CN114303955A - Breeding method of high-quality chrysanthemum morifolium ramat - Google Patents
Breeding method of high-quality chrysanthemum morifolium ramat Download PDFInfo
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Abstract
The invention relates to a breeding method of high-quality chrysanthemum morifolium ramat, which sequentially comprises the following steps: sampling explants, and cutting plant stem sections with growing points in the optimal time; culturing aseptic seedlings, flushing the surface of a plant stem segment with 75% ethanol for 0.5min, sterilizing with 0.1% mercuric chloride for 12min, stripping a 0.3mm stem tip meristem, and inoculating the stem tip meristem in a stem tip culture medium; culturing tissue culture bottle seedlings, namely, when an aseptic plant is cultured in a stem tip culture medium to 2.0cm, selecting a stem tip meristem with the diameter of 0.3mm, inoculating the stem tip meristem into the stem tip culture medium, and inoculating the aseptic plant into a rooting culture medium when the stem tip culture medium grows to 5cm, so as to obtain tissue culture bottle seedlings; tissue culture bottle seedling rapid breeding; hardening seedlings by using a plug; performing plug propagation; preparing a seedbed; and (5) planting original seedlings. According to the method, the seedlings are detoxified in a multi-step and step-by-step mode, the chrysanthemum morifolium ramat tissue culture bottle seedlings with CVB removed are obtained, the chrysanthemum morifolium ramat tissue culture bottle seedlings are bred quickly, the chrysanthemum morifolium ramat tissue culture bottle seedlings are planted in a large batch mode in a mode of overhead seedbed planting, the yield of the chrysanthemum morifolium ramat can be effectively improved, and the quality of the produced chrysanthemum morifolium ramat is high.
Description
Technical Field
The invention relates to the technical field of plant cultivation, in particular to a breeding method of high-quality chrysanthemum morifolium ramat.
Background
Hangzhou white chrysanthemum, also known as chamomile and white chrysanthemum, is a special product in Kangxiang city in Jiang city, Zhejiang province, and is a geographical sign product in China. The chrysanthemum morifolium ramat has strong adaptability, is cold-resistant and not high-temperature resistant, has the growth temperature of 15-25 ℃, is planted in loose, fertile and moist loam or sandy loam for optimal growth, is a dry head-shaped inflorescence of chrysanthemum morifolium ramat of Compositae, has long cultivation history, is recorded by related historical materials, has at least more than 360 years so far, and belongs to one of Zhe eight-flavor genuine medicinal materials. Hangzhou white chrysanthemum contains a plurality of flavonoid active ingredients such as luteolin-7-beta-D-glucoside, apigenin-7-beta-D-glucoside, acacetin-7-beta-D-glucoside and the like, has excellent antioxidation effect and has the efficacies of clearing heat, dispelling wind, calming liver and improving eyesight. More researches show that the chrysanthemum morifolium ramat has the effects of clearing heat, inhibiting bacteria, resisting influenza viruses, resisting cardiovascular diseases, resisting inflammation and the like, and meanwhile, researches at home and abroad in recent years show that the chrysanthemum morifolium ramat has the effects of resisting oxidation, resisting tumors, resisting AIDS and the like.
In recent years, the outbreak of virus disease seriously affects the yield and quality of Hangzhou white chrysanthemum, and through investigation of Hangzhou white chrysanthemum professional cooperative society in main production areas of Tongxiang, the virus infection of important planting bases of several Hangzhou white chrysanthemum is found to be serious, the main symptoms of diseased plants are that leaf stripes are obvious, flower leaves and branches are few, seriously, the leaves of the region are brownish spots and withered, and plants are withered, so that the yield and the quality of chrysanthemum are reduced gradually. The existing breeding methods suitable for Hangzhou white chrysanthemum cannot recover the yield and the quality of Hangzhou white chrysanthemum under the condition that the virus is abused, so that a breeding method capable of improving the yield of Hangzhou white chrysanthemum under the condition of ensuring the quality of Hangzhou white chrysanthemum is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a high-quality chrysanthemum morifolium ramat breeding method.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a breeding method of high-quality Hangzhou white chrysanthemum comprises the following steps:
s1: sampling explants, selecting plants with pure variety characteristics, good quality and no plant diseases and insect pests in the optimal time, and cutting plant stem segments with growing points to be 2-3 cm;
s2: sterile seedling cultivation, namely cleaning a plant stem segment with sterile water, placing the plant stem segment on a super-clean workbench, cutting the stem segment with a growing point by 1-2 cm, washing the surface with 70-75% of ethanol for 0.5-1 min, then sequentially sterilizing with hypochlorite and Tween-20 for 8-12 min, continuously shaking, finally washing with sterile water for 5-8 times, sucking water with sterile filter paper, stripping a stem tip meristem with the diameter of 0.2-0.3 mm, and inoculating the stem tip meristem in a stem tip culture medium I;
s3: culturing tissue culture bottle seedlings, namely culturing aseptic plants in a first stem tip culture medium to 1.5-2.0 cm, placing the aseptic plants under a dissecting mirror, selecting 0.2-0.3 mm stem tip meristem, inoculating the stem tip meristem in a second stem tip culture medium, and inoculating the aseptic plants in a rooting culture medium when the second stem tip culture medium grows to 5-6 cm to obtain tissue culture bottle seedlings;
s4: tissue culture bottle seedling rapid breeding;
s5: hardening seedlings by using a plug;
s6: preparing a seedbed;
s7: planting original seedlings;
s8: and (5) expanding propagation of the aperture disk.
In the foregoing method, the fast breeding in step S4 specifically includes the following steps:
s41: performing virus detection on the sterile plant, and inoculating the sterile plant with a qualified detection result into a culture medium;
s42: when the plant grows to 6-8 cm, cutting stem segments containing 1-2 internodes, and inoculating the stem segments into a propagation culture medium;
s43: repeating the previous step, and subculturing every 20-30 days.
In the method, the plug seedling hardening in the step S5 specifically comprises the following steps:
s51: selecting 50-hole plug trays or 72-hole plug trays;
s52: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s53: planting the bottle seedlings in a substrate of a plug tray, and shading intermittently during seedling hardening.
In the foregoing method, the bed preparation in step S6 specifically includes the following steps:
s61: arranging an elevated seedbed, taking a U-shaped foam groove as a seedbed framework, and taking coconut coir as a matrix to be paved in the U-shaped groove;
s62: and (3) paving drip irrigation equipment on the elevated seedbed.
In the foregoing method, the original seedling planting in step S7 specifically includes the following steps:
s71: planting seedlings in a single row on an elevated seedbed, wherein the distance between adjacent seedlings is 25-30 cm;
s72: and adding a high-nitrogen water-soluble fertilizer into the drip irrigation equipment for irrigation.
In the foregoing method, the expanding propagation of the plug in step S8 specifically includes the following steps:
s81: more 50-hole or 72-hole plug trays are selected;
s82: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s83: cutting propagation is adopted, and when the height of other healthy seedlings is more than 30cm, stem sections with 3-5 internodes of 8-10 cm are cut by a disinfection cutter;
s84: cutting the base of the stem section in a hole tray, watering thoroughly after planting, compacting by water, keeping the humidity at 75% -90%, shading, and removing the shading net after the base of the stem section grows roots.
In the method, the field planting method in the step S71 comprises the steps of domesticating the seedlings under the condition of normal temperature and weak illumination for 1 to 2 days, then cleaning agar, planting the seedlings on a high ridge, watering the seedlings thoroughly after planting, shading the seedlings for the first week, keeping the humidity of the seedlings to be 75 to 90 percent, and removing the shading net after the base of the stem section is rooted.
Preferably, the optimal time in step S1 is a sunny day of 10 to 11 months per year, and the specific sampling time is 10:00 to 16: 00.
Preferably, the rooting medium in step S3 has a composition of 1/2MS +0.1mg/L NAA.
Preferably, the components of the stem tip culture medium are MS +0.1mg/L6-BA +0.1 mg/LNAA.
Preferably, the culture conditions of the tissue culture bottle seedlings in the steps S2, S3 and S4 are as follows: in the daytime, the temperature is 25 +/-2 ℃, the illumination time is 12-14 h, and the illumination intensity is 30 mu mol-2 s-1-60 mu mol-2 s-1; and at night, the temperature is 18 +/-2 ℃, and the culture in a dark environment is ensured.
Preferably, the base fertilizer application ratio in step S52 is specifically: adding 200kg of decomposed organic fertilizer and 15kg of ternary compound fertilizer containing nitrogen, phosphorus and potassium into each mu of the fertilizer; wherein the contents of nitrogen, phosphorus and potassium in the ternary compound fertilizer are respectively 15%, 15% and 15%.
Compared with the prior art, the invention has the following advantages and beneficial effects: performing multi-step detoxification treatment on the seedlings to obtain chrysanthemum morifolium ramat tissue culture bottle seedlings with CVB removed, rapidly breeding the chrysanthemum morifolium ramat tissue culture bottle seedlings, and performing mass planting by matching with healthy seedlings in an overhead seedbed planting mode, so that the yield of the chrysanthemum morifolium ramat can be effectively improved, and the produced chrysanthemum morifolium ramat is high in quality; while the Hangzhou white chrysanthemum plant group planted on the elevated seedbed has good light transmission and air permeability, the Hangzhou white chrysanthemum plant has sufficient lighting, is convenient for watering and fertilizing, can save the cost of water and fertilizer, is convenient for deploying mechanical equipment in the later period to pick the Hangzhou white chrysanthemum, and has high picking efficiency.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments of the present invention are described in detail below.
Examples are given.
Determination of viral etiology
In order to determine virus pathogens influencing the yield and quality of Hangzhou white chrysanthemum, plants with serious symptoms (obvious leaf stripes, less flower and leaf branches, brown spots on leaves, withered branches and stems) caused by virus infection are selected, pathogenic bacteria of the plants are identified by comprehensively applying technologies such as virus particle morphology, DAS-ELISA detection, RT-PCR detection, sequence determination and the like, and 600-700 nm slightly bent baculovirus particles in the sap of the diseased plants are observed under a radio microscope. The DAS-ELISA detection result of the antibody of the Chrysanthemum Virus B (CVB) is positive, the positive result is subjected to molecular level detection to find that the length of a CVB coat protein coding sequence is 982bp, the sequencing result is subjected to BLAST comparison in NCBI, the highest similarity with a reported nucleotide sequence (GenBank accession number: EU 499736.1) for coding the chrysanthemum virus B coat protein is 92 percent, an amplified fragment can be determined to be a coat protein gene of the chrysanthemum virus B, and the virus pathogen infecting Hangzhou white chrysanthemum in Tungxiang of Zhejiang province is determined to be CVB.
Secondly, a breeding method is developed according to virus pathogens.
A breeding method of high-quality Hangzhou white chrysanthemum comprises the following steps:
s1: sampling the explant, and cutting the explant to obtain a high-quality original planting section which can be used for cultivation. Specifically, plants with pure variety characteristics, good quality and no plant diseases and insect pests are selected in the optimal time, and plant stem segments with growing points are cut by 3 cm.
In the steps, the optimal time is sunny day of 10-11 months per year, and the specific sampling time is 10: 00-16: 00.
: and (4) sterile seedling cultivation, namely culturing the sampled plant stem after virus elimination treatment. Specifically, the stem segments of the plants are washed by sterile water and then placed on a super-clean workbench, 1-2 cm of the stem segments with growing points are cut, the surface is washed by 70-75% of ethanol for 0.5-1 min, then the stem segments are sequentially disinfected by hypochlorite and Tween-20 for 8-12 min and are continuously shaken, finally the stem segments are washed by the sterile water for 5-8 times, the water is absorbed by sterile filter paper, and stem tip meristem of 0.2-0.3 mm is stripped and inoculated in a stem tip culture medium I.
In the above step, the used first stem tip culture medium has the composition of MS +0.1mg/L6-BA +0.1 mg/LNAA.
In the steps, the optimal disinfection treatment of the explant is to wash the surface with 75% ethanol for 0.5min and disinfect with 0.1% mercuric chloride for 12min, and under the treatment method, the survival rate of the outer plant is highest and is about 70%.
: and (5) cultivating tissue culture bottle seedlings, namely cultivating aseptic plants into the tissue culture bottle seedlings for use. Specifically, when the sterile plant is cultured to 1.5-2.0 cm in the first stem tip culture medium, a 0.2-0.3 mm stem tip meristem is selected under a dissecting mirror and inoculated in the second stem tip culture medium, and when the second stem tip culture medium grows to 5-6 cm, the sterile plant is inoculated in a rooting culture medium to obtain a group of culture bottle seedlings.
In the above steps, the used stem tip culture medium II comprises MS +0.1mg/L6-BA +0.1mg/L LNAA, the used rooting culture medium comprises 1/2MS +0.1mg/L NAA, and the rooting rate of the sterile plant reaches the highest rate of 100% under the formula.
In the above steps, the culture conditions of the tissue culture bottle seedling are as follows: in the daytime, the temperature is 25 +/-2 ℃, the illumination time is 12-14 h, and the illumination intensity is 30 mu mol-2 s-1-60 mu mol-2 s-1; and at night, the temperature is 18 +/-2 ℃, and the culture in a dark environment is ensured.
: and (4) fast breeding the tissue culture bottle seedlings, and fast breeding a small amount of tissue culture bottle seedlings to obtain a large amount of tissue culture bottle seedlings for subsequent large-scale planting. The rapid breeding method specifically comprises the following steps:
s41: performing virus detection on the sterile plant, and inoculating the sterile plant with a qualified detection result into a culture medium;
s42: when the plant grows to 6-8 cm, cutting stem segments containing 1-2 internodes, and inoculating the stem segments into a propagation culture medium;
s43: repeating the previous step, and subculturing every 20-30 days.
In the above steps, virus detection on sterile plants was performed as specified in NY/T401.
In the above steps, the culture conditions of the tissue culture bottle seedling are as follows: in the daytime, the temperature is 25 +/-2 ℃, the illumination time is 12-14 h, and the illumination intensity is 30 mu mol-2 s-1-60 mu mol-2 s-1; and at night, the temperature is 18 +/-2 ℃, and the culture in a dark environment is ensured.
The tissue culture bottle seedlings are rapidly bred by the method, the breeding time of each batch is averagely 25 days (including seedling revival), 8 generations of seedlings can be bred every year, and 512 seedlings can be propagated every 1 seedling every year.
: and (4) hardening seedlings by using a plug tray, and culturing the tissue culture bottle seedlings into healthy seedlings. The method for hardening seedlings by using the plug specifically comprises the following steps:
s51: selecting 50-hole plug trays or 72-hole plug trays;
s52: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s53: planting the bottle seedlings in a substrate of a plug tray, and shading intermittently during seedling hardening.
In the above step, the matrix used in the tray may be vermiculite: perlite: the peat soil is a matrix mixed according to the proportion of 1:1:1, or a special seedling raising matrix.
: preparing a seedbed, and building the seedbed for seedling breeding. The method for preparing the seedbed specifically comprises the following steps:
s61: arranging an elevated seedbed, taking a U-shaped foam groove as a seedbed framework, and taking coconut coir as a matrix to be paved in the U-shaped groove;
s62: and (3) paving drip irrigation equipment on the elevated seedbed.
: and (5) planting original seedlings. The method for planting the original seedlings specifically comprises the following steps:
s71: planting seedlings in a single row on an elevated seedbed, wherein the distance between adjacent seedlings is 25-30 cm;
s72: and adding a high-nitrogen water-soluble fertilizer into the drip irrigation equipment for irrigation.
In the above steps, the method for planting seedlings comprises: domesticating the seedlings under the condition of normal temperature and weak illumination for 1 to 2 days, then cleaning agar, planting the agar on a high ridge, watering thoroughly after planting, shading (arranging a shading net) in the first week, keeping the humidity at 75 to 90 percent, and removing the shading net after the base of the stem segment grows roots.
: and (4) performing plug propagation, and performing large-batch breeding on a small amount of cultured healthy seedlings. The method for expanding propagation of the plug specifically comprises the following steps:
s81: more 50-hole or 72-hole plug trays are selected;
s82: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s83: cutting propagation is adopted, and when the height of other healthy seedlings is more than 30cm, stem sections with 3-5 internodes of 8-10 cm are cut by a disinfection cutter;
s84: cutting the base of the stem section in a hole tray, watering thoroughly after planting, compacting by water, keeping the humidity at 75% -90%, shading, and removing the shading net after the base of the stem section grows roots.
Hang white chrysanthemum seedling field planting on overhead seedbed, the advantage includes: (1) the plant group has good light transmission and air permeability, and the chrysanthemum morifolium plants have sufficient lighting; (2) the watering and fertilizing are convenient, the fertilizer can be directly applied to the root position of the chrysanthemum morifolium ramat plant, water and fertilizer are saved, and the root of the plant is developed; (3) make things convenient for the later stage to carry out the harvesting of Hangzhou white chrysanthemum through mechanical equipment, it is efficient to pick.
Thirdly, confirming the actual effect
Taking the 'chrysanthemum indicum' and the 'chrysanthemum indicum' as examples, the breeding method is adopted for breeding. The detoxified 'early chrysanthemum' and the detoxified 'chrysanthemum morifolium' bred by the method have significant differences from the conventional chrysanthemum morifolium in the aspects of the parameters of the number of branches, the number of effective flowers and the relative content of chlorophyll. Wherein the acre yield of the detoxified early-American chrysanthemum can reach 490.1kg at most, and the yield is increased by 11 percent; the yield per mu of the detoxified chrysanthemum morifolium can reach 1083.3kg at most, and the yield is increased by 29 percent. For comparison, the contents of total flavonoids in the detoxified "chrysanthemum senecio" and the detoxified "chrysanthemum indicum" are respectively 70.41ng/g and 63.94mg/g, while the contents in the conventional seedlings are respectively 53.44mg/g and 43.41 mg/g; the contents of chlorogenic acid in the detoxified "chrysanthemum senecium" and the detoxified "chrysanthemum senecium" are respectively 2.71mg/g and 2.13mg/g, while the contents in the conventional seedling are respectively 1.62mg/g and 1.01 mg/g. In addition, the chlorogenic acid content in detoxified "chrysanthemum senecio" and detoxified "chrysanthemum indicum" is higher than 0.2% specified in the pharmacopoeia.
According to the breeding method of the high-quality Hangzhou white chrysanthemum, the seedlings are detoxified in a multi-step and step mode, the Hangzhou white chrysanthemum tissue culture bottle seedlings with CVB removed can be obtained, the Hangzhou white chrysanthemum tissue culture bottle seedlings can be bred quickly, the Hangzhou white chrysanthemum tissue culture bottle seedlings are planted in a large batch mode in a mode of overhead seedbed planting and matched with the Hangzhou white chrysanthemum tissue culture bottle seedlings, the yield of the Hangzhou white chrysanthemum can be effectively improved, and the produced Hangzhou white chrysanthemum is high in quality.
The above description of the present invention is intended to be illustrative. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.
Claims (10)
1. A breeding method of high-quality Hangzhou white chrysanthemum is characterized by comprising the following steps:
s1: sampling explants, selecting plants with pure variety characteristics, good quality and no plant diseases and insect pests in the optimal time, and cutting plant stem segments with growing points to be 2-3 cm;
s2: sterile seedling cultivation, namely cleaning a plant stem segment with sterile water, placing the plant stem segment on a super-clean workbench, cutting the stem segment with a growing point by 1-2 cm, washing the surface with 70-75% of ethanol for 0.5-1 min, then sequentially sterilizing with hypochlorite and Tween-20 for 8-12 min, continuously shaking, finally washing with sterile water for 5-8 times, sucking water with sterile filter paper, stripping a stem tip meristem with the diameter of 0.2-0.3 mm, and inoculating the stem tip meristem in a stem tip culture medium I;
s3: culturing tissue culture bottle seedlings, namely culturing aseptic plants in a first stem tip culture medium to 1.5-2.0 cm, placing the aseptic plants under a dissecting mirror, selecting 0.2-0.3 mm stem tip meristem, inoculating the stem tip meristem in a second stem tip culture medium, and inoculating the aseptic plants in a rooting culture medium when the second stem tip culture medium grows to 5-6 cm to obtain tissue culture bottle seedlings;
s4: tissue culture bottle seedling rapid breeding;
s5: hardening seedlings by using a plug;
s6: preparing a seedbed;
s7: planting original seedlings;
s8: and (5) expanding propagation of the aperture disk.
2. The breeding method of high-quality chrysanthemum morifolium ramat according to claim 1, wherein the rapid breeding in the step S4 specifically comprises the following steps:
s41: performing virus detection on the sterile plant, and inoculating the sterile plant with a qualified detection result into a culture medium;
s42: when the plant grows to 6-8 cm, cutting stem segments containing 1-2 internodes, and inoculating the stem segments into a propagation culture medium;
s43: repeating the previous step, and subculturing every 20-30 days.
3. The breeding method of high-quality chrysanthemum morifolium ramat according to claim 1, wherein the plug seedling hardening in the S5 specifically comprises the following steps:
s51: selecting 50-hole plug trays or 72-hole plug trays;
s52: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s53: planting the bottle seedlings in a substrate of a plug tray, and shading intermittently during seedling hardening.
4. The breeding method of high-quality chrysanthemum morifolium ramat according to claim 1, wherein the seedbed preparation in the step S6 specifically comprises the following steps:
s61: arranging an elevated seedbed, taking a U-shaped foam groove as a seedbed framework, and taking coconut coir as a matrix to be paved in the U-shaped groove;
s62: and (3) paving drip irrigation equipment on the elevated seedbed.
5. The breeding method of high-quality chrysanthemum morifolium ramat according to claim 1, wherein the original seedling planting in the S7 comprises the following steps:
s71: planting seedlings in a single row on an elevated seedbed, wherein the distance between adjacent seedlings is 25-30 cm;
s72: and adding a high-nitrogen water-soluble fertilizer into the drip irrigation equipment for irrigation.
6. The breeding method of high-quality chrysanthemum morifolium ramat according to claim 1, wherein the plug propagation in the S8 comprises the following steps:
s81: more 50-hole or 72-hole plug trays are selected;
s82: vermiculite is adopted: perlite: putting the matrix mixed by peat soil according to the proportion of 1:1:1 in an aperture disk;
s83: cutting propagation is adopted, and when the height of a planted seedling is more than 30cm, stem sections with 3-5 internodes of 8-10 cm are cut by a disinfection cutter;
s84: cutting the base of the stem section in a hole tray, watering thoroughly after planting, compacting by water, keeping the humidity at 75% -90%, shading, and removing the shading net after the base of the stem section grows roots.
7. The method for breeding high-quality chrysanthemum morifolium ramat according to claim 5, wherein the method comprises the following steps: the planting method in the S81 comprises the steps of domesticating seedlings under the condition of normal temperature and weak illumination for 1 to 2 days, then cleaning agar, planting the agar on a seedbed, watering thoroughly after planting, shading in the first week, keeping the humidity at 75 to 90 percent, and removing a shading net after the base of a stem segment grows roots.
8. The method for breeding high-quality chrysanthemum morifolium ramat according to claim 1, wherein the method comprises the following steps: the optimal time in the S1 is sunny days of 10-11 months per year, and the specific sampling time is 10: 00-16: 00.
9. The method for breeding high-quality chrysanthemum morifolium ramat according to claim 1, wherein the method comprises the following steps: the components of the rooting culture medium in the S3 are 1/2MS and 0.1mg/L NAA; the components of the stem tip culture medium are MS +0.1mg/L6-BA +0.1 mg/LNAA.
10. The method for breeding high-quality chrysanthemum morifolium ramat according to any one of claims 1 to 9, which is characterized in that: the culture conditions of the tissue culture bottle seedlings in the S2, the S3 and the S4 are as follows: in the daytime, the temperature is 25 +/-2 ℃, the illumination time is 12-14 h, and the illumination intensity is 30 mu mol-2 s-1-60 mu mol-2 s-1; and at night, the temperature is 18 +/-2 ℃, and the culture in a dark environment is ensured.
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