CN111771725A - Optimized ginger virus-free seedling regeneration propagation method and development of industrialization process thereof - Google Patents
Optimized ginger virus-free seedling regeneration propagation method and development of industrialization process thereof Download PDFInfo
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- CN111771725A CN111771725A CN202010741973.6A CN202010741973A CN111771725A CN 111771725 A CN111771725 A CN 111771725A CN 202010741973 A CN202010741973 A CN 202010741973A CN 111771725 A CN111771725 A CN 111771725A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses an optimized regeneration propagation method of ginger virus-free seedlings and development of an industrialization process thereof, which is characterized in that: the optimal explant type, sterilization method, cell classifier, induction medium formula, seedling exercising condition and culture condition are determined. The optimized ginger virus-free seedlings need 3.5-4.5 months from explants to TC0 rooting seedlings, then each successive generation needs about 1.5 months, the contamination rate in the whole process is lower than 5%, the TC0 budding rate can reach 800% at most, the successive generation differentiation efficiency can reach 300% at most, the rooting rate can reach 100%, the transplanting survival rate of tissue culture seedlings can reach more than 80%, and the rooting seedlings can be used as ginger seeds for industrialization after being expanded and propagated for 2-3 generations in field.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for propagation and industrialization of ginger virus-free seedlings.
Background
The ginger is the rhizome of a plant in the family of Zingiberaceae, belongs to a perennial herb, is suitable for growing in a mild, shady and humid environment, has various colors such as white, beige, brown, yellow, golden yellow and the like due to different varieties, is warm in nature and pungent in taste, and has high nutritional value and medicinal value. The ginger is used as a food material and a medicinal material and has a long-term origin, and the traditional Chinese medicine considers that the ginger is a good product in medicine, has the effects of relieving exterior syndrome, dispelling cold, diminishing inflammation and resisting bacteria, and can be used for treating symptoms such as cold, stomach cold and vomiting; meanwhile, the ginger has certain effects of resisting oxidation, aging and tumors and can inhibit the growth of cancer cells to a certain extent; in addition, ginger has the effects of dispelling cold, activating blood circulation and reducing blood fat, and people often use ginger wine to relieve pain of diseases such as arthritis and the like by utilizing the effects of dispelling wind cold and dredging channels and collaterals.
China is the country with the largest cultivation area and the largest total production amount of ginger in the world, and as a common seasoning plant, the ginger is planted in a wide area in China, wherein the southern China mainly plants Guangdong, Zhejiang, Hunan, Anhui, Sichuan and the like, the northern China mainly plants Shandong provinces with the largest planting area, and the planting area of the Chinese ginger in 2018 reaches 30 ten thousand hectares. China is also the first export major country of ginger in the world, ginger export is mainly based on unground ginger, wherein countries such as America, Japan, Pakistan, the Netherlands and the like are the main export countries of ginger in China, and the export quantity of the Chinese ginger (unground ginger) in 2018 reaches 48 ten thousand tons. Therefore, ginger is an important economic crop in China.
Because ginger does not bloom or blooms less, sexual reproduction cannot be carried out, and only asexual reproduction can be carried out through underground stems. Through the asexual propagation of multiple generations, viruses are easy to accumulate, which causes the adverse effects of the ginger such as the degeneration of the seed properties, the reduction of the stress resistance and the like, thereby causing the irreparable economic loss such as the yield reduction, the quality reduction and the like. Common diseases comprise ginger blast, ginger pythium root rot, ginger wilt, ginger spot, ginger anthracnose, ginger bacterial leaf blight, ginger bacterial soft rot and the like. In order to prevent the diseases, a large amount of pesticide is sprayed and irrigated to roots, which not only increases the cost, but also causes the problems of pesticide residue, environmental pollution and the like. Therefore, how to detoxify the ginger brings important significance to the comprehensive development of the ginger industry.
The tissue culture of ginger utilizes totipotency of plants, and sterile tissue culture seedlings are obtained through the stages of dedifferentiation, redifferentiation, adventitious bud elongation, rooting and the like. The tissue culture seedling of ginger not only can realize detoxification and recovery of the species property of ginger, but also has the excellent characteristics of no limitation of seasons and places, and the like. However, the existing ginger tissue culture technology has the defects of easy fungus generation, low propagation efficiency, poor growth vigor after multiple subcultures and the like, and cannot be applied in a large area.
Disclosure of Invention
Aiming at the defects of the conventional ginger tissue culture technology, the invention optimizes the selection of the explant, the explant disinfection condition, the culture medium hormone condition, the seedling hardening mode and the like, establishes a ginger virus-free seedling regeneration propagation method which is sterile, has high propagation coefficient, can be subcultured for multiple times when growing normally and has high survival rate, and develops a set of ginger virus-free seedling industrialization process.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) selecting materials: selecting ginger blocks which are fat, full, bright in skin color, fresh in meat quality, not dry, not shrunk, not rotten, not frozen and hard in texture;
(2) accelerating germination: placing the selected ginger tubers in a non-illumination room, maintaining the temperature at 25-30 ℃ and the humidity at 40-50%, accelerating germination for 25-35 days, and cutting when the ginger buds grow to 1-2 cm;
(3) detoxification regeneration and subculture: washing the ginger bud explant obtained in the step (2) with tap water, removing the outermost epidermis, treating with 75% alcohol for 1min, treating with 0.1% mercuric chloride for 10min, treating with 2.5% sodium hypochlorite for 10min, washing with sterile water for 3-5 times, stripping leaf primordium, placing on an induction culture medium, dedifferentiating, differentiating, rooting and seedling formation, culturing under the conditions of 25-28 ℃, illuminating for 16h every day, illuminating at 2500 minus one thousand of Lux, culturing for 130 days for acclimation, culturing for 60-90 days for repeated subculture of the differentiated bud, wherein the subculture period is 40-50 days.
(4) Domesticating and hardening seedlings: and (3) taking the ginger tissue culture seedlings obtained in the step (3) out of the culture medium, cleaning, putting the cleaned ginger tissue culture seedlings in 1/4MS culture solution for hardening seedlings for 1 week (covering the preservative film for moisture preservation), carrying out illumination for 16 hours every day under the culture condition of 25-28 ℃, wherein the illumination intensity is 2500-.
Wherein, as optimization, the explant morphology of the ginger bud is explored, and the dwarfness and the fatness are determined to be the best morphology for improving the budding rate.
As optimization, a ginger bud explant disinfection mode is explored, and optimal disinfection conditions for ensuring germination rate and reducing contamination rate are determined by treating with 75% alcohol for 1min, treating with 0.1% mercuric chloride for 10min, treating with 2.5% sodium hypochlorite for 10min and cleaning with sterile water for 3-5 times.
For optimization, the type of cytokinin in the induction medium was searched to determine 6-BA as the best cytokinin.
As optimization, the ratio of 6-BA and NAA is explored, and the concentration ratio mode of 6-BA: NAA of 4:1 with highest differentiation efficiency is determined.
As optimization, the formula of the induction culture medium is explored, and the formula containing 2mg6-BA, 0.5mg NAA and 8g agar per liter of MS basal culture medium is determined as the best mode.
As optimization, the culture conditions were explored, and the optimal culture conditions were determined to be 26 ℃ with 16 hours of light daily.
And optimally, exploring the hardening conditions of the tissue culture seedlings, and determining a hardening mode for improving the survival rate by taking out the tissue culture seedlings from a culture bottle, maintaining the tissue culture seedlings in 1/4MS culture solution for covering for 1 week by covering with a film, and transplanting the tissue culture seedlings into nutrient soil after 3 days of film uncovering.
And optimally, transplanting the rooted seedlings to a field for expanding propagation for 2-3 generations to serve as ginger seeds for industrialization.
The invention has the beneficial effects that:
1. determining the shape of an explant in tissue culture, disinfection conditions, culture medium components and concentrations for controlling differentiation, subculture growth, rooting and the like, an optimal seedling hardening mode and an optimal industrial process;
2. the optimized ginger virus-free seedlings need 3.5-4.5 months from explants to TC0 rooting seedlings, then each successive generation needs about 1.5 months, the contamination rate in the whole process is lower than 5%, the TC0 budding rate can reach 800% at most, the successive generation differentiation efficiency can reach 300% at most, the rooting rate can reach 100%, the transplanting survival rate of tissue culture seedlings can reach more than 80%, and the rooting seedlings can be used as ginger seeds for industrialization after being expanded and propagated for 2-3 generations in field.
Drawings
FIG. 1 is a diagram of ginger explants in different states after peeling of ginger seeds
FIG. 2 is a diagram of germination of ginger explants cultured by induction
FIG. 3 is a graph of the germination induction of TC0 ginger under different induction medium conditions
FIG. 4 is a graph of the germination induction of TC1 ginger under different induction medium conditions
FIG. 5 is a flow chart of propagation and industrialization of virus-free seedlings of ginger
Detailed description of the invention
Example 1 selection of ginger explants
Selecting ginger blocks which are fat, full, bright in skin color, fresh in meat quality, not dry, not shrunk, not rotten, not frozen and hard in texture, accelerating germination for about 30 days at the temperature of 28 ℃ and the humidity of 45%, and cutting off the ginger blocks for later use when the ginger buds grow to tender buds of 1-2 cm.
The different shapes of the ginger explants directly influence the germination rate and the growth vigor, and the traditional method does not strictly select the explants, so that the germination rate and the growth vigor of the progeny ginger virus-free seedlings are different. This study explored the selection of ginger explants. The specific method comprises the following steps:
(1) selecting the cultured rhizoma Zingiberis recens bud, treating with 75% alcohol for 1min, treating with 0.1% mercuric chloride for 10min, washing with sterile water for 3-5 times, and stripping off leaf primordium to obtain 20 rhizoma Zingiberis recens bud explants (shown in figure 1) in different states;
(2) placing the explants in different states on an induction culture medium (MS basal medium, TDZ2.0mg/L, NAA0.5mg/L, 8g agar) respectively, and culturing under the condition of illumination for 16h at 26 ℃;
(3) the germination rate was counted and showed the highest germination rate and best germination state for the C state explants (stunted) compared to the a state explants (stunted) and the B state explants (elongated) (fig. 2, table 1).
TABLE 1 explant budding Rate statistics for different states
| Explant status | Bud ratio |
| A | 15％(3/20) |
| B | 80％(16/20) |
| C | 100％(20/20) |
Example 2 ginger explant Sterilization Condition optimization
The ginger explants carry various bacteria and fungi, and incomplete disinfection can reduce the overall germination rate of the ginger explants and influence the growth of offspring. In the traditional method, the pollution rate is high due to the small using amount of the disinfectant, and the germination rate is low due to the high using amount of the disinfectant. Different disinfectant combinations are used in the research, different disinfection conditions are optimized, and a disinfection method with low pollution rate and high germination rate is determined.
(1) Stripping ginger buds, cleaning with tap water, removing the outermost epidermis, and sterilizing in a superclean bench;
(2) the different sterilization conditions were as follows:
the first disinfection mode is as follows: treating with 75% alcohol for 1min, treating with 0.1% mercuric chloride for 10min, and washing with sterile water for 3-5 times;
and (2) a second disinfection mode: treating with 75% alcohol for 1min, mercuric chloride 0.1% for 10min, sodium hypochlorite 2.5% for 10min, and washing with sterile water for 3-5 times;
and (3) a third disinfection mode: treating with 75% alcohol for 1min, treating with 2.5% sodium hypochlorite for 10min, and washing with sterile water for 3-5 times;
and (4) a fourth disinfection mode: treating with 75% alcohol for 1min, treating with 10% povidone iodine for 10min, and washing with sterile water for 3-5 times;
(3) after the sterilization treatment, the leaf primordium was peeled off, to obtain 80 explants in C state (see FIG. 1), which were divided into 4 groups on average, and then placed in an induction medium (MS basal medium, TDZ2.0mg/L, NAA0.5mg/L, 8g agar) under the condition of illumination at 26 ℃ for 16 h;
(4) and (5) counting the germination rate and the bacterial contamination rate. The results show that the second disinfection mode (1 min of 75% alcohol treatment, 10min of mercuric chloride treatment with 0.1%, 10min of 2.5% sodium hypochlorite treatment, 3-5 times of sterile water cleaning) is the best disinfection mode (as shown in Table 2).
TABLE 2 statistics of the contamination rate and germination rate for different disinfection modes
| Mode of disinfection | Bacterial contamination ratio (%) | Bud ratio |
| Disinfection mode one | 10％(2/20) | 100％(20/20) |
| Disinfection mode two | 5％(1/20) | 100％(20/20) |
| Disinfection mode three | 25％(5/20) | 100％(20/20) |
| Fourth mode of disinfection | 20％(4/20) | 100％(20/20) |
Example 3 selection of hormone types in Induction Medium
The ginger explant budding rate and the progeny differentiation rate are influenced by the variety and concentration of hormones, and the research screens the variety of cytokinins.
(1) Stripping ginger buds, cleaning with tap water, removing outermost epidermis, sterilizing in a superclean bench (sterilization mode II), and stripping leaf primordium to obtain 60C-state explants (shown in figure 1) which are divided into 3 groups on average;
(2) preparing the following induction culture media of different cytokinins and carrying out induction treatment on the explant;
a first culture medium: MS culture medium +2mg/LTDZ +0.5mg/LNAA +8g agar;
and (2) culture medium II: MS culture medium +2mg/LKT +0.5mg/LNAA +8g agar;
and (3) culture medium III: MS culture medium +2mg/L6-BA +0.5mg/LNAA +8g agar;
(3) the culture conditions were 26 ℃ for 16h under illumination, and the germination rate and rooting rate were counted, and the results showed that 6-BA in Medium III was the best cytokinin (as shown in Table 3).
TABLE 3 statistics of different cytokinins on germination and rooting rates
| Culture medium formula | Bud ratio | Rooting rate |
| Culture medium one | 100％(20/20) | 70％(14/20) |
| Culture medium II | 160％(32/20) | 78％(25/32) |
| Culture medium III | 300％(60/20) | 100％(60/60) |
Example 4 optimization of hormone concentration and formulation in Induction Medium
The present study continued to optimize the 6-BA and NAA ratios.
(1) Stripping ginger buds, cleaning with tap water, removing outermost epidermis, sterilizing in a superclean bench (sterilization mode II), and stripping leaf primordium to obtain 60C-state explants (shown in figure 1) which are divided into 3 groups on average;
(2) preparing culture media with different conditions, wherein the specific culture formula is as follows:
and (4) culture medium IV: MS culture medium +1.8mg/L6-BA +0.4mg/LNAA +8g agar;
and (5) culture medium V: MS culture medium +2mg/L6-BA +0.5mg/LNAA +8g agar;
and (5) culture medium six: MS culture medium +2.2mg/L6-BA +0.6mg/LNAA +8g agar;
(3) respectively carrying out induction and subculture on each group of C-state explants (shown in figure 1) by using the culture medium, and counting the germination rate, differentiation efficiency and rooting rate of TC0 and TC1 generations;
(4) the results showed that the induction medium was medium six (MS medium +2.2mg/L6-BA +0.6mg/L NAA +8g agar) with the highest differentiation efficiency and the best growth vigor (see FIG. 3, FIG. 4, Table 4).
TABLE 4 ratio of germination and rooting rates for different hormone ratios
| Culture medium formula | TC0 germination rate | TC0 rooting rate | TC1 differentiation Rate | TC1 rooting rate |
| Culture medium four | 180％(36/20) | 100％(36/36) | 111％(40/36) | 100％(40/40) |
| Culture medium five | 300％(60/20) | 100％(60/60) | 183％(110/60) | 100％(110/110) |
| Six culture mediums | 800％(160/20) | 100％(160/160) | 300％(480/160) | 100％(480/480) |
Example 5 optimization of ginger detoxification tissue culture seedling hardening conditions
In the traditional method, the rooted ginger tissue culture seedlings are directly taken out of the culture medium, cleaned, placed in clear water for culturing for several days and then transplanted. The method has low survival rate of the ginger tissue culture seedlings and weak cultivation time when the ginger tissue culture seedlings are transferred into soil. The study optimizes the seedling hardening conditions of the ginger tissue culture seedlings.
(1) Taking the rooted ginger tissue culture seedlings out of the culture medium, cleaning, dividing each 100 ginger tissue culture seedlings into one group, and respectively putting the groups into clear water or 1/4MS culture solution for hardening seedlings for 1 week (covering preservative film for moisture preservation);
(2) removing the preservative film for 3 days, transplanting into nutrient soil (nutrient soil/matrix is 2:1), and culturing under 26 deg.C for 16 h;
(3) and (5) counting the survival rate. The survival rate of the tissue culture seedlings obtained by exercising the seedlings in 1/4MS culture solution can reach more than 80 percent (see table 5).
TABLE 5 survival rate statistics for different seedling exercising methods
| Seedling exercising mode | Average survival rate |
| Cleaning the rooted seedlings, and putting the cleaned rooted seedlings into clean water for 1 week | 49％(49/100) |
| Cleaning the rooted seedlings, and placing the cleaned rooted seedlings in 1/4MS culture solution for 1 week | 82％(82/100) |
Example 6 ginger detoxification seedling industrialization process
The study determines the optimal regeneration and propagation method of ginger virus-free seedlings, determines the optimal explant type as C state (dwarf), the optimal disinfection method (1 min of 75% alcohol treatment, 10min of 0.1% mercuric chloride treatment, 10min of 2.5% sodium hypochlorite treatment, 3-5 times of sterile water cleaning), the optimal cell classifier as 6-BA, the optimal induction culture medium formula (MS culture medium +2.2mg/L6-BA +0.6mg/LNAA +8g agar), the optimal seedling hardening condition (ginger rooting tissue culture seedlings are placed in 1/4MS culture solution for 1 week after being cleaned from the culture medium), and the culture condition is illumination at 26 ℃ for 16 h. The optimized process of the ginger virus-free seedling requires 3.5-4.5 months from the explant to TC0 rooting seedlings, then the subculture time is about 1.5 months, the bacterial contamination rate in the whole process is lower than 5%, the TC0 budding rate can reach 800% at most, the subculture differentiation efficiency at each later time can reach 300% at most, the rooting rate can reach 100%, the transplanting survival rate of the tissue culture seedlings can reach more than 80%, and the rooting seedlings can be used as ginger seeds for industrialization after being expanded and propagated for 2-3 generations in the field (at the moment, the ginger seed state is the best).
Claims (9)
1. An optimized regeneration propagation method of ginger virus-free seedlings is characterized in that: the method comprises the following steps:
(1) selecting materials: selecting ginger blocks which are fat, full, bright in skin color, fresh in meat quality, not dry, not shrunk, not rotten, not frozen and hard in texture;
(2) accelerating germination: placing the selected ginger tubers in a non-illumination room, maintaining the temperature at 25-30 ℃ and the humidity at 40-50%, accelerating germination for 25-35 days, and cutting when the ginger buds grow to 1-2 cm;
(3) detoxification regeneration and subculture: cleaning the ginger bud explant obtained in the step (2) with tap water, removing the outermost epidermis, treating with 75% alcohol for 1min, treating with 0.1% mercuric chloride for 10min, treating with 2.5% sodium hypochlorite for 10min, cleaning with sterile water for 3-5 times, stripping leaf primordium, placing on an induction culture medium, dedifferentiating, differentiating, rooting and seedling formation, culturing under the conditions of 25-28 ℃, illuminating for 16h every day, illuminating at 2500 minus one thousand of Lux, culturing for 130 days for acclimation, culturing for 60-90 days for repeated subculture of the differentiated bud, wherein the subculture period is 40-50 days;
(4) domesticating and hardening seedlings: and (3) taking the ginger tissue culture seedlings obtained in the step (3) out of the culture medium, cleaning, putting the cleaned ginger tissue culture seedlings in 1/4MS culture solution for hardening seedlings for 1 week (covering the preservative film for moisture preservation), carrying out illumination for 16 hours every day under the culture condition of 25-28 ℃, wherein the illumination intensity is 2500-.
2. The regeneration propagation method according to claim 1, wherein the ginger bud explants in step (3) are in a dwarf form.
3. The regeneration propagation method according to claim 1, wherein the sterilization conditions in step (3) are 75% alcohol treatment for 1min, 0.1% mercuric chloride treatment for 10min, 2.5% sodium hypochlorite treatment for 10min, and sterile water washing for 3-5 times.
4. The regeneration propagation method according to claim 1, wherein the induction medium at each stage in step (3) is formulated as: MS basal medium, 2mg6-BA, 0.5mg NAA, 8g agar.
5. The regeneration and propagation method according to claim 1, wherein the cytokinin species in the induction medium is 6-BA.
6. The regeneration propagation method according to claim 1, wherein the concentration of 6-BA in the induction medium is 2mg/L, NAA and 0.5mg/L, and the concentration of agar is 8 g/L.
7. The regeneration propagation method as claimed in claim 1, wherein the culture conditions in step (3) are 25-28 ℃ and 16h light irradiation per day with illumination intensity of 2500-.
8. The regeneration propagation method according to claim 1, wherein the tissue culture plantlets in step (4) are maintained in 1/4MS culture medium at the previous stage after being taken out from the culture flask.
9. The regeneration propagation method according to claim 1, wherein the tissue culture seedling is transplanted to a field for propagation for 2-3 generations and then applied to industrialization as a ginger seed.
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