CN110558226A - In-vitro culture rapid propagation method of Ardisia gigantea - Google Patents
In-vitro culture rapid propagation method of Ardisia gigantea Download PDFInfo
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Abstract
An isolated culture rapid propagation method of a giant typhonium rhizome comprises the following steps: adding FeSO into the original MS culture medium4·7H2Adding ascorbic acid, sucrose and agar to prepare an improved MS culture medium, placing the sterilized explant in an induction culture medium for induction culture, wherein the formula of the induction culture medium is as follows: adding IBA, 6-BA and KT based on improved MS culture medium; inoculating adventitious buds of Ardisia Makino in a cluster bud multiplication culture medium, and adding IBA, 6-BA L and KT based on an improved MS culture medium; the formula of the rooting culture medium is as follows: adjusting the dosage of macroelements in the MS culture medium to half of the original dosage based on the improved MS culture medium, and adding NAA and IBA; finally, the regeneration plants of the giant typhonium rhizome are hardened and transplanted. The tissue culture rapid propagation technology can provide a large amount of high-quality rhizoma ardisiae japonicae seedlings, meet market demands, protect wild resources from being damaged, and promote better application of the rhizoma ardisiae japonicae seedlings in the traditional Chinese medicine industry.
Description
Technical Field
The invention relates to a plant tissue culture and rapid propagation technology, in particular to an in vitro culture and rapid propagation method of Ardisia gigantea.
Background
Ardisia gigantea (Ardisia gigantifolia), also known as Dactylicapnos macrophylla, is a perennial shrub plant of the genus Ardisia of the family Myrsinaceae (Myrsinaceae), and the dried roots and rhizomes thereof are traditional Chinese medicinal materials in China, have a long medicinal history and have high medicinal value. The rhizoma Ardisiae Gigantifoliae is pungent in flavor and warm in nature, has effects of dispelling pathogenic wind and removing dampness, strengthening tendons and bones, promoting blood circulation and removing scar, and can be widely used for treating traumatic injury, rheumatalgia, puerperal blood scar, superficial infection and ulcer, and has remarkable curative effect. The main medicinal chemical components of the rhizoma coptidis include three-mushroom soap cover a roof with straw, bergenin, phenols, waken, volatile oil and the like, wherein the two compounds of the three-mushroom soap cover a roof with straw and the bergenin have important significance in the aspects of tumor resistance, oxidation resistance, thrombus resistance and the like and are the hot points of the current research. At present, the giant typhonium rhizome is not limited to be used as clinical medicine, and the application of the giant typhonium rhizome in the aspects of food therapy and health care is expanded continuously. The response of the trogopterus dung to light rays, the determination of proper planting conditions and the research on population distribution are provided, and a foundation is laid for the development of artificial planting. The method has the advantages that young leaves of the giant leaf ardisia herb are used as explants, callus of the leaves is induced, adventitious bud differentiation, proliferation and rooting culture are successfully carried out, stem propagation is utilized, and industrialized production of giant leaf ardisia herb seedlings is carried out. The purity and consistency of germplasm are guaranteed through a tissue culture technology, the large-scale production of the seedlings of the Ardisia Makino can be realized through standardized production in a short period, sufficient seedlings are provided for production, and the current planting dilemma is relieved. The invention explores the factors of the rapid propagation of the stem segments of the Ardisia gigantea Makino, provides a technical system for the industrial production of the Ardisia gigantea Makino tissue culture seedlings, and meets the current market demand.
The breeding coefficient of the rhizoma coptidis hemsleyanae is low, the seedling raising time can be effectively shortened by establishing a rapid breeding technology of the rhizoma coptidis hemsleyanae seedlings, the quality of the seedlings is guaranteed, and technical support is provided for industrial production of the rhizoma coptidis hemsleyanae seedlings. The breeding of the seedlings in the human body is beneficial to the large-scale and standardized planting of the Ardisia gigantea, and the dilemma of the current shortage of raw materials is relieved. Meanwhile, the method provides guarantee for protecting the seed resources of the wild Ardisia Maultflora and is beneficial to the stability of the Ardisia Maultflora. Can continuously provide a large amount of seedlings of the trogopterus dung, and has important significance in production. At present, the technology of plant tissue culture and rapid propagation is very mature, the production of seedlings is premised on that offspring stably keeps the excellent characters of a mother plant, and the variation of an organogenesis approach is lower than that of a callus approach, so that stem section propagation is mostly adopted in production. The sterile explant obtained in production and high proliferation efficiency are main factors for carrying out tissue culture and rapid propagation, and the production cost is direct.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an isolated culture rapid propagation method of a falcate cratoxylum herb.
in order to achieve the purpose, the invention adopts the following technical scheme:
An isolated culture rapid propagation method of a giant typhonium rhizome comprises the following steps:
(1) Preparation of modified MS Medium
The formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2O, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2The concentration of O is 50-60 mg/L, the concentration of ascorbic acid is 5-10 mg/L, and the concentration of sucrose is 25-35 g/L-1The concentration of the agar is 5.0-6.0 g/L-1；
(2) Explant sterilization and adventitious bud induction:
taking young branches of healthy giant leaf horse embryo plants as explants, and cutting the young branches into single-bud stem sections; soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of tween HgCl for 9min2shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; sucking water from the single-bud stem with sterile filter paper, and contacting HgCl at two ends of the cut single-bud stem2The part of the solution, obtaining a sterilized explant for later use; placing the sterilized explant in an induction culture medium for induction culture for 15 days, and growing an adventitious bud of a horse embryo on the explant after 15 days; the formula of the induction culture medium is as follows: based on an improved MS culture medium, 0.1-0.3 mg/L IBA, 0.3-0.7 mg/L6-BA and 0.3-0.7 mg/L KT are added;
(3) Multiplication of cluster buds
Cutting adventitious buds of the Ardisia gigantea from explants, inoculating the excised adventitious buds to a cluster bud multiplication medium containing different hormone types and concentrations for subculture multiplication, wherein the formula of the cluster bud multiplication medium comprises the following components: based on an improved MS culture medium, 0.03-0.07 mg/L IBA, 0.8-1.2 mg/L6-BA and 0.8-1.2 mg/L KT are added; culturing on the cluster bud multiplication medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino;
(4) root induction
cutting stem sections with 2-3 buds from the sterile plantlets of the Ardisia gigantea, and inserting the stem sections into a rooting culture medium; the formula of the rooting culture medium is as follows: based on an improved MS culture medium, adjusting the dosage of macroelements in the MS culture medium to be half of the original dosage, and adding 0.1-0.3 mg/L NAA and 0.2-0.5 mg/L IBA; carrying out rooting induction culture for 40-45 days;
(5) Planting of trogopterus dung
Taking the regenerated plantlets of the trametes trogopterus dunn with roots of more than 0.5cm and seedlings of more than 3cm for hardening and transplanting; hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, placing in sterilized peat soil, maintaining the environmental humidity,
The culture conditions in the steps (2) to (4) are all as follows: the temperature is 28 +/-2 ℃, the illumination intensity is 1900-2100Lx, and the illumination time is 10-15 h/d; adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 5.8 + -0.2, and performing autoclaving at 121 ℃ for 20 min.
Compared with the prior art, the invention has the following beneficial effects:
In the tissue culture of Chinese herbal medicine plants, the fibrosis degree of the material, whether the surface has villus and waxiness, and especially the growth environment and physiological state can influence the disinfection effect, generally aquatic plants are difficult to obtain explants, the rhizoma Ardisiae Gigantifoliae is woody, tender stems are taken, and the content of HgCl is 0.1 percent2sterilization is suitable for treatment for 7-9 minutes. 6-BA and KT play the role of cytokinin in the proliferation process, are mainly auxin in the aspect of rooting induction, and have larger difference to the sensitivity of different stages of plant tissue culture. The invention obtains the proliferator of the Ardisia gigantea embryo by determining the optimal concentration of 6-BA and KTIs described. The NAA and IBA in the auxin are stable and are used for rooting induction, and the combination of the NAA and the IBA can greatly improve the root form and induce to obtain the robust root of the giant knotweed. The tissue culture rapid propagation technology can provide a large amount of high-quality rhizoma ardisiae japonicae seedlings, meet market demands, protect wild resources from being damaged, and promote better application of the rhizoma ardisiae japonicae seedlings in the traditional Chinese medicine industry.
Drawings
FIG. 1 shows the result of multiplying the bushy buds of a male-female-type horse.
FIG. 2 shows the first induction result of rooting in the births of the horse.
FIG. 3 shows the transplanted surviving plants of the horseshoe.
FIG. 4 shows the result of multiplying the bushy buds of the racehorse fetus.
FIG. 5 shows the rooting induction result II of the falcate fetus.
Detailed Description
the technical solution of the present invention is further illustrated by the following examples.
Example 1
1 materials and methods
1.1 materials
In 2018, in summer, wild Ardisia gigantea embryo plants which grow strongly are selected from the southern male in Guangdong, and are cultured in key laboratories for crop genetic improvement in Guangdong province.
1.2 culture Medium and culture conditions
in this example, document 1 is the "plant tissue culture rapid propagation technique" edited by Juuhuang and Changcpeng Hui published by the chemical industry publisher. Formulation of the original MS medium described in this example reference 1, page 22 of table 2-1MS medium (Murashige and Skoog, 1962); preparation of Primary MS Medium as described in this example the preparation of Medium, page 27, section seven of reference 1.
The formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2O, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2the concentration of O is 54.8mg/L, the concentration of ascorbic acid is 8mg/L, and the concentration of sucrose is 30g/L-1Agar concentration of 5.5g/L-1。
The formula of the induction culture medium is as follows: based on the improved MS culture medium, 6-BA, KT and IBA are added.
The formula of the cluster bud multiplication medium is as follows: based on the improved MS culture medium, 6-BA and KT are added.
The formula of the rooting culture medium is as follows: based on the improved MS culture medium, the dosage of macroelements in the MS culture medium is adjusted to be half of the original dosage, and NAA and IBA are added. The macroelements in the modified MS culture medium are potassium nitrate, ammonium nitrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate and calcium chloride dihydrate (see the preparation of the mother liquor of the MS culture medium on the 28 th page table 2-17 of the literature 1).
Adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 5.8 + -0.2, and performing autoclaving at 121 ℃ for 20 min.
The culture conditions are as follows: the temperature is 28 +/-2 ℃, the illumination intensity is 1900-2100Lx, and the illumination time is 10-15 h/d.
1.3 explant Disinfection
Selecting young branches of healthy giant leaf horse embryo plants as explants, and cutting the young branches into single-bud stem segments. Soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of Tween HgCl for 5, 7, 9 and 11min, respectively, as shown in Table 12Shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; sucking water from the single-bud stem with sterile filter paper, and contacting HgCl at two ends of the cut single-bud stem2The area of solution, the sterilized explant is obtained for use. Placing the sterilized explant in an induction culture medium for induction culture for 15 days, growing adventitious buds of the Ardisia gigantea on the explant after 15 days, and then counting the pollution rate, the germination rate, the death rate and the success rate of the adventitious buds of the Ardisia gigantea, wherein the formula of the induction culture medium is as follows: based on the improved MS culture medium, IBA 0.2mg/L, 6-BA 0.5mg/L and KT 0.5mg/L are added.
preparing HgCl containing tween with mass fraction of 0.1%2in solution, each one hundred milliliters of HgCl2One drop of tween was added dropwise to the solution, the volume of one drop being 0.05 ml.
TABLE 1 explant Disinfection time
1.4 Induction culture test method
Six media were prepared according to the types of hormones and their concentrations for each number in Table 1. And (3) inserting the sterilized explants with the sterilization time of 9 minutes into an induction culture medium, treating 10 buds in the induction culture medium corresponding to each serial number, growing adventitious buds of the trametes latifolia on the explants after 15 days, and counting the germination rate and the success rate.
TABLE 2 Table of the types of hormones in the Induction Medium and their concentrations
1.5 Cluster bud proliferation test method
cutting adventitious buds of the Ardisia Majorana Makino from explants, inoculating the Ardisia Majorana Makino adventitious buds to a cluster bud multiplication culture medium containing different hormone types and concentrations for subculture multiplication, wherein the hormone types and concentrations in the cluster bud multiplication culture medium are shown in table 1, and culturing the Ardisia Makino adventitious buds on the cluster bud multiplication culture medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino. And (5) comparing the effect of different hormone combinations on the multiplication of the bushy buds of the stallion and counting the multiplication conditions of 10 buds.
TABLE 3 Table of hormone types and concentrations in Cluster bud growth Medium
1.6 rooting induction test method
Cutting stem segments with 2-3 buds from sterile plantlets of Ardisia gigantea, and inserting into rooting medium to a depth of about 0.5 cm. The types of hormones and their concentrations in rooting medium are shown in Table 3. And performing rooting induction culture for 45 days, and counting the number and the length of roots of 20 plants.
TABLE 4 list of hormone types and concentrations in rooting induction medium
1.7 planting of Ardisia Majorana
Taking the regenerated plantlet of the trametes trogopterus dunn with the root length of more than 0.5cm and the seedling height of more than 3cm for hardening and transplanting. Hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, and placing in sterilized peat soil to maintain environmental humidity.
2 results and analysis
2.1 explant Disinfection test results and analysis
After the explant of the stem segment of the walking horse embryo after the disinfection treatment is placed on an induction culture medium for 7-10 days, axillary buds gradually begin to recover and sprout (figure 1).
The results of the statistics of the contamination rate, the germination rate, the death rate and the success rate are shown in table 5, and the results of the table 5 show that sterile explants can be obtained by sterilizing the explants of the Ardisia gigantea within 5-11 minutes, but the success rates are different. Fungi are a main pollution source, and fungi may exist in axillary buds of the axillary buds, but disinfectant cannot contact the axillary buds, so that the disinfection is not thorough. HgCl with prolonged disinfection time2The larger the area of the solution contacting the explant, the more thorough the disinfection, the slightly reduced pollution rate, but the rapidly increased damage to the explant, mainly shown in the less amount of fungus appearing in the explant, the gradually reduced germination rate, the delayed germination time and the continuously increased explant browning death rate.
the contamination rate of 4 treatments involved in the test was between 20-60%, with the contamination rate of 9 and 11min of disinfection being lower, 20%; the disinfection time of 11min has the most serious poison to the trotter fetus, the germination rate is 30 percent, and the germination rate is 100 percent lower than that of the trotter fetus treated for 9 min; the disinfection time of 7-9 minutes can reach 40-60% of the success rate of obtaining the sterile explant, and the requirement on production is met. Sterile explants can be obtained after 1-4 treatments in the test, and the time for sterilizing the explants of the tramadol is recommended to be 7-9 minutes by combining the comprehensive analysis of germination rate, pollution rate and the like.
TABLE 5 Effect of different Disinfection times on explants
2.2 Induction culture test results and analysis
After the explant of the stem segment of the rumex japonicus is placed on each induction culture medium for 15 days, axillary buds gradually begin to recover and sprout, statistics of the sprouting rate and success rate of adventitious bud induction of the rumex japonicus in each numbered culture medium are shown in table 6, it can be seen from table 6 that in the culture medium numbered 1, the sprouting rate and the success rate are higher than those of other groups, no IBA is added in groups 4-6, the sprouting rate is lower, and in groups 4-6, the sprouting rate of IBA added with higher concentration is lower than that of groups 1-3, so that IBA is indispensable in the adventitious bud induction process, but the concentration cannot be too high, and therefore, the recommended concentration of IBA is about 0.2 mg/L. The concentrations of 6-BA and KT have a large influence on the success rate, and after comprehensive analysis, the hormone numbered 1 and the concentration level thereof have the optimal comprehensive effect.
TABLE 6 Effect of different hormones and their concentrations on adventitious bud Induction in Mare foetuses
2.3 Cluster bud proliferation test results and analysis
The propagation culture of the cluster buds can rapidly improve the number of seedlings in a short period, and the increment efficiency determines the production cost of the seedlings, so that the propagation culture of the tissue culture seedlings of the Ardisia Makino is the key for the industrialized seedling culture of the tissue culture seedlings of the Ardisia Makino.
The test results are shown in Table 7, and it can be seen from Table 7 that each hormone combination of 6-BA, KT and IBA can induce the proliferation of adventitious buds, and the proliferation efficiency is positively correlated with the concentration of 6-BA in the culture medium. Along with the increase of the concentration of 6-BA, the multiplication efficiency of the trogopterus dung increases, and the multiplication coefficient reaches about 12 when the concentration of 6-BA is 2 mg/L. However, the tissue culture seedling has a large number of adventitious buds, is too dense and weak, has low quality and is inconvenient for the next proliferation. The addition of KT and IBA improves the uniformity of the shoots, and the combination of 1mg/L of 6-BA, 1mg/LKT and 0.05mg/L IBA is suitable for the multiplication of the equine fetuses.
TABLE 7 Effect of different hormones and their concentrations on adventitious bud proliferation
2.4 rooting culture test results and analysis
And (3) inoculating the stem segments containing 2-3 buds into a rooting culture medium to perform rooting induction on the tissue culture seedlings. After 40 days of culture, the number, length, time and quality of roots were counted.
the test results are shown in Table 8, and it can be seen from Table 8 that the hormone combination of NAA and IBA according to the present invention induced rooting of the horseshoe tires, but the horseshoe tires rooted more slowly and have more main roots and fewer lateral roots than other herbaceous plants. At low hormone levels, root length increases as the number of roots increases with increasing hormone concentration. Addition of 0.2mg/L NAA and 0.3mg/L IBA promoted the number and quality of roots, but as the concentration of NAA and IBA increased, the time for inducing roots in the tramadol was increased. As the concentration of NAA increases, the number of roots increases, but the root diameter becomes slightly smaller. The NAA treatment at 0.5mg/L induced a 1.25-fold increase in the number of roots and 46.15% increase in length over the NAA treatment at 0.1 mg/L. There was no significant difference in root diameter. The combination of the rooting time and the root quality ensures that the 0.2mg/L NAA and the 0.3mg/L IBA induced Ardisia tissue culture seedling roots are better and are suitable for later stage transplantation.
TABLE 8 Effect of different hormones and their concentrations on the induction of Ardisia gigantea fetal roots
2.5 hardening and transplanting
Rooting induction culture for 40-60 days, wherein the root of the seedling of the Ardisia gigantea is 0.7cm, and the plant height is about 3 cm. Through exercising the seedlings, the leaves of the tissue culture seedlings become thick and the texture becomes hard. After 5 days of transplantation, the plants are tall and straight, the survival rate is more than 85 percent, and the transplanted survival plants are shown in figure 3.
2.6 conclusion
through 2.1-2.5 single-factor tests and result analysis, the following key control parameters are obtained:
The soaking time in the disinfectant is preferably 7-9 min.
The formula of the induction culture medium is as follows: based on the improved MS culture medium, IBA 0.2mg/L, 6-BA 0.5mg/L and KT 0.5mg/L are added.
The formula of the cluster bud multiplication medium is as follows: based on the improved MS culture medium, IBA 0.05mg/L, 6-BA 1.0mg/L and KT 1.0mg/L are added.
The formula of the rooting culture medium is as follows: based on the improved MS culture medium, the dosage of macroelements in the MS culture medium is adjusted to be half of the original dosage, and then 0.2mg/L NAA and 0.3mg/L IBA are added.
Example 2
An isolated culture rapid propagation method of a giant typhonium rhizome comprises the following steps:
(1) Preparation of modified MS Medium
The formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2o, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2The concentration of O is 60mg/L, the concentration of ascorbic acid is 10mg/L, and the concentration of sucrose is 35g/L-1Agar concentration of 6.0g/L-1；
(2) Explant sterilization and adventitious bud induction:
Taking tender of healthy trogopterus dung plantThe branch is taken as an explant, and the young and tender branch is cut into single-bud stem segments; soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of tween HgCl for 9min2shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; sucking water from the single-bud stem with sterile filter paper, and contacting HgCl at two ends of the cut single-bud stem2The part of the solution, obtaining a sterilized explant for later use; placing the sterilized explant in an induction culture medium for induction culture for 15 days, and growing an adventitious bud of a horse embryo on the explant after 15 days; the formula of the induction culture medium is as follows: adding 0.1mg/L IBA, 0.3 mg/L6-BA and 0.3mg/L KT on the basis of an improved MS culture medium;
(3) Multiplication of cluster buds
cutting adventitious buds of the Ardisia gigantea from explants, inoculating the excised adventitious buds to a cluster bud multiplication medium containing different hormone types and concentrations for subculture multiplication, wherein the formula of the cluster bud multiplication medium comprises the following components: adding 0.03mg/LIBA, 0.8 mg/L6-BA and 0.8mg/L KT based on the improved MS culture medium; culturing on the cluster bud multiplication medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino;
(4) Root induction
Cutting stem segments with 2-3 buds from the sterile plantlets of the Ardisia gigantea, and inserting the stem segments into a rooting culture medium to a depth of about 0.5 cm; the formula of the rooting culture medium is as follows: based on an improved MS culture medium, adjusting the dosage of macroelements in the MS culture medium to be half of the original dosage, and adding 0.1mg/L NAA and 0.2mg/L IBA; performing rooting induction culture for about 40 days;
(5) Planting of trogopterus dung
Taking the regenerated plantlets of the trametes trogopterus dunn with roots of more than 0.5cm and seedlings of more than 3cm for hardening and transplanting; hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, placing in sterilized peat soil, maintaining the environmental humidity,
The culture conditions in the steps (2) to (4) are all as follows: the temperature is 26 ℃, the illumination intensity is 1900Lx, and the illumination time is 10 h/d; adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 5.6, and performing autoclaving at 121 ℃ for 20 min.
Example 3
An isolated culture rapid propagation method of a giant typhonium rhizome comprises the following steps:
(1) Preparation of modified MS Medium
The formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2O, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2The concentration of O is 54.8mg/L, the concentration of ascorbic acid is 8mg/L, and the concentration of sucrose is 30g/L-1Agar concentration of 5.5g/L-1；
(2) Explant sterilization and adventitious bud induction:
Taking young branches of healthy giant leaf horse embryo plants as explants, and cutting the young branches into single-bud stem sections; soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of tween HgCl for 8min2Shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; sucking water from the single-bud stem with sterile filter paper, and contacting HgCl at two ends of the cut single-bud stem2The part of the solution, obtaining a sterilized explant for later use; placing the sterilized explant in an induction culture medium for induction culture for 15 days, and growing an adventitious bud of a horse embryo on the explant after 15 days; the formula of the induction culture medium is as follows: adding 0.2mg/L IBA, 0.5 mg/L6-BA and 0.5mg/L KT on the basis of an improved MS culture medium;
(3) Multiplication of cluster buds
Cutting adventitious buds of the Ardisia gigantea from explants, inoculating the excised adventitious buds to a cluster bud multiplication medium containing different hormone types and concentrations for subculture multiplication, wherein the formula of the cluster bud multiplication medium comprises the following components: adding 0.03-0.07 mg/L IBA, 1.0 mg/L6-BA and 1.0mg/L KT on the basis of an improved MS culture medium; culturing on the cluster bud multiplication medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino;
(4) Root induction
cutting stem segments with 2-3 buds from the sterile plantlets of the Ardisia gigantea, and inserting the stem segments into a rooting culture medium to a depth of about 0.5 cm; the formula of the rooting culture medium is as follows: based on the improved MS culture medium, adjusting the dosage of macroelements in the MS culture medium to be half of the original dosage, and adding 0.2mg/L NAA and 0.3mg/L IBA; carrying out rooting induction culture for 42 days;
(5) Planting of trogopterus dung
taking the regenerated plantlets of the trametes trogopterus dunn with roots of more than 0.5cm and seedlings of more than 3cm for hardening and transplanting; hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, placing in sterilized peat soil, maintaining the environmental humidity,
The culture conditions in the steps (2) to (4) are all as follows: the temperature is 28 +/-2 ℃, the illumination intensity is 1900-2100Lx, and the illumination time is 14 h/d; adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 5.8 + -0.2, and performing autoclaving at 121 ℃ for 20 min.
Example 4
An isolated culture rapid propagation method of a giant typhonium rhizome comprises the following steps:
(1) Preparation of modified MS Medium
the formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2O, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2The concentration of O is 50mg/L, the concentration of ascorbic acid is 5mg/L, and the concentration of sucrose is 25g/L-1Agar concentration of 5.0g/L-1；
(2) Explant sterilization and adventitious bud induction:
Taking young branches of healthy giant leaf horse embryo plants as explants, and cutting the young branches into single-bud stem sections; soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of Tween HgCl for 7min2Shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; by usingSterile filter paper is used for sucking water on the single-bud stem segments, and two ends of the cut single-bud stem segments are contacted with HgCl2The part of the solution, obtaining a sterilized explant for later use; placing the sterilized explant in an induction culture medium for induction culture for 15 days, and growing an adventitious bud of a horse embryo on the explant after 15 days; the formula of the induction culture medium is as follows: adding 0.3mg/L IBA, 0.7 mg/L6-BA and 0.7mg/L KT on the basis of an improved MS culture medium;
(3) multiplication of cluster buds
Cutting adventitious buds of the Ardisia gigantea from explants, inoculating the excised adventitious buds to a cluster bud multiplication medium containing different hormone types and concentrations for subculture multiplication, wherein the formula of the cluster bud multiplication medium comprises the following components: adding 0.07mg/LIBA, 1.2 mg/L6-BA and 1.2mg/L KT based on the improved MS culture medium; culturing on the cluster bud multiplication medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino;
(4) Root induction
cutting stem segments with 2-3 buds from the sterile plantlets of the Ardisia gigantea, and inserting the stem segments into a rooting culture medium to a depth of about 0.5 cm; the formula of the rooting culture medium is as follows: based on the improved MS culture medium, adjusting the dosage of macroelements in the MS culture medium to be half of the original dosage, and adding 0.3mg/L NAA and 0.5mg/L IBA; performing rooting induction culture for 45 days;
(5) Planting of trogopterus dung
Taking the regenerated plantlets of the trametes trogopterus dunn with roots of more than 0.5cm and seedlings of more than 3cm for hardening and transplanting; hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, placing in sterilized peat soil, maintaining the environmental humidity,
The culture conditions in the steps (2) to (4) are all as follows: the temperature is 30 ℃, the illuminance is 2100Lx, and the illumination time is 15 h/d; adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 6.0, and performing autoclaving at 121 ℃ for 20 min.
Claims (1)
1. an isolated culture rapid propagation method of a giant typhonium rhizome is characterized by comprising the following steps:
(1) preparation of modified MS Medium
The formula of the improved MS culture medium is as follows: adding FeSO into the original MS culture medium4·7H2O, adding ascorbic acid, sucrose and agar, and keeping other components unchanged; FeSO in modified MS Medium4·7H2The concentration of O is 50-60 mg/L, the concentration of ascorbic acid is 5-10 mg/L, and the concentration of sucrose is 25-35 g/L-1The concentration of the agar is 5.0-6.0 g/L-1；
(2) Explant sterilization and adventitious bud induction:
Taking young branches of healthy giant leaf horse embryo plants as explants, and cutting the young branches into single-bud stem sections; soaking the single bud stem in 70% ethanol for 30s, washing with sterile water for 3 times, each for 1-3 min; soaking in disinfectant containing 0.1 wt% of tween HgCl for 9min2Shaking the beaker with the solution to allow the material to fully contact the disinfectant; washing with sterile water for 5min for 3-5 times; sucking water from the single-bud stem with sterile filter paper, and contacting HgCl at two ends of the cut single-bud stem2The part of the solution, obtaining a sterilized explant for later use; placing the sterilized explant in an induction culture medium for induction culture for 15 days, and growing an adventitious bud of a horse embryo on the explant after 15 days; the formula of the induction culture medium is as follows: based on an improved MS culture medium, 0.1-0.3 mg/L IBA, 0.3-0.7 mg/L6-BA and 0.3-0.7 mg/L KT are added;
(3) multiplication of cluster buds
Cutting adventitious buds of the Ardisia gigantea from explants, inoculating the excised adventitious buds to a cluster bud multiplication medium containing different hormone types and concentrations for subculture multiplication, wherein the formula of the cluster bud multiplication medium comprises the following components: adding 0.03-0.07 mg/LIBA, 0.8-1.2 mg/L6-BA and 0.8-1.2 mg/L KT based on the improved MS culture medium; culturing on the cluster bud multiplication medium for 20-25 days to obtain sterile cluster seedlings of the Ardisia Makino;
(4) root induction
Cutting stem sections with 2-3 buds from the sterile plantlets of the Ardisia gigantea, and inserting the stem sections into a rooting culture medium; the formula of the rooting culture medium is as follows: based on an improved MS culture medium, adjusting the dosage of macroelements in the MS culture medium to be half of the original dosage, and adding 0.1-0.3 mg/L NAA and 0.2-0.5 mg/L IBA; carrying out rooting induction culture for 40-45 days;
(5) Planting of trogopterus dung
taking the regenerated plantlets of the trametes trogopterus dunn with roots of more than 0.5cm and seedlings of more than 3cm for hardening and transplanting; hardening and culturing in room for 3-7 days, washing with tap water, soaking in 0.1% carbendazim solution for 10min, placing in sterilized peat soil, maintaining the environmental humidity,
The culture conditions in the steps (2) to (4) are all as follows: the temperature is 28 +/-2 ℃, the illumination intensity is 1900-2100Lx, and the illumination time is 10-15 h/d; adjusting pH values of the induction culture medium, the clumpy bud multiplication culture medium and the rooting culture medium to 5.8 + -0.2, and performing autoclaving at 121 ℃ for 20 min.
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