CN115885849A - Industrial tissue culture rapid propagation method of Wuping germplasm Mesona chinensis - Google Patents
Industrial tissue culture rapid propagation method of Wuping germplasm Mesona chinensis Download PDFInfo
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Abstract
The invention relates to an industrial tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis Benth, belonging to the technical field of plant tissue culture. The invention relates to an industrial tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis Benth, which comprises the following steps: s1: obtaining a sterile explant; s2: primary adventitious bud induction culture; s3: subculture multiplication of adventitious buds; s4: rooting culture; s5: hardening and transplanting the seedlings. The tissue culture rapid propagation method has the advantages of simple culture medium, short culture period, high success rate, high induction rate, high propagation coefficient, high genetic stability, good seedling quality and low cost, is suitable for industrial production of Wuping germplasm mesona chinensis, can meet the large-scale planting requirement of Wuping germplasm mesona chinensis, and solves the current situation that high-quality mesona chinensis resources are scarce day by day and supply is not in demand in the market.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to an industrial tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis.
Background
Mesona chinensis Benth, a plant of the genus Mesona in the family of Labiatae, is mainly distributed in tropical and subtropical humid areas such as Guangdong, taiwan, fujian and Guangxi in China, belongs to a perennial root annual herbaceous plant, is astringent, sweet and cold in nature and taste, has the effects of clearing summer heat, quenching thirst and cooling blood, can be used for treating diseases such as heatstroke, hypertension, muscle and joint pain and the like, is an important plant for both medicine and food, and is an important oriental economy and medicinal plant resource. The mesona chinensis benth as a Chinese herbal medicine and a raw material for preparing the bean jelly has a long history in China and south-east Asia regions, and researches show that the bean jelly polysaccharide has obvious lipid peroxidation resistance, the function of improving the immunity of the organism, the damage resistance and the like. In addition, the mesona chinensis benth is widely used as a raw material for preparing various health-care functional foods, such as herbal tea, health-care tea, tortoise jelly, burnt mesona chinensis benth and the like, and can also be processed into mesona chinensis benth dew, mesona chinensis benth essence and mesona chinensis benth honey.
Mesona chinensis Benth is used as a raw material plant for herbal tea and bean jelly, and the demand for the herbal tea and the bean jelly is increased year by year. The agrimony belongs to asexual propagation crops, although the agrimony can blossom and knot, the variation is easy, the seed quantity is small, the seedling emergence and the survival are not easy, the seeds are not suitable for propagation, so the agrimony is mainly based on the cutting propagation mode. However, the germplasm of the mesona chinensis benth is seriously degraded due to long-term asexual propagation, the wild resources are in short supply, the excellent seedlings are very deficient, the existing varieties are too short in growth period, easy to fall down, susceptible to diseases and insect pests and the like, and the fine variety of the mesona chinensis benth is in short supply.
Therefore, the method develops the industrial tissue culture and rapid propagation research of the mesona chinensis benth with different germplasms, establishes an industrial tissue culture and rapid propagation system of the mesona chinensis benth, and cultures a large number of high-quality, robust and high-yield mesona chinensis benth seedlings, is the most effective way for protecting excellent germplasms and solving the contradiction between supply and demand, and has remarkable social benefit and economic benefit.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides an industrial tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis.
The invention is realized by the following technical scheme:
the invention provides an industrial tissue culture and rapid propagation method of Wuping germplasm immortal grass, which comprises the following steps:
s1: picking up strong and healthy Wuping seed mesona blume stem segments without diseases, insect pests and 2-3 stem nodes, cleaning and sterilizing to obtain a sterile explant;
s2: cutting the sterile explant obtained in the step S1 into 1.0-2.5 cm stem sections with one stem node, inoculating the stem sections into a primary induction culture medium, and culturing for 5-15 days to obtain adventitious buds;
s3: cutting 0.5-1.5 cm from the base of the adventitious bud obtained in the step S2, inoculating the cut adventitious bud into a subculture proliferation induction culture medium, and culturing for 15-30 days to obtain the adventitious bud subjected to subculture proliferation;
s4: cutting 1.0-3.0 cm from the base of the adventitious bud obtained in the step S3 through subculture proliferation culture, inoculating the cut adventitious bud into a rooting culture medium, and culturing for 22-43 days to obtain a transplantable test-tube plantlet;
s5: and (4) hardening the test-tube plantlets obtained in the step (S4) for 3-5 days, transplanting the test-tube plantlets into a seedling culture medium, pouring a mixed solution of the sterilizing agent and the rooting powder, covering the test-tube plantlets with a transparent plastic film for 7-15 days, and then placing the test-tube plantlets in a small shed with proper sunshade for continuous culture for 15-25 days to obtain the Mesona chinensis seedlings.
The invention selects Wuping county seed mesona chinensis benth in Fujian province, which has the excellent characteristics of high yield, high quality, disease and pest resistance and the like, the tissue culture rapid propagation method of the invention has the advantages of simple culture medium, shorter culture period, high success rate, high induction rate, high propagation coefficient, high genetic stability, good seedling quality and low cost, is suitable for industrial production of Wuping germplasm mesona chinensis benth, can meet the large-scale planting requirement of the Wuping germplasm mesona chinensis benth, and solves the current situation that the high-quality mesona chinensis benth resource is gradually scarce and the supply is not in demand in the market.
As a preferred embodiment of the tissue culture and rapid propagation method of the present invention, in step S1, the cleaning and disinfecting method comprises: washing with tap water for 1min-2min, drying surface water with sterile absorbent paper, rinsing with 75% alcohol for 5s-10s on a clean bench, cleaning with sterile water for 1-3 times (1 min-2min each time), sterilizing with 10% chlorine dioxide solution for 13min-18min, and rinsing with sterile water for 3-4 times (1 min-2min each time).
As a preferred embodiment of the tissue culture and rapid propagation method of the present invention, in step S2, the formula of the primary induction medium is: WPM,0.2-0.8 mg/L6-BA,0.05-0.1mg/L NAA,30-50g/L banana puree, 25-40g/L sucrose, 6.0-8.0g/L agar, and pH is 5.8-6.2.
As a preferred embodiment of the tissue culture and rapid propagation method of the present invention, in step S2, the formula of the primary induction medium is: WPM,0.5 mg/L6-BA, 0.1mg/L LNAA,50g/L banana puree, 30g/L sucrose, 7.0g/L agar, pH 5.9.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in step S3, the formula of the secondary proliferation induction medium is: WPM,0.1-0.5mg/L6-BA,0.05-0.1mg/L LNAA,0.5-1.0g/L peptone, 25-40g/L sucrose, 6.0-8.0g/L agar, pH 5.8-6.2.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in the step S3, the formula of the secondary proliferation induction culture medium is: WPM,0.1mg/L6-BA,0.05mg/LNAA,1g/L peptone, 30g/L sucrose, 7.0g/L agar, pH 5.9.
As a preferred embodiment of the tissue culture and rapid propagation method of the present invention, in step S4, the formula of the rooting medium is: 1/2MS or WPM,0.2-0.5g/L peptone, 25-40g/L sucrose, 6.0-8.0g/L agar, and pH 5.8-6.2.
As a preferred embodiment of the tissue culture and rapid propagation method of the present invention, in step S4, the formula of the rooting medium is: 1/2MS or WPM,0.5g/L peptone, 30g/L sucrose, 7.6g/L agar, pH 5.9.
As a preferred embodiment of the tissue culture and rapid propagation method, in the step S5, the sterilizing agent is 0.4 to 0.6 percent by mass, and the rooting powder is 0.1 to 0.3 percent by mass; the seedling raising substrate comprises peat soil, vermiculite, perlite and river sand.
Preferably, the mass fraction of the sterilizing agent is 0.5%, and the mass fraction of the rooting powder is 0.2%; the mass ratio of peat soil, vermiculite, perlite and river sand in the seedling substrate bag is peat soil: vermiculite: perlite: river sand =1:1:1:1.
as a preferred embodiment of the tissue culture rapid propagation method, the culture temperature of the culture in the steps S2, S3 and S4 is 25-26 ℃, the illumination intensity is 1500lx-2000lx, and the illumination time is 10-12 hours/day.
Preferably, the primary generation induction culture medium, the secondary multiplication induction culture medium and the rooting culture medium are subjected to high-temperature damp-heat sterilization for later use.
The concentration fractions of 0.5 percent of carbendazim and 0.2 percent of rooting powder are mass fractions, and the rest concentration fractions are volume fractions.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention selects the stem section of Wuping germplasm Mesona chinensis as the explant, the cluster bud induction speed is high, and the rapid seedling culture of the Wuping germplasm Mesona chinensis can be realized within 20-40 days through the steps of induction, subculture, rooting, transplanting and the like by screening and optimizing the culture medium formula and the culture conditions in the processes of cluster bud primary induction, cluster bud subculture proliferation, rooting culture, hardening seedling transplanting and the like. The invention has the characteristics of high success rate, high inductivity, high propagation coefficient, high genetic stability, good seedling quality, low cost, consistent seedling growth, short breeding period and the like.
(2) The invention adopts plant tissue culture technology to establish the industrial tissue culture rapid propagation of Wuping germplasm Mesona chinensis, has universal applicability, has great significance for promoting the popularization and the planting of Wuping germplasm Mesona chinensis and the protection and the development of improved variety resources, and is beneficial to the improvement of high-quality Mesona chinensis varieties and the academic research thereof in the future.
Drawings
FIG. 1 shows the first generation of the Wuping germplasm Mesona chinensis (L.) Merr.f.in example 1 for inducing adventitious buds;
FIG. 2 shows the secondary proliferation of Wuping germplasm Mesona chinensis in example 1 to induce adventitious buds;
FIG. 3 shows the elongated tissue culture seedlings of Wuping germplasm Mesona in example 1;
FIG. 4 is the example 1, the Wuping germplasm Mesona chinensis Benth rooted tissue culture seedling;
FIG. 5 shows the tissue culture seedlings of Mesona wuping after transplantation in example 1;
FIG. 6 shows the example 1 of transplantation management of industrial seedlings of Wuping germplasm Mesona chinensis.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. Wherein, carbendazim is purchased from Jiangsu san shan pesticide Limited company, and the batch number is: HG/T3290-2016; rooting powder was purchased from Zhengzhou Shangun agriculture science and technology Limited, lot number: NY1428-2010.
Example 1
A tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis comprises the following steps:
s1, obtaining a sterile explant: selecting mesona chinensis benth plants which are transplanted to a greenhouse culture of Guangdong Hanchao Chinese medicine science and technology limited company from a mesona chinensis benth planting base in Wuping county, longyan city, fujian province, picking robust mesona chinensis benth stem sections without diseases and insect pests and with 2-3 stem sections, wherein the picking time is about 14 hours in sunny days; slowly washing the picked tender cactus stem segments with tap water for 2min, drying surface water with sterile absorbent paper, rinsing with 75% ethanol solution for 5s in a super clean bench, rinsing with sterile water for 2 times (1 min each time), sterilizing with 10% chlorine dioxide solution for 13min, and rinsing with sterile water for 3 times (1 min each time);
s2, adventitious bud primary induction culture: cutting the disinfected stem segment of Mesona chinensis Benth into 1.5cm long stem segment with one stem node, inoculating into cluster bud primary induction culture medium, culturing for about 3 days to obtain adventitious bud of Mesona chinensis Benth, culturing for 7 days, wherein the adventitious bud length is greater than 1cm, and the induction rate is 100%. The primary induction culture medium comprises: WPM culture medium, 0.5mg/L6-BA, 0.1mg/LNAA,50g/L banana puree, 30g/L sucrose and 7.0g/L agar, and pH is 5.9;
s3, carrying out subculture multiplication culture on the adventitious buds: shearing the adventitious buds subjected to primary induction culture and with the length of more than 1.0cm, inoculating the adventitious buds into a subculture multiplication culture medium, and culturing for 30 days to realize the multiplication of the adventitious buds, wherein the multiplication coefficient reaches 10.05; the subculture proliferation induction proliferation culture medium comprises: WPM medium, 0.1mg/L6-BA,0.05mg/LNAA,1g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s4, rooting culture: and (2) shearing the adventitious buds which are cultured in the subculture multiplication step S3 and have the length of about 3.0cm and good growth from the base, inoculating the adventitious buds into a rooting medium, culturing the adventitious buds for about 5 days to start rooting, and continuously culturing for 35 days to obtain the transportable test-tube plantlets, wherein the rooting rate reaches 100%, and the rooting medium is: 1/2MS culture medium, 0.5g/L peptone, 30g/L sucrose and 7.0g/L agar, and the pH is 5.9;
s5, transplanting: selecting the tissue culture seedling with better root growth in the step S4, opening a bottle cap, spraying water mist, hardening the seedling for 3 days, taking out the tissue culture seedling, washing the culture medium of the root, transplanting the tissue culture seedling into a seedling raising tray with a certain proportion of matrix placed therein, wherein the proportion of the transplanting matrix is peat soil: vermiculite: perlite: river sand =1:1:1:1, pouring 0.5 percent of sterilizing agent and 0.2 percent of rooting powder mixed solution, covering the solution for 7 days by using a transparent plastic film, placing the solution in a small shed with proper sunshade for continuously culturing for 25 days to obtain Wuping germplasm Mesona seedlings, and transplanting the seedlings for 30 days until the survival rate reaches 100 percent.
The culture conditions of the steps S2-S4 are as follows: culturing at 25 deg.C under illumination intensity of 1500lx for 12 hr/day, and sterilizing the culture medium at high temperature and humidity for 116 deg.C/20 min.
Example 2
A tissue culture and rapid propagation method of Mesona chinensis Benth comprises the following steps:
s1, obtaining a sterile explant: selecting mesona chinensis benth plants which are transplanted to a greenhouse culture of Guangdong Hanchao Chinese medicine science and technology limited company from a mesona chinensis benth planting base in Wuping county, longyan city, fujian province, picking robust mesona chinensis benth stem sections without diseases and insect pests and with 2-3 stem sections, wherein the picking time is about 14 hours in sunny days; slowly washing the picked tender cactus stem segments with tap water for 2min, drying surface water with sterile absorbent paper, rinsing with 75% ethanol solution for 5s in a super clean bench, rinsing with sterile water for 2 times (1 min each time), sterilizing with 10% chlorine dioxide solution for 14min, and rinsing with sterile water for 3 times (1 min each time);
s2, primary induction culture of adventitious buds: cutting the disinfected stem segment of the Mesona chinensis Benth into a stem segment with a length of 1.5cm and inoculating the stem segment into a cluster bud primary induction culture medium, culturing for about 3 days until adventitious buds of the Mesona chinensis Benth obviously grow at the stem segment, culturing for 7 days, wherein the length of the adventitious buds is more than 1cm, and the induction rate reaches 100%; the primary induction culture medium comprises: WPM culture medium, 0.8mg/L6-BA, 0.1mg/LNAA,50g/L banana puree, 30g/L sucrose and 7.0g/L agar, and pH is 5.9;
s3, carrying out subculture multiplication culture on the adventitious buds: shearing the adventitious bud with the length of more than 1.0cm, inoculating the adventitious bud to a subculture multiplication medium, and culturing for 40 days to realize the multiplication of the adventitious bud, wherein the multiplication coefficient reaches 11.20; the adventitious bud induction multiplication culture medium comprises: WPM medium, 0.2 mg/L6-BA,0.05mg/LNAA,1g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s4, rooting culture: scissoring the adventitious bud which is cultured in the step S3 and has the length of about 3.0cm and good growth from the base part, inoculating the scissored bud into a rooting culture medium, culturing the adventitious bud for about 3 days to start rooting, and continuously culturing for 35 days to obtain a transportable test-tube plantlet, wherein the rooting rate reaches 100%; the rooting culture medium comprises: 1/2MS culture medium, 0.5g/L peptone, 30g/L sucrose and 7.0g/L agar, and the pH is 5.9;
s5, transplanting: selecting the tissue culture seedling with better root growth in the step S4, opening the bottle cap, spraying water mist, hardening the seedling for 5 days, taking out the tissue culture seedling, washing the culture medium of the root, transplanting the tissue culture seedling into a seedling raising tray with a certain proportion of matrix placed, wherein the proportion of the transplanting matrix is peat soil: vermiculite: perlite: river sand =1:1:1:1, pouring 0.5 percent of sterilizing agent and 0.2 percent of rooting powder mixed solution, covering the solution for 7 days by using a transparent plastic film, placing the solution in a small shed with proper sunshade for continuously culturing for 25 days to obtain the Mesona chinensis Benth seedlings, and transplanting the seedlings for 30 days until the survival rate reaches 99.65 percent.
The culture conditions of the steps S2-S4 are as follows: the culture temperature was 25 ℃, the light intensity was 2000lx, and the light time was 12 hours/day. The culture medium is sterilized by high temperature and moist heat at 116 deg.C/20 min.
Comparative example 1
A tissue culture and rapid propagation method of Mesona chinensis Benth comprises selecting leaves of a plant of Mesona chinensis Benth which has been transplanted from the Zengcheng district of Guangzhou city of Guangdong province and has grown for 3 months as explant, and the rest is the same as example 1, and the result shows that the germination rate of the first generation induction culture is 53.33%, the time required for growing adventitious buds of more than 1cm is 35-50 days, and the vitrification rate is 57.14%. The reason is guessed that the induction rate and the germination rate are low and the Mesona chinensis seedling cannot be rapidly cultured and bred due to the long time required for the dedifferentiation and the redifferentiation of the leaves, the vitrification phenomenon easily occurs in the process and the browning rate of the explant is high.
Comparative example 2
The tissue culture and rapid propagation method of Mesona chinensis Benth is the same as example 1 except that NAA is not added into a primary induction culture medium, and results show that the germination rate of the primary induction culture is only 62.50%, the vitrification rate is 55.56%, and the time required for growing adventitious buds of more than 1cm is 15 days.
Comparative example 3
A tissue culture and rapid propagation method of Mesona chinensis Benth uses MS as a basic culture medium for rooting induction, the rest is the same as that in example 1, and the result shows that the time required for rooting is 13-15 days, the plant is short, the main root number is less, the root is thin and short, the stem is thin, the leaf is small, the leaf is yellow, the rooting rate is 77.50%, and the survival rate after 30 days of transplanting is only 56%.
The following is a research study of the tissue culture rapid propagation method of Mesona chinensis Benth.
Test example 1 screening of growth regulator combinations
1. Effect of different combinations of plant growth regulators on adventitious bud Primary Induction
MS is used as a basic culture medium, 30g/L of sucrose and 7.0g/L of agar are added into the culture medium, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The culture medium of the invention is prepared by carrying out high-temperature moist heat sterilization at 116 ℃ for 20min and cooling for later use (the same below). The plant growth regulator combination primary induction culture medium is inoculated into 30 bottles of 3 explants in each bottle, the explants are repeated for 3 times, the growth condition and the induction rate of the adventitious buds are recorded after 20 days of culture, and the influence of the plant growth regulator combinations with different types and concentrations on the adventitious bud primary induction is shown in Table 1.
TABLE 1 Effect of different types and concentrations of plant growth regulator combinations on adventitious bud Primary Induction
Note: different letters in the same case indicate significant differences, P < 0.05. The same applies below.
As can be seen from Table 1, when the plant growth regulator combination is 0.2-0.8 mg/L6-BA +0.05-0.1mg/L NAA, more adventitious buds are present at a rate higher than 1cm, the adventitious buds are higher, the vitrification rate is lower, the germination rate is more than 87%, the adventitious bud leaves are wider, the color is verdure, the adventitious buds are strong and grow well.
2. Effect of different combinations of plant growth regulators on adventitious bud induced proliferation
MS is used as a basic culture medium, 30g/L of sucrose and 7.0g/L of agar are added into the culture medium, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The plant growth regulators are combined with the adventitious bud subculture multiplication induction culture mediums and are inoculated into 30 bottles respectively, 2 adventitious buds are added into each bottle, and the process is repeated for 3 times. And recording the growth condition of the adventitious buds after culturing for 40 days, and counting the number of the adventitious buds higher than 1cm in each bottle, the average length of the highest new buds, the vitrification rate and the proliferation coefficient. The effect of different types and concentrations of plant growth regulator combinations on adventitious bud subculture proliferation is shown in table 2.
TABLE 2 Effect of different types and concentrations of plant growth regulator combinations on adventitious bud subculture proliferation
As shown in Table 2, when the plant growth regulator is 0.1-0.5mg/L6-BA +0.05-0.1mg/LNAA, the vitrification rate of the adventitious buds is low, the germination rate is high, the number of the adventitious buds is more than 1cm, the average length of the buds is longer, the leaves of the adventitious buds are wider, the buds are green or light green, the stems are thicker, the clumpy buds are more, and the growth is better.
Test example 2 screening of minimal Medium
1. Effect of different basic Medium on Primary Induction
MS, 1/2MS or WPM are respectively used as basic culture media, the plant growth regulator is 0.5mg/L6-BA +0.1mg/LNAA, 30g/L of cane sugar and 7.0g/L of agar are added into the culture media, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The different minimal media were inoculated into 30 flasks each, 2 explant shoots were inoculated per flask, and repeated 3 times. And recording the bud growth condition of the explant after 20 days of culture, and counting the bud emergence days of stem segments of each bottle of the explant, wherein the number of the adventitious buds is more than 1cm, the length of the adventitious buds, the vitrification rate and the bud emergence rate. The effect of different basal media on primary induction is shown in table 3.
TABLE 3 Effect of different basic media on Primary Induction
As can be seen from Table 3, when the minimal medium was WPM, the germination was fast, the number of adventitious buds was large and the number of adventitious buds was high, the leaves of adventitious buds was wide and large, the color was verdant, the stems were strong and the growth was good.
2. Effect of different basal Medium on subculture Induction
MS, 1/2MS or WPM are respectively used as basic culture media, the plant growth regulator is 0.1mg/L6-BA +0.05mg/LNAA, 30g/L of cane sugar and 7.0g/L of agar are added into the culture media, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. 30 bottles of different minimal media were inoculated, each bottle was inoculated with 2 adventitious buds, and the process was repeated 3 times. And recording the growth condition of the adventitious buds after 40 days of culture, and counting the number of the adventitious buds higher than 1cm in each bottle, the maximum sprout length, the vitrification ratio and the proliferation coefficient. The effect of different basal media on subculture induction is shown in table 4.
TABLE 4 Effect of different basic media on subculture induction
As can be seen from Table 4, when the minimal medium was WPM, more adventitious buds were present at a height of more than 1cm, higher adventitious buds were present, higher multiplication factor, strong adventitious buds, and better growth.
3. Effect of different basic media on rooting
MS, 1/2MS or WPM are respectively used as basic culture media, 30g/L of sucrose and 7.0g/L of agar are added into the culture media, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The combined rooting medium was inoculated into 40 flasks, 2 adventitious shoots were transferred per flask, and the procedure was repeated 3 times. And recording the growth condition of adventitious buds after culturing for 35 days, and counting the initial rooting days, plant height, main root number, average main root length and rooting rate of each bottle of adventitious buds. The effect of different basic media on adventitious bud rooting is shown in Table 5.
TABLE 5 Effect of different basic media on rooting
As can be seen from Table 5, when the minimal medium is 1/2MS or WPM, the roots of the adventitious buds of Mesona chinensis Benth grow faster, the main roots are thicker and longer, the leaves are wide and large, the leaves are dark green, the stems are thicker and healthy, the plants grow better, and the seedlings survive more easily after being transplanted, while the adventitious buds of MS, the minimal medium is more in the number of the main roots, the plants are higher, but the roots grow slower, the main roots are thicker and shorter, the side roots are fewer, the leaves are smaller, the leaves are more yellow, the stems are slender, and the transplanting survival rate is lower. By integrating various indexes, the optimal basic culture medium for Wuping germplasm Mesona chinensis rooting is 1/2MS or WPM.
Test example 3 screening of organic additives
1. Effect of different organic additives on Primary Induction
MS is used as a basic culture medium, a plant growth regulator is 0.5mg/L6-BA +0.1mg/LNAA, banana puree, potato puree, peptone and coconut juice organic additives are respectively added, 30g/L sucrose and 7.0g/L agar are added into the culture medium, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The different media were inoculated in 30 flasks each, with 2 explant shoots inoculated per flask, and repeated 3 times. And recording the bud growth condition of the explant after 20 days of culture, and counting the bud emergence days of each bottle of explant stem segments, wherein the number of the adventitious buds is more than 1cm, the length of the adventitious buds, the vitrification rate and the bud emergence rate. The effect of different organic additive media on primary induction is shown in table 6.
TABLE 6 Effect of different organic additives on Primary Induction
As can be seen from Table 6, when 50g/L of banana puree was added, the adventitious buds germinated rapidly, more adventitious buds higher than 1cm, higher adventitious buds, no vitrified seedlings, a germination rate of 100%, strong adventitious buds and good growth were observed.
2. Effect of different organic additives on subculture Induction
MS is used as a basic culture medium, a plant growth regulator is 0.1mg/L6-BA +0.05mg/LNAA, banana puree, potato puree, peptone and coconut juice organic additives are respectively added, 30g/L sucrose and 7.0g/L agar are added into the culture medium, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The culture medium was inoculated into 30 flasks each, and each flask was inoculated with 2 adventitious buds, which were repeated 3 times. And recording the growth condition of the adventitious buds after culturing for 40 days, and counting the budding days of each bottle of the adventitious buds, wherein the number of the adventitious buds is more than 1cm, the length of the adventitious buds, the vitrification rate and the budding rate. The effect of different organic additive media on subculture induction is shown in table 7.
TABLE 7 Effect of different organic additives on subculture induction
As can be seen from Table 7, when 1g/L peptone was added, more adventitious buds higher than 1cm were present, higher adventitious buds were present, no vitrified seedlings were present, the proliferation coefficient was higher, the adventitious buds were robust, and the growth was better.
3. Effect of different organic additives on rooting
1/2MS is used as a basic culture medium, no plant growth regulator is added, banana puree, potato puree, peptone and coconut juice organic additives are respectively added, 30g/L of sucrose and 7.0g/L of agar are added into the culture medium, the pH value is 5.9, the culture temperature is 25 ℃, the continuous illumination is 12h/d, and the illumination intensity is 1500-2000Lx. The combined rooting medium was inoculated into 40 flasks, 2 adventitious shoots were transferred per flask, and the procedure was repeated 3 times. And recording the growth condition of adventitious buds after culturing for 35 days, and counting the initial rooting days, plant height, main root number, average main root length and rooting rate of each bottle of adventitious buds. The effect of different basic media on adventitious bud rooting is shown in Table 8.
TABLE 8 Effect of different organic additives on rooting
As can be seen from Table 8, when 0.5g/L peptone was added, the adventitious bud roots grew faster, the plants were higher, the number of main roots was larger, the main roots were thicker and longer, the leaves were wide and large, the leaf color was dark green, the stems were thick and healthy, and the plants were robust.
Test example 4 screening of seedling substrate
Taking peat soil, vermiculite, perlite and river sand as seedling hardening and transplanting substrates, selecting plants which grow consistently and well after rooting induction, firstly opening the plants at room temperature for 3 days, covering the plants with a transparent plastic film in the period, then flushing a culture medium on the surface of a root system with running water, transplanting the plants into the mixed substrate, pouring 0.5% of sterile ethyl and 0.2% of rooting powder mixed solution, treating 100 plants each, putting the plants into a small shed with proper sunshade for hardening seedlings, watering the plants regularly, counting the survival rate after 30 days, and showing the influence of different substrate mixing ratios on the transplanting survival rate of the tissue-cultured seedlings of the mesona chinensis in a table 9.
TABLE 9 influence of different seedling-hardening media on the survival rate of Mesona chinensis Benth tissue culture seedlings
As can be seen from table 9, when the mass ratio of peat soil, vermiculite, perlite and river sand is peat soil: vermiculite: perlite: river sand =1:1:1: when 1 hour, the survival rate of the tissue culture seedlings of the Mesona chinensis Benth is the highest and reaches 100 percent.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. An industrial tissue culture and rapid propagation method of Wuping germplasm Mesona chinensis is characterized by comprising the following steps:
s1: picking up strong and healthy Wuping seed mesona blume stem segments without diseases, insect pests and 2-3 stem nodes, cleaning and sterilizing to obtain a sterile explant;
s2: cutting the sterile explant obtained in the step S1 into 1.0-2.5 cm stem sections with one stem node, inoculating the stem sections into a primary induction culture medium, and culturing for 5-15 days to obtain adventitious buds;
s3: cutting 0.5-1.5 cm from the base of the adventitious bud obtained in the step S2, inoculating the cut adventitious bud into a subculture proliferation induction culture medium, and culturing for 15-30 days to obtain the adventitious bud subjected to subculture proliferation;
s4: 1.0cm-3.0cm of the base part of the adventitious bud subjected to the subculture multiplication obtained in the step S3 is cut and inoculated into a rooting culture medium, and the rooting culture medium is cultured for 22-43 days to obtain a transplantable test-tube plantlet;
s5: and (4) hardening the test-tube plantlets obtained in the step (S4) for 3-5 days, transplanting the test-tube plantlets into a seedling culture medium, pouring a mixed solution of the sterilizing agent and the rooting powder, covering the test-tube plantlets with a transparent plastic film for 7-15 days, and then placing the test-tube plantlets in a small shed with proper sunshade for continuous culture for 15-25 days to obtain the Mesona chinensis seedlings.
2. The tissue culture rapid propagation method according to claim 1, wherein in the step S1, the cleaning and disinfecting method comprises: washing with tap water for 1min-2min, drying surface water with sterile absorbent paper, rinsing with 75% alcohol for 5s-10s on a clean bench, cleaning with sterile water for 1-3 times (1 min-2min each time), sterilizing with 10% chlorine dioxide solution for 13min-18min, and rinsing with sterile water for 3-4 times (1 min-2min each time).
3. The tissue culture rapid propagation method according to claim 1, wherein in the step S2, the formula of the primary induction medium is: WPM,0.2-0.8 mg/L6-BA,0.05-0.1mg/L NAA,30-50g/L banana puree, 25-40g/L sucrose, 6.0-8.0g/L agar, and pH is 5.8-6.2.
4. The tissue culture rapid propagation method according to claim 3, wherein in the step S2, the formula of the primary induction medium is as follows: WPM,0.5 mg/L6-BA, 0.1mg/L LNAA,50g/L banana puree, 30g/L sucrose, 7.0g/L agar, pH 5.9.
5. The tissue culture rapid propagation method according to claim 1, wherein in the step S3, the formula of the secondary proliferation induction culture medium is: WPM,0.1-0.5mg/L6-BA,0.05-0.1mg/LNAA,0.5-1.0g/L peptone, 25-40g/L sucrose, 6.0-8.0g/L agar, pH 5.8-6.2.
6. The tissue culture rapid propagation method according to claim 5, wherein in the step S3, the formula of the subculture proliferation induction medium is as follows: WPM,0.1mg/L6-BA,0.05mg/LNAA,1g/L peptone, 30g/L sucrose, 7.0g/L agar, pH 5.9.
7. The tissue culture rapid propagation method according to claim 1, wherein in the step S4, the formula of the rooting medium is as follows: 1/2MS or WPM,0.2-0.5g/L peptone, 25-40g/L sucrose, 6.0-8.0g/L agar, and pH 5.8-6.2.
8. The tissue culture rapid propagation method according to claim 7, wherein in the step S4, the formula of the rooting medium is as follows: 1/2MS or WPM,0.5g/L peptone, 30g/L sucrose, 7.6g/L agar, pH 5.9.
9. The tissue culture rapid propagation method according to claim 1, wherein in the step S5, the mass fraction of the sterilizing agent is 0.4% -0.6%, and the mass fraction of the rooting powder is 0.1% -0.3%; the seedling raising substrate comprises peat soil, vermiculite, perlite and river sand.
10. The tissue culture rapid propagation method according to claim 1, wherein the culture temperature of the culture in the steps S2, S3 and S4 is 25-26 ℃, the illumination intensity is 1500lx-2000lx, and the illumination time is 10-12 hours/day.
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