CN117158322B - Tissue culture breeding rapid propagation seedling method based on ginger root - Google Patents
Tissue culture breeding rapid propagation seedling method based on ginger root Download PDFInfo
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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 root, which comprises the following steps: step one, selecting a root section of a parent ginger rapid propagation seedling of 0.2-1 cm, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 3-4 weeks, and inducing to form a root section callus; transferring the root section callus to a root section cluster bud proliferation culture medium, and culturing for 3-4 weeks to generate cluster buds; dividing the cluster buds into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain a daughter ginger rapid propagation seedling; according to the invention, root systems of the parent ginger cut seedlings in the ginger passage process are utilized to carry out root section callus culture, root section cluster buds are proliferated and cultured, and the number of the rapid propagation seedlings is increased; the method has the advantages of high efficiency, low pollution rate of rapid propagation seedlings, high propagation speed and large propagation quantity without using a stereoscopic microscope, and can provide a large number of receptors for transgenic ginger and new varieties of mutagenesis.
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 roots.
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 ginger tissue culture mainly uses stem tips, propagation and proliferation mainly depend on stem segments of detoxification rapid propagation seedlings, the growth capacity of the rapid propagation seedlings is weakened along with the increase of the number of passages, and the survival rate of the rapid propagation seedlings in later transplanting is reduced. The root of each seedling is required to be removed during the subculture, the stem is sheared, only the base part (stem section with the length of 0.5-1 cm) of the seedling stem for rapid propagation is left, the multiplication factor is about 4-5 times, germination acceleration is required in advance during material drawing, the fresh ginger bud grows to 2-3 cm, and about 2-3 weeks is required, and the technique requires professionals to finely draw materials by means of a stereoscopic vision, so that the operation is complicated, the efficiency is low, the sterilization is difficult to complete, and the pollution rate of the seedling for rapid propagation is high; meanwhile, the fresh 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, the wide popularization and the use are not facilitated, the propagation rate of fresh ginger cultivation is reduced, and in the tissue culture passage process of the stem tip, the root system of the fresh ginger is a abandoned part and the due value of the fresh ginger is not exerted.
Disclosure of Invention
The invention aims to solve the technical problem of providing a tissue culture breeding rapid propagation seedling method based on ginger roots, which takes ginger roots as explants, has the advantages of more convenient material taking, changing waste into valuable, no need of a stereoscopic microscope, simple operation, high efficiency, low pollution rate of rapid propagation seedlings, high propagation speed and large propagation quantity.
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 root comprises the following steps:
step one, selecting a root section of a parent ginger rapid propagation seedling of 0.2-1 cm, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 3-4 weeks, and inducing to form a root section callus;
the root primary culture medium takes Ms as a basic culture medium, and is added with 1-2 mg/L of 2,4-D, 0.5-1 mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar;
step two, transferring the root section callus in the step one into a root section cluster bud proliferation culture medium, and culturing for 3-4 weeks to generate cluster buds;
the root section cluster bud proliferation culture medium takes Ms as a basic culture medium, and is added with 1.5-2.5 mg/L of 6-BA, 0.1-0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar for 3-4 weeks;
and thirdly, dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain the ginger rapid propagation seedlings as the daughter.
As a preferable technical scheme, the root segments of the parent ginger rapid propagation seedlings in the first step are obtained by the following steps:
step 11, selecting full, massive and pest-free ginger, washing the ginger with tap water, if the ginger is newly harvested, placing the ginger in a shade place for 15 days, washing the soil on the surface of the ginger with tap water, placing the ginger into a mesh bag, continuously airing the ginger in the sun for 3-5 days, accelerating germination, and when the ginger bud grows to 2-3 cm, treating the ginger bud at an ultra-high temperature of 55 ℃ for 5 minutes, and placing the ginger bud in an ultra-clean workbench;
step 12, soaking the ginger buds selected in the step 11 with 70% ethanol for 30-60 s, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 10min, washing with sterile water for 10 times, sucking water on the surfaces of the stem tips with sterile filter paper, stripping the stem tips to only leave 1-2 leaf primordia, inoculating in a stem tip primary culture medium, and culturing stem tip callus;
step 13, inoculating the stem tip callus cultured in the step 12 into a stem tip clustered bud proliferation culture medium, and culturing clustered buds;
and 14, dividing the cluster buds cultured in the step 13 into single plants, transferring the single plants into a rooting culture medium, and culturing a complete parent ginger rapid propagation seedling plant so as to obtain the root system of the parent ginger rapid propagation seedling plant.
As a preferable technical scheme, the stem tip primary culture medium in the step 12 takes Ms as a basic culture medium, 1-1.5 mg/L of 6-BA, 0.1-0.2 mg/L of NAA, 30g/L of sucrose and 5g/L of agar are added, the pH value is regulated to 5.8 by NaOH or HCl, the culture is carried out for 4-5 weeks, the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, and the relative humidity is kept at 60% -80%.
As a preferable technical scheme, the stem tip cluster bud multiplication medium in the step 13 is prepared by taking Ms as a basic medium, adding 2-3 mg/L of 6-BA, 0.2-0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar, and culturing for 8-9 weeks.
As a preferable technical scheme, the rooting medium in the third step and the step 14 is based on Ms, and NAA of 0.1mg/L, sucrose of 30g/L and agar of 5g/L are added, and the rooting medium is cultured for 15d at 28 ℃.
In the step 14, after the parent ginger rapid propagation seedling cultivates the root system, cutting off the root system and leaves to leave stem segments with a stem base of about 1cm, inoculating the stem segments into a stem tip cluster bud propagation medium for propagation, and selecting the root segments with a length of 0.2-1 cm from the cut root system as the explant of the step one.
As a preferable technical scheme, the culture medium of the root of the rapid propagation seedling of the daughter ginger obtained in the step three 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 of the ginger 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 method for tissue culture breeding and rapid propagation of seedlings based on ginger roots comprises the following steps: step one, selecting a root section of a parent ginger rapid propagation seedling of 0.2-1 cm, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 3-4 weeks, and inducing to form a root section callus; the root primary culture medium takes Ms as a basic culture medium, and is added with 1-2 mg/L of 2,4-D, 0.5-1 mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar; step two, transferring the root section callus in the step one into a root section cluster bud proliferation culture medium, and culturing for 3-4 weeks to generate cluster buds; the root section cluster bud proliferation culture medium takes Ms as a basic culture medium, and is added with 1.5-2.5 mg/L of 6-BA, 0.1-0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar for 3-4 weeks; dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting medium, and culturing to obtain a daughter ginger rapid propagation seedling; the beneficial effects of the invention are as follows: the invention fully utilizes the root system excised from the parent ginger rapid propagation seedling in the ginger passage process to carry out root callus culture and root cluster bud proliferation culture, and can obviously improve the number of the child ginger rapid propagation seedlings in a certain time; on the other hand, the same number of rapid propagation seedlings are required to be obtained at the same time, the roots of the parent ginger rapid propagation seedlings are utilized, the passage number of the ginger rapid propagation seedlings can be reduced, the number of times of stem tip material drawing can be reduced, and the operation difficulty is reduced. The invention uses the root of ginger as an explant, changes waste into valuable, does not need a stereoscopic microscope, has high efficiency, low pollution rate of rapid propagation seedlings, high propagation speed and large propagation quantity, and can provide a large number of receptors for transgenic ginger and new varieties of mutagenesis due to the formation of root callus.
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 picture of a root section selected from a tissue-cultured and propagated seedling based on ginger root;
FIG. 2 (1) is one of the pictures of the tissue culture propagation rapid propagation Miao Genduan callus based on ginger root of the present invention;
FIG. 2 (2) is a second picture of the tissue culture and propagation of the rapid propagation Miao Genduan callus based on ginger root in the present invention;
FIG. 3 is a picture of the generation of cluster buds by tissue culture propagation and rapid propagation Miao Genduan based on ginger roots;
FIG. 4 is a picture of a rapid propagation seedling of a daughter ginger generated by tissue culture propagation of a rapid propagation seedling based on a ginger root;
FIG. 5 is a cultivation flow chart of tissue culture and propagation seedlings based on ginger roots.
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.
Example 1
The tissue culture breeding and rapid propagation seedling method based on ginger root comprises the following steps:
step one, selecting a root section of 0.2cm of a parent ginger rapid propagation seedling, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 3 weeks, and inducing to form a root section callus;
transferring the root section callus in the first step into a root section cluster bud proliferation culture medium, and culturing for 3 weeks to generate cluster buds;
and thirdly, dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain the ginger rapid propagation seedlings as the daughter.
The root segments of the parent ginger rapid propagation seedlings in the first step are obtained by the following steps:
step 11, selecting full, massive and pest-free ginger, washing the ginger with tap water, if the ginger is newly harvested, placing the ginger in a shade place for 15 days, washing the soil on the surface of the ginger with tap water, placing the ginger into a mesh bag, continuously airing the ginger in the sun for 3-5 days, accelerating germination, and when the ginger bud grows to 2-3 cm, treating the ginger bud at an ultra-high temperature of 55 ℃ for 5 minutes, and placing the ginger bud in an ultra-clean workbench;
step 12, soaking the ginger buds selected in the step 11 with 70% ethanol for 30-60 s, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 10min, washing with sterile water for 10 times, sucking water on the surfaces of the stem tips with sterile filter paper, stripping the stem tips to only leave 1-2 leaf primordia, inoculating in a stem tip primary culture medium, and culturing stem tip callus;
step 13, inoculating the stem tip callus cultured in the step 12 into a stem tip clustered bud proliferation culture medium, and culturing clustered buds;
and 14, dividing the cluster buds cultured in the step 13 into single plants, transferring the single plants into a rooting culture medium, and culturing a complete parent ginger rapid propagation seedling plant so as to obtain the root system of the parent ginger rapid propagation seedling plant.
The primary culture medium of the stem tip in the step 12 is based on Ms, 1mg/L of 6-BA, 0.1mg/L of NAA, 30g/L of sucrose and 5g/L of agar are added, the pH value is regulated to 5.8 by NaOH or HCl, the culture is carried out for 4 to 5 weeks, the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, and the relative humidity is kept at 60 to 80 percent.
The stem tip cluster bud multiplication medium in the step 13 is cultured for 8-9 weeks by taking Ms as a basic medium and adding 2.5mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 5g/L of agar.
The rooting medium in the third step and the step 14 is based on Ms, and NAA of 0.1mg/L, sucrose of 30g/L and agar of 5g/L are added for 15d at 28 ℃.
The root section cluster bud proliferation culture medium in the second step is based on Ms, and is added with 1.5mg/L of 6-BA, 0.1mg/L of NAA, 30g/L of sucrose and 5g/L of agar for 3-4 weeks.
In the step 14, after the parent ginger rapid propagation seedling cultivates a root system, cutting off the root system and leaves to leave a stem section with about 1cm of the stem base, inoculating the stem section into a stem tip cluster bud propagation medium for propagation, and taking the root section with the length of 0.2-1 cm as the explant of the step one.
And (3) cleaning the culture medium of the root of the quick-propagation seedling of the daughter ginger obtained in the step (III), 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.
The rapid propagation seedling of the ginger is irrigated in the mixed matrix in a micro-wetting mode, so that the wettability of the mixed matrix is maintained.
In the first step, the primary culture medium of the root section is based on Ms, and 1mg/L of 2,4-D, 0.5mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar are respectively added; and 1mg/L NAA, 0.5 mg/L6-BA, 30g/L sucrose and 5g/L agar were added for comparison.
Table 1 shows the effect of the primary culture medium of root segments with different hormone ratios on the formation of callus of ginger root segments.
| Group of culture media | Seed stem tip number | Pollution number (number) | Callus induction derivative (individual) | Callus induction rate (%) |
| C1 | 60 | 5 | 41 | 68.3% |
| C2 | 60 | 3 | 52 | 86.6% |
TABLE 1 Effect of Primary culture Medium of root segments with different hormone ratios on formation of callus of ginger root segments
Wherein,
the ratio of the culture medium groups C1 and C2 is respectively as follows:
c1: MS+1. mg/L NAA+0. mg/L6-BA+30 g/L sucrose+5 g/L agar;
c2: MS+1.0 mg/L of 2,4-D+0.5 mg/L of 6-BA+30g/L of sucrose+5 g/L of agar; as can be seen from Table 1, the hormone ratio callus induction rate of the culture medium group C2 is 86.6%, so that the ginger root section callus preferably adopts the formula of a root section primary culture medium: ms-based medium, 1mg/L of 2,4-D, 0.5mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar were added.
Example two
The tissue culture breeding and rapid propagation seedling method based on ginger root comprises the following steps:
firstly, selecting 1cm of a root section of a parent ginger rapid propagation seedling, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 4 weeks, and inducing to form a root section callus;
transferring the root section callus in the first step into a root section cluster bud proliferation culture medium, and culturing for 4 weeks to generate cluster buds;
and thirdly, dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain the ginger rapid propagation seedlings as the daughter.
The root segments of the parent ginger rapid propagation seedlings in the first step are obtained by the following steps:
step 11, selecting full, massive and pest-free ginger, washing the ginger with tap water, if the ginger is newly harvested, placing the ginger in a shade place for 15 days, washing the soil on the surface of the ginger with tap water, placing the ginger into a mesh bag, continuously airing the ginger in the sun for 3-5 days, accelerating germination, and when the ginger bud grows to 2-3 cm, treating the ginger bud at an ultra-high temperature of 55 ℃ for 5 minutes, and placing the ginger bud in an ultra-clean workbench;
step 12, soaking the ginger buds selected in the step 11 with 70% ethanol for 30-60 s, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 10min, washing with sterile water for 10 times, sucking water on the surfaces of the stem tips with sterile filter paper, stripping the stem tips to only leave 1-2 leaf primordia, inoculating in a stem tip primary culture medium, and culturing stem tip callus;
step 13, inoculating the stem tip callus cultured in the step 12 into a stem tip clustered bud proliferation culture medium, and culturing clustered buds;
and 14, dividing the cluster buds cultured in the step 13 into single plants, transferring the single plants into a rooting culture medium, and culturing a complete parent ginger rapid propagation seedling plant so as to obtain the root system of the parent ginger rapid propagation seedling plant.
The primary culture medium of the stem tip in the step 12 is based on Ms, 1.5mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 5g/L of agar are added, the pH value is regulated to 5.8 by NaOH or HCl, the culture is carried out for 4 to 5 weeks, the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, and the relative humidity is kept at 60 to 80 percent.
The stem tip cluster bud multiplication medium in the step 13 is cultured for 8-9 weeks by taking Ms as a basic medium and adding 3mg/L of 6-BA, 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar.
The rooting medium in the third step and the step 14 is based on Ms, and NAA of 0.1mg/L, sucrose of 30g/L and agar of 5g/L are added for 15d at 28 ℃.
In the first step, the primary culture medium of the root section is based on Ms, and 1.5mg/L of 2,4-D, 0.8mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar are added.
In the step 14, after the parent ginger rapid propagation seedling cultivates a root system, cutting off the root system and leaves to leave a stem section with about 1cm of the stem base, inoculating the stem section into a stem tip cluster bud propagation medium for propagation, and taking the root section with the length of 0.2-1 cm as the explant of the step one.
And (3) cleaning the culture medium of the root of the quick-propagation seedling of the daughter ginger obtained in the step (III), 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.
The rapid propagation seedling of the ginger is irrigated in the mixed matrix in a micro-wetting mode, so that the wettability of the mixed matrix is maintained.
The root section cluster bud multiplication medium in the second step is based on Ms, and is respectively added with 1.5mg/L of 6-BA, 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar, and is cultured for 3-4 weeks; and 3-4 weeks of addition of KT 3mg/L, NAA 1mg/L, sucrose 30g/L and agar 5 g/L; a comparative test was performed.
Table 2 shows the effect of the proliferation culture medium of root section cluster buds with different hormone ratios on the induction of buds and rooting of ginger roots.
| Group of culture media | Seed number (number) | Survival rate (%) | Expansion ratio (%) | Expansion multiple | Browning rate (%) | Bud ratio (%) | Rooting percentage (%) |
| S1 | 30 | 80 | 100 | 5-6 | 30% | 60% | 40% |
| S2 | 30 | 100 | 100 | 2-3 | 15% | 75% | 80% |
TABLE 2 Effect of different hormone ratios of root section Concatenation bud propagation Medium on ginger root induced bud formation and rooting
Wherein,
the ratio of the culture medium groups S1 and S2 is respectively as follows:
s1: MS+3 mg/L KT+1 mg/L NAA+30g/L sucrose+5 g/L agar;
s2: MS+1.5 mg/L6-BA+0.3 mg/L NAA+30g/L sucrose+5 g/L agar;
as is clear from Table 2, the hormone ratio of the culture medium group S2 can reach 75% and the rooting rate can reach 80%, so that the ginger root section cluster buds are preferably cultured by adopting the root section cluster bud proliferation culture medium as a basic culture medium and adding 1.5mg/L of 6-BA, 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar.
Embodiment III: as shown in fig. 1 to 5,
the tissue culture breeding and rapid propagation seedling method based on ginger root comprises the following steps:
step one, selecting a root section of a parent ginger rapid propagation seedling of 0.5cm, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 4 weeks, and inducing to form a root section callus;
transferring the root section callus in the first step into a root section cluster bud proliferation culture medium, and culturing for 4 weeks to generate cluster buds;
and thirdly, dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain the ginger rapid propagation seedlings as the daughter.
The root segments of the parent ginger rapid propagation seedlings in the first step are obtained by the following steps:
step 11, selecting full, massive and pest-free ginger, washing the ginger with tap water, if the ginger is newly harvested, placing the ginger in a shade place for 15 days, washing the soil on the surface of the ginger with tap water, placing the ginger into a mesh bag, continuously airing the ginger in the sun for 3-5 days, accelerating germination, and when the ginger bud grows to 2-3 cm, treating the ginger bud at an ultra-high temperature of 55 ℃ for 5 minutes, and placing the ginger bud in an ultra-clean workbench;
step 12, soaking the ginger buds selected in the step 11 with 70% ethanol for 30-60 s, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 10min, washing with sterile water for 10 times, sucking water on the surfaces of the stem tips with sterile filter paper, stripping the stem tips to only leave 1-2 leaf primordia, inoculating in a stem tip primary culture medium, and culturing stem tip callus;
step 13, inoculating the stem tip callus cultured in the step 12 into a stem tip clustered bud proliferation culture medium, and culturing clustered buds;
and 14, dividing the cluster buds cultured in the step 13 into single plants, transferring the single plants into a rooting culture medium, and culturing a complete parent ginger rapid propagation seedling plant so as to obtain the root system of the parent ginger rapid propagation seedling plant.
The primary culture medium of the stem tip in the step 12 is based on Ms, 1.5mg/L of 6-BA, 0.2mg/L of NAA, 30g/L of sucrose and 5g/L of agar are added, the pH value is regulated to 5.8 by NaOH or HCl, the culture is carried out for 4 to 5 weeks, the illumination intensity is 4000lx, the illumination time is 16h/d, the culture temperature is 25 ℃, and the relative humidity is kept at 60 to 80 percent.
The stem tip cluster bud multiplication medium in the step 13 is cultured for 8-9 weeks by taking Ms as a basic medium and adding 3mg/L of 6-BA, 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar.
The rooting medium in the third step and the step 14 is based on Ms, and NAA of 0.1mg/L, sucrose of 30g/L and agar of 5g/L are added for 15d at 28 ℃.
In the first step, the primary culture medium of the root section is based on Ms, and 1mg/L of 2,4-D, 0.5mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar are added.
The root section cluster bud proliferation culture medium in the second step is based on Ms, and is added with 1.5mg/L of 6-BA, 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar for 3-4 weeks.
In the step 14, after the parent ginger rapid propagation seedling cultivates a root system, cutting off the root system and leaves to leave a stem section with a stem base of about 1cm, inoculating the stem section into a stem tip cluster bud propagation medium for propagation, and taking the root section with a length of 0.2-1 cm as the explant of the step one.
And (3) cleaning the culture medium of the root of the quick-propagation seedling of the daughter ginger obtained in the step (III), 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.
The rapid propagation seedling of the ginger is irrigated in the mixed matrix in a micro-wetting mode, so that the wettability of the mixed matrix is maintained.
The invention fully utilizes the root system excised in the ginger passage process to carry out root callus culture and root propagation and rooting culture of the cluster buds of the root section, can obviously improve the total number of the ginger rapid propagation seedlings within 6 months, simultaneously obtains the same number of rapid propagation seedlings, utilizes the root, can reduce the passage number of the ginger rapid propagation seedlings, can also reduce the number of times of stem tip material taking, and reduces the operation difficulty.
As shown in fig. 5, in the process of producing rapid propagation seedlings by using a common stem tip material taking 10 stem tips of 1 jin of ginger seeds as an example, the conventional stem tip cultivation can produce about 2500 rapid propagation seedlings within 6 months, after the combination of root segments cultivation, one parent ginger rapid propagation seedling is cut into 70 root segments, at least one daughter ginger rapid propagation seedling is produced in each root segment, at least 40 rapid propagation seedlings can be obtained by the stem tip primary cultivation, about 2800 daughter ginger rapid propagation seedlings can be produced in total within 6 months, and at least 5300 (2500+2800) daughter ginger rapid propagation seedlings can be produced by combining with the conventional stem tip cultivation. If the seedling is obtained only through the stem tip, at least 20 stem tips are needed to be obtained, at least 2 jin of ginger seeds are needed, meanwhile, the requirement on the material taking personnel is high, the operation difficulty is increased, the pollution rate is increased, the survival rate is reduced, the root section is obtained, the operation is simple and convenient, the time can be saved by combining the two materials, the requirement on the operator is reduced, a large number of sub-body ginger seedlings can be obtained in a short time, and a convenient and easy-to-operate technical scheme is provided for industrial production.
The invention has the following advantages:
(1) The invention selects root segments from the ginger root system as explants, and fully utilizes raw materials;
(2) The materials are easy to obtain, and a stereoscopic microscope is not needed;
(3) In the same time, a large number of ginger detoxification and rapid propagation seedlings can be obtained by combining with stem tip tissue culture;
(4) Callus formed by root segments can provide a large number of receptors for transgenic ginger and new varieties generated by mutagenesis;
(5) The water is saved, the effective utilization rate of the water is improved, the micro-irrigation is a water-saving irrigation technology, and the water consumption is about 40% -50% of that of drip irrigation;
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 tissue culture breeding and rapid propagation seedling method based on ginger root is characterized by comprising the following steps:
step one, selecting a root section of 0.2cm of a parent ginger rapid propagation seedling, inoculating the parent ginger rapid propagation seedling into a root section primary culture medium, culturing for 3-4 weeks, and inducing to form a root section callus;
the root primary culture medium takes MS as a basic culture medium, and is added with 1mg/L of 2,4-D, 0.5mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar; step two, transferring the root section callus in the step one into a root section cluster bud proliferation culture medium, and culturing for 3-4 weeks to generate cluster buds;
the root section cluster bud proliferation culture medium takes MS as a basic culture medium, is added with 1.5mg/L of 6-BA, 0.1mg/L of NAA, 30g/L of sucrose and 5g/L of agar, and is cultured for 3-4 weeks;
and thirdly, dividing the cluster buds in the second step into single plants, transferring the single plants into a rooting culture medium, and culturing to obtain the ginger rapid propagation seedlings as the daughter.
2. The method for tissue culture propagation of rapid propagation seedlings based on ginger roots as claimed in claim 1, wherein the root segments of the parent ginger rapid propagation seedlings in the step one are obtained by:
step 11, selecting full, massive and pest-free ginger, washing the ginger with tap water, if the ginger is newly harvested, placing the ginger in a shade place for 15 days, washing the soil on the surface of the ginger with tap water, placing the ginger into a mesh bag, continuously airing the ginger in the sun for 3-5 days, accelerating germination, and when the ginger bud grows to 2-3 cm, treating the ginger bud at an ultra-high temperature of 55 ℃ for 5 minutes, and placing the ginger bud in an ultra-clean workbench;
step 12, soaking the ginger buds selected in the step 11 with 70% ethanol for 30-60 s, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 10min, washing with sterile water for 10 times, sucking water on the surfaces of the stem tips with sterile filter paper, stripping the stem tips to only leave 1-2 leaf primordia, inoculating in a stem tip primary culture medium, and culturing stem tip callus;
step 13, inoculating the stem tip callus cultured in the step 12 into a stem tip clustered bud proliferation culture medium, and culturing clustered buds;
and 14, dividing the cluster buds cultured in the step 13 into single plants, transferring the single plants into a rooting culture medium, and culturing a complete parent ginger rapid propagation seedling plant so as to obtain the root system of the parent ginger rapid propagation seedling plant.
3. The method for tissue culture and propagation of rapid propagation seedlings based on ginger root as claimed in claim 2, wherein the stem tip primary culture medium in the step 12 is based on MS, 1-1.5 mg/L of 6-BA, 0.1-0.2. 0.2mg/L of NAA, 30g/L of sucrose and 5g/L of agar are added, pH value is adjusted to 5.8 by NaOH or HCl, culturing is carried out for 4-5 weeks, illumination intensity is 4000lx, illumination time is 16h/d, culturing temperature is 25 ℃, and relative humidity is kept at 60% -80%.
4. The method for tissue culture and propagation of seedlings based on ginger root as claimed in claim 2, wherein the propagation medium of shoot tip cluster buds in the step 13 is based on MS, and is added with 2-3 mg/L of 6-BA, 0.2-0.3. 0.3 mg/L of NAA, 30g/L of sucrose and 5g/L of agar, and cultured for 8-9 weeks.
5. The method for tissue culture propagation of seedlings based on ginger root as claimed in claim 2, wherein the rooting medium in the third step and the step 14 is based on MS, and NAA of 0.1mg/L, sucrose of 30g/L and agar of 5g/L are added thereto, and the culture is carried out at 28 ℃ for 15d.
6. The method for tissue culture and rapid propagation of seedlings based on ginger root as claimed in claim 2, wherein in the step 14, after the parent ginger rapid propagation seedlings cultivate root systems, cutting off the root systems and leaves to leave stem segments of about 1cm of the stem base, inoculating the stem segments into a propagation medium of shoot tips clustered buds for propagation, and taking root segments with the length of 0.2-1 cm as explants of the step one.
7. The method for tissue culture propagation of seedlings based on ginger roots as set forth in any one of claims 1 to 6, wherein the culture medium of the ginger root of the daughter obtained in the third step is cleaned, transplanted to a mixed matrix with volume ratio vermiculite: perlite=1:1, and simultaneously added with a slow release fertilizer with mass ratio nitrogen: phosphorus: potassium=1:1:1, properly irrigated, cultured for one month and transplanted to a field for planting.
8. The method for tissue culture propagation of rapid propagation seedlings based on ginger roots as claimed in claim 7, wherein the rapid propagation seedlings of the daughter ginger are irrigated in a mixed matrix in a micro-wetting manner to maintain the wettability of the mixed matrix.
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