CN113287519B - Culture medium for tissue culture of physochlaina dwarfii and tissue culture method - Google Patents
Culture medium for tissue culture of physochlaina dwarfii and tissue culture method Download PDFInfo
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Abstract
The invention is suitable for the technical field of agricultural production, and provides a culture medium for tissue culture of short-growing clitocybe and a tissue culture method, wherein the culture medium comprises a seedling breeding culture medium, a clustered bud induction culture medium, a multiplication culture medium and a rooting culture medium, each culture medium is based on a cheap and easily-obtained MS culture medium and is formed by adding proper exogenous hormone or raw materials, the formula of each culture medium is scientific and reasonable, the culture medium can be respectively used for each stage of tissue culture of the short-growing clitocybe, the breeding speed in the culture process is high, the period is short, the survival rate of plants is high and can reach about 90 percent, the excellent genetic characteristic of the variety of the short-growing clitocybe can be well maintained, and the market popularization and the garden application of the variety are facilitated.
Description
Technical Field
The invention relates to the technical field of agricultural production, in particular to a culture medium for tissue culture of short-growing bowl column grass and a tissue culture method.
Background
In recent years, african ornamental plants have been widely used in modern landscaping. The short-growing Boschniakia herb is a perennial small herb of Boschniakia of the family of mother grass, and is mainly distributed in the south of Africa, the south of Arabian peninsula and India. The delicate and lovely dwarf bowl column grass has higher ornamental value, is suitable for various purposes such as potted plants, garden flower bed landscaping, rock slope protection, under-forest land covering, plant micro-landscape manufacturing and the like, and has good garden application prospect.
The dwarf pillworm grass introduced from Africa to south China can bear a large amount of seeds under natural conditions, but seedlings grow slowly after seeding and seedling emergence; the plant division propagation has the defects that the root system is easy to damage, the plant activity is reduced, and the propagation expansion coefficient is low, so that the popularization and the application are limited.
Therefore, the propagation period of the existing propagation mode of the short-growing lysimachia verticillata is long, the propagation coefficient is low, and the current popularization and application requirements are difficult to meet.
Disclosure of Invention
Aiming at the problems that the propagation period of the propagation mode of the short-growing lysimachia sikokiana in the prior art is long, the propagation expansion coefficient is low, and the current popularization and application requirements are difficult to meet, the invention provides a culture medium for tissue culture of the short-growing lysimachia sikokiana and a tissue culture method.
The invention provides a culture medium for tissue culture of short-growing bowl-shaped pillar grass, which comprises a seedling breeding culture medium, a cluster bud induction culture medium, a multiplication culture medium and a rooting culture medium; the seedling breeding culture medium is an MS culture medium added with agar and sucrose; the cluster bud induction culture medium is an MS culture medium added with agar, sucrose, kinetin and naphthylacetic acid; the multiplication culture medium is an MS culture medium containing agar, sucrose, 6-benzylamino adenine and naphthylacetic acid; the rooting culture medium is 1/2MS culture medium or MS culture medium.
The invention also provides a tissue culture method of the short-growing bowl column grass, which comprises the following steps:
inoculating the sterilized seeds of the physalis pubescens in the seedling breeding culture medium for culturing to obtain sterile physalis pubescens seedlings;
inoculating the aseptic seedlings of the short-growing lysimachia sikokiana into the cluster bud induction culture medium for culture to obtain cluster buds;
inoculating the cluster buds into the enrichment culture medium for enrichment culture to obtain an enrichment seedling;
inoculating the proliferated seedling into the rooting culture medium for culture to obtain a rooting seedling;
and (4) hardening the rooted seedlings to obtain replanting seedlings.
The culture medium for the tissue culture of the dwarf pillared grass comprises a seedling breeding culture medium, a cluster bud induction culture medium, a proliferation culture medium and a rooting culture medium, wherein each culture medium is based on a cheap and easily-obtained MS culture medium and is formed by adding proper exogenous hormone or raw materials, the formula of each culture medium is scientific and reasonable, the culture medium can be respectively used for each stage of the tissue culture of the dwarf pillared grass, the breeding speed in the breeding process is high, the breeding period is short, the survival rate of plants is high and can reach about 90 percent, the excellent genetic characteristic of the dwarf pillared grass variety can be well maintained, and the market popularization and the garden application of the variety are facilitated.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.
FIG. 1 is a germination diagram of a dwarf pillared grass seed in a sterile environment provided by an embodiment of the invention;
FIG. 2 is a graph of the differentiation of bush-grass clumps provided by an embodiment of the present invention;
FIG. 3 is a diagram showing the proliferation of the seedling of the short-growing bowl column grass provided by the embodiment of the present invention;
FIG. 4 is a diagram of the rooting of the short-grown bowl-column grass sprouts according to the embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a culture medium for tissue culture of short-growing clitocybe maxima, and particularly provides a culture medium formula for each stage of tissue culture of the short-growing clitocybe maxima.
The embodiment of the invention provides a culture medium for tissue culture of short-growing clitocybe herb, which comprises a seedling breeding culture medium, a cluster bud induction culture medium, a multiplication culture medium and a rooting culture medium; the seedling breeding culture medium is an MS culture medium added with agar and sucrose; the cluster bud induction culture medium is an MS culture medium added with agar, sucrose, kinetin and naphthylacetic acid; the multiplication culture medium is an MS culture medium containing agar, sucrose, 6-benzylamino adenine and naphthylacetic acid; the rooting culture medium is 1/2MS culture medium or MS culture medium.
MS culture medium is a basic culture medium commonly used in plant tissue culture. In practical application, the culture medium can be obtained in a commercial mode and can also be prepared according to the existing MS culture medium formula.
In the embodiment of the invention, the seedling breeding culture medium is an MS culture medium containing 6g/L of agar and 30g/L of sucrose. The cluster bud induction culture medium is an MS culture medium containing 6g/L agar, 30g/L sucrose, 0.1-1.0 mg/L Kinetin (KT) and 0-0.1 mg/L naphthylacetic acid (NAA). The enrichment medium is MS medium containing 6g/L agar, 30g/L sucrose, 0.1-1.0 mg/L6-benzylamino adenine (6-BA) and 0.05-0.25 mg/L naphthylacetic acid (NAA).
In a preferred embodiment of the present invention, the ratio of the kinetin to the naphthylacetic acid in the culture medium for inducing the multiple shoots is 25:1, 10:1 or 1: 1.
More preferably, the content of kinetin in the cluster bud induction culture medium is 0.5mg/L, the content of naphthylacetic acid is 0.05mg/L, namely the compounding ratio of kinetin to naphthylacetic acid is 10:1, which is beneficial to induction of cluster buds, and the induction rate can reach 100% after 35 days.
In a preferred embodiment of the invention, in the proliferation medium, the compound ratio of the 6-benzylaminopurine to the naphthylacetic acid is 10: 3-5.
More preferably, in the proliferation culture medium, the content of 6-benzylamino adenine is 0.5mg/L, the content of naphthylacetic acid is 0.20mg/L, namely the compounding ratio of 6-benzylamino adenine to naphthylacetic acid is 10:4, which is beneficial to the proliferation of tissue culture seedlings, and the proliferation coefficient can reach 5.46 after 30 days.
As a preferred embodiment of the invention, the rooting medium is 1/2MS medium, which is favorable for seedling rooting, and the rooting rate can reach 100% after 21 d.
The culture medium for the tissue culture of the dwarf pillared grass provided by the embodiment of the invention comprises a seedling breeding culture medium, a cluster bud induction culture medium, a proliferation culture medium and a rooting culture medium, wherein each culture medium is based on a cheap and easily-obtained MS culture medium and is formed by adding proper exogenous hormone or raw materials, the formula of each culture medium is scientific and reasonable, the culture medium can be respectively used for each stage of the tissue culture of the dwarf pillared grass, the breeding speed in the culture process is high, the breeding period is short, the survival rate of plants is high and can reach about 90 percent, the excellent genetic characteristic of the dwarf pillared grass variety can be well maintained, and the market popularization and the garden application of the variety are facilitated.
The embodiment of the invention also provides a tissue culture method of the short-growing bowl column grass, which comprises the following steps:
and 101, inoculating the sterilized seeds of the short-stalked physalis pubescens in the seedling breeding culture medium for culturing to obtain the sterile seedlings of the short-stalked physalis pubescens.
In the embodiment of the invention, firstly, ripe and full seeds of the dwarf pillbug are selected and collected in the current year, and preferably selected in sunny days, then washed by tap water for 1-2 h, then soaked in clear water for 30min, empty and shrunken seeds at the upper layer are removed, the bottom-sinking seeds are sterilized by 2% NaClO (sodium hypochlorite) solution for 15min, washed by sterile water for 4-6 times, and the surface water of the seeds is sucked by sterile filter paper for standby. Then, MS is used as a basic culture medium, cane sugar is added, agar is used for melting and then subpackaged in a culture dish, the pH value is adjusted to 6.0-6.2, sterilization is carried out at 121 ℃ for 20min, and then cooling is carried out for standby application; inoculating the sterilized seeds to MS +6 g.L-1Agar +30 g. L-1Culturing and germinating on a sucrose culture medium, inoculating 10 seeds to each culture dish, inoculating 15 culture dishes, and culturing at 25 +/-2 ℃ under the illumination intensity of 37-40 mu mol/m2And s, the illumination time is 16h/d, the seeds germinate after being cultured for 30d (the germination chart of the dwarf pillared grass seeds in the sterile environment is shown in figure 1), the pollution rate of the seed disinfection treatment is 1.5 percent, the germination rate is 82.35 percent, and after germination, 2-4 full-spread leaves grow for later use. Finally, the fully extended, dark green leaves were cut into small leaflet pieces 0.5cm x 0.5cm in size, which were used as explants for future use.
102, inoculating the sterile seedlings of the short-growing bowl column grass into the cluster bud induction culture medium for culture to obtain cluster buds.
In an embodiment of the invention, after sterilizing the seed room, the superclean bench, the inoculating instrument and the hands, the superclean benchOpening a culture medium sterilized at high temperature according to the requirement of aseptic operation, inoculating the processed explant of the dwarf pillworm grass leaf into a cluster bud induction culture medium, and culturing at the temperature of 25 +/-2 ℃ and the illumination intensity of 37-40 mu mol/m2S, culturing under the condition that the illumination time is 16h/d to obtain cluster buds.
103, inoculating the cluster buds into the enrichment culture medium for enrichment culture to obtain the enrichment seedlings.
In the embodiment of the invention, the differentiated robust cluster buds are selected from the obtained cluster buds (wherein the cluster buds are differentiated as shown in figure 2), cut into single seedlings and transferred into a multiplication culture medium at the culture temperature of 25 +/-2 ℃ and the illumination intensity of 37-40 mu mol/m2S, culturing for 30d under the condition of the illumination duration of 16h/d, and proliferating the cluster buds to obtain proliferated seedlings (shown in figure 3).
And 104, inoculating the proliferated seedling into the rooting culture medium for culture to obtain a rooted seedling.
In the embodiment of the invention, robust plantlets with 4-6 leaves are selected from the obtained proliferated plantlets and inoculated in a rooting culture medium, the culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, culturing for 21-42 d under the condition that the illumination time is 16h/d, and inducing to root to obtain rooted seedlings (shown in figure 4).
And 105, hardening the rooted seedlings to obtain replanting seedlings.
In the embodiment of the invention, the bottle cap of a rooted tissue culture seedling culture bottle is uncovered, the seedling is hardened for 2-3 days under the indoor natural illumination condition, then the seedling is carefully taken out, a root culture medium is washed away, and the seedling is planted in a substrate which is sterilized in advance, wherein the substrate is peat soil according to the volume ratio: perlite: argil is 3:1: 1. The matrix is subpackaged by using nutrition pots, 1 plant is planted in each pot, watering is performed to thoroughly leach, the culture is performed in an outdoor environment with the culture temperature of 25 +/-2 ℃ and the relative humidity of more than 75%, and then watering is performed for 1 time every 2-3 days for conventional management. After 30 days, the survival rate of the seedlings can reach about 90 percent.
The technical effects of the culture medium for tissue culture of cymbidium dwarfii according to the invention will be further illustrated below by means of specific examples, in which all the components not specifically illustrated are commercially available. For example, MS media may be purchased from jonan guerite instruments ltd; agar is commercially available from Beijing sub-Mi Biotech, Inc.
Example 1
The formula of the culture medium for tissue culture of the short-growing cymbopogon japonicus in the embodiment is as follows:
seedling breeding culture medium: 6g/L agar +30g/L sucrose + MS medium. Cluster bud induction medium: 6g/L agar, 30g/L sucrose, 0.1mg/L kinetin, 0.1mg/L naphthylacetic acid and MS culture medium. Proliferation culture medium: 6g/L agar +30g/L sucrose +0.1 mg/L6-benzylamino adenine +0.05mg/L naphthylacetic acid + MS culture medium. The rooting culture medium is 1/2MS culture medium.
Example 2
The formula of the culture medium for tissue culture of the short-growing cymbopogon japonicus in the embodiment is as follows:
seedling breeding culture medium: 6g/L agar +30g/L sucrose + MS medium. Cluster bud induction medium: 6g/L agar, 30g/L sucrose, 0.1mg/L kinetin, 0mg/L naphthylacetic acid and MS culture medium. Proliferation culture medium: 6g/L agar +30g/L sucrose +0.1 mg/L6-benzylamino adenine +0.1mg/L naphthylacetic acid + MS culture medium. The rooting culture medium is 1/2MS culture medium.
Example 3
The formula of the culture medium for tissue culture of the short-growing cymbopogon japonicus in the embodiment is as follows:
seedling breeding culture medium: 6g/L agar +30g/L sucrose + MS medium. Cluster bud induction medium: 6g/L agar, 30g/L sucrose, 0.5mg/L kinetin, 0.05mg/L naphthylacetic acid and MS culture medium. Proliferation culture medium: 6g/L agar +30g/L sucrose +0.5 mg/L6-benzylamino adenine +0.2mg/L naphthylacetic acid + MS culture medium. The rooting culture medium is 1/2MS culture medium.
Example 4
The formula of the culture medium for tissue culture of the short-growing cymbopogon japonicus in the embodiment is as follows:
seedling breeding culture medium: 6g/L agar +30g/L sucrose + MS medium. Cluster bud induction medium: 6g/L agar, 30g/L sucrose, 1.0mg/L kinetin, 0.1mg/L naphthylacetic acid and MS culture medium. Proliferation culture medium: 6g/L agar +30g/L sucrose +0.5 mg/L6-benzylamino adenine +0.25mg/L naphthylacetic acid + MS culture medium. The rooting culture medium is 1/2MS culture medium.
Example 5
The formula of the culture medium for tissue culture of the short-growing cymbopogon japonicus in the embodiment is as follows:
seedling breeding culture medium: 6g/L agar +30g/L sucrose + MS medium. Cluster bud induction medium: 6g/L agar, 30g/L sucrose, 0.5mg/L kinetin, 0.05mg/L naphthylacetic acid and MS culture medium. Proliferation culture medium: 6g/L agar +30g/L sucrose +0.5 mg/L6-benzylamino adenine +0.15mg/L naphthylacetic acid + MS culture medium. The rooting culture medium is 1/2MS culture medium.
The culture medium for the tissue culture of the short-growing bowl-shaped pillar grass provided by the embodiment 1-5 is adopted to carry out the tissue culture of the short-growing bowl-shaped pillar grass, and the specific culture method is as follows: inoculating the sterilized dwarf pillworm grass seeds in a seedling breeding culture medium, and culturing at 25 deg.C under the illumination intensity of 37 μmol/m2S, culturing for 30d under the condition that the illumination time is 16h/d, and after the seeds germinate, growing 2-4 full-spread leaves of seedlings; taking a dwarf pillared seedling of the pillared dwarfed clover with unfolded leaves and dark green leaves, cutting the fully unfolded leaves into small leaf blocks with the size of 0.5cm multiplied by 0.5cm, and taking the small leaf blocks as explants for later use; inoculating the processed explant of the leaves of the short-growing lysimachia sikokiana in a cluster bud induction culture medium, and controlling the temperature at 25 ℃ and the illumination intensity at 40 mu mol/m2S, culturing under the condition that the illumination duration is 16h/d to obtain cluster buds; selecting strong cluster buds, cutting into single plantlet, transferring into proliferation culture medium at 25 deg.C under illumination intensity of 37 μmol/m2S, culturing for 30d under the condition that the illumination time is 16h/d, and proliferating the cluster buds to obtain proliferated seedlings; selecting strong plantlets with 4-6 leaves from the obtained proliferated plantlets, inoculating the robust plantlets in a rooting culture medium, and culturing at 25 ℃ under the illumination intensity of 37 mu mol/m2S, duration of light irradiation 16h/d, 21d, inducing to root to obtain rooted seedlings; uncovering the bottle cap of a rooted tissue culture seedling culture bottle, hardening the seedling for 2d under indoor natural illumination conditions, then carefully taking out the seedling, washing off a root culture medium, and planting the seedling in a substrate which is sterilized in advance, wherein the substrate is peat soil according to the volume ratio: perlite: argil is 3:1: 1. The matrix is separately packaged by using nutrition pots, 1 plant is planted in each pot, watering and drenching are carried out, the culture is carried out in an outdoor environment with the culture temperature of 25 ℃ and the relative humidity of more than 75%, and then watering is carried out for 1 time every 3 days for conventional management.
In the stage of seedling breeding and culturing, the germination rate of the seeds is observed and counted, and the result shows that the germination rate of the seeds in the embodiments 1-5 is 80% -82.35%.
In the induction culture stage of the cluster buds, the sprouting time, the form and the number of the cluster buds, the growth speed and the induction rate are observed and recorded, and the result shows that the dwarf bowl-pillar grasses of the examples 1 to 5 have early sprouting, more buds and fast growth, and the cluster buds have the form of extended leaves, dark green leaves and less vitrified seedlings.
In the proliferation culture stage, the number and the form of the proliferated seedlings are observed and recorded, and the result shows that the seedlings of the embodiments 1 to 5 are thick, the leaves are stretched and can root in the proliferation process.
In the rooting culture stage, the rooting rate, the average root length and the average root number are observed and recorded, and the result shows that the seedlings of the embodiments 1-5 are rooted early (21d), and the root systems and the seedlings grow strongly.
In addition, a large number of experiments show that the variety and the proportion of hormones added into the cluster bud induction culture medium and the proliferation culture medium and the variety of the rooting culture medium have obvious influence on the culture effect. The method comprises the following specific steps:
in the cluster bud induction culture stage, the processed explant of the dwarf bowl-column grass leaf is inoculated into 12 culture mediums added with different exogenous hormone ratios, each treatment is inoculated into 8 bottles, each bottle is inoculated with 3 pieces, and the cluster buds are obtained by culture under the same culture conditions. The sprouting time, the form and number of cluster buds, the growth speed and the induction rate are observed and researched, and the specific results are shown in table 1.
TABLE 1 Effect of different hormone combinations on the induction of dwarf bowl-post plantlet shoots
Leaves and tender tips of the vitrified seedlings of the plant tissue culture are in crystal transparent or semitransparent water stain shapes; dwarfing, swelling and greening of the whole plant; the blade is shrunk to be longitudinally curled, and is fragile and fragile; the leaf surface lacks cuticle waxy substance, has no functional air hole, has no palisade tissue and only has sponge tissue. The vitrified seedlings are difficult to survive due to high water content in vivo, reduced activity of dry matters, chlorophyll, proteins, cellulose and enzyme, tissue malformation, incomplete organ function and reduced differentiation capability, and thus the improvement of the propagation rate of tissue culture seedlings is seriously influenced.
From the data in table 1, under the culture condition of the invention, when the compound proportion of KT and NAA added in the culture medium for inducing cluster bud differentiation is 25:1, 10:1 or 1:1, the bud emergence of the dwarf bowl-pillar grass cluster is earliest, the bud number is the largest, the growth is the fastest, the cluster bud form shows that the leaves are spread, the leaf color is dark green, the occurrence of vitrified seedlings is few, and the survival rate and the reproduction rate of the cultured seedlings are higher; when the compounding ratio of the added KT and the added NAA is 50:1, 5:1 or 10:1, the dwarf bowl column grass cluster buds grow well and a small amount of vitrified seedlings appear; when NAA is not added or the compounding ratio of KT and NAA is 5:1 or 20:1, the growth of the dwarf bowl column grass clump buds is general, more than half of seedlings are vitrified, and the survival rate and the propagation rate of the cultured seedlings are lower.
And the selection formula is MS +6 g.L-1Agar +30 g. L-1Sucrose +0.5 mg. L-1Kinetin (KT) +0.05 mg. L-1In the presence of naphthylacetic acid (NAA), the buds of the dwarf bowl-pillar grass clumps grow earliest, the number of the buds is the largest, the growth is the fastest, the clump buds are shown to be in the form of extended leaves, dark green leaves, little vitrified seedlings and the survival rate of the cultured seedlingsAnd the reproduction rate is higher.
In the propagation culture stage, the differentiated robust multiple shoots are selected from the obtained multiple shoots, and the strong multiple shoots are divided into individual seedlings and transferred into propagation culture media added with different exogenous hormones for propagation of the shoots, as shown in Table 2 below. And (3) inoculating 8 bottles into each treatment, inoculating 3 uniformly cut plantlets into each bottle, and culturing for 30d under the same condition to obtain the proliferated plantlets. The number and the form of the proliferated seedlings were observed and studied, and the specific results are shown in Table 2.
TABLE 2 Effect of different hormone combinations on the proliferation of dwarf Symphytum
From the data in table 2, it can be seen that under the culture conditions of the present invention, when the compounding ratio of 6-BA and NAA added to the culture medium for inducing tissue culture seedling subculture proliferation is 0.5: 0.15-0.25, the seedling is strong and the leaf is extended, and can root during proliferation. And the selected formula is MS +6 g.L-1Agar +30 g. L-1Sucrose +0.5 mg. L-16-benzylaminoadenine (6-BA) +0.20 mg. L-1Naphthylacetic acid (NAA), the multiplication coefficient reaches 5.46 after culturing for 30 days, the bud seedling is strong and can root in the multiplication process.
In the rooting culture stage, robust plantlets with 4-6 leaves are selected and inoculated into rooting culture media with different ionic strengths to induce rooting, as shown in table 3 below. And inoculating 8 bottles to each treatment, inoculating 3 strong seedlings to each bottle, and culturing for the same days under the same conditions to obtain rooted seedlings. And observing and recording the rooting conditions under the action of different ionic strengths, wherein the rooting conditions comprise rooting rate, average root length and average root number, and the specific results are shown in a table 3.
TABLE 3
As can be seen from the data in Table 3, under the culture conditions of the present invention, the rooting medium is selected from the medium with the ionic strength of 1/2MS or MS, 100% of roots can be rooted after 21 days of culture, the roots are rooted early, and the roots and the seedlings grow strongly; because the later period (21 d-42 d) can still keep a higher growth speed and continuously provide nutrition for the bud seedlings, and the invention can preferably adopt 1/2MS culture medium as the culture medium in the rooting culture stage by comprehensively considering the economic cost.
As described above, MS +0.5 mg. L-1KT+0.05mg·L-1NAA is beneficial to the induction of cluster buds, and the induction rate reaches 100% after 35 d; MS +0.5 mg. L-1 6-BA+0.20mg·L-1NAA is beneficial to the proliferation of tissue culture seedlings, and the proliferation coefficient can reach 5.46 after 30 days; 1/2MS is beneficial to the rooting of the seedling, and the rooting rate reaches 100% after 21 d; hardening and transplanting the seedlings, wherein after 30 days, the survival rate of the seedlings reaches about 90 percent, the plants grow well, and the plants can blossom and fruit after 3 months. The dwarf pillared grass seeds are large in quantity, easy to pick up and easy to disinfect, the tissue culture method adopts the seeds to culture aseptic seedlings, takes full-spread leaves of the aseptic seedlings as explants to induce germination, is convenient to obtain materials and has low pollution rate. And the culture medium for the tissue culture of the short-growing bowl column grass is simple in formula and remarkable in effect, can well overcome the defect of low survival rate of the division propagation of the short-growing bowl column grass, realizes rapid propagation, can keep the excellent genetic characteristics of the variety, and is beneficial to market popularization and garden application of the variety.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A culture medium for tissue culture of short-growing clitocybe herb is characterized by consisting of a seedling breeding culture medium, a cluster bud induction culture medium, a multiplication culture medium and a rooting culture medium;
the seedling breeding culture medium comprises: MS culture medium, 6g/L agar and 30g/L cane sugar, wherein the seedling breeding culture medium is used for culturing the dwarf pillared grass seeds;
the cluster bud induction culture medium comprises: MS culture medium, 6g/L agar, 30g/L cane sugar, 0.1-1.0 mg/L kinetin and 0.02-0.1 mg/L naphthylacetic acid;
the proliferation culture medium is as follows: MS culture medium, 6g/L agar, 30g/L cane sugar, 0.1-1.0 mg/L6-benzylamino adenine and 0.05-0.25 mg/L naphthylacetic acid;
the rooting culture medium is 1/2MS culture medium or MS culture medium.
2. The culture medium for tissue culture of short-growing bowl column grass according to claim 1, wherein the compounding ratio of the kinetin to the naphthylacetic acid is 25:1, 10:1 or 1: 1.
3. The culture medium for tissue culture of short-rooted bowl pillar grass according to claim 2, wherein the concentration of kinetin in the clump shoot induction medium is 0.5mg/L and the concentration of naphthylacetic acid in the clump shoot induction medium is 0.05 mg/L.
4. The culture medium for tissue culture of short-growing bowl column grass as claimed in claim 1, wherein the compounding ratio of 6-benzylamino adenine and naphthylacetic acid is 10: 3-5.
5. The culture medium for tissue culture of short-growing bowl grass according to claim 4, wherein the content of 6-benzylamino adenine and the content of naphthylacetic acid in the propagation medium are 0.5mg/L and 0.20mg/L, respectively.
6. A tissue culture method of short-growing clitocybe herb is characterized by comprising the following steps:
inoculating the sterilized seeds of the short-stalked physalis pubescens in the seedling breeding culture medium as claimed in any one of claims 1 to 5 for culture to obtain sterile seedlings of the short-stalked physalis pubescens;
inoculating the aseptic seedlings of the lysimachia sikokiana into the cluster bud induction culture medium as claimed in any one of claims 1 to 5 for culture to obtain cluster buds;
inoculating the cluster buds into a propagation culture medium as defined in any one of claims 1 to 5 for propagation culture to obtain propagated seedlings;
inoculating the proliferated seedling into the rooting culture medium of any one of claims 1 to 5 for culture to obtain rooted seedling;
and (4) hardening the rooted seedlings to obtain replanting seedlings.
7. The tissue culture method of short-rooted bowl grass according to claim 6, wherein the conditions for obtaining the multiple shoots comprise:
the culture temperature is as follows: 25 +/-2 ℃;
the illumination intensity is as follows: 37 to 40 μmoL/m2·s；
Illumination duration: 16 h/d.
8. The tissue culture method of short-rooted bowl grass according to claim 6, wherein the conditions for obtaining the multiple shoots comprise:
the culture temperature is as follows: 25 +/-2 ℃;
the illumination intensity is as follows: 37 to 40 μmoL/m2·s；
Illumination duration: 16 h/d;
culture days: and 21-42 days.
9. The method for tissue culture of short-rooted pistachio herb according to claim 6, further comprising, after the step of exercising the rooted shoots to obtain transplantable shoots:
planting the transplantable seedling in a matrix, and performing outdoor culture under the conditions that the temperature is 25 +/-2 ℃ and the humidity is more than 75%, wherein the matrix is a mixture of peat soil, perlite and argil according to a volume ratio of 3:1: 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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