CN106386492B - Induction method of loose embryonic callus of potato - Google Patents

Abstract

The invention discloses an induction method of loose embryonic callus of potato, which comprises the following steps of firstly taking potato tubers as explants, and inducing the loose embryonic callus of potato on a low-osmotic MS culture medium containing 2, 4-D; then transferring the soft callus to a normal osmotic pressure MS culture medium containing the mitogen and 2,4-D to induce the loose callus, and finally obtaining the loose potato callus through multiple subculture and selective inoculation of the explant; transferring the obtained potato loose callus onto a culture medium containing a certain concentration of the mitogen and 2,4-D, carrying out subculture for multiple times, selecting a proper culture temperature, and combining selective inoculation on the callus during the subculture to finally obtain the potato callus with loose structure and embryogenic characteristics. The invention establishes a set of induction system of loose embryonic callus of potatoes and lays a foundation for the virus-free culture of potatoes.

Description

Induction method of loose embryonic callus of potato

Technical Field

The invention belongs to the technical field of tissue culture, and relates to an induction method for obtaining loose embryonic callus of potatoes by utilizing a tissue culture technology.

Background

The potato is commonly called as potato in the vast crop seed area of China, and the potato is edible ' can be used as a dish ' and can be used for raising ' processable grain crops, and is a grain economic crop which has high yield and high benefit and is suitable for being cultivated in the vast crop area of China. In recent years, extensive researches on cultivation and breeding of potatoes are carried out in a wide area in China, so that more breakthroughs are made, and particularly, the potato tissue culture is remarkably carried out.

Tubers are mostly used for potato production for planting, after the new species is popularized, viruses are accumulated in the tubers along with the increase of vegetative generation propagation of the seed potatoes along with the increase of the planting age, so that the plants are often shortened and clustered in the field, the stems are thin and weak, and the branches are reduced; the blades are wrinkled, curled and reduced; leaves are alternate yellow and green, lose green, are abnormal in color and decline in growth; the tuber becomes small, deforms and cracks, and is necrotic inside; the yield is reduced year by year, the excellent characteristics of the variety are continuously lost, the production potential and the commodity value are seriously influenced, the variety is rapidly increased along with the increase of the service life, and finally the planting value is lost, which is the phenomenon that the potato seeds are degraded. At present, the potato seed degeneration is generally accepted to be caused by virus hazards in the world.

The core technology of potato detoxification is a stem tip tissue culture (called meristem culture) detoxification technology. The characteristic that the stem tip has no virus is utilized, under the aseptic operation condition, the virus disease, fungus and bacterial disease which mainly harm the potato are removed through stem tip stripping, and the nontoxic test-tube plantlet is cultivated by using the artificially prepared culture medium (the stem tip tissue culture plantlet is subjected to virus detection, and the stem tip plantlet still carrying the virus is eliminated). Then the test-tube plantlet is used for breeding the miniature potato breeder seeds in a greenhouse or a net shed, and the breeder seeds are used for breeding the breeder seeds and the improved seeds are used for production under the isolation condition.

At present, the potato detoxification culture mainly depends on a stem tip detoxification technology, but reports of tissue detoxification by using loose embryogenic callus are few, wherein the dispute about whether detoxification can be carried out by using the technology also has a difficult obstacle to establishing the loose embryogenic callus of the potato. There have been some reports on the studies of plant tissue detoxification using callus culture, and success has been achieved in some plant studies, but there has been no report on potato, and it can be preliminarily judged that this may be due to the difficulty of induction of potato embryogenic callus.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an induction method of loose embryonic callus of potatoes. The invention aims to establish a set of loose embryonic callus induction system for potatoes, lays a foundation for virus-free culture of the potatoes, can be used for research on somatic cell mutation breeding, and widens the approach for a breeding method of the potatoes.

The technical scheme for realizing the purpose of the invention is as follows:

a method for inducing loose embryogenic callus of potato comprises the following steps:

preparation of potato explant

Selecting a part with an outer skin; selecting the part with buds to perform disinfection treatment;

inducing soft callus of potato

Inoculating the potato explants which are sterilized to a culture medium which is prepared in advance and contains 1/2-1/4MS +1.0-2.0mg/L2 and 4-D +15-20g/L sucrose; carrying out induction culture on the soft callus; adding 30-50ml of culture medium into each container; the culture conditions were: irradiating at 24-25 deg.C for 24 hr; the illumination intensity is 1000 Lux; culturing for 30-40 days; soft and light white callus can be induced on the periphery of the potato slices;

induction of potato loose callus

Transferring the induced soft callus of the potato to a culture medium of MS +0.5-1.0mg/LBA +0.25-0.5mg/L2,4-D +30g/L sucrose to induce the loose callus; care is taken during the transfer of the callus; otherwise, soft callus tissue is injured; the callus is too small to die; subculturing for 10-14 days in culture; selecting hard callus as much as possible in the subculture process; gradually eliminating the color-changing and soft callus in the process of subculture; adding 20-40ml of culture medium into each container; the culture conditions were: irradiating at 23-24 deg.C for 24 hr; the illumination intensity is 2000-; performing 3-5 times of subculture; the light green callus with moderate growth speed, hard texture and loose structure can be induced;

fourth step of induction of loose embryonic callus of potatoes

Taking out the potato loose callus induced in the step three in a super clean bench; transferring to a culture medium of MS +1.0-1.5mg/LBA +0.5-1.0mg/L2,4-D +30g/L sucrose to induce the loose embryonic callus; during the callus culture process; subculturing every 15-20 days; selecting a callus group which has a loose structure and a light green color and is granular during subculture and reserving the callus group; simultaneously, microscope examination is carried out in combination with a microscope; further determining the embryogenic property of the callus; performing 5-8 times of subculture; about 100-; then loose embryonic callus of potato can be induced; a 100ml triangular flask is adopted during induction; due to the long subculture time; this step requires the addition of 40ml of medium to each flask; the culture conditions were: irradiating at 21-22 deg.C for 24 h; the light intensity was 2500-3000 Lux.

Moreover, the detailed operation of the step is as follows:

washing potato tubers in tap water repeatedly; cutting potato tubers with a scalpel; selecting a skin-bearing part; selecting a part with buds; placing the cut potato tubers in a clean bench; cutting into small pieces with diameter of 5-10mm with scalpel; soaking tubers for 30S by using 70% ethanol; then carrying out surface disinfection treatment by using 40% antipuritic water solution; the disinfection time is 20 Min; after the disinfection is finished; washing the tuber in sterile water repeatedly for 5 times; 10Min each time; then, absorbing the excess water by using sterile absorbent paper; and the outermost layer of tissue of the tuber was excised with a scalpel.

The application has the advantages and positive effects as follows:

the invention aims to establish a potato non-toxic seedling culture system by adopting the new way of detoxification, and a high-quality non-toxic potato seed source is obtained for production. Compared with other methods relying on callus detoxification, the method adopting the loose embryonic callus for detoxification culture has the following advantages: firstly, the loose embryonic callus has less intercellular communication, which makes barriers for the transfer of virus between cells; secondly, the loose embryonic callus has a higher growth speed than that of the common callus, so that the propagation of virus among cells can be inhibited, and nontoxic cells can be obtained after multi-generation differentiation; thirdly, the embryogenic callus can easily obtain the regeneration seedling, and once the callus is detoxified, the regeneration seedling can be obtained by an embryoid regeneration mode; fourthly, the regeneration mode of the loose embryonic callus is mostly single cell regeneration, so that the regenerated seedling is not infected and infected again due to the fact that the regenerated bud is not affected by the virus-free cells, the virus-free step is simplified, the time is saved, and the efficiency is improved. Therefore, the high-efficiency and high-quality nontoxic material can be obtained in the whole view.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

Example 1

A method for inducing loose embryogenic callus of potato comprises the following steps:

preparation of potato callus culture explant

The "Zhongshu No. 5" potato was repeatedly washed in tap water, and the potato tuber was cut with a scalpel, with the selection of the part with the outer skin and not the part with the bud. The cut potato tubers were placed in a clean bench and cut into small pieces of approximately 5mm in diameter with a scalpel. Then soaking tuber 30S with 70% ethanol, and then performing surface disinfection treatment with 40% antipurimeans aqueous solution. The disinfection time is 20Min, after the disinfection is finished, tubers are repeatedly washed in sterile water for 5 times, 10Min each time, then excess water is sucked to be dry by sterile absorbent paper, and the outermost layer of tissues of the tubers are cut off by a scalpel, because the tissues are easy to brown and die under the action of disinfectant, the callus induction effect is influenced. The processed potato tuber can be used as an explant for the primary induction of the callus.

Inducing soft callus of potato

The sterilized potato tuber explants were inoculated on previously prepared soft callus induction medium of 1/4MS +1.0mg/L2,4-D +15g/L sucrose. A100 ml flask was used as a culture vessel, and 30ml of a medium was added to each flask. The culture conditions were: the illumination is carried out for 24 hours at the temperature of 25 ℃, and the illumination intensity is 1000 Lux. After 40 days of culture, soft and light white callus can be induced.

Induction of potato loose callus

The induced potato soft callus is transferred to a culture medium of MS +0.5mg/LBA +0.25mg/L2,4-D +30g/L sucrose to induce the loose callus. Care was taken during transfer of the callus, otherwise soft callus would be injured, causing the callus to die too small. In the culture, the callus with hard texture is selected as much as possible after one subculture for 10 days, and the callus with color change and fine and soft texture is gradually eliminated in the subculture process. A100 ml flask was used as a culture vessel, and 20ml of the medium was added to each flask. The culture conditions were: the illumination is carried out for 24 hours at 24 ℃, and the illumination intensity is 2000 Lux. After 3-5 times of subculture, the light green callus with moderate growth speed, hard texture and loose structure can be induced.

Fourth step of induction of loose embryonic callus of potatoes

The potato loose callus induced in the previous step was taken out in a clean bench, cut into small pieces with a diameter of 5mm in the clean bench with a scalpel, and carefully transferred to a loose embryogenic callus induction medium which was transferred to MS +1.0mg/LBA +0.5mg/L2,4-D +30g/L sucrose for subculture. In the process of callus culture, subculture is carried out once every 20d, and callus groups which are loose in structure, light green in color and granular in shape are selected and retained during subculture. And simultaneously, microscopic examination is carried out in combination with a microscope to further determine the embryogenic property of the callus. After 5-8 subcultures, the experiment was stopped when embryogenic callus appeared in the treatment, about 120 days. The callus has larger change from the appearance, wherein some callus has tumor-shaped particles on the surface, the structure is loose, the texture is compact, the callus has smaller cell individual, the cytoplasm concentration is increased, the cell nucleus is larger, the cell is in a vigorous division state and shows a high embryogenic state under a microscope. Induction experiments were performed using 100ml flasks, and 40ml of medium was added to each flask. The culture conditions were: at 22 deg.c and 24 hr in illumination at 2500 Lux.

Claims (2)

1. A method for inducing loose embryonic callus of potatoes is characterized by comprising the following steps: the method comprises the following steps:

preparation of potato explant

Selecting a part with an outer skin; selecting the part with buds to perform disinfection treatment;

inducing soft callus of potato

Inoculating the potato explants which are sterilized to a culture medium which is prepared in advance and contains 1/2-1/4MS +1.0-2.0mg/L2 and 4-D +15-20g/L sucrose; carrying out induction culture on the soft callus; adding 30-50ml of culture medium into each container; the culture conditions were: irradiating at 24-25 deg.C for 24 hr; the illumination intensity is 1000 Lux; culturing for 30-40 days; soft and light white callus can be induced on the periphery of the potato slices;

induction of potato loose callus

Transferring the induced soft callus of the potato to a culture medium of MS +0.5-1.0mg/LBA +0.25-0.5mg/L2,4-D +30g/L sucrose to induce the loose callus; care is taken during transferring the callus, otherwise soft callus is injured; the callus is too small to die; subculturing for 10-14 days in culture; selecting hard callus as much as possible in the subculture process; gradually eliminating the color-changing and soft callus in the process of subculture; adding 20-40ml of culture medium into each container; the culture conditions were: irradiating at 23-24 deg.C for 24 hr; the illumination intensity is 2000-; performing 3-5 times of subculture; the light green callus with moderate growth speed, hard texture and loose structure can be induced;

fourth step of induction of loose embryonic callus of potatoes

Taking out the potato loose callus induced in the step three in a super clean bench; transferring to a culture medium of MS +1.0-1.5mg/LBA +0.5-1.0mg/L2,4-D +30g/L sucrose to induce the loose embryonic callus; during the callus culture process; subculturing every 15-20 days; selecting a callus group which has a loose structure and a light green color and is granular during subculture and reserving the callus group; simultaneously, microscope examination is carried out in combination with a microscope; further determining the embryogenic property of the callus; performing 5-8 times of subculture; the time is 100-120 d; then loose embryonic callus of potato can be induced; a 100ml triangular flask is adopted during induction; due to the long subculture time; this step requires the addition of 40ml of medium to each flask; the culture conditions were: irradiating at 21-22 deg.C for 24 h; the light intensity was 2500-3000 Lux.

2. The method for inducing potato embryogenic callus according to claim 1, wherein: the concrete operation of the step is as follows:

washing potato tubers in tap water repeatedly; cutting potato tubers with a scalpel; selecting a skin-bearing part; selecting a part with buds; placing the cut potato tubers in a clean bench; cutting into small pieces with diameter of 5-10mm with scalpel; soaking tubers for 30S by using 70% ethanol; then carrying out surface disinfection treatment by using 40% antipuritic water solution; the disinfection time is 20 Min; after the disinfection is finished; washing the tuber in sterile water repeatedly for 5 times; 10Min each time; then, absorbing the excess water by using sterile absorbent paper; and the outermost layer of tissue of the tuber was excised with a scalpel.