CN113973717A - Germination accelerating and seedling raising method for bulbil konjak tissue culture micro-corms - Google Patents

Germination accelerating and seedling raising method for bulbil konjak tissue culture micro-corms Download PDF

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CN113973717A
CN113973717A CN202111434758.2A CN202111434758A CN113973717A CN 113973717 A CN113973717 A CN 113973717A CN 202111434758 A CN202111434758 A CN 202111434758A CN 113973717 A CN113973717 A CN 113973717A
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tissue culture
culture
seedling
bulbil
bud
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李金威
岩香甩
田耀华
魏丽萍
龚燕雄
黄菁
原慧芳
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Yunnan Institute of Tropical Crops
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Yunnan Institute of Tropical Crops
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
    • A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
    • A01G24/25—Dry fruit hulls or husks, e.g. chaff or coir
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
    • A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
    • 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

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  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
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  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides a method for accelerating germination and seedling raising of bulbil konjak tissue culture micro-corms, belonging to the technical field of vegetative propagation of plants. The seedling culture method comprises the processes of tissue culture seedling culture, seedling hardening and tissue culture microsphere stem germination acceleration seedling culture. The bulbil konjak tissue culture micro-corm can be cultivated in each time period within one year and is used as a propagation material for planting in the next year; the surface of the tissue culture microsphere stem is provided with a plurality of germination points, the buds have growing points and cell totipotency, and a complete plant can be formed by taking down the cuttage and can grow normally. According to the characteristics, the bud is taken for planting after the bud is accelerated to germinate the bulbil konjak tissue culture micro-bulb, so that the bulb propagation coefficient can be effectively improved, the seed consumption and seed cost are reduced, the yield and economic benefit are increased, and the development of konjak industry is accelerated.

Description

Germination accelerating and seedling raising method for bulbil konjak tissue culture micro-corms
Technical Field
The invention belongs to the technical field of vegetative propagation of plants, and particularly relates to a method for accelerating germination and seedling culture of bulbil konjac tissue culture corms.
Background
The bulbil konjak is a general name of perennial herb plants in the genus of konjak of the family of araceae, and a group of konjak with aerial bulblets growing on the leaf surface, is the only plant containing a large amount of glucomannan in nature so far, and is widely applied to the fields of food, medical treatment, industry and the like. Because of high yield and strong disease resistance, the fertilizer is suitable for planting in high-temperature and high-humidity environment and is popular with growers; in recent years, the rapid enlargement of planting area and the lack of germplasm become main restriction factors for development. At present, propagation materials adopted in the planting industry mainly comprise bulbs, bulbels and a small part of seedling seeds, but a conventional planting mode is usually adopted, only one bulb is still harvested after one bulb (bulbels and seedling seeds) is sown, the yield is higher than that of the white konjak and the flower konjak, but the growth characteristics of multiple seedlings are not fully utilized.
Kunming plant research institute of Chinese academy of sciences discloses a bulbil konjak seedling breeding method (application number 200410040723.0), which comprises the steps of collecting bulbil konjak in the field, slicing leaves, petioles, roots, tuber buds, scale leaves and tubers of the plant as explants, and completing tissue culture of the bulbil konjak for the first time through bud induction and proliferation, induction of similar tubers, rooting and transplantation; wufuping and the like carry out tissue culture propagation and rapid propagation of the amorphophallus bulbifer, and the coleoptiles of the amorphophallus bulbifer are taken as explants, and callus induction, adventitious bud formation, rooting, seedling hardening and transplanting are carried out to obtain the rooting seedlings of the amorphophallus bulbifer, and the survival rate reaches 90 percent; kunming academy (application No. 201611000366.4) changes the induction development and growth path of explants by tissue culture technology, guides the growth and development path from the organ generation path to the embryoid generation path by adding corresponding growth regulating substances, directly obtains a large amount of mature bulbil konjac balls by in vitro culture, is used for sowing and further grows and expands, and then harvests and improves the yield of the bulbil konjac. Obviously, the prior art does not describe the large-scale propagation and seedling raising by utilizing the vegetative propagation characteristics of the bulbil konjak tissue culture seedling bulbodium.
Disclosure of Invention
In view of the above, the present invention provides a technique for accelerating germination and seedling raising by using bulbil konjac tissue culture microsphere stems, and researches on a tissue culture technique and a seedling hardening mechanism of bulbil konjac to obtain a large amount of bulbil konjac seedlings, accelerate industrial development and expand large-scale planting area, aiming at the problems of rapid development, insufficient germplasm, long natural propagation period and low yield of the current bulbil konjac.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for accelerating germination and seedling culture of bulbil konjak tissue culture microspheres comprises the following steps:
taking a bulbil konjak explant, and performing callus induction, adventitious bud proliferation and rooting culture to obtain an aseptic tissue culture seedling; transplanting and hardening the aseptic tissue culture seedlings, and cultivating and harvesting the microspheres; accelerating germination of the microsphere stems, and taking off bud heads for transplantation and culture after the microsphere stems grow bud heads.
Preferably, the callus induction and adventitious bud induction medium consists of: MS +6-BA1-2mg/L + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 25-32 g/L.
Preferably, the adventitious bud propagation medium consists of: MS +6-BA1-2mg/L + IAA 0.3-0.6mg/L + agar 6.5-8g/L + sucrose 25-32 g/L.
Preferably, the rooting medium consists of: 1/2MS + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 13-17 g/L.
Preferably, the seedling hardening matrix consists of 2-4 parts of peat soil, 0-2 parts of laterite and 1-2 parts of coconut coir by volume, and the water content of the matrix is 65% -82%.
Preferably, the seedling exercising process comprises the following steps: shading degree is 60% -80%, temperature is 25-32 ℃, and air humidity is 65% -92%.
Preferably, the weight of the micro-bulbs is 1-3 g.
Preferably, theThe pregermination method comprises the following steps: covering a layer of paper on the harvested microsphere, and spraying clear water or 40-60mg/L GA3Or 40-60mg/L IAA, until the paper surface is wet but no water drops.
Preferably, the bud picking comprises: and (3) when the micro-corm grows to have a bud head with the length of more than 1cm, taking a bud at the joint of the bud root and the surface of the micro-corm.
Preferably, the micro-bulbs are repeatedly subjected to pregermination, bud taking and transplanting culture until the bulbs are shrunk and aggravated, have soft texture and are transplanted and sown.
Compared with the prior art, the invention has the following beneficial effects:
(1) the bulbil konjak tissue culture micro-corm can be cultivated in each period of one year, as a propagation material planted in the next year, the surface of the bulbil konjak tissue culture micro-corm is provided with a plurality of sprouting points, the sprouts have growing points and cell totipotency, and a complete plant can be formed by taking down cuttage and can grow normally.
(2) The callus induction rate, the adventitious bud induction rate, the proliferation rate and the rooting rate of the tissue culture seedling cultivation method are high, and the method is simple and easy to operate.
(3) The seedling hardening method of the amorphophallus bulbifer can effectively improve the survival rate of the amorphophallus bulbifer tissue culture seedlings to 92% or above, and is beneficial to promoting the development of the amorphophallus bulbifer industry.
(4) The growth cycle of the tissue culture micro-bulb is short, under the condition that the tissue culture seedling is sufficiently supplied, the micro-bulb of the amorphophallus bulbifer with the single weight of about 1-3g can be harvested in 1-2 months, 3-4 rounds can be harvested in one year, the propagation is carried out for 2-3 times, the volume is small, and the storage and the transportation are convenient.
(5) The germination accelerating seedling method can effectively improve the sprouting rate of the tissue culture microsphere stems, expand the bulbil konjak seedlings, double the propagation coefficient, reduce the seed consumption and seed consumption cost, improve the economic benefit, and is simple and easy to operate.
Detailed Description
The invention provides a method for accelerating germination and seedling culture of bulbil konjak tissue culture microspheres, which comprises the following steps: taking a bulbil konjak explant, and performing callus induction, adventitious bud proliferation and rooting culture to obtain an aseptic tissue culture seedling; transplanting and hardening the aseptic tissue culture seedlings, and cultivating and harvesting the microspheres; accelerating germination of the microsphere stems, and taking off bud heads for transplantation and culture after the microsphere stems grow bud heads.
The invention preferably selects the bulbil konjak explant as underground corms or leaf surface bulbils, and further preferably selects the healthy and full explant material without mechanical damage, diseases and mildewing. After the explant is collected, cleaning impurities on the surface of the explant by using clear water, and cutting off the epidermis for later use.
The present invention preferably performs a sterilization treatment on the explant material, and further preferably the sterilization treatment comprises: washing the peeled explant with flowing water for 30min, taking out, washing with sterile water for 2 times, transferring to a sterile operating table, soaking with 75% alcohol for 20min, and soaking with 0.1% mercuric chloride solution for 20min and washing with sterile water for 3 times.
After the explant is sterilized, the explant is cut into small pieces by being transferred to a sterile operating table. Preferably the volume of the small block is 0.5-1.0cm3(ii) a Further preferably, the volume of the small block is 1 cm. times.1 cm.
And inoculating the small explant blocks into a callus induction culture medium to induce the formation of the callus. The preferable callus induction culture medium of the invention comprises the following components: MS +6-BA1-2mg/L + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 25-32 g/L; further preferred compositions are: MS +6-BA 1.5mg/L + NAA0.1mg/L + agar 7g/L + sucrose 30 g/L. By using the callus induction culture medium, a large amount of callus can be formed after 1 month.
Transferring the explants forming the callus to an adventitious bud induction culture medium to induce the adventitious bud. The preferred adventitious bud induction culture medium of the invention comprises the following components: MS +6-BA1-2mg/L + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 25-32 g/L; further preferred compositions are: MS +6-BA1mg/L + NAA0.5mg/L + agar 7g/L + sucrose 30 g/L. By using the adventitious bud induction culture medium, a large number of cluster buds are formed on the explant after 20-25 days of culture under illumination.
Taking out the cluster buds on an aseptic operating platform, and inoculating the cluster buds to an adventitious bud multiplication culture medium for culture after plant division. The preferred adventitious bud propagation culture medium comprises the following components: MS +6-BA1-2mg/L + IAA 0.3-0.6mg/L + agar 6.5-8g/L + sucrose 25-32 g/L; further preferred compositions are: MS +6-BA2mg/L + IAA 0.4mg/L + agar 7g/L + sucrose 30 g/L. The adventitious bud multiplication culture medium can be used for obtaining the bulbil konjak cluster seedlings of about 5cm after being cultured for 25 days under illumination.
Dividing the cluster seedlings into single plants, inoculating the single plants to a rooting culture medium to culture and take roots. The preferred rooting culture medium of the invention comprises the following components: 1/2MS + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 13-17 g/L; further preferred compositions are: 1/2MS + NAA 0.3mg/L + agar 7g/L + sucrose 15 g/L. The rooting culture medium is used for culturing tissue culture seedlings with the height of 8-12cm and the root length of 2-3cm for about 25-30 days, and the tissue culture seedlings can be transplanted to a greenhouse for hardening seedlings.
The invention preferably selects the stages of callus induction, adventitious bud multiplication and rooting culture, the pH of each culture medium is 5.6-6.2, the culture conditions are that the illumination is 12h every day, the temperature is 22-28 ℃, and the illumination intensity is 1000-2000 lx.
Transplanting the rooted tissue culture seedling into the mixed matrix, and hardening the seedling under a proper environment. The optimized seedling hardening environment of the invention is that the shading degree is 60-80%, the temperature is 25-32 ℃, and the air humidity is 65-92%; further preferably, the degree of shading is 75%, the temperature is 28 ℃ and the air humidity is 80%. Preferably, the rooting culture bottle is transferred to a seedling hardening greenhouse, the bottle is opened overnight at the first night, the seedling is taken out the next day, the root culture medium is cleaned by clear water and is transplanted into a seedling raising tray filled with a seedling hardening matrix.
The optimized seedling hardening matrix consists of 2-4 parts of peat soil, 0-2 parts of laterite and 1-2 parts of coconut coir by volume; more preferably, peat soil, red soil, coconut chaff, 2:1: 2.
The invention preferably disinfects the seedling exercising matrix, and further preferably disinfects the disinfection mixed solution by spraying the mixed spraying matrix with the carbendazim and the thievone in the volume ratio of 1: 1.
The method preferably sprays clear water every 2-3h by using a spraying facility 10 days before hardening off the seedlings, the time length is controlled within 1min, and the leaves of the tissue culture seedlings are kept moist at any time because the tissue culture seedlings are tender, particularly at 11-15 pm; keeping the water content of the hardening-seedling substrate between 65 and 82 percent; further preferably, the water content of the hardening-seedling substrate is kept 80%.
In the early stage of hardening seedlings, a layer of sunshade net is built above the shed, the shading degree in the shed is kept to be 60% -80%, after 10 days, new roots of the tissue culture seedlings grow out, and the sunshade net is removed; spraying clear water once in the morning and evening to keep the substrate moist. According to the invention, nitrogen fertilizer or NPK compound fertilizer is preferably applied twice a week in the early stage of seedling hardening, and the concentration of the nitrogen fertilizer is further preferably 1-5 per mill, and the ratio of N to P to K of the compound fertilizer is 15:5: 30.
After hardening off the seedlings for 45-60 days, cutting off water and fertilizer for the tissue culture seedlings, and collecting tissue culture micro-bulbs after pouring the seedlings. And cleaning the collected surface impurities of the tissue culture micro-bulbs, accelerating germination and obtaining bud heads.
Preferably selecting 1-3g of tissue culture micro-corms for accelerating germination to obtain bud heads; further preferably, 2g of tissue culture micro-bulbs are selected.
The preferred pregermination process of the invention comprises: covering a layer of paper on the harvested microsphere, and spraying clear water or 40-60mg/L GA3Or 40-60mg/L IAA, until the paper surface is wet and no water drops drop; further preferably spraying 50mg/L GA3Or IAA.
And (3) taking buds at the joint of the bud roots and the surfaces of the microsphere stems when the microsphere stems grow to form bud heads with the lengths of more than 1 cm. As an implementable way, the bud extraction tool needs to be sterilized with 75% -100% alcohol.
The bud heads which are preferably taken down are sterilized by 800-1000 mixed solution of 1-2 parts by volume of carbendazim and 1-2 parts by volume of thievone, dried in the air and dried in a ventilated place and then transplanted for planting; further, it is preferable to sterilize the mixture solution by a volume ratio of 1000 times of carbendazim and thievone of 1: 1.
The optimized bud transplantation substrate consists of 2-4 parts of peat soil, 0-2 parts of laterite and 1-2 parts of coconut coir by volume; further preferably, the volume ratio of the peat soil to the laterite to the coconut husk is 2:2: 1. Preferably, the moisture content of the substrate is kept between 20 and 45 percent; further preferably at 30%. After 10 days, the root of the bud grows new roots, normal watering and fertilization can be carried out, and the humidity of the matrix is kept to be 80% or more.
After the preferable corm takes buds, 1-2 parts by volume of carbendazim and 1-2 parts by volume of thievone are used for sterilizing 800-1000 mixed solution, and then germination accelerating, bud taking and transplanting culture are repeatedly carried out until the corm is shrunken and aggravated and has soft texture, and transplanting and sowing are carried out; further, it is preferable to sterilize the mixture solution by a volume ratio of 1000 times of carbendazim and thievone of 1: 1.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Cultivation of amorphophallus bulbifer tissue culture seedling
1.1, selecting healthy, plump, non-mechanical damage, non-disease and non-mildew leaf surface bulbils as tissue culture explants.
1.2, cleaning impurities on the surface of the explant by using clear water, and peeling off the epidermis.
1.3, washing the peeled explants with running water for 30min, taking out, washing with sterile water for 2 times, transferring to a sterile operating platform, soaking with 75% alcohol for 20min, then respectively soaking with 0.1% mercuric chloride solution for 20min and washing with sterile water for 3 times, and finally transferring to the sterile operating platform and cutting into small blocks of 1cm × 1cm × 1 cm.
1.4, callus induction: the small blocks are inoculated into a callus induction culture medium with the mixture ratio of MS +6-BA 1.5mg/L + NAA0.1mg/L + agar 7g/L + sucrose 30g/L, 5 blocks are inoculated into each bottle, and a large amount of callus is formed after 1 month.
1.5, adventitious bud induction: transferring the explant forming the callus into an adventitious bud induction culture medium with the mixture ratio of MS +6-BA1mg/L + NAA0.5mg/L + agar 7g/L + sucrose 30 g/L; after 20-25 days of culture under light, a large number of clumpy buds form on the explants.
1.6, adventitious bud multiplication culture: taking out the cluster buds on an aseptic operation platform, inoculating the cluster buds into a multiplication culture medium with the mixture ratio of MS +6-BA2mg/L + IAA 0.4mg/L + agar 7g/L + sucrose 30g/L after plant division, inoculating 20 plants in each bottle, culturing under the same illumination, and obtaining the bulbil konjac cluster seedlings with the length of about 5cm after 25 days.
1.7, finally, dividing the cluster seedlings into single plants, inoculating the single plants into rooting culture media with the mixture ratio of 1/2MS, NAA 0.3mg/L, agar 7g and cane sugar 15g/L, and transplanting the seedlings to a greenhouse for hardening the seedlings after the seedlings are 8-12cm high and 2-3cm long in roots after 25-30 days.
Wherein the pH of each culture medium is 6.0, and the culture conditions are illumination for 12h every day, temperature is 26 deg.C, and illumination intensity is 1500 lx.
2. Hardening off seedlings
2.1, mixing the seedling hardening matrix with peat soil in a volume ratio: laterite: uniformly mixing coconut chaff-2: 1:2 mixed matrix, filling the mixture into a seedling culture disc, and spraying 1000 times of mixed solution of carbendazim and thiabendazole in a volume ratio of 1:1 for disinfection; the water holding capacity of the matrix is kept at 80% in the early growth stage, and the tissue culture seedling can be watered and fertilized normally after new roots germinate.
2.2, moving the bulbil konjak tissue culture seedlings with strong plants and root systems into a seedling hardening greenhouse with the periphery being rainproof, the shading degree being 75%, the room temperature being 28 ℃ and the humidity being 70%, and opening a bottle cover for staying overnight; taking out the seedlings the next day, cleaning the root culture medium with clear water, and transplanting the seedlings into a seedling culture tray filled with the matrix.
2.3, spraying clear water every 2-3h in the first 10 days, controlling the time length within 1min, keeping the leaf surface of the tissue culture seedling moist at any time due to the tender plant of the tissue culture seedling, especially at the time interval of 11-15 pm; keeping the water content of the matrix at 80%;
2.4, building a layer of sunshade net above the shed in the early stage, keeping the shading degree in the shed at 75 percent, and removing the sunshade net after 10 days when a new root system of the tissue culture seedling grows out; spraying clear water once in the morning and at night to keep the substrate moist; nitrogen fertilizer of 1% per thousand is applied twice a week.
After 2.5 and 45-60 days, the water and fertilizer of tissue culture seedlings are cut off, and 1-3g of tissue culture microspheres can be collected after the seedlings are poured.
3. Propagating seedling by tissue culture micro-corm
3.1, selecting 2g of bulbil konjac tissue culture microspheres, cleaning surface impurities, sequentially placing the microspheres in a clean rectangular seedling culture tray, then covering a layer of newspaper on the microspheres, spraying a small amount of clear water on the newspaper every day, keeping the newspaper moist, but no drop, and carrying out germination acceleration without accumulated water in the tray.
3.2 when the buds are longer than 1cm, taking the buds at the joint of the bud root and the surface of the bulb by using a blade sterilized by 75% alcohol.
3.3, sterilizing the tissue culture micro-corms after sprouting by using 1000 times of mixed solution of carbendazim and thiacetone in a ratio of 1:1, putting the sterilized tissue culture micro-corms back to the original seedling culture plate, accelerating sprouting again, and taking sprouts.
3.4, disinfecting the taken buds by using 1000 times of mixed solution of carbendazim and thiacetone in a ratio of 1:1, airing the buds in a ventilated and dried place, and transplanting the buds to a container filled with peat soil: laterite: in the seedling raising plate of the coconut husk seedling raising matrix with the volume ratio of 2:2:1, the water content of the matrix is kept between 20 and 45 percent in the early stage.
After 3.5 days and 10 days, after new roots grow out from the root parts of the buds, normal watering and fertilizing can be carried out, and the humidity of the matrix is kept at 80 percent or above; repeating the steps until the bulbs shrink and the texture is soft, and transplanting the bulbs to a field.
The method for propagating the seedlings by the tissue culture micro-bulbs comprises the following steps: when the bulbodium konjac tissue culture bulbodium is subjected to germination acceleration for the first time, the bulbodium germination rate is 100%, the survival rate of transplanting after bud taking reaches 85%, and the root system of the base part of the main bud gradually germinates after 10 days; the bulbs are slightly shrunken at this time, and secondary pregermination can be carried out. Gradually sprouting for the second time after 20 days, wherein the sprouting rate of the bulbs for the second time is 90 percent, the sprouting number of each bulb is 1-2, the sprouts can be taken for the second time and transplanted in about 30 days, and the transplanting survival rate is 81 percent; and (3) taking the corm after the germination to be shrunken and aggravated, ensuring that the corm is soft, stopping accelerating the germination, sowing the corm into a field, and after 45 days, germinating and breaking the soil of the corm, wherein the germination rate is 78%.
Example 2
1. Cultivation of amorphophallus bulbifer tissue culture seedling
1.1, selecting healthy, plump and underground bulbs without mechanical damage, diseases and mildew as tissue culture explants.
1.2, cleaning impurities on the surface of the explant by using clear water, and peeling off the epidermis.
1.3, washing the peeled explants with running water for 30min, taking out, washing with sterile water for 2 times, transferring to a sterile operating platform, soaking with 75% alcohol for 20min, then respectively soaking with 0.1% mercuric chloride solution for 20min and washing with sterile water for 3 times, and finally transferring to the sterile operating platform and cutting into small blocks of 0.8cm × 0.8cm × 0.8 cm.
1.4, callus induction: the small blocks are inoculated into a callus induction culture medium with the mixture ratio of MS +6-BA1mg/L + NAA 0.2mg/L + agar 6.5g/L + sucrose 25g/L, and a large amount of callus is formed after 1 month.
1.5, adventitious bud induction: transferring the explant forming the callus into an adventitious bud induction culture medium with the mixture ratio of MS +6-BA1.2mg/L + NAA0.1mg/L + agar 6.5g/L + sucrose 25 g/L; after 20-25 days of culture under light, a large number of clumpy buds form on the explants.
1.6, adventitious bud multiplication culture: taking out the cluster buds on an aseptic operation platform, inoculating the cluster buds into a multiplication culture medium with the mixture ratio of MS +6-BA1mg/L + IAA 0.3mg/L + agar 6.5g/L + sucrose 25g/L after the plant division, and culturing under the illumination, wherein the cluster buds of the bulbifer can obtain the cluster buds of the bulbifer which are about 5cm after 25 days.
1.7, finally dividing the cluster seedlings into single plants, inoculating the single plants into rooting culture media with the mixture ratio of 1/2MS, NAA0.1mg/L, agar 6.5g/L and sucrose 13g/L, and transplanting the single plants into a greenhouse for hardening seedlings after 30 days.
Wherein the pH of each culture medium is 5.6, and the culture conditions are that the culture medium is irradiated for 12h every day at 22 ℃ and the illumination intensity is 1000 lx.
2. Hardening off seedlings
2.1, uniformly mixing a mixed matrix of peat soil and coconut chaff in a volume ratio of 2:1, filling the mixed matrix into a seedling tray, and spraying 800 times of mixed solution of carbendazim and thiabendazole in a volume ratio of 2:1 for disinfection; the water holding capacity of the matrix is kept 65% in the early growth stage, and the tissue culture seedling can be watered and fertilized normally after new roots germinate.
2.2, moving the bulbil konjak tissue culture seedlings with strong plants and root systems into a seedling hardening greenhouse with the periphery being rainproof, the shading degree being 60%, the room temperature being 25 ℃ and the humidity being 92%, and opening a bottle cover for staying overnight; taking out the seedlings the next day, cleaning the root culture medium with clear water, and transplanting the seedlings into a seedling culture tray filled with the matrix.
2.3, spraying clear water every 2-3h in the first 10 days, controlling the time length within 1min, keeping the leaf surface of the tissue culture seedling moist at any time due to the tender plant of the tissue culture seedling, especially at the time interval of 11-15 pm; maintaining the substrate moisture content at 82%;
2.4, building a layer of sunshade net above the shed in the early stage, keeping the shading degree in the shed at 60 percent, and removing the sunshade net after 10 days when a new root system of the tissue culture seedling grows out; spraying clear water once in the morning and at night to keep the substrate moist; nitrogen fertilizer of 5% per thousand is applied twice a week.
After 2.5 and 45-60 days, the water and fertilizer of tissue culture seedlings are cut off, and 1-3g of tissue culture microspheres can be collected after the seedlings are poured.
3. Propagating seedling by tissue culture micro-corm
3.1, selecting 1-2g of bulbil konjak tissue culture microspheres, cleaning surface impurities, sequentially placing the microspheres in a clean rectangular seedling culture plate, covering the microspheres with a layer of newspaper, and spraying 50mg/L GA on the newspaper every day3Keeping the newspaper moist but without water drop and no water accumulation in the tray, and accelerating germination.
3.2 when the buds grow to more than 1.5 cm, taking the buds at the joint of the bud root and the surface of the bulb by using a blade sterilized by 100 percent alcohol.
3.3, sterilizing the tissue culture micro-corms after sprouting by using a mixed solution of carbendazim and thievone which is 800 times of 2:1, putting the sterilized tissue culture micro-corms back to the original seedling culture plate, accelerating sprouting again, and taking sprouts.
3.4, disinfecting the taken buds by using 800 times of mixed solution of carbendazim and benziothiazolinone at the ratio of 2:1, airing the buds in a ventilated and dried place, transplanting the buds into a seedling raising tray filled with a seedling raising matrix of peat soil and coconut husk in the volume ratio of 2:1, and keeping the water content of the matrix at 20-45% in the early stage.
After 3.5 days and 10 days, after new roots grow out from the root parts of the buds, normal watering and fertilizing can be carried out, and the humidity of the matrix is kept at 80 percent or above; repeating the steps until the bulbs shrink and the texture is soft, and transplanting the bulbs to a field.
Example 3
1. Cultivation of amorphophallus bulbifer tissue culture seedling
1.1, selecting healthy, plump and underground bulbs without mechanical damage, diseases and mildew as tissue culture explants.
1.2, cleaning impurities on the surface of the explant by using clear water, and peeling off the epidermis.
1.3, washing the peeled explants with running water for 30min, taking out, washing with sterile water for 2 times, transferring to a sterile operating platform, soaking with 75% alcohol for 20min, then respectively soaking with 0.1% mercuric chloride solution for 20min and washing with sterile water for 3 times, and finally transferring to the sterile operating platform and cutting into small blocks of 0.5cm × 0.5cm × 1 cm.
1.4, callus induction: the small blocks are inoculated into a callus induction culture medium with the mixture ratio of MS +6-BA2mg/L + NAA0.5mg/L + agar 8g/L + sucrose 32g/L, and a large amount of callus is formed after 1 month.
1.5, adventitious bud induction: transferring the explant forming the callus into an adventitious bud induction culture medium with the mixture ratio of MS +6-BA2mg/L + NAA 0.4mg/L + agar 8g/L + sucrose 32 g/L; after 20-25 days of culture under light, a large number of clumpy buds form on the explants.
1.6, adventitious bud multiplication culture: taking out the cluster buds on an aseptic operation platform, inoculating the cluster buds into a multiplication culture medium with the mixture ratio of MS +6-BA 1.5mg/L + IAA 0.6mg/L + agar 8g/L + sucrose 32g/L after the plant division, and culturing under the illumination, wherein the cluster buds of the bulbifer can obtain the cluster buds of the bulbifer which are about 5cm after 25 days.
1.7, finally dividing the cluster seedlings into single plants, inoculating the single plants into rooting culture media with the mixture ratio of 1/2MS, NAA0.5mg/L, agar 8g/L and sucrose 17g/L, and transplanting the single plants into a greenhouse for hardening seedlings after 30 days.
Wherein the pH of each culture medium is 6.2, and the culture conditions are 12h per day under 28 deg.C and illumination intensity of 2000 lx.
2. Hardening off seedlings
2.1, uniformly mixing a mixed substrate of peat soil and laterite and coco coir in a volume ratio of 4:1:2, filling the mixed substrate into a seedling raising tray, and spraying 900 times of mixed solution of carbendazim and thiacetone in a volume ratio of 1:2 for disinfection; the water holding capacity of the matrix in the early growth stage is kept at 82%, and the tissue culture seedling can be watered and fertilized normally after new roots germinate.
2.2, moving the bulbil konjak tissue culture seedlings with strong plants and root systems into a seedling hardening greenhouse with the periphery being rainproof, the shading degree being 80%, the room temperature being 32 ℃ and the humidity being 65%, and opening a bottle cover for staying overnight; taking out the seedlings the next day, cleaning the root culture medium with clear water, and transplanting the seedlings into a seedling culture tray filled with the matrix.
2.3, spraying clear water every 2-3h in the first 10 days, controlling the time length within 1min, keeping the leaf surface of the tissue culture seedling moist at any time due to the tender plant of the tissue culture seedling, especially at the time interval of 11-15 pm; maintaining the water content of the matrix at 65%;
2.4, building a layer of sunshade net above the shed in the early stage, keeping the shading degree in the shed at 80 percent, and removing the sunshade net after 10 days when a new root system of the tissue culture seedling grows out; spraying clear water once in the morning and at night to keep the substrate moist; compound fertilizer with N: P: K ═ 15:5:30 was applied twice a week.
After 2.5 and 45-60 days, the water and fertilizer of tissue culture seedlings are cut off, and 1-3g of tissue culture microspheres can be collected after the seedlings are poured.
3. Propagating seedling by tissue culture micro-corm
3.1, selecting 2-3g of bulbil konjac tissue culture microspheres, cleaning surface impurities, sequentially placing the microspheres in a clean rectangular seedling culture tray, covering a layer of newspaper on each microsphere, spraying 50mg/L IAA on the newspaper every day, keeping the newspaper moist, but not dripping water drops, and accelerating germination without accumulated water in the tray.
3.2 when the buds are 2cm long, taking the buds at the joint of the bud root and the surface of the bulb by using a blade sterilized by 85% alcohol.
3.3, sterilizing the tissue culture micro-corms after sprouting by using a mixed solution of carbendazim and thiacetone which is 900 times of the ratio of 1:2, putting the sterilized tissue culture micro-corms back to the original seedling culture plate, accelerating sprouting again, and taking sprouts.
3.4, disinfecting the taken buds by using a mixed solution of carbendazim and benziothiazolinone which is 900 times of 1:2, airing the buds in a ventilated and dried place, transplanting the buds into a seedling raising tray filled with peat soil, red soil and coconut husk in a volume ratio of 4:1:2, and keeping the water content of the substrate at 20-45% in the early stage.
After 3.5 days and 10 days, after new roots grow out from the root parts of the buds, normal watering and fertilizing can be carried out, and the humidity of the matrix is kept at 80 percent or above; repeating the steps until the bulbs shrink and the texture is soft, and transplanting the bulbs to a field.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for accelerating germination and seedling culture of a bulbil konjak tissue culture microsphere stem is characterized by comprising the following steps:
taking a bulbil konjak explant, and performing callus induction, adventitious bud proliferation and rooting culture to obtain an aseptic tissue culture seedling; transplanting and hardening the aseptic tissue culture seedlings, and cultivating and harvesting the microspheres; accelerating germination of the microsphere stems, and taking off bud heads for transplantation and culture after the microsphere stems grow bud heads.
2. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the callus induction and adventitious bud induction culture medium comprises: MS +6-BA1-2mg/L + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 25-32 g/L.
3. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the adventitious bud proliferation medium comprises: MS +6-BA1-2mg/L + IAA 0.3-0.6mg/L + agar 6.5-8g/L + sucrose 25-32 g/L.
4. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the rooting medium comprises: 1/2MS + NAA 0.1-0.5mg/L + agar 6.5-8g/L + sucrose 13-17 g/L.
5. The method for accelerating germination and raising seedlings of the bulbiferous konjac tissue culture corms as claimed in claim 1, wherein the seedling hardening matrix comprises 2-4 parts by volume of peat soil, 0-2 parts by volume of red soil and 1-2 parts by volume of coconut coir, and the water content of the matrix is 65% -82%.
6. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the seedling hardening process comprises the following steps: shading degree is 60% -80%, temperature is 25-32 ℃, and air humidity is 65% -92%.
7. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the weight of the bulbil konjac is 1-3 g.
8. The method for accelerating germination and raising seedlings of tissue culture bulbodium of amorphophallus bulbifer according to claim 1, wherein the accelerating germination comprises: covering the harvested microsphere with a layer of paper, spraying clear water or 40-60mg/LGA3Or 40-60mg/LIAA, until the paper surface is wet and no water drops.
9. The method for accelerating germination and seedling culture of the bulbil konjac tissue culture bulbodium according to claim 1, wherein the bud picking comprises: and (3) when the micro-corm grows to have a bud head with the length of more than 1cm, taking a bud at the joint of the bud root and the surface of the micro-corm.
10. The method for accelerating germination and raising seedlings of the bulbil konjac tissue culture bulbodium as claimed in claim 1, wherein the bulbil is repeatedly subjected to accelerating germination, picking up buds, transplanting culture until the bulbil is shrunk and aggravated, the texture is soft, and transplanting seeding is performed.
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