CN102893866B - Strawberry root tip detoxification and tissue culture method - Google Patents

Abstract

The invention discloses a strawberry root tip detoxification and tissue culture method, which comprises the following steps: 1) preparing a medium; 2) preprocessing a material and selecting and sterilizing a root tip explants; 3) culturing a root tip induced regenerated plant; 4) performing bud multiplication culture; 5) performing subculture growth culture; and 6) performing rooting culture. The strawberry root tip detoxification and tissue culture method can solve problems of low efficiency, not thorough disinfection, high contamination rate of tissue culture seedlings and the like in the traditional stem tip detoxification technology for strawberry tissue culture, the operating efficiency is high, the disinfection is thorough, and the contamination rate of the tissue culture seedlings is low.

Description

A kind of strawberry tip of a root detoxification and tissue culture method

Technical field

The present invention relates to plant toxic and tissue culture technology field, the detoxification of especially a kind of strawberry tip of a root and tissue culture method.

Background technology

Strawberry is as a kind of economic crops, and the stem apex detoxify tissue-culturing rapid propagation of its seedling, is a kind of conventional technology adopting.But in the process of strawberry stem tip tissue-culturing rapid propagation, have that operation easier is large, inefficiency (under the microscope, with operation tool peel off 0.02mm stem apex), be difficult to thorough disinfection, tissue culture seedling pollution rate is high.To at present, about adopting the tip of a root take strawberry as the technology that explant carries out detoxification and group training, there is not yet the reported in literature of mature technology.

Summary of the invention

Technical problem to be solved by this invention is to provide the detoxification of a kind of strawberry tip of a root and tissue culture method, can solve the problems such as the existing efficiency of Strawberry Plantlets tradition Shoot-tip Grafting In Vitro is low, sterilization is difficult thoroughly, tissue culture seedling pollution rate height, operating efficiency is high, sterilization is thorough, tissue culture seedling pollution rate is low.

For solving the problems of the technologies described above, the technical solution adopted in the present invention is: the detoxification of a kind of strawberry tip of a root and tissue culture method, and the method comprises the following steps:

1) preparation of medium: take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, 2,4 –after dichlorphenoxyacetic acid 0.3～0.5mg/L, make pH value and be 5.8～6.0 tip of a root inducing culture;

Take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, a –after methyl α-naphthyl acetate 0.2～0.5mg/L, make pH value and be 5.8～6.0 shoot proliferation medium;

Take 1/2MS medium as minimal medium, after additional heteroauxin 0.2～0.5mg/L, make pH value and be 5.8～6.0 root media;

2) the choosing of pretreatment and tip of a root explant, sterilizing: strawberry is put into sterilizing fine sand heat-treat 4～6 thoughtful pumpings root system that makes new advances at 38 ℃ ± 2 ℃; Get the long main root tip of a root of 1～2cm, blot surface moisture with aseptic filter paper, under the microscope the tip of a root of the long 1～2mm of main root tip of a root front end is cut as explant;

3) tip of a root regeneration induction plant is cultivated: explant is seeded on tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, be strengthened to 1600lux to the constant light intensity of all the other conditions after surrounding, after six weeks, light intensity is strengthened to 3200lux, after eight weeks, light intensity decreasing continues cultivation until induce regeneration plant young shoot from the tip of a root under 1200lux, and this stage needs to cultivate 2～3 months altogether;

4) young shoot propagation is cultivated: plant young shoot is seeded on shoot proliferation medium, at 28 ± 2 ℃, under light intensity 2000～3000lux, 12 hours/d of illumination, is cultured to and grows up to the long seedling of 8～10cm;

5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm and is seeded on root media, at 28 ± 2 ℃, be cultured to and grow up to the long seedling of 8～10cm under light intensity 2000～3000lux, 12 hours/d of illumination; Every 3～4 weeks, seedling is carried out to a subculture expansion by same said method numerous later, until meet seedling, count requirement;

6) culture of rootage: the subculture seedling that increases is seeded on root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, 12 hours/d of illumination, send out roots after 1 week and be tied to form as complete test-tube plantlet.

Step 2) in, the method for long main root tip of a root sterilization is soaked 30 seconds for putting into 70% alcohol, then be that 10.5% liquor natrii hypochloritis sterilizes 8 minutes and aseptic washing 6 times by mass percentage concentration.

The composition of MS medium is (mg/L):

ⅰ）NH ₄NO ₃?33000、KNO ₃?38000、CaCl ₂·2H ₂O?8800、MgSO ₄·7H ₂O?7400、KH ₂PO ₄?3400；

ⅱ）KI?166、H ₃BO ₃?1240、MnSO ₄·4H ₂O?4460、ZnSO ₄·7H ₂O?1720、Na ₂·MoO ₄·2H ₂O?50、CuSO ₄·5H ₂O?5、CaCl ₂·6H ₂O?5；

ⅲ）FeSO ₄·7H ₂O?5560、Na ₂·EDTA·2H ₂O?7460；

To after the MS medium dilute with water twice preparing, obtain 1/2MS medium.

A kind of strawberry tip of a root provided by the invention detoxification and tissue culture method, beneficial effect is as follows:

1, processing ease, efficiency significantly improves: because existing strawberry adopts the stem apex detoxify group culturation rapid propagating technology of seedling, but in the fast numerous process of Shoot-tip Culture, with operation tool, peeling off 0.02mm stem apex is the larger and ineffective work of difficulty, and outside the tip of a root of strawberry is directly exposed to, the tip of excising under the microscope its tip of a root is just very easy, and efficiency improves greatly.

2, sterilization is thorough, tissue culture seedling pollution rate is low: stem apex detoxify is due to not easy-clear of its peripheral stem sheet, and sterilization is difficult to thoroughly, cause tissue culture seedling pollution rate often up to 60% left and right; And tip of a root detoxification is because it is peripheral without any parcel, to sterilize easily thoroughly, tissue culture seedling pollution rate is low.

3, simplified operation, improved operating efficiency, and inoculum breaks up, test-tube plantlet reproduction speed is fast, and detoxification is thorough, and strawberry grows fine.

Embodiment

A kind of strawberry tip of a root detoxification and tissue culture method, the method comprises the following steps:

1) preparation of medium: take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, 2,4 –after dichlorphenoxyacetic acid 0.3～0.5mg/L, make pH value and be 5.8～6.0 tip of a root inducing culture;

Take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, a –after methyl α-naphthyl acetate 0.2～0.5mg/L, make pH value and be 5.8～6.0 shoot proliferation medium;

Take 1/2MS medium as minimal medium, after additional heteroauxin 0.2～0.5mg/L, make pH value and be 5.8～6.0 root media;

2) the choosing of pretreatment and tip of a root explant, sterilizing: strawberry is put into sterilizing fine sand heat-treat 4～6 thoughtful pumpings root system that makes new advances at 38 ℃ ± 2 ℃; Get the long main root tip of a root of 1～2cm, blot surface moisture with aseptic filter paper, under the microscope the tip of a root of the long 1～2mm of main root tip of a root front end is cut as explant;

3) tip of a root regeneration induction plant is cultivated: explant is seeded on tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, be strengthened to 1600lux to the constant light intensity of all the other conditions after surrounding, after six weeks, light intensity is strengthened to 3200lux, after eight weeks, light intensity decreasing continues cultivation until induce regeneration plant young shoot from the tip of a root under 1200lux, and this stage needs to cultivate 2～3 months altogether;

4) young shoot propagation is cultivated: plant young shoot is seeded on shoot proliferation medium, at 28 ± 2 ℃, under light intensity 2000～3000lux, 12 hours/d of illumination, is cultured to and grows up to the long seedling of 8～10cm;

5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm and is seeded on root media, at 28 ± 2 ℃, be cultured to and grow up to the long seedling of 8～10cm under light intensity 2000～3000lux, 12 hours/d of illumination; Every 3～4 weeks, seedling is carried out to a subculture expansion by same said method numerous later, until meet seedling, count requirement;

6) culture of rootage: the subculture seedling that increases is seeded on root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, 12 hours/d of illumination, send out roots after 1 week and be tied to form as complete test-tube plantlet.

Step 2) in, the method for long main root tip of a root sterilization is soaked 30 seconds for putting into 70% alcohol, then be that 10.5% liquor natrii hypochloritis sterilizes 8 minutes and aseptic washing 6 times by mass percentage concentration.

The composition of MS medium is (mg/L):

ⅰ）NH ₄NO ₃?33000、KNO ₃?38000、CaCl ₂·2H ₂O?8800、MgSO ₄·7H ₂O?7400、KH ₂PO ₄?3400；

ⅱ）KI?166、H ₃BO ₃?1240、MnSO ₄·4H ₂O?4460、ZnSO ₄·7H ₂O?1720、Na ₂·MoO ₄·2H ₂O?50、CuSO ₄·5H ₂O?5、CaCl ₂·6H ₂O?5；

ⅲ）FeSO ₄·7H ₂O?5560、Na ₂·EDTA·2H ₂O?7460；

To after the MS medium dilute with water twice preparing, obtain 1/2MS medium.

Claims (1)

1. the detoxification of the strawberry tip of a root and a tissue culture method, is characterized in that the method comprises the following steps:

1) preparation of medium: take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, 2,4 –after dichlorphenoxyacetic acid 0.3～0.5mg/L, make pH value and be 5.8～6.0 tip of a root inducing culture;

Take MS medium as minimal medium, additional 6 –benayl aminopurine 5～7.5mg/L, a –after methyl α-naphthyl acetate 0.2～0.5mg/L, make pH value and be 5.8～6.0 shoot proliferation medium;

Take 1/2MS medium as minimal medium, after additional heteroauxin 0.2～0.5mg/L, make pH value and be 5.8～6.0 root media;

2) the choosing of pretreatment and tip of a root explant, sterilizing: strawberry is put into sterilizing fine sand heat-treat 4～6 thoughtful pumpings root system that makes new advances at 38 ℃ ± 2 ℃; Get the long main root tip of a root of 1～2cm, after sterilization, blot surface moisture, the tip of a root of the long 1～2mm of main root tip of a root front end is cut as explant;

3) tip of a root regeneration induction plant is cultivated: explant is seeded on tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, be strengthened to 1600lux to the constant light intensity of all the other conditions after surrounding, after six weeks, light intensity is strengthened to 3200lux, after eight weeks, light intensity decreasing continues cultivation until induce regeneration plant young shoot from the tip of a root under 1200lux, and this stage needs to cultivate 2～3 months altogether;

4) young shoot propagation is cultivated: plant young shoot is seeded on shoot proliferation medium, at 28 ± 2 ℃, under light intensity 2000～3000lux, 12 hours/d of illumination, is cultured to and grows up to the long seedling of 8～10cm;

5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm and is seeded on root media, at 28 ± 2 ℃, be cultured to and grow up to the long seedling of 8～10cm under light intensity 2000～3000lux, 12 hours/d of illumination; Every 3～4 weeks, seedling is carried out to a subculture expansion by same said method numerous later, until meet seedling, count requirement;

6) culture of rootage: the subculture seedling that increases is seeded on root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, 12 hours/d of illumination, send out roots after 1 week and be tied to form as complete test-tube plantlet;

Step 2) in, the method for long main root tip of a root sterilization is soaked 30 seconds for putting into 70% alcohol, then be that 10.5% liquor natrii hypochloritis sterilizes 8 minutes and aseptic washing 6 times by mass percentage concentration.