CN110122335B - Induction method of salt-tolerant mutant of slow-fragrant kiwi fruit tissue culture seedling - Google Patents
Induction method of salt-tolerant mutant of slow-fragrant kiwi fruit tissue culture seedling Download PDFInfo
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- CN110122335B CN110122335B CN201910566729.8A CN201910566729A CN110122335B CN 110122335 B CN110122335 B CN 110122335B CN 201910566729 A CN201910566729 A CN 201910566729A CN 110122335 B CN110122335 B CN 110122335B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention belongs to the technical field of agricultural breeding, and particularly relates to an induction method of salt-tolerant mutants of slow-fragrance kiwi fruit tissue culture seedlings. The salt-tolerant slow-fragrance kiwi fruit plant obtained by the method is suitable for the environment of saline-alkali soil, and has important significance for improving the land utilization rate and economic benefit, and for improving the productivity of agriculture and forestry and the sustainability thereof.
Description
Technical Field
The invention belongs to the technical field of agricultural breeding, and particularly relates to an induction method of salt-tolerant mutants of slow-fragrant kiwi fruit tissue culture seedlings.
Background
'Xuxiang' kiwi fruit is a perennial deciduous vine plant of the genus Actinidia of Actinidiaceae, and is one of the best comprehensive quality varieties of delicious kiwi fruit. The 'xuxiang' kiwi fruit is mainly planted in Jiangsu regions, and is also introduced and planted in Shaanxi, Henan and other places, and the salt tolerance of the kiwi fruit needs to be improved.
China is one of the most serious countries affected by land salinization, and the prevention and treatment of the salinization soil can be realized by cultivating salt-tolerant varieties; the technology of screening plant somatic cell salt-tolerant mutants by using a tissue culture technology becomes an important subject of research in the field of salt-tolerant plant breeding, but the research on the induction scheme of the salt-tolerant mutants of the 'slow-fragrance' kiwi fruit tissue culture seedlings still belongs to a blank at present.
Disclosure of Invention
Aiming at the problem that the conventional 'slow-fragrance' kiwi fruit variety is poor in salt tolerance, the invention provides an induction method of a slow-fragrance kiwi fruit tissue culture seedling salt-tolerant mutant.
The invention discloses an induction method of salt-tolerant mutants of slow-fragrant kiwi fruit tissue culture seedlings, which is characterized by comprising the following steps of:
1) establishing an aseptic system:
picking healthy and exuberant-growing slow-fragrance kiwi fruit leaves, cleaning the surfaces of the leaves, disinfecting a superclean workbench, a culture medium and other experimental articles, treating disinfectant combination by using 75% ethanol for 15 seconds, and treating mercuric chloride for 4 minutes; fully rinsing the explant by using sterile water for four to five times after disinfection, wherein the dosage of the sterile water is used for submerging the explant each time;
different disinfection treatments have obvious difference on the disinfection effect of the slow-fragrance kiwi fruit explant. Compared with the mortality rate, the mortality rate is lowest 0 in the case of treating with 75% ethanol for 15 seconds and treating with mercuric chloride for 4 minutes, but the survival rate and the pollution rate of the optimal disinfectant combination are considered, namely treating with 75% ethanol for 15 seconds and treating with mercuric chloride for 4 minutes, the mean value of the survival rate is 95.00%, the mean value of the pollution rate is 0, and the mean value of the mortality rate is 5.00%.
2) Callus induction:
cutting leaves into square blocks of 1cm multiplied by 1cm, and inoculating the square blocks into a culture medium with the leaf surfaces upward, wherein the culture medium is MS + TDZ1.0mg/L + IBA0.15mg/L; lightly pressing to make it adhere to the culture medium, inoculating, culturing in a culture chamber, performing shading and dark treatment for 5 days, and then culturing under light for 25 days to form callus;
the induction of the 'xuxiang' kiwifruit callus is closely related to cytokinin and auxin, and balance adjustment between the cytokinin and the auxin is needed; by adopting the culture medium disclosed by the patent through experiments, the callus is green in color, is in a compact block shape, grows fast, has good callus quality and can induce 100% of callus.
3) EMS induction:
inoculating the surviving callus into a culture medium by adopting a mixed culture method, wherein the culture medium is agar 4.5g/L + sucrose 30.0g/L, and the pH = 5.8-6.0; filtering and sterilizing EMS solution prepared by phosphate buffer solution, wherein the concentration of the EMS solution is 0.4g/L, adding the EMS solution into a culture medium by an addition method, and growing for 20 days to obtain a plant induced by EMS;
EMS concentration is one of the core problems of the invention: EMS with the concentration is added into a proliferation culture medium which is not solidified at the temperature of about 50 ℃ and is uniformly vibrated, callus with good growth potential is inoculated after the culture medium is solidified, the average lethality of the callus is 53.33 percent after 20 days, and the survival callus is almost half browned and dead, so the culture medium is added by an addition method, and the survival callus can be half killed after 20 days.
4) Adventitious bud induction and differentiation:
inoculating the callus materials which grow well into a culture medium, wherein the culture medium is MS +6-BA1.0mg/L + NAA0.2mg/L, and placing the inoculated callus materials into a culture chamber for culture;
the adventitious bud induced differentiation result of the slow fragrant kiwi fruit needs to consider the bud number, the increment coefficient, the average seedling height and the average bud number, the conditions of the seedling height and the hormone dosage are integrated, the combined hormone dosage for the treatment is less, the average bud number of the adventitious bud is 6.43, and the average bud height is 1.37 cm.
5) Screening salt-tolerant plants:
after callus survived under EMS induction is cultured into a complete plant, the plant is inoculated into a salt-containing culture medium with the salt concentration of 1.0 percent, the culture medium is MS culture medium + TDZ1mg/L + IBA0.15mg/L + sucrose 25g/L + agar 4.5g/L, the pH value is 5.8, after the growth of the salt-containing culture medium for 15 days, the plant is transferred into a salt-free proliferation culture medium for 15 days, and salt-resistant screening is carried out in this way; alternately culturing for 3 periods, transferring the survived seedlings to a salt-free proliferation culture medium for rejuvenation, and obtaining the salt-tolerant plants of the actinidia arguta seedlings.
After the kiwi fruit is inoculated into the salt culture medium with the salt concentration for 20 days, the lethality rate of the 'slow-fragrance' kiwi fruit plant reaches 95%, the concentration is critical concentration, and the salt tolerance of the screened and survived mutant is stronger;
at present, the area of saline-alkali soil in China is large, and the development space of saline-alkali soil is wide. The 'slow fragrance' kiwi fruit has good comprehensive properties and high economic value, but the salt tolerance of the kiwi fruit has a space for improving. EMS induction is carried out in a tissue culture system, NaCl is used as a salt-tolerant selection agent, and salt-tolerant mutants are further screened to breed the slow-fragrance kiwi fruit seedlings with higher salt-tolerant capability. Tests prove that the mutant plant obtained by the induction method of the invention and a normal plant are placed in a culture medium with the lethal concentration of 95% and the salt content of 1.0%, the normal plant is obviously damaged by salt after one week, and the mutant plant grows normally, so that the mutant plant with salt tolerance is preliminarily obtained. After the method is applied, the popularization and planting area of the 'slow-fragrance' kiwi fruit can be increased, the saline-alkali soil can be effectively developed and utilized, the land utilization rate and the economic benefit can be increased, and the method has important significance on the productivity of agriculture and forestry and the sustainability of the agriculture and forestry.
6) Rooting induction:
selecting a tissue culture seedling with good growth potential and inoculating the tissue culture seedling into a culture medium, wherein the culture medium is MS + IBA0.75mg/L + AC0.30g/L, removing callus during inoculation, cutting a stem part, and placing the stem part into a culture chamber for culture after inoculation;
the rooting of the 'slow fragrant' kiwi fruit tissue culture seedling is mainly auxin, the auxin IBA with strong action, long action time and many and long induction roots is selected, simultaneously, the rooting induction is carried out by combining active carbon, the average rooting number is 8.91, the average root length reaches 9.38cm, and the average rooting rate reaches 98.33 percent by adopting the culture medium.
7) Hardening and transplanting seedlings:
selecting humus soil: garden soil: transplanting tissue culture seedlings which grow vigorously and normally take roots to natural light according to a mixed matrix of turfy soil in a ratio of 1:1:1, moving tissue culture bottle caps after the tissue culture seedlings grow for 10 days, loosening the tissue culture bottle caps, opening the tissue culture bottle caps and covering the tissue culture bottle caps with gauze after 5 days, taking out the tissue culture seedlings after 5 days, washing the culture medium attached to roots with running water, disinfecting and soaking the culture medium with a carbendazim solution for 5 minutes, drying surface water in the shade, planting the tissue culture seedlings into different soil matrixes subjected to disinfection treatment in advance by using a carbendazim bactericide, placing the soil matrixes in the shade, covering flowerpots with plastic covers, keeping the temperature at 26 +/-3 ℃, and controlling the spraying water mist humidity to be more than 80%;
the average survival rate of the substrate combination reaches 83.33 percent, and the substrate combination is the optimal substrate combination; the matrix can be one or more of humus soil, garden soil, turfy soil or vermiculite: the vermiculite is loose and porous, has light weight, can absorb a large amount of water, and has strong water retention, fertilizer retention, heat absorption and heat preservation capabilities; the turfy soil is formed by rotting plant remains, and has good water retention property and strong fertilizer storage capacity; the humus is formed by rotting fallen leaves and contains a large amount of mineral nutrients and organic substances; the garden soil has good viscosity and certain fertility. Tests prove that when the humus soil and the turfy soil are mixed for use, the method is more suitable for the survival of the tissue culture seedlings of the 'slow fragrance' kiwi fruit. The hardening seedling transplanting effects of the four matrixes are sequenced: turfy soil, humus soil, garden soil and vermiculite.
Drawings
FIG. 1 shows the growth of ` slow fragrance ` kiwi mutant plants on medium containing 1% salt.
FIG. 2 shows the growth of both 'slow fragrance' kiwi normal and mutant plants in 1.0% salt medium.
Detailed Description
Example 1: picking healthy and exuberant-growing slow-fragrance kiwi fruit leaves, cleaning the surfaces of the leaves, disinfecting a superclean workbench, a culture medium and other experimental articles, treating disinfectant combination by using 75% ethanol for 15 seconds, and treating mercuric chloride for 4 minutes; fully rinsing the explant by using sterile water for four to five times after disinfection, wherein the dosage of the sterile water is used for submerging the explant each time;
cutting leaves into square blocks of 1cm multiplied by 1cm, and inoculating the square blocks with the leaf surfaces upward into a culture medium, wherein the culture medium is MS + TDZ1.0mg/L + IBA0.15IBAmg/L; lightly pressing to make it adhere to the culture medium, inoculating, culturing in a culture chamber, performing shading and dark treatment for 5 days, and then culturing under light for 25 days to form callus;
inoculating the callus materials which grow well into a culture medium, wherein the culture medium is MS +6-BA1.0mg/L + NAA0.2mg/L, and placing the inoculated callus materials into a culture chamber for culture;
inoculating the surviving callus into a culture medium, wherein the culture medium is agar 4.5g/L + sucrose 30.0g/L, and the pH = 5.8-6.0; filtering and sterilizing EMS solution prepared by phosphate buffer solution, wherein the concentration of the EMS solution is 0.4g/L, adding the EMS solution into a culture medium by an addition method, and growing for 20 days to obtain a plant induced by EMS;
after callus survived under EMS induction is cultured into a complete plant, the plant is inoculated into a salt-containing culture medium with the salt concentration of 1.0 percent, the culture medium is MS culture medium + TDZ1mg/L + IBA0.15mg/L = + sucrose 25g/L + agar 4.5g/L, the pH is 5.8, after the growth of the salt-containing culture medium for 15 days, the plant is transferred into a salt-free proliferation culture medium for 15 days, and salt-tolerant screening is carried out in this way; alternately culturing for 3 periods, transferring the survived seedlings to a salt-free proliferation culture medium for rejuvenation to obtain salt-tolerant plants of the actinidia arguta seedlings;
selecting a tissue culture seedling with good growth potential and inoculating the tissue culture seedling into a culture medium, wherein the culture medium is MS + IBA0.75mg/L + AC0.30g/L, removing callus during inoculation, cutting a stem part, and placing the stem part into a culture chamber for culture after inoculation;
selecting humus soil: garden soil: transplanting tissue culture seedlings which grow vigorously and normally and take roots to natural light according to a matrix mixed by turfy soil in a ratio of 1:1:1 for growing for 10 days, loosening a tissue culture bottle cap, opening the tissue culture bottle cap and covering with gauze after 5 days, taking out the tissue culture seedlings after 5 days, washing the culture medium attached to the roots with running water, disinfecting and soaking for 5 minutes with a carbendazim solution, drying surface water in the air in a shade and cool environment, planting the surface water into different soil matrixes subjected to disinfection treatment in advance with a carbendazim bactericide, placing the soil matrixes in the shade and cool place, covering a flowerpot with a plastic cover, keeping the temperature at 26 +/-3 ℃, and controlling the spraying water mist humidity to be more than 80%.
Claims (1)
1. The induction method of the slow-release kiwi fruit tissue culture seedling salt-tolerant mutant is characterized by comprising the following steps:
1) establishing an aseptic system:
picking healthy and exuberant-growing slow-fragrance kiwi fruit leaves, cleaning the surfaces of the leaves, disinfecting a superclean workbench, a culture medium and other experimental articles, treating disinfectant combination by using 75% ethanol for 15 seconds, and treating mercuric chloride for 4 minutes; fully rinsing the explant by using sterile water for four to five times after disinfection, wherein the dosage of the sterile water is used for submerging the explant each time;
2) callus induction:
cutting leaves into square blocks of 1cm multiplied by 1cm, and inoculating the square blocks into a culture medium with the leaf surfaces upward, wherein the culture medium is MS + TDZ1.0mg/L + IBA0.15mg/L; lightly pressing to make it adhere to the culture medium, inoculating, culturing in a culture chamber, performing shading and dark treatment for 5 days, and then culturing under light for 25 days to form callus;
3) EMS induction:
inoculating the surviving callus into a culture medium, wherein the culture medium is agar 4.5g/L + sucrose 30.0g/L, and the pH = 5.8-6.0; filtering and sterilizing EMS solution prepared by phosphate buffer solution, wherein the concentration of the EMS solution is 0.4g/L, adding the EMS solution into a culture medium by an addition method, and growing for 20 days to obtain a plant induced by EMS;
4) adventitious bud induction and differentiation:
inoculating the callus materials which grow well into a culture medium, wherein the culture medium is MS +6-BA1.0mg/L + NAA0.2mg/L, and placing the inoculated callus materials into a culture chamber for culture;
5) screening salt-tolerant plants:
after callus survived by EMS induction is cultured into a complete plant, the plant is inoculated into a salt-containing culture medium with the salt concentration of 1.0 percent, the culture medium is an MS culture medium, 1mg/LTDZ, 0.15mg/LIBA, 25g/L of cane sugar and 4.5g/L of agar, the pH value is 5.8, after the salt-containing culture medium grows for 15 days, the plant is transferred into a salt-free proliferation culture medium to grow for 15 days, and salt-resistant screening is carried out in this way; alternately culturing for 3 periods, transferring the survived seedlings to a salt-free proliferation culture medium for rejuvenation to obtain salt-tolerant plants of the slow-fragrant kiwi fruit seedlings;
6) rooting induction:
selecting a tissue culture seedling with good growth potential and inoculating the tissue culture seedling into a culture medium, wherein the culture medium is MS + IBA0.75mg/L + AC0.30g/L, removing callus during inoculation, cutting a stem part, and placing the stem part into a culture chamber for culture after inoculation;
7) hardening and transplanting seedlings:
selecting humus soil: garden soil: transplanting tissue culture seedlings which grow vigorously and normally and take roots to natural light according to a matrix mixed by turfy soil in a ratio of 1:1:1 for growing for 10 days, loosening a tissue culture bottle cap, opening the tissue culture bottle cap and covering with gauze after 5 days, taking out the tissue culture seedlings after 5 days, washing the culture medium attached to the roots with running water, disinfecting and soaking for 5 minutes with a carbendazim solution, drying surface water in the air in a shade and cool environment, planting the surface water into different soil matrixes subjected to disinfection treatment in advance with a carbendazim bactericide, placing the soil matrixes in the shade and cool place, covering a flowerpot with a plastic cover, keeping the temperature at 26 +/-3 ℃, and controlling the spraying water mist humidity to be more than 80%.
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