CN102860259A - High-efficiency industrial production method of virus-free tissue culture seedling of sweet potato - Google Patents

Abstract

The invention discloses a high-efficiency industrial production method of virus-free tissue culture seedling of sweet potatoes. The method comprises the following steps: (1) culturing materials; (2) disinfecting materials; (3) inducing an explant bud; (4) performing subculture multiplication culture; (5) detecting viruses; (6) performing rooting culture; and (7) exercising seedling and transplanting. According to a principle that the number of viruses on a grow point at the top end of a plant is far less than that on a mature position of the lower part of the plant, the algebra of taking the grow point is increased, and the aim of removing virus is achieved. According to the method, a method of low-hormone concentration, improved MS (Murashige and Skoog) basic culture medium and three-day dark culture is adopted, so that the aberration rate of virus-free tissue culture seedling can be greatly reduced, the growing speed of tissue culture seedlings can be accelerated, the multiplication cycle is shortened, the expanding propagation speed is high, and the production cost is low.

Description

The method of sweet potato tissue cultural seedlings of free is produced in a kind of efficient industrialization

Technical field

The invention belongs to agricultural biological technical field, be specifically related to the method that the sweet potato tissue cultural seedlings of free is produced in a kind of efficient industrialization.

Background technology

Sweet potato (Ipomoea batatas Lam.) is a kind of important grain, feed and industrial crop, has the advantages such as output is high, nutritious, yield stability is good.Yet, because the extensive existence of sweet potato virus disease, and sweet potato carries out vegetative propagation with the potato piece in producing, and causes spreading of virus disease, thus cause sweet potato yield reducation, quality to descend, plant sexual involution.Main by stem apex cultured in vitro acquisition virus-elimination seedlings in the at present scientific research, but applying to industrialization, the method produces in enormous quantities, and reproductive speed is slow, and aberration rate is high, and production cost is high.

Summary of the invention

The purpose of this invention is to provide that a kind of method is simple, cost is low, aberration rate is low, reproductive speed is fast, shortened the method that the sweet potato tissue cultural seedlings of free is produced in the efficient industrialization of breeding cycle.

To achieve these goals, technical scheme of the present invention is: provide a kind of efficient industrialization to produce the method for sweet potato tissue cultural seedlings of free, may further comprise the steps:

(1) material is cultivated

Fetch the long stem section of 20～30 ㎝ for the treatment of the detoxification sweet potato from the land for growing field crops, cuttage allows its growth shoot on the booth medium; In the process, jede Woche waters one time of nutrition liquid, and every night was with surrounding environment of 75% alcohol spray, with clean environment in guarantor's canopy with 50% carbendazim or tpn 500 times of sprays leaf and surrounding environment in per 5 days; The branch for the treatment of newly to grow is long during to 8～10 ㎝, cuts shoot, and cuttage is to new medium again, and using the same method allows it send forth branches;

(2) materials disinfection

Treat that the shoot of secondary cuttage seeding is long when above to 8 ㎝, get the long stem section of two or three joint, put into the sealed glass jars of sterilizing after cutting and seal, take superclean bench, soaked 19～20 seconds with 75% alcohol, with 0.1% mercuric chloride sterilization 5～7 minutes, use at last aseptic water washing 4～5 times again;

(3) the explant bud is induced

The armpit joint position that cuts the explant that 1 ㎝ sterilized is inoculated into explant bud inducing culture, carries out the dark cultivation of three day time after the inoculation, and 28 ± 2 ℃ of temperature settings are in photoperiod more than 16 hours, more than the light intensity 2000LX;

(4) the subculture increment is cultivated

Explant was cultivated 25 days, when the potato seedling grows to 3 ㎝～6 ㎝, get every strain growing point 2～4mm and be incubated at separately the shoot proliferation medium, residue stem section single-unit is incubated at the shoot proliferation medium, carry out three days dark cultivations after the inoculation, 28 ± 2 ℃ of temperature settings are in photoperiod more than 16 hours, more than the light intensity 2000LX; Just can take root after three days, in lower generation, get growing point and single stem segment again and cultivates, 12～15 days or 20 days subcultures once, growth coefficient can reach 4～5 times;

(5) virus detects

Just carry out the virus detection after breeding third and fourth generation, at first adopt ocular estimate, eliminate weak seedling and aobvious disease seedling, then random sampling adopts the indicator plant method to identify, the random sampling virus elimination rate is lower than 100%, then strengthens and cultivates algebraically, until the random sampling virus elimination rate reaches 100%;

(6) culture of rootage

Get every strain growing point 2～4mm and receive root media, strengthen light application time and intensity of illumination, or be placed on to cultivate under the natural daylight and take root;

(7) hardening, transplanting

Seedling after taking root well is being placed on 1 week of natural daylight lower refining seedling, open again bottleneck and put 3～5 days, then clean medium, cut off long root, putting into 0.2% potassium permanganate soaked 1～2 minute, after taking out airing, plant compost, and then lid layer arch film, all to allow its vented exhaust every day, keep compost moistening, humidity treats that 80～85% it is long to 5～8 young leaves transplanting lands for growing field crops.

Growing the culture medium prescription that the body bud induces outward is: MS+6-BA 0～0.5mg/L+IBA 0～0.5mg/L+ sugar 30g+ activated carbon 3～6g+ agar 6g;

The culture medium prescription that shoot proliferation is cultivated is: improvement MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g;

The culture medium prescription of culture of rootage is: improvement 2/3MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g+ activated carbon 3～6g.

The present invention in the quantity of the plant apical growing point principle far less than the ripe position of plant bottom, strengthens the algebraically of getting growing point according to virus, reaches the purpose of detoxification.The present invention adopts the method for the dark cultivation of low hormone concentration, improvement MS minimal medium and three days, greatly reduces the aberration rate of tissue cultural seedlings of free, has accelerated the growth rate of group training seedling, has shortened the breeding cycle, and reproductive speed is high, and production cost is low.

Embodiment

The method that the sweet potato tissue cultural seedlings of free is produced in the efficient industrialization of the present invention may further comprise the steps:

1, material is cultivated

Fetch the long stem section of 20～30 ㎝ for the treatment of the detoxification sweet potato from the land for growing field crops, cuttage allows its growth shoot on booth matrix.In the process, jede Woche waters one time of nutrition liquid, and per 5 days with 500 times of 50% carbendazim (or tpn) spray leaf and surrounding environment, surrounding environment preferably every day before leaving offices with 75% alcohol spray every night once, with clean environment in guarantor's canopy.

When treating shoot length to 8～10 ㎝, cut shoot, cuttage is to new matrix again, and using the same method allows it send forth branches.

2, materials disinfection

Treat that the shoot of secondary cuttage seeding is long when above to 8 ㎝, get the long stem section of two or three joint, put into the sealed glass jars good seal of sterilizing after cutting, before taking workbench, soak about 20 seconds with 75% alcohol, with 0.1% mercuric chloride sterilization 5～7 minutes, use at last aseptic water washing 4～5 times again.

3, the explant bud is induced

The armpit joint position that cuts the explant of the sterilization about 1 ㎝ is inoculated into explant bud inducing culture, carries out the dark cultivation of three day time after the inoculation, and temperature setting (28 ± 2) ℃ is in photoperiod more than 16 hours, more than the light intensity 2000LX.

4, shoot proliferation is cultivated

Explant was cultivated about 25 days, when the potato seedling grows to 3 ㎝～6 ㎝, get about every strain growing point 2～4mm and be incubated at separately the shoot proliferation medium, residue stem section single-unit is incubated at the shoot proliferation medium, carry out three days dark cultivations after the inoculation, temperature setting (28 ± 2) ℃ is in photoperiod more than 16 hours, more than the light intensity 2000LX.Just can take root after three days,

In lower generation, get growing point and single stem segment again and cultivates, general 12～15 days or 20 days subcultures once all can, growth coefficient can reach 4～5 times.

5, virus detects

General propagation just can be carried out virus and be detected after third and fourth generation.At first adopt ocular estimate, eliminate weak seedling and aobvious disease seedling, then random sampling adopts the indicator plant method to identify.The random sampling virus elimination rate is lower than 100%, then strengthens and cultivates algebraically, until the random sampling virus elimination rate reaches 100%.

6, culture of rootage

Get about every strain growing point 2～4mm and receive root media, strengthen light application time and intensity of illumination, or be placed on to cultivate under the natural daylight and take root.

7, hardening, transplanting

Seedling after taking root well is being placed on 1 week of natural daylight lower refining seedling, opens bottleneck to put 3～5 days again, then cleans medium, cuts off long root, puts into 0.2% potassium permanganate and soaks 1～2 minute, behind the taking-up airing, plants compost.And then lid layer arch film, but all to allow its vented exhaust every day, keep compost moistening, humidity can not be excessive about 80～85%, otherwise easily rot, and treats that it longly can transplant the land for growing field crops to 5～8 young leaves.

Culture medium prescription

Growing the body bud outward induces: MS+6-BA 0～0.5mg/L+IBA 0～0.5mg/L+ sugar 30g+ activated carbon 3～6g+ agar 6g

Shoot proliferation is cultivated: improvement MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g

Culture of rootage: improvement 2/3MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g+ activated carbon 3～6g.

The comparison of table 1. method of the present invention and common Shoot Tip Culture

Method of the present invention is through practice: 1. all again draw materials in ground every year, in general three, four generations, just can detoxification, even the peasant reserves the kind that the potato piece is planted for a long time for one's own use, viral level is higher, and also general 14 generations just can detoxification; 2. just begin to occur variation after method of the present invention will arrive for 30 generations, general industrialization production, the sterilization of all again drawing materials every year does not reach generation more than 30, does not have the variation seedling and occurs.

Above disclosed only is preferred embodiment of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to claim of the present invention still belongs to the scope that the present invention is contained.

Claims (2)

1. method that the sweet potato tissue cultural seedlings of free is produced in efficient industrialization is characterized in that may further comprise the steps:

(1) material is cultivated

Fetch the long stem section of 20～30 ㎝ for the treatment of the detoxification sweet potato from the land for growing field crops, cuttage allows its growth shoot on the booth medium; In the process, jede Woche waters one time of nutrition liquid, and every night was with surrounding environment of 75% alcohol spray, with clean environment in guarantor's canopy with 50% carbendazim or tpn 500 times of sprays leaf and surrounding environment in per 5 days; The branch for the treatment of newly to grow is long during to 8～10 ㎝, cuts shoot, and cuttage is to new medium again, and using the same method allows it send forth branches;

(2) materials disinfection

Treat that the shoot of secondary cuttage seeding is long when above to 8 ㎝, get the long stem section of two or three joint, put into the sealed glass jars of sterilizing after cutting and seal, take superclean bench, soaked 19～20 seconds with 75% alcohol, with 0.1% mercuric chloride sterilization 5～7 minutes, use at last aseptic water washing 4～5 times again;

(3) the explant bud is induced

The armpit joint position that cuts the explant that 1 ㎝ sterilized is inoculated into explant bud inducing culture, carries out the dark cultivation of three day time after the inoculation, and 28 ± 2 ℃ of temperature settings are in photoperiod more than 16 hours, more than the light intensity 2000LX;

(4) the subculture increment is cultivated

Explant was cultivated 25 days, when the potato seedling grows to 3 ㎝～6 ㎝, get every strain growing point 2～4mm and be incubated at separately the shoot proliferation medium, residue stem section single-unit is incubated at the shoot proliferation medium, carry out three days dark cultivations after the inoculation, 28 ± 2 ℃ of temperature settings are in photoperiod more than 16 hours, more than the light intensity 2000LX; Just can take root after three days, in lower generation, get growing point and single stem segment again and cultivates, 12～15 days or 20 days subcultures once, growth coefficient can reach 4～5 times;

(5) virus detects

Just carry out the virus detection after breeding third and fourth generation, at first adopt ocular estimate, eliminate weak seedling and aobvious disease seedling, then random sampling adopts the indicator plant method to identify, the random sampling virus elimination rate is lower than 100%, then strengthens and cultivates algebraically, until the random sampling virus elimination rate reaches 100%;

(6) culture of rootage

Get every strain growing point 2～4mm and receive root media, strengthen light application time and intensity of illumination, or be placed on to cultivate under the natural daylight and take root;

(7) hardening, transplanting

Seedling after taking root well is being placed on 1 week of natural daylight lower refining seedling, open again bottleneck and put 3～5 days, then clean medium, cut off long root, putting into 0.2% potassium permanganate soaked 1～2 minute, after taking out airing, plant compost, and then lid layer arch film, all to allow its vented exhaust every day, keep compost moistening, humidity treats that 80～85% it is long to 5～8 young leaves transplanting lands for growing field crops.

2. a kind of efficient industrialization as claimed in claim 1 method of producing the sweet potato tissue cultural seedlings of free is characterized in that:

Growing the culture medium prescription that the body bud induces outward is: MS+6-BA 0～0.5mg/L+IBA 0～0.5mg/L+ sugar 30g+ activated carbon 3～6g+ agar 6g;

The culture medium prescription that shoot proliferation is cultivated is: improvement MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g;

The culture medium prescription of culture of rootage is: improvement 2/3MS+IBA 0～0.5mg/L+ sugar 30g+ agar 6g+ activated carbon 3～6g.