentiation and rooting induction.Specifically production process is：

(1) explant is chosen

Choose the sweet sorghum seed 100 of mature and plump.

(2) callus induction and bud differentiation

Seed is placed in culture dish, using 10ml 10% (v/v) NaClO surface sterilization 2h, is during which placed on shaking table Do not stop to stir；

With the mass fraction hypochlorite disinfectant 20min of 10ml 10%, sterile water wash 3~5 times, it is inoculated with aseptic condition Into calli induction media.Culture medium prescription is

2,4-D 3mg/L, 6-BA 1.0mg/L, sucrose 30g/L, 0.7~1g/L of proline are added on MS basal mediums, KH₂PO₄1~2g/L, CuSO₄1~3 μM, pH 5.8.

25 DEG C, 15~20d inductions are cultivated in illumination box and obtain callus；

Differentiation culture, during 30~40d, can regenerate substantial amounts of Multiple Buds from callus.

By statistics, 100 sugar grass mature seed induction inductivities can only achieve 65% or so.

(3) Multiple Buds culture of rootage

Shift and rooting induction culture is carried out on callus to the root media of differentiation and bud formation, 25 DEG C, under illumination condition 20~30d is cultivated, root induction, obtains regeneration plant.Wherein, prescription of rooting medium is to add IAA on MS basal mediums 2.0mg/L, sucrose 30g/L, proline 0.7~1g/L, KH₂PO₄1~2g/L, CuSO₄0.15~0.3mg/L, pH 5.8.

(4) transplant to greenhouse

Culture bottle closure is opened, regeneration plant 4~6d of hardening is transplanted to greenhouse.

Claims

1. one kind uses sweet sorghum seed tissue culturing fast seedling-cultivating method, it is characterised in that carries out as steps described below：

（1）Seed treatment

A, sweet sorghum seed is soaked into 2 h in 10%v/v NaClO, does not during which stop to stir；

C, 20 min are soaked with 10%v/v NaClO；

D, in superclean bench, with sterile water wash 3 ~ 5 times, it is seeded on callus inducing medium and is cured Hinder Fiber differentiation；

（2）Callus induction

By the media transfer of inoculation to incubator, the daily h of illumination 16, intensity of illumination 1400-1500 lux（Lux）, temperature 25 DEG C ± 1 DEG C of degree, after cultivating 3 ~ 5 d under aseptic condition, seed starts to sprout, and beginning with callus after 1 week forms, and companion With the formation for having sprouting or young leaves, callus induction is MS basal mediums from culture medium, 2,4-D of supplement 1 ~ 3mg/L, 6- 1 ~ 3mg/L of BA, sucrose 30g/L, proline 0.7 ~ 1g/L, KH₂PO₄ 1~2g/L ,CuSO₄1 ~ 3 μM, pH 5.8；

（3）Sprout culture of rootage

After 4 weeks, aseptically the callus newly grown up to is transferred on root media, 25 DEG C ± 1 DEG C of temperature, Start to take root after the h of illumination 16, light intensity 1500 ~ 2000 lux, 7 ~ 10 d, and speed, after 2 weeks, the sugar grass that will take root Seedling carries out transiting cultivation；

Step（3）Root media selects MS basal mediums, supplements 1 ~ 3mg/L of IAA, sucrose 30g/L, and proline 0.7 ~ 1g/L, KH₂PO₄ 1~2g/L ,CuSO₄1 ~ 3 μM, pH 5.8.