CN117158323B - Tissue culture breeding and rapid propagation seedling method based on ginger tubers - Google Patents
Tissue culture breeding and rapid propagation seedling method based on ginger tubers Download PDFInfo
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- 239000008223 sterile water Substances 0.000 claims description 16
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- 238000005406 washing Methods 0.000 claims description 4
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Abstract
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture breeding and rapid propagation seedling method based on ginger tubers, which comprises the following steps: step one, selecting N tubers around the pretreated ginger terminal buds, wherein N is more than or equal to 1; step two, cleaning and sterilizing tubers treated in the step one; step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus; transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds; step five, dividing the cluster buds after the culture in the step four into single plants, and transferring the single plants into a rooting culture medium to obtain quick propagation seedlings; the invention takes materials from tubers around the top buds of the ginger, has simple and convenient operation, greatly saves the time of early germination, has simpler material taking process, and has the quantity of the rapid propagation seedlings obtained in the same time by each jin of ginger seeds at least three times as large as that of stem tips.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture breeding and rapid propagation seedling method based on ginger tubers.
Background
As the ginger takes the rhizomes as propagation materials, the propagation coefficient is low, diseases are easy to spread through the ginger, and the seed degeneration is caused by long-term asexual propagation. Stem rot and ginger fever are main diseases in ginger production, and a typical transmission path is seed transmission, and serious diseases can be destructive diseases, namely, ginger withers in a large range. At present, various physical and chemical approaches are performed for pathogen prevention and treatment, the effect is not ideal, and irreparable harm is caused to the ecological system of soil and the surrounding environment, thus impeding the development of ecological agriculture.
The rapid propagation of ginger seedlings can solve various pathogenic bacteria in ginger seeds, and an effective biological control method is provided for ginger planting. Meanwhile, after pathogenic bacteria in the ginger are eliminated, the original excellent properties of the ginger can be recovered, the growth performance of the ginger is enhanced, and the growth index and economic index of the ginger are effectively improved. If the management is proper after transplanting, the ginger rapid propagation seedlings have better capability of resisting re-infection, thereby greatly reducing the occurrence of various diseases.
The existing explant for tissue culture of ginger is mainly stem tip, and can be obtained when the germination accelerating is needed and the ginger buds grow to 2-3 cm, and the time is about 2-3 weeks, and the technique requires professional personnel to finely obtain materials by means of a stereoscopic microscope, so that the operation is complicated, the efficiency is low, the sterilization is difficult to complete, and the rapid propagation seedling pollution rate is high; meanwhile, the ginger stem tip is relatively difficult to obtain, the stem tip is too large in material, pollution is easy to cause, the material is too small, the survival rate is reduced, and the popularization and the use in a large range are not facilitated.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the tissue culture breeding and rapid propagation seedling method based on the ginger tubers, which takes the tubers around the ginger terminal buds as explants, has the advantages of more convenient material taking, no need of a stereoscopic microscope, simple operation, high efficiency, low pollution rate of the rapid propagation seedling, high propagation speed and large propagation quantity, and saves the germination accelerating time.
In order to solve the technical problems, the technical scheme of the invention is as follows: the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
firstly, selecting N tubers around the pretreated ginger terminal buds, wherein N is more than or equal to 1, and the size of the tubers is 0.2-0.8 cm;
step two, cleaning and sterilizing tubers treated in the step one;
the cleaning and disinfecting treatment comprises the following steps: firstly, treating tubers obtained in the first step with 70% alcohol for 1min, then washing with sterile water for five times, then treating with 0.1% mercuric chloride or 5% sodium hypochlorite for 10-15 min, then washing with sterile water for ten times, and then removing the part, which contacts with disinfectant, around the tubers;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
the cluster bud proliferation culture medium takes Ms as a basic culture medium, and 0.1-0.2 mg/L KT, 0.5-1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar are added;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
In the first step, the pretreatment operation is to select full, massive and pest-free ginger, wash the ginger clean with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15d, wash the soil on the surface clean with tap water, then put the ginger into a mesh bag, sun-shine the ginger continuously for 3-5 d under the sun, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud is required to be determined, the ginger bud does not need to wait for growing to 2-3 cm, and N ginger tubers with the circumference of the terminal bud within 0.2-0.8 cm are cut.
As a preferable technical scheme, the primary culture medium is based on Ms, and is added with 1-2 mg/L of 6-BA, 0.2-0.3 mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is subjected to dark culture for 30d.
As a preferable technical scheme, the culture conditions of the cluster buds in the fourth step are as follows: the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, the relative humidity is kept between 60 and 80 percent, and the culture is carried out for 5 to 6 weeks.
As a preferable technical scheme, the rooting culture medium takes Ms as a basic culture medium, and NAA of 0.1-0.2 mg/L, sucrose of 30g/L and agar of 4g/L are added.
As a preferable technical scheme, the culture conditions of the rapid propagation seedlings in the fifth step are as follows: the culture temperature was 28℃and the culture time was 15d.
As a preferable technical scheme, the culture medium of the root of the rapid propagation seedling obtained in the step five is cleaned, transplanted into a mixed matrix with the volume ratio of vermiculite to perlite=1:1, and simultaneously, a slow release fertilizer with the mass ratio of nitrogen to phosphorus to potassium=1:1:1 is added, irrigation is carried out properly, and after one month of culture, transplanting to a field for planting is carried out.
As an optimal technical scheme, the rapid propagation seedlings are irrigated in a mixed matrix in a micro-wetting mode, so that the wettability of the mixed matrix is maintained.
Due to the adoption of the technical scheme, the invention has the beneficial effects that: the method is simple and convenient to operate, does not need to use a stereoscopic microscope, does not need to wait until the ginger buds grow to 2-3 cm, only needs to determine the positions of the terminal buds, greatly saves the time of early germination, omits complicated procedures of germination and bud taking, has low technical requirements on operators, has simpler and more convenient material taking process compared with stem tip material taking, saves time, and has the quantity of rapid propagation seedlings obtained per jin of ginger seeds in the same time at least three times that of stem tip material taking.
Drawings
The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention. Wherein:
FIG. 1 is a drawing of tuber material from step one of the present invention;
FIG. 2 is a picture of callus formation in step three of the present invention;
FIG. 3 is a photograph showing the formation of multiple shoots in step four of the present invention;
FIG. 4 is a picture of the generation of the rapid propagation seedling in step five of the present invention;
fig. 5 is a cultivation flow chart of a fifth embodiment of the present invention.
Detailed Description
The invention is further illustrated in the following, in conjunction with the accompanying drawings and examples. In the following detailed description, certain exemplary embodiments of the present invention are described by way of illustration only. It is needless to say that the person skilled in the art realizes that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive in scope.
In the first embodiment, the selection of the material size of tubers around the top buds of ginger:
the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
step one, selecting 20 tubers around the pretreated ginger terminal buds, wherein 3 tubers are preferably selected around each terminal bud, and 20 tubers around a plurality of terminal buds can be selected;
step two, cleaning and sterilizing tubers treated in the step one;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
In the first step, the pretreatment operation is to select full, massive and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then put the ginger into a mesh bag, sun-stand for 3-5 days, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud is required to be determined, the ginger bud does not need to wait for growing to 2-3 cm, and 20 ginger tubers within 0.5-1.5 cm around the terminal bud are cut.
In the second step, the cleaning and disinfecting treatment comprises the following steps of firstly treating the tubers obtained in the first step with 70% alcohol for 1min, then cleaning the tubers with sterile water for five times, then treating the tubers with 0.1% mercuric chloride for 10 min, then cleaning the tubers with sterile water for ten times, and then removing the part, which contacts with the disinfectant, around the tubers.
The primary culture medium is based on Ms, and is added with 1mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is dark-cultured for 30d.
The cluster bud proliferation medium is based on Ms, and is added with 0.2mg/L KT, 1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar.
The effect of the size of the tubers surrounding the terminal buds of ginger on the callus induction rate is shown in Table 1.
| Numbering of drawing materials | Seed number (block) | Days of culture (d) | Pollution number (block) | Pollution rate (%) | Number of calli (blocks) | Callus formation rate (%) |
| M1 | 20 | 7 | 16 | 80 | 0 | 0 |
| M2 | 20 | 7 | 10 | 50 | 0 | 0 |
| M3 | 20 | 7 | 6 | 30 | 7 | 50 |
| M4 | 20 | 7 | 0 | 0 | 14 | 70 |
TABLE 1 influence of tuber size around ginger terminal buds on callus induction rate
Wherein,
tuber size (length x width x height) of material number M1 is: 1.5cm×1.5cm×0.5cm;
tuber size (length x width x height) of material number M2 is: 1cm by 0.5cm;
tuber size (length x width x height) of material number M3 is: 0.8cm by 0.5cm;
tuber size (length x width x height) of material number M4 is: 0.5cm by 0.2cm;
as shown in Table 1, the callus induction rate was highest under the same culture conditions with the tuber size of M4, and it was found that the callus induction rate was 70% by cutting ginger tubers 0.5 cm.times.0.5 cm.times.0.2 cm around the terminal buds as explants, which was the most preferable technique.
In a second embodiment, a sterilization mode is selected:
the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
step one, selecting 20 tubers around the pretreated ginger terminal buds, wherein 3 tubers are preferably selected around each terminal bud, and 20 tubers around a plurality of terminal buds can be selected;
step two, cleaning and sterilizing tubers treated in the step one;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
In the first step, the pretreatment operation is to select full, large and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then put the ginger into a mesh bag, sun-stand for 3-5 days, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud is required to be determined, the ginger bud does not need to wait for growing to 2-3 cm, and 20 ginger tubers with the size of 0.5cm multiplied by 0.2cm around the terminal bud are cut.
In the second step, the cleaning and disinfecting treatment comprises the following steps of firstly treating the tubers obtained in the first step with 70% alcohol for 1min, then cleaning with sterile water for five times, then treating with 0.1% mercuric chloride or 5% sodium hypochlorite for 10-20 min, then cleaning with sterile water for ten times, and then removing the part, which is in contact with the disinfectant, around the tubers.
The primary culture medium is based on Ms, and is added with 1mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is dark-cultured for 30d.
The cluster bud proliferation medium is based on Ms, and is added with 0.2mg/L KT, 1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar.
The effect of different disinfection modes on the survival rate of the callus is shown in Table 2.
| Disinfection number | Seed number (block) | Days of culture (d) | Pollution number (block) | Pollution rate (%) | Survival rate (%) |
| D1 | 20 | 30 | 2 | 10 | 90 |
| D2 | 20 | 30 | 1 | 5 | 60 |
| D3 | 20 | 30 | 0 | 0 | 0 |
| D4 | 20 | 30 | 4 | 20 | 30 |
TABLE 2 Effect of different disinfection modes on callus survival rates
Wherein,
the disinfection modes of the disinfection numbers D1 to D4 are respectively as follows:
d1: alcohol treatment at 70% concentration for 1 min+0.1% mercuric chloride treatment for 10 min;
d2: alcohol treatment at 70% concentration for 1 min+0.1% mercuric chloride treatment for 15 min;
d3: alcohol treatment at 70% concentration for 1 min+0.1% mercuric chloride treatment for 20 min;
d4: alcohol treatment of 70% concentration for 1 min+5% sodium hypochlorite for 10 min;
as can be seen from Table 2, the sterilization mode with the sterilization number D1 has the highest callus survival rate under the same culture conditions, and thus, it can be seen that the tuber is treated with 70% alcohol for 1min, then washed with sterile water for five times, then with 0.1% mercuric chloride for 10 min, then washed with sterile water for ten times, and then the part of the periphery of the tuber contacted with the sterilization agent is removed.
Example three, effect of different hormone ratios on ginger tuber callus formation:
the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
step one, selecting 20 tubers around the pretreated ginger terminal buds, wherein 3 tubers are preferably selected around each terminal bud, and 20 tubers around a plurality of terminal buds can be selected;
step two, cleaning and sterilizing tubers treated in the step one;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
In the first step, the pretreatment operation is to select full, large and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then put the ginger into a mesh bag, sun-stand for 3-5 days, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud is required to be determined, the ginger bud does not need to wait for growing to 2-3 cm, and 20 ginger tubers with the size of 0.5cm multiplied by 0.2cm around the terminal bud are cut.
In the second step, the cleaning and disinfecting treatment comprises the following steps of firstly treating the tubers obtained in the first step with 70% alcohol for 1min, then cleaning the tubers with sterile water for five times, then treating the tubers with 0.1% mercuric chloride for 10 min, then cleaning the tubers with sterile water for ten times, and then removing the part, which contacts with the disinfectant, around the tubers.
One group of the primary culture mediums is based on Ms, and is added with 1-2 mg/L of 6-BA, 0.2-0.3 mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is subjected to dark culture for 30d. The proportions of the other groups are shown in the following table 3.
The cluster bud proliferation medium is based on Ms, and is added with 0.2mg/L KT, 1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar.
The effect of different hormone ratios on the callus induction rate of ginger tubers is shown in Table 3.
| Primary Medium numbering | Cultivation time (d) | Seed number (block) | Callus formation number (block) | Callus formation rate (%) |
| C1 | 30 | 20 | 10 | 50 |
| C2 | 30 | 20 | 4 | 20 |
| C3 | 30 | 20 | 5 | 25 |
| C4 | 30 | 20 | 8 | 40 |
| C5 | 30 | 20 | 9 | 45 |
| C6 | 30 | 20 | 14 | 70 |
| C7 | 30 | 20 | 6 | 30 |
| C8 | 30 | 20 | 4 | 20 |
TABLE 3 influence of different hormone ratios on callus induction rate of ginger tubers
Wherein,
the hormone ratios of the primary culture medium numbers C1 to C8 are respectively as follows:
c1: MS+0.2mg/L KT+1.0 mg/L2, 4-D+30g/L sucrose+4 g/L agar;
c2: MS+1.0 mg/L6-BA+2.0 mg/L2, 4-D+30g/L sucrose+4 g/L agar;
and C3:1/2MS+1.0 mg/L6-BA+1.0 mg/L2, 4-D+30g/L sucrose+4 g/L agar;
and C4: MS+0.2mg/L of 2,4-D+30g/L of sucrose+4 g/L of agar;
c5: MS+0.2 mg/L6-BA+1.0 mg/L NAA+30g/L sucrose+4 g/L agar;
c6: MS+1.0 mg/L6-BA+0.2 mg/L NAA+30g/L sucrose+4 g/L agar;
c7: MS+2.0 mg/L6-BA+0.5 mg/L NAA+30g/L sucrose+4 g/L agar;
and C8: MS+0.5mg/L NAA+30g/L sucrose+4 g/L agar;
as shown in Table 3, the hormone ratio of the primary culture medium No. C6 was the highest in callus induction rate under the same culture conditions, and it was found that the primary culture medium was the most preferable technical scheme in which 1mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 4g/L of agar were added to the primary culture medium based on Ms, and the callus induction rate was the highest, up to 70%.
In the fourth embodiment, the influence of different hormone ratios on the induction of cluster buds from ginger tubers is shown:
the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
step one, selecting 20 tubers around the pretreated ginger terminal buds, wherein 3 tubers are preferably selected around each terminal bud, and 20 tubers around a plurality of terminal buds can be selected;
step two, cleaning and sterilizing tubers treated in the step one;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
In the first step, the pretreatment operation is to select full, large and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then put the ginger into a mesh bag, sun-stand for 3-5 days, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud is required to be determined, the ginger bud does not need to wait for growing to 2-3 cm, and 20 ginger tubers with the size of 0.5cm multiplied by 0.2cm around the terminal bud are cut.
In the second step, the cleaning and disinfecting treatment comprises the following steps of firstly treating the tubers obtained in the first step with 70% alcohol for 1min, then cleaning the tubers with sterile water for five times, then treating the tubers with 0.1% mercuric chloride for 10 min, then cleaning the tubers with sterile water for ten times, and then removing the part, which contacts with the disinfectant, around the tubers.
The primary culture medium is based on Ms, and is added with 1mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is dark-cultured for 30d.
One group of the cluster bud proliferation culture mediums is based on Ms, and 0.1-0.2 mg/L KT, 0.5-1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar are added; the composition and ratios of the other two comparative groups are shown in Table 4 below.
The culture conditions of the cluster buds in the fourth step are as follows: the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, the relative humidity is kept between 60 and 80 percent, and the culture is carried out for 5 to 6 weeks. The effect of different hormone ratios on the induction of the formation of cluster buds from ginger tubers is shown in Table 4.
| Cluster bud proliferation culture medium numbering | Seed number (block) | Bud number (block) | Bud ratio (%) |
| S1 | 20 | 0 | 0 |
| S2 | 20 | 9 | 45 |
| S3 | 20 | 15 | 75 |
TABLE 4 influence of different hormone ratios on the induction of clustered shoots from ginger tubers
Wherein,
the hormone ratios of cluster bud proliferation culture medium numbers S1 to S3 are respectively as follows:
s1: MS+2 mg/L6-BA+0.5 mg/L NAA+30g/L sucrose+4 g/L agar;
s2: MS+0.5 mg/L6-BA+1 mg/L IAA+30 g/L sucrose+4 g/L agar;
s3: MS+0. mg/L KT+1 mg/L2, 4-D+30g/L sucrose+4 g/L agar;
as is clear from Table 4, the hormone ratio of S3, which is the cluster bud multiplication medium, was the most preferable in that the culture medium was based on Ms, and 0.2. 0.2mg/L KT, 1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar were added thereto, and the bud ratio was the highest, up to 75%.
Fifth embodiment, as shown in fig. 1 to 5, the optimal technical solution is as follows:
the tissue culture breeding and rapid propagation seedling method based on ginger tubers comprises the following steps:
step one, selecting 30 tubers around the pretreated ginger terminal buds, wherein 3 tubers are preferably selected around each terminal bud, and 30 tubers around a plurality of terminal buds can be selected; FIG. 1 shows 3 tubers selected around one of the terminal buds.
Step two, cleaning and sterilizing tubers treated in the step one;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus; see fig. 2;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds; see fig. 3;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings. See fig. 4.
In the first step, the pretreatment operation is to select full, large and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then put the ginger into a mesh bag, sun-stand for 3-5 days, then carry out constant temperature treatment for 5min at 55 ℃ to carry out germination acceleration, only the growing position of the terminal bud needs to be determined, the ginger bud does not need to wait for growing to 2-3 cm, 3 ginger tubers with the size of 0.5cm multiplied by 0.2cm around the terminal bud are cut, and the total number of ginger tubers is sampled for 30.
In the second step, the cleaning and disinfecting treatment comprises the following steps of firstly treating the tubers obtained in the first step with 70% alcohol for 1min, then cleaning the tubers with sterile water for five times, then treating the tubers with 0.1% mercuric chloride for 10 min, then cleaning the tubers with sterile water for ten times, and then removing the part, which contacts with the disinfectant, around the tubers.
The primary culture medium is based on Ms, and is added with 1mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is dark-cultured for 30d.
The cluster bud proliferation medium is based on Ms, and is added with 0.2mg/L KT, 1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar.
The culture conditions of the cluster buds in the fourth step are as follows: the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, the relative humidity is kept between 60 and 80 percent, and the culture is carried out for 5 to 6 weeks.
The rooting culture medium takes Ms as a basic culture medium, and NAA with the concentration of 0.1-0.2 mg/L, sucrose with the concentration of 30g/L and agar with the concentration of 4g/L are added.
The culture conditions of the rapid propagation seedlings in the fifth step are as follows: the culture temperature was 28℃and the culture time was 15d.
And D, cleaning the culture medium of the root of the rapid propagation seedling obtained in the step five, transplanting the culture medium into a mixed matrix with the volume ratio of vermiculite to perlite=1:1, adding a slow release fertilizer with the mass ratio of nitrogen to phosphorus to potassium=1:1:1, properly irrigating, culturing for one month, and transplanting to a field for planting.
Irrigation is carried out on the rapid propagation seedlings in the mixed matrix in a micro-wetting mode, and the wettability of the mixed matrix is maintained. Instead of adopting an intermittent irrigation mode of drip irrigation which is 'water stored in soil' for the rapid propagation seedling domestication stage in the prior art, the invention adopts a micro-wetting mode for irrigation, the water consumption is about 40-50% of that of drip irrigation, the effective utilization rate of water is improved, and the problem of water loss caused by matrix evaporation and matrix leakage in the prior art in the sprinkling irrigation mode is solved.
The invention has the following advantages:
(1) The method has the advantages of simple and quick material taking, no need of waiting for ginger buds to grow to 2-3 cm, no need of a stereoscopic microscope, low professional technical requirements on operators, and simpler material taking process and more time saving compared with stem tip material taking.
(2) The number of the rapid propagation seedlings obtained in the same time is at least 3 times that of the stem tip materials of each jin of ginger seeds, as only one callus is obtained from each ginger bud, at least more than three tubers can be obtained from each ginger bud at present, and one callus can be obtained from each tuber, so that the number of the rapid propagation seedlings obtained in the same time is at least 3 times that of the stem tip materials of each jin of ginger seeds, and the propagation rate is greatly increased.
(3) The micro-irrigation is adopted, so that water is saved, the effective utilization rate of water is improved, and the water consumption is about 40-50% of that of drip irrigation.
FIG. 1 shows a drawing of tuber material according to a fifth embodiment of the present invention; FIG. 2 is a drawing showing the generation of calli according to the fifth embodiment of the present invention; FIG. 3 is a diagram showing the generation of five cluster buds according to the embodiment of the present invention; fig. 4 shows a picture generated by the fifth rapid propagation seedling according to the present embodiment of the invention; fig. 5 shows a cultivation flow chart of a fifth embodiment of the present invention.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that modifications may be made to the described embodiments in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive of the scope of the invention, which is defined by the appended claims.
Claims (8)
1. The method for tissue culture breeding and rapid propagation of seedlings based on ginger tubers is characterized by comprising the following steps:
firstly, selecting N tubers around the pretreated ginger terminal buds, cutting the N tubers within 0.2-0.8 cm around the terminal buds, wherein N is not less than 1 and not more than 3, and the length, width and height of the tubers are (0.5-0.8) cm (0.2-0.5) cm;
step two, cleaning and sterilizing tubers treated in the step one;
the cleaning and disinfecting treatment comprises the following steps: firstly, treating tubers obtained in the first step with 70% alcohol for 1min, then washing with sterile water for five times, then treating with 0.1% mercuric chloride or 5% sodium hypochlorite for 10 min, then washing with sterile water for ten times, and then removing the part, which contacts with disinfectant, around the tubers;
step three, inoculating the tubers treated in the step two into a primary culture medium for culture, and inducing to generate callus;
transferring the callus subjected to the culture in the third step to a cluster bud proliferation culture medium for continuous culture to obtain cluster buds;
the cluster bud proliferation culture medium takes MS as a basic culture medium, and 0.1-0.2 mg/L KT, 0.5-1 mg/L2, 4-D, 30g/L sucrose and 4g/L agar are added;
and fifthly, dividing the cluster buds after the culture in the fourth step into single plants, and transferring the single plants into a rooting culture medium to obtain the rapid propagation seedlings.
2. The method for tissue culture and propagation of seedlings based on ginger tubers according to claim 1, wherein in the first step, the pretreatment operation is to select full, massive and pest-free ginger, wash the ginger with tap water, if the ginger is newly harvested, place the ginger in a shade place for 15 days, wash the soil on the surface with tap water, then place the ginger in a mesh bag, sun the ginger for 3-5 days continuously, and then carry out constant temperature treatment at 55 ℃ for 5min for germination acceleration, and only the growing position of terminal buds is required to be determined, without waiting for the growth of the ginger buds to 2-3 cm.
3. The method for tissue culture and propagation of seedlings based on ginger tubers as claimed in claim 1, wherein the primary culture medium is based on MS, and is added with 1-2 mg/L of 6-BA, 0.2-0.3 mg/L of NAA, 30g/L of sucrose and 4g/L of agar, and is dark-cultured for 30d.
4. The method for tissue culture propagation of rapid propagation seedlings based on ginger tubers as claimed in claim 1, wherein the culture conditions of the cluster buds in the fourth step are as follows: the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, the relative humidity is kept between 60 and 80 percent, and the culture is carried out for 5 to 6 weeks.
5. The method for tissue culture propagation of rapid propagation seedlings based on ginger tubers as claimed in claim 1, wherein the rooting medium is based on MS, and NAA of 0.1-0.2 mg/L, sucrose of 30g/L and agar of 4g/L are added.
6. The method for tissue culture propagation of rapid propagation seedlings based on ginger tubers as claimed in claim 1, wherein the culture conditions of the rapid propagation seedlings in the fifth step are as follows: the culture temperature was 28℃and the culture time was 15d.
7. A method for tissue culture propagation of rapid propagation seedlings based on ginger tubers as claimed in any one of claims 1 to 6, characterized in that: and D, cleaning the culture medium of the root of the rapid propagation seedling obtained in the step five, transplanting the culture medium into a mixed matrix with the volume ratio of vermiculite to perlite=1:1, adding a slow release fertilizer with the mass ratio of nitrogen to phosphorus to potassium=1:1:1, properly irrigating, culturing for one month, and transplanting to a field for planting.
8. The method for tissue culture propagation of rapid propagation seedlings based on ginger tubers as claimed in claim 7, wherein: irrigation is carried out on the rapid propagation seedlings in the mixed matrix in a micro-wetting mode, and the wettability of the mixed matrix is maintained.
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