CN114766367A - Kiwi fruit tissue culture method - Google Patents
Kiwi fruit tissue culture method Download PDFInfo
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- CN114766367A CN114766367A CN202210537618.6A CN202210537618A CN114766367A CN 114766367 A CN114766367 A CN 114766367A CN 202210537618 A CN202210537618 A CN 202210537618A CN 114766367 A CN114766367 A CN 114766367A
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- 235000009436 Actinidia deliciosa Nutrition 0.000 title claims abstract description 85
- 244000298697 Actinidia deliciosa Species 0.000 title claims abstract description 84
- 238000012136 culture method Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 23
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 15
- 229930006000 Sucrose Natural products 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000005286 illumination Methods 0.000 claims description 24
- 230000035755 proliferation Effects 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 22
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- 229910052902 vermiculite Inorganic materials 0.000 claims description 12
- 235000019354 vermiculite Nutrition 0.000 claims description 12
- 239000010455 vermiculite Substances 0.000 claims description 12
- 239000012883 rooting culture medium Substances 0.000 claims description 10
- 235000009434 Actinidia chinensis Nutrition 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 230000003203 everyday effect Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- 238000012364 cultivation method Methods 0.000 claims description 3
- 239000012882 rooting medium Substances 0.000 claims description 3
- 229960004793 sucrose Drugs 0.000 abstract description 13
- 241000196324 Embryophyta Species 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000004161 plant tissue culture Methods 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 230000029553 photosynthesis Effects 0.000 abstract description 3
- 238000010672 photosynthesis Methods 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 230000000243 photosynthetic effect Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000004659 sterilization and disinfection Methods 0.000 description 16
- 238000005406 washing Methods 0.000 description 14
- 238000005520 cutting process Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000009423 ventilation Methods 0.000 description 7
- 206010020649 Hyperkeratosis Diseases 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 238000005057 refrigeration Methods 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 239000005708 Sodium hypochlorite Substances 0.000 description 4
- 239000012459 cleaning agent Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000002420 orchard Substances 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 244000298715 Actinidia chinensis Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241000722923 Tulipa Species 0.000 description 1
- 241000722921 Tulipa gesneriana Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
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- 235000021028 berry Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 230000004069 differentiation Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
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- 239000007921 spray Substances 0.000 description 1
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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
- A01H4/002—Culture media for tissue culture
-
- 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
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of plant tissue culture, and provides a kiwi fruit tissue culture method. The invention adopts photoautotrophic micropropagation technology, fully utilizes the rapidity of plant tissue culture technology and the integrity of environmental engineering regulation, does not use sugar as a plant carbon source in the culture process, and adopts CO supply2Inducing the tissue culture seedling to carry out photosynthesis, solving the problems of longer rooting period and lower efficiency of the traditional tissue culture, reducing the production cost and supplying CO2The method changes the previous heterotrophic culture mode powered by cane sugar by making the tissue culture seedling carry out photoautotrophic culture, makes the growth of the tissue culture seedling in a state close to the natural state, is more beneficial to the tissue culture seedling to adapt to the field environment, and hardly needs transplanting and domesticating time. The method solves the problems of high pollution rate of the tissue culture seedlings of the kiwi fruit, difficult rooting, long rooting period, weak photosynthetic capacity and poor seedling quality, and promotes the industrial production of kiwi fruit seedling breeding.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a kiwi fruit tissue culture method.
Background
The kiwi fruit (Actinidia Chinensis) is a woody vine plant with higher economic value, the fruit of the kiwi fruit is thick in pulp, has more juice, moderate sweetness and sourness and rich nutrition, becomes a treasure in the current fruit due to the extremely high nutritional value of vitamin C, and becomes a new cultivated fruit tree in the world nowadays.
With the increasing demand of the consumption market, the kiwi fruit industry in China enters a high-speed development stage, and high-quality and high-efficiency seedling breeding becomes a key link which is urgently needed to be broken through in the development of the whole industry. The traditional tissue culture technology can effectively obtain the clonal kiwi seedlings, but the rooting efficiency is low, the rooting period is long, and microbial pollution is easy to occur in the rooting process, thereby seriously affecting the quality of tissue culture seedlings.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for tissue culture of kiwi fruits, which does not use sugar as a plant carbon source during the rooting culture process, but adopts supply of CO2Promoting the photosynthesis of the tissue culture seedlings, being beneficial to inducing the rapid development of root systems, and solving the technical problems of easy microbial pollution, long rooting period and low rooting rate in the rooting process of the kiwi fruit tissue culture seedlings.
The invention provides a kiwi fruit tissue culture method, which comprises the following steps:
inoculating the tissue culture seedling of the kiwi fruit to a rooting culture medium, and performing rooting culture in a culture container under the illumination condition to obtain the seedling of the kiwi fruit; the rooting culture medium does not contain sugar; introducing CO into the culture container in the rooting culture process2。
Preferably, said introduction of CO2The method comprises the following steps: introducing CO on the 3 rd day of the rooting culture2(ii) a In 3 to 7 days of the rooting culture, the CO2The flow rate of (A) is 0.8 to 1.2 L.min-1(ii) a At the beginning of 8d of the rooting culture, the CO2The flow rate of (A) is 1.8 to 2.2 L.min-1(ii) a CO in the environment where the rooting culture was maintained at the beginning of the culture at 3d2The concentration is 800 to 1200. mu. mol/mol-1。
Preferably, the CO is2The introducing frequency is 1 time at intervals of 15-30 min, and the introducing time is 3-8 min each time.
Preferably, the lighting conditions include: in 0-7 d of the rooting culture, illuminating for 8 hours and for 16 hours in darkness every day, wherein the illumination intensity is lower than 1000 lx; and starting from the 8 th day of the rooting culture, illuminating for 14 hours and darkness for 10 hours every day, wherein the illumination intensity is 2500-3000 lx.
Preferably, the cultivation method of the kiwi fruit tissue culture seedling comprises the following steps:
1) placing the kiwi fruit stem section in an explant culture medium for explant culture until the stem section germinates axillary buds with the length more than 2 cm;
2) and transplanting the axillary buds to a proliferation culture medium for proliferation culture to obtain the kiwi fruit tissue culture seedling.
Preferably, the explant medium is water as a solvent and comprises the following components in concentrations: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L.
Preferably, the illumination intensity of the explant culture is 2500-3000 lx; the temperature for culturing the explant is 22-26 ℃.
Preferably, the proliferation medium uses water as a solvent and comprises the following components in concentration: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L and agar 6-7 g/L.
Preferably, the illumination intensity of the propagation culture is 2500-3000 lx; the temperature of the proliferation culture is 22-26 ℃.
Preferably, the rooting medium comprises a vermiculite matrix and a nutrient solution; the humidity of the rooting culture medium is more than or equal to 70 percent; the nutrient solution takes 1/2MS as a basic nutrient solution and also comprises a mass concentration of 0.5-2.0 mg.L-1IBA of (1).
The invention provides a kiwi fruit tissue culture method. The invention utilizes photoautotrophic micropropagation technology, makes full use of rapidity and environmental regulation integrity of plant tissue culture technology, does not use sugar as plant carbon source in the rooting culture process of kiwi fruit tissue culture seedlings, and supplies CO2Inducing the tissue culture seedling to carry out photosynthesis. The invention does not use sugar as a plant carbon source, solves the pollution problem in the rooting culture process, improves the rooting rate, and reduces the production cost at the same timeSupply of CO2The method changes the previous heterotrophic culture mode powered by cane sugar by making the tissue culture seedling carry out photoautotrophic culture, makes the growth of the tissue culture seedling in a state close to the natural state, is more beneficial to the tissue culture seedling to adapt to the field environment, and hardly needs transplanting and domesticating time. The method solves the problems of high pollution rate of the tissue culture seedlings of the kiwi fruits, difficult rooting, long rooting period, weak photosynthetic capacity and poor seedling quality, and promotes the industrialized production of kiwi fruit seedling breeding.
The rooting period is obviously shortened by utilizing the photoautotrophic micropropagation technology, the root system can be observed in 7d, the growth and development of plants are facilitated due to the huge root system, and the huge root system can be formed in 28 days.
Drawings
FIG. 1 shows the rooting of ` cord red ` in two culture modes, wherein g-i: performing photoheterotrophy (a traditional tissue culture rooting way CK); j-l: photoautotrophic rooting mode; g, j: culturing the 7 th strain; h, k: culturing the 14 th d; i, l: culturing at 28 d;
FIG. 2 is the rooting condition of the Yuxiang under two culture modes; wherein, a-c: performing photoheterotrophy (a traditional tissue culture rooting way CK); d-f: photoautotrophic rooting mode; a, d: culturing the 7 th strain; b, e: culturing at 14 d; c, f: culture No. 28 d.
Detailed Description
The invention provides a kiwi fruit tissue culture method, which comprises the following steps:
inoculating the tissue culture seedling of the kiwi fruit to a rooting culture medium, and carrying out rooting culture in a culture container under the illumination condition to obtain the kiwi fruit seedling; the rooting culture medium does not contain sugar; introducing CO into the culture container during the rooting culture process2。
In the invention, before the kiwi fruit tissue culture seedling is inoculated to the rooting culture medium, the callus at the bottom of the kiwi fruit tissue culture seedling is preferably cut off, so that the wound is favorable for the formation of the root.
In the present invention, the rooting medium preferably comprises a vermiculite matrix and a nutrient solution; the humidity of the rooting culture medium is more than or equal to 70 percent; the nutrient solution takes 1/2MS as a basic nutrient solution and also comprises a mass concentration of 0.5-2.0 mg.L-1IB of (1)A. In the present invention, the vermiculite is preferably subjected to a sterilization treatment; the temperature of the sterilization is preferably 121 ℃; the time for the sterilization is preferably 20 min.
In the invention, the culture container adopted by the rooting culture is preferably a photoautotrophic culture box; the photoautotrophic culture box preferably has the specification of length × width × height: the number of the tissue culture seedlings inoculated in each photoautotrophic culture box is preferably 30-35.
The vermiculite is loose and porous, and the invention takes the vermiculite as a substrate and is matched with photoautotrophic rooting culture, so that the underground environment of tissue culture seedlings can be obviously improved, the growth of the root system of the kiwi fruit is more facilitated, and the well-developed root system is more beneficial to the transplantation of the tissue culture seedlings.
In the present invention, the introduction of CO is performed2Preferably, the method comprises the following steps: introducing CO on the 3 rd day of the rooting culture2(ii) a In 3 to 7 days of the rooting culture, the CO2The flow rate of (A) is 0.8 to 1.2 L.min-1(ii) a At the beginning of 8d of the rooting culture, the CO2The flow rate of (A) is 1.8-2.2 L.min-1(ii) a CO in the environment where the rooting culture was maintained at the beginning of the culture at 3d2The concentration is 800 to 1200. mu. mol/mol-1。
In the present invention, when the variety of kiwi fruit is 'Tulipa', the CO is2The concentration of (b) is preferably 1200. mu. mol/mol-1When the variety of kiwi fruit is 'navel red', the CO2The concentration of (b) is preferably 800. mu. mol-1。
In the present invention, the CO is2The introducing frequency is preferably 1 time at intervals of 15-30 min, and when the variety of the kiwi fruit is 'Yuxiang', the CO is introduced2The introduction frequency of (2) is preferably 1 time at intervals of 15 min; when the variety of kiwi fruit is 'navel red', the CO2The introduction frequency of (2) is preferably 1 time per 30min interval; each time CO is introduced2The time for introducing (2) is preferably 3 to 8min, and more preferably 5 min.
The invention selects different CO according to different kiwi fruit varieties2The concentration and the ventilation frequency can obviously improve the rooting rate of the kiwi fruit seedlings.
In the present invention, the illumination conditions include: in 0-7 d of the rooting culture, preferably illuminating for 8h and 16h in darkness every day, wherein the illumination intensity is preferably lower than 1000 lx; preferably, the light intensity is 2500-3000 lx at the 8 th day of the rooting culture, and the light intensity is 14h and 10h in darkness every day.
In the invention, the rooting culture temperature is preferably 22-26 ℃, and more preferably 24 ℃. In the present invention, the rooting culture is preferably performed in a photoautotrophic culture chamber.
The invention solves the pollution problem in the rooting culture process without using sugar as a plant carbon source, improves the rooting rate, reduces the production cost and supplies CO2The method changes the previous heterotrophic culture mode powered by cane sugar by photoautotrophic culture of the tissue culture seedlings, enables the growth of the tissue culture seedlings to be close to a natural state, is more favorable for the tissue culture seedlings to adapt to the field environment, and almost does not need transplanting and domesticating time.
In the invention, the preferable cultivation method of the kiwi fruit tissue culture seedling comprises the following steps:
1) placing the kiwi fruit stem section in an explant culture medium for explant culture until the stem section germinates axillary buds with the length more than 2 cm;
2) transplanting the axillary buds to a proliferation culture medium, and performing proliferation culture to obtain the kiwi fruit tissue culture seedling.
Firstly, placing the kiwi fruit stem section in an explant culture medium for explant culture until the stem section germinates axillary buds with the length of more than 2 cm.
In the present invention, the variety of kiwifruit is preferably 'tulip' and 'navel red'; the kiwi fruit stem is preferably taken from a kiwi fruit young sprout; the preferable collection time of the kiwi fruit young shoots is 4-5 months in spring; the kiwi fruit young shoots are preferably subjected to first disinfection, refrigeration, cleaning and second disinfection treatment in sequence; the first disinfection is preferably to spray the young kiwi fruit shoots by using 75% alcohol by volume percentage; the refrigerating time is preferably 5-8 h; the refrigerating temperature is preferably 0 ℃; after the cold storage, the preferable method also comprises the step of cutting the cold-stored kiwi fruit young shoots into small sections with axillary buds; the length of the small section is preferably 3-5 cm; preferably, the cleaning is carried out by soaking and washing with an aqueous solution containing a cleaning agent and then washing with clear water; the time of the rinsing is preferably > 3 h; preferably, the second disinfection is that alcohol with the volume percentage of 75% is sequentially adopted to wash the washed kiwi fruit young shoots, and then the washed kiwi fruit young shoots are soaked in 1% sodium hypochlorite water solution, wherein the washing time is preferably 10-20 s, and the soaking time is preferably 5-7 min; after the second disinfection, preferably washing the second disinfected kiwi fruit stem, preferably washing with sterilized purified water, and preferably washing 5-7 times. After the kiwi fruit young shoots subjected to the second disinfection are washed, preferably, after water on the surfaces of the kiwi fruit young shoots subjected to the second disinfection is sucked, the top ends and the bottom ends are cut off, and the completeness of axillary buds is guaranteed.
In the present invention, the explant medium is water as a solvent, and preferably comprises the following components in the following concentrations: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L. In the invention, the illumination intensity of the explant culture is preferably 2500-3000 lx; the temperature of the explant culture is preferably 22-26 ℃, and more preferably 24 ℃.
After axillary buds germinate at the stem section and have the length larger than 2cm, the axillary buds are transplanted to a multiplication culture medium for multiplication culture to obtain the kiwi fruit tissue culture seedling.
In the invention, the proliferation culture is preferably carried out in 320ml culture bottles, the number of axillary buds in each culture bottle is preferably 3-5, and 3-5 tissue culture seedlings can be obtained by proliferation and differentiation of each axillary bud.
In the present invention, the proliferation medium uses water as a solvent, and preferably comprises the following components in the following concentrations: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L and agar 6-7 g/L.
In the invention, the illumination intensity of the proliferation culture is preferably 2500-3000 lx; the temperature of the proliferation culture is preferably 22-26 ℃, and more preferably 24 ℃.
In the present invention, the height of the tissue culture seedling is preferably > 3 cm. In the present invention, after cutting out the callus at the bottom of the tissue culture seedling, it is preferable that the bottom of the tissue culture seedling from which the callus at the bottom is cut out is washed, and the reagent used for washing is preferably sterile water.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1.1. Explant establishment
In spring for 4-5 months, collecting 'navel red' and 'Yuxiang' young shoots of kiwi fruits in an orchard, spraying 75% of the cut young shoots to sterilize, putting the sterilized young shoots into a refrigerator at 0 ℃ for refrigeration for 5-8 hours, taking out the young shoots after refrigeration, cutting the young shoots into small segments with leaf buds of 3-5 cm, putting the small segments into a container, squeezing 5 drops of cleaning agent into the container, adding clear water, fully shaking the small segments uniformly, and then putting the small segments under a faucet to wash the small segments with running water for more than 3 hours. And (3) putting the washed kiwi fruit stem segments on an ultra-clean workbench for disinfection treatment, washing the kiwi fruit stem segments for 10-20 s by using 75% alcohol by volume, soaking the kiwi fruit stem segments for 5-7 min by using 1% sodium hypochlorite, and washing the kiwi fruit stem segments for 5-7 times by using sterilized purified water. Placing the sterilized kiwi fruit stem segments on sterilized filter paper to absorb water, cutting off the top end and the bottom end, ensuring the integrity of axillary buds, and placing the axillary buds in an explant culture medium: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L, wherein the illumination intensity is 2500-3000 lx; the culture temperature is 24 +/-2 ℃.
1.2. Proliferation culture
The axillary buds of the stem section begin to germinate about 7 days, when the axillary buds grow to be more than 2cm, the stem section is cut off on an ultra-clean workbench, and the axillary buds are transplanted to a proliferation culture medium. After propagation culture, 3-5 seedlings are planted in one bottle, and each seedling can be propagated and differentiated by 3-5 seedlings. Proliferation culture medium: culture medium: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L and agar 6-7 g/L, wherein the illumination intensity is 2500-3000 lx; the culture temperature was 24. + -. 2 ℃.
1.3. Rooting culture
Packaging 450g vermiculite matrix in sterilization bag, sterilizing in autoclave at 121 deg.C for 20min, and packaging into photoautotrophA culture box, which is matched with 1/2MS + 0.5-2.0 mg.L-1The IBA solution keeps the humidity of the vermiculite matrix to be more than 70 percent. And (3) cutting off callus at the bottom of the tissue culture seedling with the height of more than 3cm, cleaning the bottom of the tissue culture seedling with sterile water, inoculating 30-35 seedlings in each box, and performing photoautotrophic rooting culture.
In the photoautotrophic culture chamber, CO is arranged by a controller2The concentration is 800 to 1200 mu mol/mol-1And setting time by using a controller and adopting ventilation for 15-30 min/time. Ventilation was started from day 3, and ventilation flow (1.0 + -0.2) L.min-1And in the later stage of rooting culture: ventilation flow (2.0 +/-0.2) L.min-1. The photoperiod at the early rooting stage is 8h/16h, the photoperiod at the later rooting stage is 14h/10h, the illumination intensity at the early rooting stage is lower than 1000lx, and the illumination intensity at the later rooting stage is 2500-3000 lx; the culture temperature is 24 +/-2 ℃.
Example 2
2.1 explant establishment
Collecting 'navel red' kiwi fruit young shoots in an orchard in 4-5 months in spring, spraying 75% of the cut young shoots to sterilize, putting the disinfected young shoots into a0 ℃ refrigerator for refrigeration for 5-8 hours, taking out the young shoots after refrigeration, cutting the young shoots into 3-5 cm small segments with leaf buds, putting the small segments into a container, extruding 5 drops of a cleaning agent into the container, adding clear water, fully shaking the small segments uniformly, and then putting the small segments under a faucet to wash the small segments with running water for more than 3 hours. And (3) putting the washed kiwi fruit stem segments on an ultra-clean workbench for disinfection treatment, washing the kiwi fruit stem segments for 10-20 s by using 75% alcohol by volume, soaking the kiwi fruit stem segments for 5-7 min by using 1% sodium hypochlorite, and washing the kiwi fruit stem segments for 5-7 times by using sterilized purified water. Putting the sterilized kiwi fruit stem into sterilized filter paper, sucking water, cutting off the top end and the bottom end, ensuring the integrity of axillary buds, and placing the axillary buds in an explant culture medium, wherein the culture medium comprises the following components in parts by weight: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L, wherein the illumination intensity is 2500-3000 lx; the culture temperature was 24. + -. 2 ℃.
2.2 enrichment culture
The axillary buds of the stem section begin to germinate about 7 days, when the axillary buds grow to be more than 2cm, the stem section is cut off on an ultra-clean workbench, and the axillary buds are transplanted to a proliferation culture medium. 3-5 tissue culture seedlings can be planted in each 350ml tissue culture bottle, and after proliferation culture, each seedling can proliferate and differentiate 3-5 seedlings. Proliferation culture medium: culture medium: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L and agar 6-7 g/L, wherein the illumination intensity is 2500-3000 lx; the culture temperature was 24. + -. 2 ℃.
2.3 rooting culture
Packaging 450g vermiculite matrix in sterilization bag, placing in autoclave at 121 deg.C for 20min, packaging in photoautotrophic culture box, and matching with 1/2MS + 0.5-2.0 mg.L-1IBA solution keeps the humidity of vermiculite matrix to be more than 70%. And (3) cutting off callus at the bottom of the tissue culture seedling with the height of more than 3cm, cleaning the bottom of the tissue culture seedling with sterile water, inoculating 30-35 seedlings in each box, and performing photoautotrophic rooting culture. The process of rooting by navel red is shown in figure 1.
In the photoautotrophic culture chamber, CO is arranged by a controller2The concentration is 800 to 1200 mu mol/mol-1And setting time by using a controller and ventilating for 15-30 min/time. Ventilating from day 3, and ventilating flow (1.0 + -0.2) L.min-1And in the later stage of rooting culture: ventilation flow (2.0 +/-0.2) L.min-1. The photoperiod at the early rooting stage is 8h/16h, the photoperiod at the later rooting stage is 14h/10h, the illumination intensity at the early rooting stage is lower than 1000lx, and the illumination intensity at the later rooting stage is 2500-3000 lx; the culture temperature was 24. + -. 2 ℃.
TABLE 1 influence of different culture modes on the root system index of kiwi fruit tissue culture seedlings
Example 3
3.1 explant establishment
Collecting 'Yuxiang' kiwi fruit young shoots in an orchard in 4-5 months in spring, spraying 75% of the cut young shoots to sterilize, putting the sterilized young shoots into a refrigerator with the temperature of 0 ℃ for cold storage for 5-8 hours, taking out the young shoots after cold storage, cutting the young shoots into small segments with leaf buds of 3-5 cm, putting the small segments into a container, extruding 5 drops of a cleaning agent into the container, adding clear water, fully shaking up, and then putting the small segments under a faucet and washing the small segments with running water for more than 3 hours. And (3) putting the washed kiwi fruit stem segments on an ultra-clean workbench for disinfection treatment, washing the kiwi fruit stem segments with 75% alcohol by volume for 10-20 s, soaking the kiwi fruit stem segments with 1% sodium hypochlorite for 5-7 min, and washing the kiwi fruit stem segments with sterilized purified water for 5-7 times. Putting sterilized filter paper into the sterilized kiwi fruit stem segments, sucking dry water, cutting off the top end and the bottom end, ensuring the integrity of axillary buds, and placing the axillary buds in an explant culture medium, wherein the culture medium comprises: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L
3.2 enrichment culture
The axillary buds of the stem section begin to germinate about 7 days, when the axillary buds grow to be more than 2cm, the stem section is cut off on an ultra-clean workbench, and the axillary buds are transplanted to a proliferation culture medium. 3-5 tissue culture seedlings can be planted in each 350ml tissue culture bottle, and after proliferation culture, each seedling can be proliferated and differentiated by 3-5 seedlings. Proliferation culture medium: culture medium: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L, agar 6-7 g/L
3.3 rooting culture
Packaging 450g of vermiculite into sterilization bags, placing into an autoclave at 121 ℃ for 20min, packaging into photoautotrophic culture boxes, and matching with 1/2MS + 0.5-2.0 mg.L-1The IBA solution keeps the humidity of the vermiculite matrix to be more than 70 percent. And (3) cutting off callus at the bottom of the tissue culture seedling with the height of more than 3cm, cleaning the bottom of the tissue culture seedling with sterile water, inoculating 30-35 seedlings in each box, and performing photoautotrophic rooting culture. A picture of the Yuxiang rooting process is shown in figure 2.
In the photoautotrophic culture chamber, CO is arranged by a controller2The concentration is 800 to 1200. mu. mol/mol-1And setting time by using a controller and ventilating for 15-30 min/time. Ventilating from day 3, and ventilating flow (1.0 + -0.2) L.min-1And in the later stage of rooting culture: ventilation flow (2.0 +/-0.2) L.min-1. The photoperiod at the early rooting stage is 8h/16h, the photoperiod at the later rooting stage is 14h/10h, the illumination intensity at the early rooting stage is lower than 1000lx, and the illumination intensity at the later rooting stage is 2500-3000 lx; the culture temperature was 24. + -. 2 ℃.
TABLE 2 influence of different culture modes on the root indexes of tissue culture seedlings of Kiwi berry
Although the present invention has been described in detail with reference to the above embodiments, it is to be understood that the present invention is not limited to the details of the embodiments, and that other embodiments may be devised without departing from the spirit and scope of the present invention.
Claims (10)
1. A kiwi fruit tissue culture method comprises the following steps:
inoculating the tissue culture seedling of the kiwi fruit to a rooting culture medium, and carrying out rooting culture in a culture container under the illumination condition to obtain the kiwi fruit seedling; the rooting culture medium does not contain sugar; introducing CO into the culture container during the rooting culture process2。
2. The method of claim 1, wherein the introducing CO is performed in a tissue culture system2The method comprises the following steps: introducing CO starting on the 3 rd day of the rooting culture2(ii) a In 3 to 7 days of the rooting culture, the CO2The flow rate of (A) is 0.8 to 1.2 L.min-1(ii) a At the beginning of 8d of the rooting culture, the CO2The flow rate of (A) is 1.8 to 2.2 L.min-1(ii) a CO in the environment where the rooting culture is maintained at the beginning of the culture at 3d2The concentration is 800 to 1200 mu mol/mol-1。
3. The kiwi tissue culture method of claim 2 or 3, wherein said CO is2The introducing frequency is 1 time at intervals of 15-30 min, and the introducing time is 3-8 min each time.
4. The kiwi tissue culture method of claim 1, wherein the lighting conditions comprise: in 0-7 d of the rooting culture, illuminating for 8 hours and for 16 hours in darkness every day, wherein the illumination intensity is lower than 1000 lx; and starting from the 8 th day of the rooting culture, illuminating for 14 hours and darkness for 10 hours every day, wherein the illumination intensity is 2500-3000 lx.
5. The tissue culture method of kiwi fruit of claim 1, wherein said cultivation method of kiwi fruit tissue culture seedling comprises the following steps:
1) placing the kiwi fruit stem section in an explant culture medium for explant culture until the stem section germinates axillary buds with the length more than 2 cm;
2) transplanting the axillary buds to a proliferation culture medium, and performing proliferation culture to obtain the kiwi fruit tissue culture seedling.
6. The kiwi tissue culture method of claim 5, wherein the explant media is water as a solvent, comprising the following components in concentrations: MS4.43g/L, sucrose 30g/L, NAA0.2mg/L and agar 6-7 g/L.
7. The kiwi tissue culture method of claim 5 or 6, wherein the illumination intensity of the explant culture is 2500-3000 lx; the temperature for culturing the explant is 22-26 ℃.
8. The tissue culture method of kiwi fruit of claim 5, wherein said proliferation medium is water as solvent, comprising the following components in concentration: MS4.43g/L, sucrose 30g/L, NAA 0.1.1-0.6 mg/L, 6-BA0.1mg/L and agar 6-7 g/L.
9. The kiwi tissue culture method according to claim 5 or 6, wherein the light intensity of the propagation culture is 2500-3000 lx; the temperature of the proliferation culture is 22-26 ℃.
10. The kiwi tissue culture method of claim 5, wherein the rooting medium comprises a vermiculite matrix and a nutrient solution; the humidity of the rooting culture medium is more than or equal to 70 percent; the nutrient solution is based on 1/2MSThe nutrient solution also comprises 0.5-2.0 mg/L of mass concentration-1The IBA of (1).
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