CN115885849B - Industrial tissue culture and rapid propagation method of Mesona chinensis Benth - Google Patents
Industrial tissue culture and rapid propagation method of Mesona chinensis Benth Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a method for industrial tissue culture and rapid propagation of Mesona chinensis Grandis, belonging to the technical field of plant tissue culture. The invention discloses a method for the industrialized tissue culture and rapid propagation of Wu Ping germplasm mesona, which comprises the following steps: s1: obtaining a sterile explant; s2: primary induction culture of adventitious buds; s3: carrying out subculture multiplication culture on adventitious buds; s4: rooting culture; s5: hardening and transplanting 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 Wu Ping germplasm mesona, can meet the large-scale planting requirement of the Wuping germplasm mesona, and solves the current situation that high-quality mesona resources are gradually scarce and the market is not required.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a factory tissue culture and rapid propagation method of Mesona chinensis Grandiflor.
Background
Mesona chinensis benth is a plant of Mesona of Labiatae, is mainly distributed in tropical subtropical humid areas such as Guangdong, taiwan, fujian, guangxi, etc., belongs to perennial annual herb plants, has astringent, sweet and cold nature, has effects of clearing summer heat, quenching thirst, cooling blood, can be used for treating heatstroke, hypertension, muscle and joint pain, etc., is an important medicinal and edible dual-purpose plant, and is an important eastern economy and medicinal plant resource. The mesona chinensis benth has long history in China and even southeast Asia as a raw material for preparing mesona chinensis benth, and researches show that mesona chinensis benth polysaccharide has remarkable lipid peroxidation resistance, organism immunity improvement, injury resistance and the like. In addition, the mesona chinensis is widely used as raw materials for preparing various health-care and functional foods, such as herbal tea, health-care tea, tortoise jelly, mesona chinensis and the like, can be processed into mesona chinensis syrup, mesona chinensis essence and mesona chinensis honey, and can be used for preparing mesona chinensis gum through polymer separation instead of imported endocarp, so that the mesona chinensis has a broad market prospect and becomes an important agricultural product for foreign exchange in export in some places in China.
The demand of mesona chinensis as a raw material plant of herbal tea and mesona chinensis is increasing year by year. Although the plant of Mesona can bloom and stick, the plant is easy to mutate, the seed quantity is not large, the plant is not easy to survive, and the plant is not suitable to propagate by seeds, so the Mesona is mainly in a cutting propagation mode. However, the germplasm of the mesona chinensis is seriously degenerated due to long-term asexual propagation, and the supply of wild resources is short, the excellent seedlings are very short, the existing variety has short growing period, is easy to lodge and is easy to infect diseases and insect pests, and the supply of fine variety mesona chinensis is short.
Therefore, the method develops the industrial tissue culture and rapid propagation research of the mesona chinensis benth of different germplasm, establishes an industrial tissue culture and rapid propagation system of the mesona chinensis benth, cultivates a large number of high-quality, robust and high-yield mesona chinensis benth seedlings, is the most effective way for protecting the excellent germplasm and solving the contradiction between supply and demand, and has obvious social benefit and economic benefit.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for the industrialized tissue culture and rapid propagation of Mesona with Wuping germplasm.
The invention is realized by the following technical scheme:
the invention provides a method for the industrialized tissue culture and rapid propagation of Mesona chinensis Grandis, which comprises the following steps:
s1: picking up Wu Ping germplasm mesona stems with 2-3 stems which are robust and free of plant diseases and insect pests, cleaning and sterilizing to obtain a sterile explant;
s2: cutting the sterile explant obtained in the step S1 into stem segments with a stem node of 1.0cm-2.5cm, inoculating the stem segments into a primary induction culture medium, and culturing for 5-15 days to obtain adventitious buds;
s3: shearing 0.5cm-1.5cm from the base of the adventitious bud obtained in the step S2, inoculating into a secondary proliferation induction culture medium, and culturing for 15-30 days to obtain a secondary proliferation cultured adventitious bud;
s4: shearing 1.0cm-3.0cm from the base of the adventitious bud obtained in the step S3 for inoculation into a rooting medium, and culturing for 22-43 days to obtain a transplantable test tube seedling;
s5: hardening seedlings of the test tube seedlings obtained in the step S4 for 3-5 days, transplanting the test tube seedlings into a seedling substrate, pouring mixed solution of sterilizing agent and rooting powder, covering the mixed solution with a transparent plastic film for 7-15 days, and then placing the test tube seedlings in a small shed with moderate sunshade for continuous culture for 15-25 days to obtain the mesona chinensis 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 Wu Ping germplasm mesona, can meet the large-scale planting requirement of the Wuping germplasm mesona, and solves the current situation that high-quality mesona resources are gradually scarce and the market is not required.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in the step S1, the method for cleaning and disinfecting is as follows: washing with tap water for 1-2 min, absorbing surface water with sterile absorbent paper, placing on an ultra-clean workbench, rinsing with 75% alcohol for 5s-10s, washing with sterile water for 1-3 times each for 1min-2min, sterilizing with 10% chlorine dioxide solution for 13min-18min, and rinsing with sterile water for 3-4 times each for 1min-2min.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in the step S2, the formulation of the primary induction medium is as follows: 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 5.8-6.2.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in the step S2, the formulation 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.
As a preferred embodiment of the tissue culture rapid propagation method of the present invention, in the step S3, the formulation of the subculture propagation induction medium is as follows: 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, and 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 formulation of the subculture propagation induction medium is as follows: WPM,0.1mg/L6-BA,0.05mg/L LNAA,1g/L peptone, 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 the step S4, the rooting medium comprises the following formula: 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 rapid propagation method of the present invention, in the step S4, the rooting medium comprises the following formula: 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 rapid propagation method, 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 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 package is that the 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 induction culture medium, the secondary proliferation induction culture medium and the rooting culture medium are subjected to high-temperature damp-heat sterilization treatment for later use.
In the invention, the concentration fractions of 0.5% carbendazim and 0.2% 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 segments of the Mesona chinensis Benth as the explants, the cluster bud induction speed is high, and the rapid seedling culture of the Mesona chinensis Benth can be realized within 20-40d through the steps of induction, subculture, rooting, transplanting and the like by screening and optimizing the culture medium formula and culture conditions in the processes of cluster bud primary induction, cluster bud subculture proliferation, rooting culture, seedling hardening, transplanting and the like. The invention has the characteristics of high success rate, high induction rate, high propagation coefficient, high genetic stability, good seedling quality, low cost, consistent seedling growth, short propagation period and the like.
(2) The invention establishes the industrial tissue culture and rapid propagation of Wu Ping germplasm mesona chinensis benth by adopting a plant tissue culture technology, has universal applicability, has great significance for promoting the popularization and the planting of the Wuping germplasm mesona chinensis benth and the protection and the development of improved variety resources, and is favorable for the improvement of quality varieties of mesona chinensis benth and academic research thereof in the future.
Drawings
FIG. 1 is a drawing of the initial induction of adventitious buds of Mesona chinensis Grandiflora in example 1;
FIG. 2 is a drawing of the adventitious bud induced by the secondary proliferation of Mesona chinensis Grandis of example 1;
FIG. 3 is an elongated tissue culture seedling of Mesona chinensis Grandiflorae of example 1;
FIG. 4 is a tissue culture seedling of Mesona chinensis benth rooting in example 1;
FIG. 5 shows a Wu Ping germplasm mesona tissue culture seedling after transplanting according to example 1;
FIG. 6 shows the management of the industrial seedling test tube plantlet transplantation of Mesona chinensis Grandiflorae in example 1.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available. Wherein, carbendazim is purchased from Jiangsu Sanshan pesticide Co., ltd., batch number: HG/T3290-2016; rooting powder is purchased from Zhengzhou Shi Kanong agricultural technologies, inc., lot number: NY1428-2010.
Example 1
A tissue culture and rapid propagation method of Mesona chinensis Benth comprises the following steps:
s1, acquiring a sterile explant: selecting a plant of Mesona chinensis cultivated in a greenhouse of Chinese medicine science and technology limited company of Guangdong, transplanting the plant of Mesona chinensis from a planting base of Mesona chinensis in Wu Ping county of Longyan, fujian, and picking a stem section of Mesona chinensis with 2-3 stem knots, wherein the picking time is about 14 hours on sunny days; slowly washing picked young grass stem segments with tap water for 2min, absorbing surface moisture by using sterile absorbent paper, placing the young grass stem segments in an ultra-clean workbench, rinsing with 75% ethanol solution for 5s, rinsing with sterile water for 2 times and 1min each time, and then sterilizing with 10% chlorine dioxide solution for 13min and rinsing with sterile water for 3 times and 1min each time;
s2, primary induction culture of adventitious buds: the sterilized mesona chinensis benth stem is cut into stem sections with the length of 1.5cm and one stem node is inoculated into a primary induction culture medium of cluster buds, adventitious buds of mesona chinensis benth are obviously grown at the stem node for about 3 days, the adventitious buds are cultured for 7 days, the length of the adventitious buds is more than 1cm, and the induction rate reaches 100%. The primary induction culture medium is as follows: WPM medium, 0.5mg/L6-BA, 0.1mg/L LNAA,50g/L banana puree, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s3, subculturing and proliferation culture of adventitious buds: shearing the adventitious buds with the length of more than 1.0cm through primary induction culture, inoculating the adventitious buds into a secondary proliferation culture medium, and culturing for 30 days to realize proliferation of the adventitious buds, wherein the proliferation coefficient reaches 10.05; the secondary proliferation induction proliferation culture medium is as follows: WPM medium, 0.1mg/L6-BA,0.05mg/L LNAA,1g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s4, rooting culture: and (3) cutting off adventitious buds which grow well and are subjected to subculture in the step (S3) from a basal part, inoculating the adventitious buds into a rooting culture medium, culturing for about 5 days, and continuously culturing for 35 days to obtain movable test tube seedlings, wherein the rooting rate reaches 100%, and the rooting culture medium is: 1/2MS culture medium, 0.5g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s5, transplanting: selecting a 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 a culture medium of the root, and transplanting the tissue culture seedling into a seedling raising tray in which a substrate with a certain proportion is placed, wherein the proportion of the transplanting substrate is peat soil: vermiculite: perlite: river sand = 1:1:1:1, pouring mixed solution of 0.5% sterilizing agent and 0.2% rooting powder, covering with transparent plastic film for 7 days, placing in a small shed with moderate sunshade, and culturing for 25 days to obtain Wuping germplasm Mesona chinensis seedling, and transplanting for 30 days to obtain 100% survival rate.
The culture conditions of the steps S2-S4 are as follows: the culture temperature is 25 ℃, the illumination intensity is 1500lx, the illumination time is 12 hours/day, the culture medium is Gao Wenshi and the heat sterilization is 116 ℃/20min.
Example 2
A tissue culture and rapid propagation method of Mesona chinensis comprises the following steps:
s1, acquiring a sterile explant: selecting a plant of Mesona chinensis cultivated in a greenhouse of Chinese medicine science and technology limited company of Guangdong, transplanting the plant of Mesona chinensis from a planting base of Mesona chinensis in Wu Ping county of Longyan, fujian, and picking a stem section of Mesona chinensis with 2-3 stem knots, wherein the picking time is about 14 hours on sunny days; slowly washing picked young grass stem segments with tap water for 2min, absorbing surface moisture by using sterile absorbent paper, placing the young grass stem segments in an ultra-clean workbench, rinsing with 75% ethanol solution for 5s, rinsing with sterile water for 2 times and 1min each time, and then sterilizing with 10% chlorine dioxide solution for 14min and then rinsing with sterile water for 3 times and 1min each time;
s2, primary induction culture of adventitious buds: cutting the sterilized mesona chinensis benth stem into stem segments with the length of 1.5cm and one stem node, inoculating the stem segments into a cluster bud primary induction culture medium, culturing for about 3 days until mesona chinensis benth adventitious buds obviously grow out, 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 is as follows: WPM medium, 0.8mg/L6-BA, 0.1mg/L LNAA,50g/L banana puree, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s3, subculturing and proliferation culture of adventitious buds: shearing the adventitious buds with the length of more than 1.0cm through primary induction culture, inoculating the adventitious buds into a secondary proliferation culture medium, and culturing for 40 days to realize proliferation of the adventitious buds, wherein the proliferation coefficient reaches 11.20; the adventitious bud induction proliferation culture medium is as follows: WPM medium, 0.2 mg/L6-BA,0.05mg/L LNAA,1g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s4, rooting culture: cutting off adventitious buds which grow well and are grown up by 3.0cm from a basal part and performing subculture multiplication in the step S3, inoculating the adventitious buds into a rooting culture medium, culturing for about 3 days, and continuously culturing for 35 days to obtain movable test tube seedlings, wherein the rooting rate reaches 100%; the rooting culture medium is as follows: 1/2MS culture medium, 0.5g/L peptone, 30g/L sucrose and 7.0g/L agar, pH 5.9;
s5, transplanting: selecting a tissue culture seedling with better root growth in the step S4, opening a bottle cap, spraying water mist, hardening the seedling for 5 days, taking out the tissue culture seedling, washing a culture medium of the root, and transplanting the tissue culture seedling into a seedling raising tray in which a substrate with a certain proportion is placed, wherein the proportion of the transplanting substrate is peat soil: vermiculite: perlite: river sand = 1:1:1:1, pouring mixed solution of 0.5% sterilizing agent and 0.2% rooting powder, covering with transparent plastic film for 7 days, placing in a small shed with moderate sunshade, and culturing for 25 days to obtain seedling of Mesona chinensis, and transplanting for 30 days to obtain survival rate of 99.65%.
The culture conditions of the steps S2-S4 are as follows: the culture temperature was 25℃and the illumination intensity was 2000lx, and the illumination time was 12 hours/day. The medium Gao Wenshi was heat sterilized at 116℃for 20min.
Comparative example 1
A tissue culture rapid propagation method of Mesona chinensis Benth adopts leaves of a potted Mesona chinensis Benth plant transplanted from the city-increasing area of Guangzhou, guangdong province and grown for 3 months as explants, and the other parts are the same as in example 1, and the result shows that the germination rate of primary induction culture is 53.33%, the time required for growing adventitious buds higher than 1cm is 35-50 days, and the vitrification rate is 57.14%. The reason is hypothesized that the leaf dedifferentiation and redifferentiation require longer time, vitrification is easy to occur in the process, the browning rate of the explant is high, the induction rate and germination rate are low, and the mesona chinensis seedlings cannot be quickly bred through tissue culture.
Comparative example 2
The method for tissue culture and rapid propagation of Mesona chinensis Benth is characterized in that NAA is not added into a primary induction culture medium, and the rest is the same as in example 1, and the result shows 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 higher than 1cm is 15 days.
Comparative example 3
The method for tissue culture and rapid propagation of Mesona chinensis Benth adopts MS as a basic culture medium for rooting induction, and the rest is the same as that of example 1, and results show that the time required for rooting is 13-15 days, the plants are short, the number of main roots is less, the roots are thin and short, the stems are thin, the leaves are small, the leaves are yellow, the rooting rate is 77.50%, and the survival rate after transplanting for 30 days is only 56%.
The following is a test study of the tissue culture and rapid propagation method of Mesona chinensis Benth of the present invention.
Test example 1 screening of growth regulator combinations
1. Effect of different plant growth regulator combinations on adventitious bud Primary Induction
MS is taken as a basic culture medium, 30g/L sucrose, 7.0g/L agar, pH of 5.9 and culture temperature of 25 ℃ are added into the culture medium, continuous illumination is carried out for 12h/d, and illumination intensity is 1500-2000Lx. The culture medium of the invention is sterilized by high temperature damp heat at 116 ℃ for 20min and then cooled for standby (the same applies below). The primary induction culture medium of each plant growth regulator combination is inoculated with 30 bottles, 3 pieces of explants are inoculated in each bottle, the culture is repeated for 3 times, the growth condition and the induction rate of the adventitious bud are recorded after 20 days of culture, and the influence of the plant growth regulator combinations of different types and concentrations on the primary induction of the adventitious bud is shown in Table 1.
TABLE 1 Effect of different types and concentrations of plant growth regulator combinations on adventitious bud Primary induction
Note that: the same case of different letters indicates significant differences, P < 0.05. The following is the same.
As is clear from Table 1, when the plant growth regulator composition was 0.2-0.8 mg/L6-BA+0.05-0.1mg/L NAA, the number of adventitious buds was large, the adventitious buds were high, the vitrification rate was low, the germination rate was more than 87%, the adventitious bud leaves were wide, the color was emerald green, the adventitious buds were strong, and the growth was good.
2. Effects of different plant growth regulator combinations on adventitious bud induced proliferation
MS is taken as a basic culture medium, 30g/L sucrose, 7.0g/L agar and pH of 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. Each plant growth regulator was inoculated with 30 flasks of adventitious bud subculture proliferation induction medium, 2 adventitious buds per flask, and repeated 3 times. After culturing for 40 days, recording the growth condition of adventitious buds, and counting the number of adventitious buds, the average length of the highest buds, the vitrification rate and the proliferation coefficient of each bottle, wherein the number of adventitious buds is higher than 1 cm. The effect of different types and concentrations of plant growth regulator combinations on adventitious bud proliferation is shown in Table 2.
TABLE 2 Effect of different types and concentrations of plant growth regulator combinations on adventitious bud proliferation
As shown in Table 2, when the plant growth regulator is 0.1-0.5mg/L6-BA+0.05-0.1mg/LNAA, the adventitious bud vitrification rate is low, the bud yield is high, the number of adventitious buds higher than 1cm is large, the average length of buds is long, the adventitious bud leaves are wide, the adventitious buds are green or light green, the stems are thicker, the cluster buds are more, and the growth is good.
Test example 2 screening of minimal Medium
1. Effect of different minimal Medium on Primary Induction
MS, 1/2MS or WPM is used as a basic culture medium, a plant growth regulator is 0.5mg/L6-BA+0.1mg/LNAA, 30g/L sucrose, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. The different minimal media were inoculated with 30 flasks, each with 2 explant stem segments, and repeated 3 times. Recording the bud growth condition of the explant after culturing for 20 days, and counting the bud emergence days of each bottle of the stem section of the explant, wherein the bud emergence days are higher than 1cm of adventitious bud number, adventitious bud length, vitrification rate and bud emergence rate. The effect of different minimal media on primary induction is shown in table 3.
TABLE 3 Effect of different minimal Medium on Primary Induction
As shown in Table 3, when the minimal medium was WPM, the buds were rapid, the number of adventitious buds higher than 1cm was large, the adventitious buds were high, the leaves of the adventitious buds were wide, the color was emerald green, the stems were strong, and the growth was good.
2. Effect of different minimal Medium on the subculture Induction
MS, 1/2MS or WPM is used as a basic culture medium, a plant growth regulator is 0.1mg/L6-BA+0.05mg/LNAA, 30g/L sucrose, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. The different minimal media were inoculated in 30 flasks, each with 2 adventitious buds, and repeated 3 times. After culturing for 40 days, recording the growth condition of the adventitious buds, and counting the number of the adventitious buds, the length of the highest new buds, the vitrification rate and the proliferation coefficient of each bottle of adventitious buds which are higher than 1 cm. The effect of different minimal media on the secondary induction is shown in table 4.
TABLE 4 Effect of different minimal Medium on the secondary Induction
As shown in Table 4, when the minimal medium was WPM, the number of adventitious buds was larger than 1cm, the proliferation factor was larger, and the adventitious buds were robust and grew better.
3. Effect of different minimal Medium on rooting
MS, 1/2MS or WPM is used as basic culture medium, 30g/L sucrose, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and illumination intensity is 1500-2000Lx. Each combined rooting medium was inoculated with 40 flasks, each flask was removed with 2 adventitious buds and repeated 3 times. Recording the rooting growth condition of the adventitious buds after culturing for 35 days, and counting the starting rooting days, plant height, main root number, average main root length and rooting rate of each bottle of adventitious buds. The effect of different minimal media on adventitious bud rooting is shown in Table 5.
TABLE 5 Effect of different minimal Medium on rooting
As shown in Table 5, when the minimal medium is 1/2MS or WPM, the adventitious bud root system of Mesona chinensis Benth grows faster, the main root is thicker and longer, the leaf is wide and big, the leaf color is dark green, the stem is thick and healthy, the plant grows better and survives more easily after transplanting, while the adventitious bud of MS in the minimal medium has more main roots and higher plant, but the root system grows slower, the main root is thicker and shorter, the lateral root is fewer, the leaf color is yellow, the stem is slender, and the transplanting survival rate is lower. The best basic culture medium for Wu Ping mesona blume rooting is 1/2MS or WPM by combining various indexes.
Test example 3 screening for organic additives
1. Effect of different organic additives on Primary Induction
MS is taken 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, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. The different media were inoculated with 30 flasks, 2 explant stems per flask, and repeated 3 times. Recording the bud growth condition of the explant after culturing for 20 days, and counting the bud emergence days of each bottle of the stem section of the explant, wherein the bud emergence days are higher than 1cm of adventitious bud number, adventitious bud length, vitrification rate and 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 is clear from Table 6, when 50g/L banana puree was added, the adventitious buds were sprouted faster, more adventitious buds higher than 1cm, higher adventitious buds, no vitrified seedlings, 100% of sprouting rate, strong adventitious buds and better growth.
2. Effect of different organic additives on the subsequent Induction
MS is taken 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, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. The different media were inoculated 30 flasks, 2 adventitious buds per flask, and repeated 3 times. Recording the growth condition of the adventitious buds after culturing for 40 days, and counting the number of bud emergence days of each bottle of adventitious buds, which is higher than 1cm, the length of the adventitious buds, the vitrification rate and the bud emergence rate. The effect of different organic additive media on the secondary induction is shown in table 7.
TABLE 7 Effect of different organic additives on the subsequent Induction
As is clear from Table 7, when 1g/L peptone was added, more adventitious buds were present, the adventitious buds were high, the vitrified seedlings were not present, the proliferation factor was high, and the adventitious buds were strong and grew well.
3. Influence of different organic additives on rooting
1/2MS is taken 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 sucrose, 7.0g/L agar and pH 5.9 are added into the culture medium, the culture temperature is 25 ℃, continuous illumination is carried out for 12h/d, and the illumination intensity is 1500-2000Lx. Each combined rooting medium was inoculated with 40 flasks, each flask was removed with 2 adventitious buds and repeated 3 times. Recording the rooting growth condition of the adventitious buds after culturing for 35 days, and counting the starting rooting days, plant height, main root number, average main root length and rooting rate of each bottle of adventitious buds. The effect of different minimal media on adventitious bud rooting is shown in Table 8.
TABLE 8 influence of different organic additives on rooting
As shown in Table 8, when 0.5g/L peptone was added, the adventitious bud root system grew faster, the plant was higher, the number of main roots was more, the main roots were thicker and longer, the leaf width was large, the leaf color was dark green, the stem was thick and healthy, and the plant was strong.
Test example 4 screening of seedling hardening matrix
The peat soil, vermiculite, perlite and river sand are used as seedling hardening and transplanting matrixes, plants which grow consistently and have good growth vigor after rooting induction are selected, the plants are firstly opened for 3 days at room temperature, the plants are covered by transparent plastic films during the period, then the root surface culture medium is flushed by running water, the plants are transplanted into the mixed matrixes, 0.5% of folpet and 0.2% of rooting powder mixed solution are poured into the mixed matrixes, 100 plants are treated each, the plants are placed into a small shed with moderate sunshade for hardening, the seedlings are regularly watered, the survival rate is counted after 30 days, and the influence of different matrix mixing ratios on the transplanting survival rate of the mesona chinensis tissue culture seedlings is shown in table 9.
TABLE 9 Effect of different seedling hardening substrates on the survival rate of Mesona chinensis tissue culture seedlings
As can be seen from table 9, when the mass ratio of peat soil, vermiculite, perlite and river sand is: vermiculite: perlite: river sand = 1:1:1:1, the survival rate of the mesona chinensis benth tissue culture seedlings is highest and reaches 100%.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. The industrial tissue culture and rapid propagation method of Mesona chinensis Benth is characterized by comprising the following steps:
s1: picking up Wu Ping germplasm mesona stems with 2-3 stems which are robust and free of plant diseases and insect pests, cleaning and sterilizing to obtain a sterile explant;
s2: cutting the sterile explant obtained in the step S1 into stem segments with a stem node of 1.0cm-2.5cm, inoculating the stem segments into a primary induction culture medium, and culturing for 5-15 days to obtain adventitious buds; the formula of the primary induction culture medium is as follows: 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, pH 5.8-6.2;
s3: shearing 0.5cm-1.5cm from the base of the adventitious bud obtained in the step S2, inoculating into a secondary proliferation induction culture medium, and culturing for 15-30 days to obtain a secondary proliferation cultured adventitious bud;
s4: shearing 1.0cm-3.0cm from the base of the adventitious bud obtained in the step S3 for inoculation into a rooting medium, and culturing for 22-43 days to obtain a transplantable test tube seedling;
s5: hardening seedlings of the test tube seedlings obtained in the step S4 for 3-5 days, transplanting the test tube seedlings into a seedling substrate, pouring mixed solution of sterilizing agent and rooting powder, covering the mixed solution with a transparent plastic film for 7-15 days, and then placing the test tube seedlings in a small shed with moderate 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 method of cleaning and sterilizing comprises the steps of: washing with tap water for 1-2 min, absorbing surface water with sterile absorbent paper, placing on an ultra-clean workbench, rinsing with 75% alcohol for 5s-10s, washing with sterile water for 1-3 times each for 1min-2min, sterilizing with 10% chlorine dioxide solution for 13min-18min, and rinsing with sterile water for 3-4 times each for 1min-2min.
3. A tissue culture rapid propagation method according to claim 1, wherein in the step S2, the formulation of the primary induction medium is: WPM,0.5 mg/L6-BA, 0.1mg/L NAA,50g/L banana puree, 30g/L sucrose, 7.0g/L agar, pH 5.9.
4. A tissue culture rapid propagation method according to claim 1, wherein in the step S3, the formulation of the secondary proliferation induction medium is: WPM,0.1-0.5mg/L6-BA,0.05-0.1mg/L NAA,0.5-1.0g/L peptone, 25-40g/L sucrose, 6.0-8.0g/L agar, and pH 5.8-6.2.
5. The method according to claim 4, wherein in the step S3, the formulation of the secondary proliferation induction medium is: WPM,0.1mg/L6-BA,0.05mg/L NAA,1g/L peptone, 30g/L sucrose, 7.0g/L agar, pH 5.9.
6. The method according to claim 1, wherein in the step S4, the rooting medium comprises the following formula: 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.
7. The method according to claim 6, wherein in the step S4, the rooting medium comprises the following formula: 1/2MS or WPM,0.5g/L peptone, 30g/L sucrose, 7.6g/L agar, pH 5.9.
8. The 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 substrate comprises peat soil, vermiculite, perlite and river sand.
9. A 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 ℃ to 26 ℃, the illumination intensity is 1500lx to 2000lx, and the illumination time is 10 to 12 hours/day.
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