阅读说明：本技术 高茎尖诱导率和组培质量的多头菊脱毒培养基质及方法 (Chrysanthemum multocida detoxification culture medium with high stem tip induction rate and tissue culture quality and method ) 是由 郭方其 吴超 袁峰 黑银秀 柴金甫 刘君 丁晓瑜 徐智豪 黎侠 于 2019-07-15 设计创作，主要内容包括：本发明公开一种高茎尖诱导率和组培质量的多头菊脱毒培养基质及方法,属于植物培育栽培技术领域,本发明的组培基质由分阶段依次顺序使用的茎段芽诱导培养基组合、脱毒茎尖诱导培养基组合、增殖培养基组合、生根培养基组合组成,诱导培养基组合包括外植体诱导与增值培养基+继代培养基、外植体初代诱导培养基+继代培养基；脱毒茎尖诱导培养基组合包括外植体诱导与增值培养基+续代培养基、茎尖诱导培养基Ⅰ+茎尖诱导基本培养基I、茎尖诱导培养基Ⅱ+续代培养基：本发明解决菊花品种退化和生长势减弱,影响切花品质,同时茎尖培养诱导率低,组培培育质量较差,种苗无法高效大规模化生产的问题。(The invention discloses a detoxification culture medium and a detoxification culture method for a multi-headed chrysanthemum with high stem tip induction rate and tissue culture quality, belonging to the technical field of plant cultivation, wherein the tissue culture medium consists of a stem section bud induction medium combination, a detoxification stem tip induction medium combination, a proliferation medium combination and a rooting medium combination which are sequentially used in stages, and the induction medium combination comprises an explant induction and increment culture medium + a subculture medium, an explant primary induction culture medium + a subculture medium; the combination of the detoxification stem tip induction culture medium comprises an explant induction and proliferation culture medium, a subculture medium, a stem tip induction culture medium I, a stem tip induction basic culture medium I, a stem tip induction culture medium II and a subculture medium: the invention solves the problems that the chrysanthemum varieties are degenerated and the growth vigor is weakened, the cut flower quality is influenced, meanwhile, the stem tip culture induction rate is low, the tissue culture quality is poor, and the seedlings cannot be efficiently produced in a large scale.)

1. The multi-head chrysanthemum detoxification culture medium with high stem tip induction rate and tissue culture seedling quality is characterized in that the tissue culture medium consists of a stem segment bud induction culture medium combination, a detoxification stem tip induction culture medium combination, a proliferation culture medium combination and a rooting culture medium combination which are sequentially used by stages,

the induction culture medium combination comprises an explant primary induction and proliferation culture medium, a subculture medium, an explant primary induction culture medium and a subculture medium;

the detoxified stem tip induction culture medium combination comprises an explant induction and proliferation culture medium, a secondary culture medium, a stem tip induction culture medium I, a stem tip induction basic culture medium I, a stem tip induction culture medium II and a secondary culture medium, wherein the formula of the stem tip induction culture medium I is 1/2B 5+ Ca (NO 3) 2500 mg/L +6-BA 0.01 ~ 0.2mg/L + NAA 0.1 ~ 0.2.2 mg/L + AC 0.5 ~ 1.0.0 mg/L, and the formula of the stem tip induction culture medium II is MS +6-BA 0.1 ~ 0.2.2 mg/L + IBA 0.1 ~ 0.2.2 mg/L;

combining strong seedling culture media: it comprises a strong seedling culture medium and a continuous culture medium;

combining a rooting culture medium: which comprises a rooting culture medium and a rooting basic culture medium.

2. The detoxification culture medium of multi-headed chrysanthemum with high stem tip induction rate and tissue culture seedling quality according to claim 1, wherein the formulation of the stem tip induction minimal medium I comprises sucrose 20 ~ 30g/L, agar powder 4 ~ 5g/L, 1/2B 5+ Ca (NO 3) 2500 mg/L culture medium with pH5.6 ~ 5.8.8, and the formulation of the subculture medium comprises sucrose 25 ~ 35g/L, agar powder 4 ~ 5g/L, MS culture medium with pH5.6 ~ 5.8.8.

3. The multi-stem chrysanthemum detoxification culture medium with high stem tip induction rate and tissue culture seedling quality according to claim 1, wherein the formula of the strong seedling culture medium is as follows: 3/4MS +6-BA0.2 mg/L + IBA 0.1 mg/L.

4. The multi-headed chrysanthemum detoxification culture medium with high stem tip induction rate and tissue culture seedling quality as claimed in claim 1, wherein the formula of the rooting culture medium is 1/2MS + IBA 0.1 ~ 0.5.5 mg/L + AC 0 ~ 0.5mg/L, the formula of the rooting culture medium I is a 1/2MS culture medium with sucrose 30g/L, agar powder 4 ~ 5g/L and pH5. 5.6 ~ 6.0.0, and the formula of the explant primary induction and proliferation culture medium is MS +6-BA0.2 ~ 1.0.0 mg/L + IBA 0.1 ~ 0.5 mg/L.

5. A multi-headed chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality, characterized in that the method uses the matrix of claim 1 or 2 or 3 or 4, and the method comprises the following steps:

(1) preparing a culture medium: preparing a section bud induction culture medium combination, a detoxified stem tip induction culture medium combination, a proliferation culture medium combination and a rooting culture medium combination which are required by tissue culture;

(2) seed source treatment and sterilization: taking the winter solstice buds back after natural low temperature, soaking the winter solstice buds for 10min by using 1% detergent semen, washing the winter solstice buds by using clear water, and then performing seed stem sterilization treatment for later use;

(3) taking sterilized seed stem segments, cutting the seed stem segments into segments with one axillary bud as one segment, taking the segments as explants, inoculating the segments on an explant induction and proliferation culture medium, culturing until the stem segment buds are induced, cutting the stem segment buds, transferring the cut stem segment buds to an explant primary generation induction and proliferation culture medium, cutting the seedlings into stem segments with 1 ~ 2 axillary buds when the seedlings grow to 6cm ~ 7cm, transferring the stem segments to the explant induction and proliferation culture medium, and repeating inoculation culture until the number of the stem segments is enough for the induction culture of the detoxified stem tips;

(4) performing heat treatment virus-free culture after the stem section bud is propagated for a certain base number, inhibiting the propagation of chrysanthemum virus by using high-temperature culture conditions, and stripping the stem tip for induction culture until forming normal-growing seedlings with 1 ~ 2 unfolded leaves;

(5) proliferation culture, transferring the cultured seedlings with 1 piece of ~ 2 pieces of unfolded leaves to strong seedling culture medium and proliferation culture medium for repeated proliferation culture;

(6) adopting a high-throughput Small RNA sequencing technology to rapidly identify viruses, carrying out high-throughput Small RNA analysis on virus diseases of cut chrysanthemum, carrying out comparison analysis on sequencing data of a virus-free seedling experimental sample and an NCBI virus genomic database to find that some sequencing sequences are similar to virus sequences, then carrying out assembly analysis on each virus sequence of the detection sample, but not assembling the virus sequences to a whole complete virus sequence, and determining that no virus is found in the tissue culture seedlings cultured by the virus-free stem tip induction;

(7) rooting culture, cutting the tissue culture seedling without chrysanthemum virus detected by the method into stem segments with 1 ~ 2 axillary buds, transferring the stem segments to a rooting culture medium, and culturing for 25d ~ 30d under the environmental conditions of 24 +/-1 ℃, 12h/d illumination and 1000 ~ 3000Lx illumination intensity to form the tissue culture seedling with 5 ~ 7 leaves and 4 ~ 6 complete roots;

(8) selecting from the last ten days of the month to the middle ten days of the month from 2 to 4 as the transplanting time of the tissue culture seedlings, and transferring the tissue culture bottle seedlings to an overhead seedbed substrate with an insect-proof net isolation greenhouse.

6. The multi-head chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality according to claim 1, which is characterized in that after the method in the step (3) is inoculated on an explant induction and proliferation culture medium, the method comprises the following steps of culturing 20 ~ 30d under the environment conditions of temperature of 24 +/-1 ℃, illumination of 12h/d and illumination intensity of 1000 ~ 3000Lx until stem segment buds are induced, cutting the stem segment buds and transferring the stem segment buds to the explant primary generation induction and proliferation culture medium, cutting the seedlings into stem segments with 1 ~ 2 axillary buds when the seedlings grow to 6cm ~ 7cm, transferring the stem segments to the explant induction and proliferation culture medium, culturing for 30 ~ 40d under the environment conditions for proliferation culture, and repeating inoculation culture until the sufficient number is used for detoxification stem tip induction culture.

7. The multi-head chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality according to claim 6 is characterized in that in step (4), a pre-cultured tissue culture seedling is cut into stem sections with 1 ~ axillary buds when the seedling grows to 6cm ~ cm, the seedling is transferred to an explant induction and proliferation culture medium, the tissue culture bottle seedling is cultured for 10d 36015 d under the same environmental conditions, when a newly germinated seedling grows to 1cm ~.5 cm, the tissue culture bottle seedling is placed into an illumination culture box, the tissue culture seedling is gradually adapted to a high-temperature growth environment every 3d according to the sequence of temperature of 28 ℃ ~ ℃ at 28 ℃, 30 ℃ 539335 ℃ at 33 ℃ ~ ℃ and is transferred to a medium under the condition of shifting to one grade every 3d, then the tissue culture seedling is continuously cultured for 25d under the heat treatment of ~ ℃ at 33 ℃ and 538 ℃ for 25d, the tissue culture seedling grows gradually, the leaf color becomes light, when the tissue culture seedling necrosis is about 50%, the tissue culture seedling has a certain vitality, the vigor is selected, the seedling is transferred to a detoxification culture medium under the condition of 7372 mm, the normal stem tip induction culture medium, the tissue culture platform is transferred to a stem tip induction culture medium under the illumination induction, the normal conditions of 1000 mm, the tissue culture medium, the tissue culture platform is transferred to 1000 mm, the tissue culture medium, the tissue culture seedling growth induction, the tissue culture bottle seedling growth is transferred to induce the tissue culture bottle seedling growth is transferred to the tissue culture under the condition of 1000 mm, the seedling under the normal conditions, the stem tip induction, the normal stem tip induction, the stem tip induction culture medium, the stem tip induction culture platform is transferred to the stem culture medium under the normal conditions of 1000 mm, the stem tip induction and the stem tip induction culture medium, the stem culture platform is transferred to induce the stem culture medium, the stem culture under the stem tip.

8. The multi-headed chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality according to claim 6, wherein the specific method in the proliferation culture (5) is that the cultured seedlings with 1 piece of ~ 2 unfolded leaves are transferred to a strong seedling culture medium, the cultured seedlings are cultured for 25d ~ 30d under the environmental conditions of 24 +/-1 ℃, 12h/d of illumination and 1000 ~ 3000Lx of illumination intensity to form tissue culture seedlings with 5 pieces of ~ 7 leaves, the tissue culture seedlings are cut into stem segments with 1 piece of ~ 2 axillary buds, the stem segments are transferred to the strong seedling culture medium, the cultured seedlings are cultured for 30 ~ 40d to carry out proliferation culture under the environmental conditions, the transfer culture is repeated until the requirement of further propagation of the tissue culture seedlings after virus detection sampling and virus retention can be met, only the stem segments sprout and grow, the tissue culture seedlings have no morphological variation and no vitrification seedlings occur in the whole culture process.

9. The method for multi-headed chrysanthemum detoxification cultivation with high stem tip induction rate and tissue culture seedling quality according to claim 6, wherein the specific method in the step (8) comprises the steps of transferring tissue culture bottle seedlings onto an overhead seedling bed with an insect-proof net isolation greenhouse, covering the bottle seedlings with 70% shading net, placing 15d in the greenhouse to enable the tissue culture seedlings to adapt to natural illumination conditions, opening the caps of the tissue culture seedlings without shading in the morning and evening, covering the tissue culture bottle seedlings with 70% shading net during the strong light period at noon, bottling and calcining the tissue culture seedlings for 3d ~ 5d, carefully taking out the tissue culture seedlings, washing off culture medium in clear water, transplanting the tissue culture seedlings into the seedling bed with prepared coconut chaff as the substrate, paving coconut chaff blocks on the seedling bed one week before the seedling transplantation, watering to loosen the coconut chaff blocks, applying N-P-K, 20 kg/mu, 6cm in thickness, ~ 8cm, removing the coconut chaff blocks in the depth of one week, covering the medium to cover the medium, transplanting the root blocks to the root blocks, removing the root blocks, and transplanting the root blocks after the root blocks are covered with shading net, the root blocks are covered with 70% shading net, the root blocks are covered with shading net, the root blocks are suitable for seedling transplanting, the root blocks are removed, the root blocks are transplanted after the root blocks are carefully, the root blocks are transplanted, the root blocks are planted for.

10. The multi-stem chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality according to claim 6, which is characterized in that: the method further comprises the steps of:

(9) and (3) long-day treatment: the bottle seedlings are moved to a greenhouse and then start long-day treatment, 150w high-light-efficiency sodium lamps are adopted, one lamp is hung every 56 square meters, the lowest illumination intensity of the greenhouse far away from the lamp light reaches more than 70lux, and the number of days of sunshine is more than 16 hours each day;

(10) timely applying water soluble fertilizer formulation, namely pouring 1000 ~ 1500 times of water soluble fertilizer for 1 time every 7 ~ 10 days, wherein the water soluble fertilizer formulation is a full-mineral nutrient liquid fertilizer and contains major elements of nitrogen, phosphorus and potassium and trace elements of calcium, magnesium, sulfur, iron, manganese, zinc, copper, boron, molybdenum and the like;

n: P: K is 20: 20;

(11) and (3) pest control: fusarium, erwinia, rhizoctonia and other soil-borne diseases are harmful to rhizome parts, and the main symptoms are as follows: the pesticide composition is used for controlling aphids, mites and other pests by using pesticides such as rhizoctonia solani, cyanotheca, rot and the like, and spraying 25% of azoxystrobin 1000 times and 58% of wettable powder such as methoxam manganese zinc 800 times or enoyl manganese zinc 1000 times, 10% of nitenpyram soluble liquid agent 1500 times, or 20% of acetamiprid missible oil 1500 times or 5% of pyriproxyfen EC800 times, or 20% of pyrazofenapyr SC750 times.

Technical Field

The invention belongs to the technical field of plant cultivation and cultivation, and particularly relates to a detoxification cultivation medium and a detoxification cultivation method for a chrysanthemum polycephalum with high stem tip induction rate and tissue culture seedling quality.

Background

The existing planting area of cut chrysanthemum in Zhejiang province is 4165 mu, 1 hundred million branches are sold in the year, the sales amount reaches 0.7 hundred million yuan, and the situation of rapid growth is continuously maintained. The production areas of the cut chrysanthemum in Zhejiang province are mainly distributed in the places of Xinchang county, Kyoho county, Pingyang city, Jiaojiang county, Changxing county and the like, production experiences are accumulated in all places through years of cultivation, the product quality is improved, a large amount of produced cut chrysanthemum is exported to Japan and Korea, and the cut chrysanthemum produced in the Zhejiang province has higher occupation rate in the Japan market at present.

The chrysanthemum polycephalum is one of the internationally popular cut-flower chrysanthemum varieties, the chrysanthemum polycephalum is mainly from Europe and America, part of the chrysanthemum polycephalum is from Japan, particularly, the market popular cut-flower variety almost comes from Europe and America breeders, and in recent years, Nanjing agriculture university and Zhejiang agricultural academy of sciences develop new variety breeding work of the chrysanthemum polycephalum, and part of the variety is applied to production. However, the multi-head chrysanthemum is not the traditional single chrysanthemum for multi-flower production, but a new variety with stronger inflorescence branching and smaller flowers is cultivated, is influenced by lower planting management level of farmers, and has unstable product quality.

In addition, most of the species characteristics of the chrysanthemum polycephalum belong to autumn chrysanthemum and Han chrysanthemum, and a small part of the chrysanthemum polycephalum also belongs to summer chrysanthemum and summer autumn chrysanthemum, in production, the mother parent of farmers is reserved for cutting propagation, because the female parent for the propagation of the multi-headed chrysanthemum is subjected to long-term asexual propagation and is susceptible to viral diseases and fungal diseases such as fusarium, rhizoctonia and pythium, in addition, the field has the pests such as aphids, thrips and the like which transmit the viruses such as chrysanthemum B virus, tomato sterility virus and the like, so that the variety degeneration and the growth vigor decline are easily caused, because the stem tip is easy to be damaged in the induction culture, the induction rate of the stem tip culture is low, the technical difficulty is higher, and the matched detoxification culture technology of the stem tip of the multi-headed chrysanthemum is lacked in China, the seedlings need to be imported from foreign countries every year, therefore, the induction rate of the first generation of the multi-head chrysanthemum stem tip detoxification culture is improved, and the method has great significance for accelerating the production of the good multi-head chrysanthemum variety detoxification seedlings and promoting the large-scale production of high-quality seedlings.

Disclosure of Invention

The invention aims to provide a multiheaded chrysanthemum detoxification culture medium and a multiheaded chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture quality, which solve the problems that due to long-term asexual propagation, a large amount of viruses are accumulated in a plant body, chrysanthemum varieties are degraded and growth vigor is weakened, and cut flower quality is influenced, meanwhile, the stem tip culture induction rate is low, the tissue culture quality is poor, a multiheaded chrysanthemum stem tip detoxification culture method and a matrix are lacked, so that seedlings need to be imported from abroad every year, and the seedlings cannot be efficiently produced in a large scale.

The first purpose of the invention is to provide a multi-head chrysanthemum detoxification culture medium with high stem tip induction rate and tissue culture seedling quality, the tissue culture medium consists of a stem segment bud induction culture medium combination, a detoxification stem tip induction culture medium combination, a proliferation culture medium combination and a rooting culture medium combination which are sequentially used by stages,

the induction culture medium combination comprises an explant induction and proliferation culture medium, a subculture medium, an explant primary induction culture medium and a subculture medium;

the detoxified stem tip induction culture medium combination comprises an explant induction and proliferation culture medium, a secondary culture medium, a stem tip induction culture medium I, a stem tip induction basic culture medium I, a stem tip induction culture medium II and a secondary culture medium, wherein the formulation of the stem tip induction culture medium I is 1/2B 5+ Ca (NO 3) 2500 mg/L +6-BA 0.01 ~ 0.2mg/L + NAA 0.1 ~ 0.2.2 mg/L + AC 0.5 ~ 1.0.0 mg/L, and the formulation of the stem tip induction culture medium II is MS +6-BA 0.1 ~ 0.2.2 mg/L + IBA 0.1 ~ 0.2.2 mg/L;

combining strong seedling culture media: it comprises a strong seedling culture medium and a continuous culture medium;

combining a rooting culture medium: which comprises a rooting culture medium and a rooting basic culture medium.

Preferably, the matrix provided by the invention is further set in such a way that the formula of the stem tip induction minimal medium I is sucrose 20 ~ 30g/L, agar powder 4 ~ 5g/L, and 1/2B 5+ Ca (NO 3) 2500 mg/L medium with pH5.6 ~ 5.8, and the formula of the subculture medium is sucrose 25 ~ 35g/L, agar powder 4 ~ 5g/L, and MS medium with pH5.6 ~ 5.8.8.

Preferably, the substrate provided by the invention is further configured such that the formula of the strong seedling culture medium is as follows: 3/4MS +6-BA0.2 mg/L + IBA 0.1 mg/L.

Preferably, the matrix provided by the invention is further set as that the formula of the rooting medium is 1/2MS + IBA 0.1 ~ 0.5.5 mg/L + AC 0 ~ 0.5mg/L, and the formula of the rooting medium I is 30g/L of sucrose, 4 ~ 5g/L of agar powder and 1/2MS medium with the pH value of 5.6 ~ 6.0.0.

Preferably, the matrix provided by the invention is further configured in such a way that the formula of the explant primary induction and proliferation medium is MS +6-BA0.2 ~ 1.0.0 mg/L + IBA 0.1 ~ 0.5.5 mg/L.

The second purpose of the invention is to provide a detoxification culture method of multi-head chrysanthemum with high stem tip induction rate and tissue culture seedling quality, the method uses the matrix as above, and the method comprises the following steps:

(1) preparing a culture medium: preparing a section bud induction culture medium combination, a detoxified stem tip induction culture medium combination, a proliferation culture medium combination and a rooting culture medium combination which are required by tissue culture;

(2) seed source treatment and sterilization: taking the winter solstice buds back after natural low temperature, soaking the winter solstice buds for 10min by using 1% detergent semen, washing the winter solstice buds by using clear water, and then performing seed stem sterilization treatment for later use;

(3) taking sterilized seed stem segments, cutting the seed stem segments into segments with one axillary bud as one segment, taking the segments as explants, inoculating the segments on an explant induction and proliferation culture medium, culturing until the stem segment buds are induced, cutting the stem segment buds, transferring the cut stem segment buds to an explant primary generation induction and proliferation culture medium, cutting the seedlings into stem segments with 1 ~ 2 axillary buds when the seedlings grow to 6cm ~ 7cm, transferring the stem segments to the explant induction and proliferation culture medium, and repeating inoculation culture until the number of the stem segments is enough for the induction culture of the detoxified stem tips;

(4) performing heat treatment virus-free culture after the stem section bud is propagated for a certain base number, inhibiting the propagation of chrysanthemum virus by using high-temperature culture conditions, and stripping the stem tip for induction culture until forming normal-growing seedlings with 1 ~ 2 unfolded leaves;

(5) proliferation culture, transferring the cultured seedlings with 1 piece of ~ 2 pieces of unfolded leaves to strong seedling culture medium and proliferation culture medium for repeated proliferation culture;

(6) adopting a high-throughput Small RNA sequencing technology to rapidly identify viruses, carrying out high-throughput Small RNA analysis on virus diseases of cut chrysanthemum, carrying out comparison analysis on sequencing data of a virus-free seedling experimental sample and an NCBI virus genomic database to find that some sequencing sequences are similar to virus sequences, then carrying out assembly analysis on each virus sequence of the detection sample, but not assembling the virus sequences to a whole complete virus sequence, and determining that no virus is found in the tissue culture seedlings cultured by the virus-free stem tip induction;

(7) rooting culture, cutting the tissue culture seedling without chrysanthemum virus detected by the method into stem segments with 1 ~ 2 axillary buds, transferring the stem segments to a rooting culture medium, and culturing for 25d ~ 30d under the environmental conditions of 24 +/-1 ℃, 12h/d illumination and 1000 ~ 3000Lx illumination intensity to form the tissue culture seedling with 5 ~ 7 leaves and 4 ~ 6 complete roots;

(8) selecting from the last ten days of the month to the middle ten days of the month from 2 to 4 as the transplanting time of the tissue culture seedlings, and transferring the tissue culture bottle seedlings to an overhead seedbed substrate with an insect-proof net isolation greenhouse.

6. The multi-head chrysanthemum detoxification culture method with high stem tip induction rate and tissue culture seedling quality according to claim 1, wherein after the method in the step (3) is inoculated on an explant induction and proliferation culture medium, the method comprises the following steps of culturing 20 ~ 30d under the environment conditions of temperature of 24 +/-1 ℃, illumination of 12h/d and illumination intensity of 1000 ~ 3000Lx until stem segment buds are induced, cutting the stem segment buds and transferring the stem segment buds to the explant primary generation induction and proliferation culture medium, cutting the seedlings into stem segments with 1 ~ 2 axillary buds when the seedlings grow to 6cm ~ 7cm, transferring the stem segments to the explant induction and proliferation culture medium, culturing for 30 ~ 40d under the environment conditions for proliferation culture, and repeating inoculation culture until the sufficient number is used for detoxification stem tip induction culture.

Preferably, the method provided by the invention is further set as the step (4), in the specific method, pre-cultured tissue culture seedlings are cut into stem sections with 1 ~ axillary buds when the seedlings grow to 6cm 637 cm, the seedlings are transferred to an explant induction and proliferation culture medium, 10d ~ d is cultured under the same environmental conditions, when the sprouts of the new buds grow to 1cm 3611.5 cm, tissue culture bottle seedlings are placed into a light culture box, the tissue culture seedlings are gradually adapted to a high-temperature growth environment according to the sequence of temperature of 28 ℃ 53925 ℃ ~ ℃ and 33 ℃ ~ ℃ every 3d, the grade is changed once, then the tissue culture seedlings are continuously subjected to heat treatment and detoxification culture at 33 ℃ ~ ℃ for 25d, the tissue culture seedlings gradually weaken and become light leaf color, when the tissue culture seedlings are necrotic by about 50% and 60%, the tissue culture seedlings are selected to have certain activity, the tissue culture seedlings are placed on an aseptic operation table, the tissue culture medium is placed under the temperature of 1000 mm, the light culture medium is placed under the temperature of No. 7 mm, the stem sections are placed under the same environmental conditions that the stem sections are changed, the stem sections are changed to the stem sections, the stem sections are used as the stem sections, the stem sections are placed under the stem sections, the stem sections are inoculated to the stem sections with the intensity of 1000 mm of the stem sections with.

Preferably, the method provided by the invention is further configured that in the propagation culture of the step (5), the cultured seedling with 1 ~ 2 unfolded leaf is transferred to a strong seedling culture medium, the seedling is cultured for 25d ~ 30d under the environmental conditions of 24 +/-1 ℃, 12h/d of illumination and 1000 ~ 3000Lx of illumination intensity to form a tissue culture seedling with 5 ~ 7 leaves, the tissue culture seedling is cut into stem segments with ~ 2 axillary buds of 1, the stem segments are transferred to the strong seedling culture medium, the seedling is cultured for 30 ~ 40d under the environmental conditions for propagation culture, the transfer culture is repeated until the number of the tissue culture seedling is further expanded and propagated after virus detection sampling and virus detection reservation can be carried out, only the stem segments sprout and grow because no clumpy buds occur in the whole culture process, the tissue culture seedling does not show morphological variation and does not have vitrification seedlings.

Preferably, the method provided by the invention is further configured that in the step (8), the specific method comprises the following steps of moving the tissue culture bottle seedlings to an overhead seedling bed with an insect-proof net isolation greenhouse, covering the bottle seedlings with a shading net of 70%, applying 15d in the greenhouse to enable the tissue culture seedlings to adapt to natural illumination conditions, opening the covers of the tissue culture seedlings without shading in the morning and evening, covering the bottle seedlings with the shading net of 70% in a strong light period at noon, opening the bottles of the tissue culture seedlings, calcining for 3d 855 d, carefully taking out the tissue culture seedlings, washing the culture medium in clear water, transplanting the tissue culture seedlings to a seedling bed with prepared coconut chaff as the matrix, placing coconut chaff blocks on the seedling bed one week before the seedling transplantation, watering thoroughly loosening the coconut blocks, applying N-P-K (15-15-15) 20 kg/mu of compound fertilizer into the matrix, removing the coconut chaff with the thickness of 6cm 358 cm, covering the matrix to a planting depth, spreading the shading net as a shade area, spreading the root blocks, transplanting the root blocks to a survival rate of the tissue culture seedlings, carefully removing the root blocks, and transplanting the root systems after the root systems are carefully pressed for 3 weeks, and removing the root systems, and the root systems are carefully transplanted to recover the root systems.

Preferably, the method provided by the present invention is further configured such that the method further comprises the following steps:

(9) and (3) long-day treatment: the bottle seedlings are moved to a greenhouse and then start long-day treatment, 150w high-light-efficiency sodium lamps are adopted, one lamp is hung every 56 square meters, the lowest illumination intensity of the greenhouse far away from the lamp light reaches more than 70lux, and the number of days of sunshine is more than 16 hours each day;

(10) timely applying water soluble fertilizer formulation, namely pouring 1000 ~ 1500 times of water soluble fertilizer every 7 ~ 10 days, wherein the water soluble fertilizer formulation is a full-mineral nutrient liquid fertilizer and contains major elements of nitrogen, phosphorus and potassium and trace elements such as calcium, magnesium, sulfur, iron, manganese, zinc, copper, boron, molybdenum and the like, and N: P: K is 20: 20;

(11) and (3) pest control: fusarium, erwinia, rhizoctonia and other soil-borne diseases are harmful to rhizome parts, and the main symptoms are as follows: the pesticide composition is used for controlling aphids, mites and other pests by using pesticides such as rhizoctonia solani, cyanotheca, rot and the like, and spraying 25% of azoxystrobin 1000 times and 58% of wettable powder such as methoxam manganese zinc 800 times or enoyl manganese zinc 1000 times, 10% of nitenpyram soluble liquid agent 1500 times, or 20% of acetamiprid missible oil 1500 times or 5% of pyriproxyfen EC800 times, or 20% of pyrazofenapyr SC750 times.

Advantageous effects

The detoxification culture medium and the method for the callistemon chrysanthemi with high stem tip induction rate and tissue culture seedling quality provided by the invention have the following advantages:

(1) the stem tip with detoxified function in the step (4) of the invention is induced and cultured by 1/2B 5+ Ca (NO)₃）₂500mg/L is a stem tip induction basic culture medium I, the tissue culture seedlings cultured by stem section induction are subjected to detoxification culture treatment by adjusting the dosage of 6-BA, NAA and AC in the culture medium, the detoxification culture is carried out on the tissue culture seedlings cultured by stem section induction, the grade is changed every 3d according to the sequence of day and night temperatures of ~ 32 ℃, ~ 35 ℃ and ~ 38 ℃ at 28 ℃, the heat treatment is continuously carried out at the temperature of ~ 38 ℃ for 25 ~ 30d to inhibit the propagation of chrysanthemum virus, but the growth potential of the tissue culture seedlings is gradually weakened and the leaf color is lightened due to the influence of high temperature, when the tissue culture seedlings are about 50 to ~ 60 percent necrotic, the tissue culture seedlings with certain activity are selected to carry out detoxification stem tip induction culture, the initial generation induction rate of the stem tips reaches more than 30 percent, and is higher than 20 percent compared with the induction rate of directly taking tips from field sampling.

(2) In the step (5) of the invention, when the propagation culture is carried out, the propagation culture of the tissue culture seedling adopts a strong seedling culture medium, as no cluster buds are generated in the whole culture process, only axillary buds of stem segments sprout and grow, the tissue culture seedling has no morphological variation and no vitrified seedling, while the incision base part is easy to generate to form obviously enlarged callus in the conventional propagation culture, and the vitrified seedling accounts for more than 10 percent in the propagation process of the tissue culture seedling.

(3) According to the method, in the step (8), the transplanting time of the tissue culture seedlings is selected from the last ten days of 2 months to the middle ten days of 4 months, the tissue culture bottle seedlings are moved to an overhead seedling bed with an insect-proof net isolation greenhouse, 70% of shading nets cover the bottle seedlings, the greenhouse is used for 15 days to enable the tissue culture seedlings to adapt to natural illumination conditions, the tissue culture seedling bottle covers are opened and are not shaded in the morning and evening, 70% of shading nets cover the tissue culture bottle seedlings in the strong light period of noon, the tissue culture seedlings are opened and are smelted for 3d ~ 5d, then transplanting is carried out, and the transplanting survival rate is more than 95%.

(4) The method provided by the invention can greatly reduce the accumulation of a large amount of viruses in the plant body, does not cause variety degeneration and growth vigor weakening, and effectively ensures the cultivation quality and quality of the multi-headed chrysanthemum.

(5) The comprehensive use of the method can efficiently finish the cultivation of the multi-headed chrysanthemum, has higher economic value and higher tissue culture cultivation quality, can completely replace imported seedlings, and reduces the production cost.

(6) The method provided by the invention can promote the large-scale production of high-quality seedlings of the multi-headed chrysanthemum, and lays a foundation for the market promotion of the multi-headed chrysanthemum and an important large-scale marketization application guarantee.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments.