CN113598000B - Method for one-step formation of ginger rootless tissue culture seedlings into field seedlings - Google Patents
Method for one-step formation of ginger rootless tissue culture seedlings into field seedlings Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
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- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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- A—HUMAN NECESSITIES
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- 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
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to the field of plant cultivation, and particularly discloses a method for growing a ginger rootless tissue culture seedling into a field seedling in one step. The invention simplifies the breeding steps of the ginger tissue culture seedling: the tissue culture seedling is simplified into a non-root tissue culture seedling, which is fixedly planted in a container with a substrate and forms a field seedling in a culture room by the traditional procedures of rooting in a tissue culture seedling bottle, hardening the seedling in the bottle, cleaning a root culture medium and fixedly planting in a seedling bed and strictly controlling temperature, light and water. Firstly, the production link is simplified, secondly, the production cost is reduced by 40 percent, thirdly, the seedling rate reaches more than 98 percent, and fourthly, the culture time is shortened by more than 20 days. The technical method is simple, low in production cost and high in seedling rate, and the direct economic benefit is realized in production.
Description
Technical Field
The invention belongs to the field of plant cultivation, and particularly relates to a method for one-step formation of ginger rootless tissue culture seedlings into field seedlings.
Background
In recent years, with the rapid development of science and technology, plant tissue culture technology has entered the production and application stage, and is widely applied to large-scale in vitro rapid propagation of flowers and fruit trees. The quick propagation of the plant tissue culture virus-free nursery stock can be produced all the year round under the condition of manual control, and a large amount of tissue culture virus-free nursery stock can be obtained in a short time. However, from the tissue culture rooted seedlings to the transplanting field, the environment conditions suitable for illumination, temperature, near saturation humidity and sterility in the bottle gradually change to naturally variable environment conditions from indoor to outdoor, the process has high requirements on production environment conditions, and the control difficulty of temperature, humidity, illumination and the like is high, so that the process is a bottleneck for restricting the large-scale and industrialized production of the tissue culture detoxified seedlings.
The method is a key part in the biological tissue culture technology from tissue culture bottle seedlings to field seedlings, and is directly related to whether the tissue culture seedlings can be applied to field production as soon as possible. The test-tube plantlet transplanting process is complicated, and blind transplanting or slight carelessness can cause death of a large number of tissue culture plantlets (equine novelty, Wan hong, Zhao hong Liu, Shenfei, Zhang Shu, Dujunxing. the seedling hardening technique of tissue culture plantlets Anhui agricultural science, 2000, (04):420 and 421.). In plant tissue culture, the survival rate of field seedlings is a key ring for limiting whether an industrial seedling culture production line can be successfully operated, and the survival rate directly influences the production scale of products and the seedling cost (Longbing wild goose, plum fortification, seedling hardening technology of tissue culture seedlings in the shallow theory [ J ] Anhui agronomy report, 2006 (06):82+ 47). In the production process of tissue culture seedlings, the cost of transplanting the seedlings into a field accounts for 40-60% of the total cost, how to improve the survival rate of the seedlings from the tissue culture seedlings to the field and reduce the production cost is one of the keys of whether the rapid propagation technology can be applied to production (Zhang Xiaohong, Kangbing, Caoning, etc., named as the study on the transplanting conditions of the test tube seedlings of the kiwi fruit, northwest agriculture, 2002,11(3): 83-86.).
The ginger (zingiber officinale Rosc.) tissue culture test-tube plantlet is tender, weak in root system and poor in regeneration capacity, and the difference between the microenvironment in a bottle and the environment conditions of a field before and after the test-tube plantlet is transplanted is large, so that the survival rate of the test-tube plantlet transplanted to the field is often low. The ginger tissue culture seedling becomes a field seedling, and conventionally, the four stages of rooting in a bottle, hardening off the seedling in the bottle, planting in a greenhouse and rooting into the field seedling are required.
Firstly, rooting in a bottle: cutting ginger tissue culture clumps into small seedlings, transferring the seedlings into a rooting culture medium (1/2MS + IBA0.3mg/L +1mg/L activated carbon + sucrose 20mg/L (Qiuxufeng, Yubixia, ginger tissue culture seedling production technology research, academic of Mianyang academy of academic sciences, 2020,39(05):68-71) or 1/2MS + NAA0.04mg/L (chrysanthemum Pan, Sagittaria, ginger tissue culture seedling factory production technology, Hunan agricultural machinery, 2012,39(07):231), culturing for 15-20d in the rooting culture medium, growing 4-5 white roots on the tissue culture seedlings in the culture bottle, performing seedling hardening in a secondary bottle, covering the tissue culture seedlings with the grown roots, hardening the seedlings, putting a little water into the culture bottle to prevent the tissue culture seedlings from being dehydrated and the culture medium from being polluted, moving the seedlings out of the culture chamber after 3d, and hardening the seedlings under the conditions of natural illumination and temperature of about 20-28 ℃, taking out seedling from bottle when the tissue culture seedling has 6-7 leaves and the length of the leaf is 5-8cm, cleaning culture medium, soaking root in rooting agent with certain concentration for 20-30min, and air drying. And (5) secondary greenhouse planting: the tissue culture seedlings are planted on a seedbed according to the specification of 5cm multiplied by 10cm (the seedbed is required to be 10m long, 1.2-1.3m wide and 0.05m high, perlite and humus are mixed according to the proportion of 3:1 to prepare a planting substrate, the substrate is screened and strictly disinfected by soil to be filled on the seedbed, the bed surface is kept flat and loose, and the substrate is watered thoroughly. The temperature in the shed is 20-28 ℃, the relative humidity is 80%, and the shed roof is covered with a sunshade net. Finally, rooting to form field seedlings: after 5-7 days of planting, the ginger seedling begins to grow new root, needs 30-40 days, and is watered according to soil humidity, usually 1 time every 2-3 days, and plastic film is covered to keep temperature and moisture, so that the temperature is kept at 20-28 ℃ and the humidity is above 80%. Meanwhile, attention is paid to shading to avoid strong sunlight irradiation, and the illumination is controlled to be in a natural light scattering state (ginger seedlings are favored by weak light). If high temperature and strong light weather are met, the film is uncovered, the temperature is reduced, and a sunshade net is covered for sunshade; and (2) covering a double-layer film or adopting other related measures to increase the seedbed temperature in the cooling weather (Yangyingying, Qiao Xiaofeng, Li Qinghong, Yang Yongsheng. ginger seedling tissue culture and cultivation technology [ J ]. Guizhou agricultural science, 2009,37(09): 210-.
XuKun and the like invent a ginger tissue culture test tube seedling hardening method capable of being applied to production and popularization, 2 links of hardening in bottles and hardening in an extra-bottle matrix are performed, the cap of the ginger test tube seedling is opened for 3-5d in a closed environment, then the ginger test tube seedling is taken out and planted in a prepared substrate seedbed in a hardening room, the substrate formula is grass carbon, vermiculite and clay are 1:1:1, hardening in the extra-bottle matrix is completed within 25-30d, and the seedling is transplanted to a field (XuKun, Zhengyong, a ginger tissue culture test tube seedling hardening method, an authorization notice number CN 1951177B). A method for hardening off the tissue cultured ginger seedlings includes such steps as uncovering the ginger seedlings in culture bottle to harden the ginger seedlings to 4-5 white roots, moving the culture bottle out of culture room after 5 days, hardening off the ginger seedlings for 7 days under natural light and 24-26 deg.C, taking out the ginger seedlings from the culture bottle when 5-6 leaves and 5-7cm leaves are grown, washing culture medium, transplanting it in the matrix containing pearlite and vermiculite (2: 1: 1), and conventional management of fertilizer and water until the ginger seedlings grow into field seedlings, and taking out the field seedlings (Xuzhou hua, Luoyongcheng, a method for hardening off the ginger seedlings, No. CN 104585027B). The contents of the two patents are very similar to those of Yangyu Ying et al (2009), and both patents are trained and planted in a matrix. The operation procedure is complex, 50-60 days are needed from the root growth in the bottle to the field seedling, and the whole growth environment has strict requirements on temperature, humidity and illumination.
In the conventional cultivation, the germinated seed ginger is cut into pieces for planting, and the seed ginger is 4500- 2 The consumption of the planted ginger is very large; and long-term asexual propagation causes various germs and viruses such as cucumber mosaic virus-CMV, tobacco mosaic virus-TMV and the like to be accumulated in the ginger, the accumulation of the viruses causes the seed nature degradation, the yield of the ginger is reduced by 5 to 45 percent, and simultaneously, the quality of the ginger is reduced, and the disease resistance and the stress resistance are reduced; meanwhile, ginger bacteria are planted, so that ginger blast, stem rot and the like frequently occur in the planting process. The average yield reduction of the areas affected by the ginger blast and the like reaches more than 30 percent every year, and the areas are respectivelyThe zones are even as high as 50%. The tissue culture technology can be used for obtaining the detoxified and sterile healthy seedlings. Therefore, the tissue culture technology for producing ginger seedlings is an inevitable trend for healthy development of the ginger industry. However, the current relevant data show that the ginger tissue culture seedling is still required to be subjected to the procedures of rooting, seedling hardening, field cultivation management and the like.
The invention provides a method for one-step formation of ginger rootless tissue culture seedlings into field seedlings, which aims at solving the problems, enables the rootless tissue culture seedlings to root in a relatively closed matrix with nutrient solution, gradually removes a covering material, and directly moves into a field for planting after management of dryness and wetness. The method enables the rootless tissue culture plantlets to directly take root in the transplanting matrix, simplifies the production link, reduces the production cost, shortens the growth time and improves the seedling rate. The technical method is simple, low in production cost and high in seedling rate, and the direct economic benefit is realized in production.
Disclosure of Invention
The invention aims to provide a method for one-step formation of a ginger rootless tissue culture seedling into a field seedling, which solves the problem that the ginger rootless tissue culture seedling needs complex procedures of rooting, seedling hardening, seedling washing, transplanting and the like in a bottle, can directly grow the tissue culture seedling into the field seedling, has high survival rate of the field seedling, saves cost and manpower, and is suitable for large-scale popularization.
In order to achieve the purpose, the invention adopts the following technical measures:
the method for one-step formation of the ginger rootless tissue culture seedling into the field seedling comprises the following steps:
1. preparing a substrate: mixing vermiculite and turf according to the volume ratio of 1:1-2, preparing MS +0.5-1.0mg/L NAA +0.05-0.15% (mass percent) metalaxyl hymexazol, and adding the mixture into a matrix to ensure that the humidity of the matrix is 70-90%;
2. preparing an out-of-bottle rooting facility: putting the substrate into a container to flatten the surface of the substrate;
3. preparation of rooting materials: cutting off callus at the base of the ginger subculture seedling;
4. and (3) field planting of tissue culture seedlings: planting the cut single seedlings into a container filled with a matrix, wherein the bottom of the container is provided with a water suction hole, the bottom of the container is provided with a tray, and a transparent covering is covered above the container to ensure that the seedlings are in a moisture-preserving environment;
5. and (3) management of a rooting period: after culturing for 12-15 days, growing roots at the base of the plantlet; lifting the covering; when the substrate is dry, adding 1-2cm of water into the tray, and transplanting the substrate to a field after wet culture for 15-20 days.
In the above method, preferably, the environment of the culture chamber during the culture of the tissue culture seedling is: the temperature is 20-30 deg.C, the humidity is 70-90%, and the light intensity is 25-30 μmol · m -2 ·s -1 Photoperiod 14-16 h.d -1 ；
In the method, the standard of the single-plant plantlet is as follows: the plant height is 3-5cm, and the stem thickness is more than or equal to 0.2 cm.
Compared with the conventional planting technology, the invention has the following advantages and effects:
1. the rootless tissue culture seedling is directly planted in the matrix without being inoculated into a sterilized rooting culture medium, the matrix is not sterilized at high temperature and high pressure, the steps of preparing the culture medium, sterilizing, switching an operating platform, rooting in a bottle, opening a bottle cover to harden the seedling and the like are omitted, the purpose of forming the field seedling in one step is achieved, the step of forming the tissue culture seedling into the field seedling is simplified, and the culture time of more than 20 days is shortened. Therefore, the production cost is saved by nearly 40%.
2. The bacteriostatic agent added into the matrix can inhibit the contamination of the tissue culture seedlings and has the function of enhancing the rooting capacity.
3. By optimizing each parameter, the invention greatly shortens the production period from the tissue culture seedling to the field transplantation, and the seedling rate of the rootless tissue culture seedling reaches more than 98 percent. In the traditional method, the tissue culture seedling needs to be hardened after rooting in a culture medium, and because the culture medium exists in the hardening process and the hardening process is in an open environment, part of bottle seedlings can be polluted; when the tissue culture seedling with roots is taken out of the culture medium and cleaned, part of the root system is damaged or broken, and the seedling rate is only 90% at the highest even if the field management is strict in the later period.
4. The culture room is 5 layers, the ginger rootless seedlings of the invention are planted according to the row spacing of 3cm multiplied by 5cm, the area required for producing 10000 plants is 10000 multiplied by 3 multiplied by 5-150000 cm 2 ＝15m 2 . Because the culture chamber is three-dimensional with 5-6 layers, the culture chamber is counted by 5 layersIn the case of 15/5 ═ 3m 2 (ii) a Planting in greenhouse at row interval of 8cm × 10cm, wherein the area required for producing 10000 plants is 10000 × 8 × 10 ═ 800000cm 2 ＝80m 2 (XuKun, Zhengyongqiang, a ginger tissue culture test-tube seedling hardening method, No. CN 1951177B). From the data, the space saving reaches 96.25%, and the greenhouse cultivation also needs strict control of temperature, light and water. Therefore, the invention not only saves space, has good control of environmental factors, but also greatly reduces the cost of seedling.
Drawings
FIG. 1 shows that the ginger rootless tissue culture seedlings are planted in a prepared matrix and covered with a transparent cover for culture.
FIG. 2 shows that the root of the ginger seedling grows in the matrix, and the transparent cover is removed for dry and wet rooting period management.
FIG. 3 shows the growth of a field seedling in a culture vessel by the rooting period management.
FIG. 4 shows field seedlings formed by culturing ginger rootless tissue culture seedlings.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available. In this embodiment, the example of the ginger is explained, and the method of the present invention can also be used for the one-step formation of field seedlings from rootless tissue culture seedlings of other varieties of ginger.
Example 1:
the experiment in the embodiment of the invention is carried out in an important laboratory for vegetable germplasm innovation and genetic improvement in Hubei province
The method for one-step formation of the ginger rootless tissue culture seedling into the field seedling comprises the following steps:
1. preparing a substrate: vermiculite and turf are mixed according to the volume ratio of 1:1, MS +0.5mg/LNAA + 0.1% hymexazol (mass percent) is prepared and added into the substrate, and the humidity of the substrate is 80%.
2. Preparing an out-of-bottle rooting facility: filling a stirred substrate with the thickness of 8.0-10.0cm into a rectangular flowerpot (the length is 35-50cm, and the width is 15-20cm), and flattening the surface of the substrate;
3. preparation of rooting materials: taking cluster seedlings of ginger subculture tissue culture seedlings which are propagated for more than 30 days out of the culture medium by using forceps, and cutting off base callus on an operation table (standard seedling: plant height is 3-5cm, stem thickness is more than or equal to 0.2 cm);
4. and (3) field planting of tissue culture seedlings: planting the cut single seedlings into flowerpots filled with substrates at the row spacing of 3cm multiplied by 5cm, wherein trays are arranged below the flowerpots, white plastic films or plastic covers are required to cover the mouths of the flowerpots on the flowerpots, and the seedlings are placed in a closed space in the flowerpots (figure 1);
5. and (3) management of rooting period: after culturing for 12-15 days, the base of the plantlet grows cluster roots; lifting the thin film or the plastic cover on the flowerpot; adding enough water into the tray when the substrate is dry, allowing the substrate to absorb water through the holes at the bottom of the flowerpot, and allowing the water in the tray to be 1-2cm deep, and allowing the substrate to be cultured for 18 days; the biological data of plant height, stem thickness, root number, leaf weight, stem weight, root weight, fresh weight, dry weight and the like are counted, and the seedling rate is 98 percent (figure 2).
The seedling rate is equal to the number of field seedlings/the number of tissue culture seedlings multiplied by 100 percent;
6. and (5) transplanting field management: transplanting the rooting seedling matrix into the field, and managing according to the field seedling method (fig. 3-4) (Tang scholar, Huangsheng, Jiangyume, Tang Zhongguo, Panglong, Liu autumn Han, ginger detoxification tissue culture seedling breeding technology [ J ]. southern gardening, 2014,25(03): 47-48.).
The environment of the culture room has a temperature of 20-30 deg.C and a humidity of 80%, and has a light intensity of 25 μmol/m -2 ·s -1 Photoperiod 16h d -1 . Control test 1 based on the method described above:
the substrate was replaced with a different formulation from the table below based on the above method, and the rest was the same as the above method. And performing data statistics before transplanting the field.
In table 1, the ratio of vermiculite to turf is 1:1 by volume; the ratio of perlite to vermiculite is 1: 1. Table 1 shows that different matrixes have influence on the growth of ginger
Table 1 shows that when vermiculite and turf are used as a substrate, the plant height is 9.94cm, the stem thickness is nearly 4mm, the number of roots is 5, the leaf weight is 1.377g, the stem weight is 1.926g, the root weight is 1.46g, the fresh weight is 4.1g, and the dry weight is 0.312 g. Probably, vermiculite has better air permeability and water absorption, and grass carbon is rich in organic matters and humic acid. Although there are many substrates for cultivating ginger tissue culture seedlings, for example, there are formulas of peat, vermiculite, silt, 1:1:1 (qiong xufeng, yubi xia. research on ginger tissue culture seedling production technology, academic press of mianyang faculty academy of teachers, 2020,39(05):68-71), soil to perlite ratio 2:1 (panJu, Huying. industrial production technology of ginger tissue culture seedling, Hunan agricultural machinery, 2012,39(07):231.), and Xunkun taught grass carbon, vermiculite, taught as vermiculite: 1:1 (Xunkun, Zhengyong, a ginger tissue culture test tube seedling hardening method, grant publication No. CN 1951177B). But of the 6 selected substrates of the invention, vermiculite + turf is most suitable.
based on the above method, nutrient solution and hormone (MS +0.5mg/L NAA) are replaced by different formulas in the following table, and data statistics is performed before transplanting into field.
TABLE 2 growth effects of ginger on nutrient solutions and hormones at different concentrations
Table 2 we can see that the addition of different solutions and hormones to the substrate has a significant impact on its phytological properties, as in MS +0.5mg/L NAA, except for the stem thickness of the fine spots compared to 0.5MS solution and the root weight of the light spots compared to 0.5MS +1.0mg/L NAA, the other criteria are superior to the other treatments, which may be that the rootless tissue culture seedlings need photosynthesis during their growth, which needs to provide nutrients. Therefore, water is weaker in growth as a solution than the MS solution is added in the treatment. NAA is used as a common hormone for plant rooting, but the higher the concentration is, the better the NAA concentration is, and the NAA concentration of 0.5mg/L is better than that of 1.0mg/L in the invention.
based on the method, the humidity during the preparation of the matrix is replaced by the humidity in the following table, and data statistics is performed before transplanting the matrix into a field in the same way as the method.
TABLE 3 growth Effect of varying humidity Supports on ginger
In table 3, it can be seen that the index except the stem thickness is not optimal when the humidity of the substrate is 80%, the other indexes are better, the indexes such as plant height and stem thickness are lower but the number of roots is more when the humidity is 60%, which is the principle of 'wet leaf growing and dry root growing', but the humidity is too high, and the root system is weak when the humidity reaches 90%. Therefore, a substrate humidity of 80% is most preferable in the present invention.
based on the method, 0.1% hymexazol is replaced by different bacteriostats in the following table, and the rest is the same as the method, and data statistics is carried out before transplanting the hymexazol into a field.
TABLE 4 Effect of different bacteriostats and amounts on ginger growth
The rootless ginger tissue culture seedlings are planted in the unsterilized matrix, and the culture environment is high in temperature and humidity and is closed. Under such circumstances, some microorganisms inevitably grow. In order to avoid the harm of microorganisms to ginger seedlings, a part of bacteriostatic agent is added into a matrix, and the added bacteriostatic agent can inhibit the microorganisms 100% from the growth stage of rootless tissue culture seedlings to field seedlings. However, as can be seen from table 4, as the concentration of sodium hypochlorite increases, the plant height, root number, fresh weight, etc. of the ginger seedlings decrease, and thus it is known that the bacteriostatic agent may inhibit the growth of the ginger seedlings when inhibiting microorganisms. The most suitable of the invention is 0.1 percent of the hymexazol, probably because the hymexazol not only has wide sterilization surface, but also has the function of enhancing the rooting capacity.
Claims (2)
1. The method for one-step formation of the ginger rootless tissue culture seedling into the field seedling comprises the following steps:
1) preparing a substrate: mixing vermiculite and turf according to the volume ratio of 1:1-2, preparing MS +0.5-1.0mg/L NAA +0.05-0.15% hymexazol, and adding into the matrix to ensure that the humidity is 70-90%;
2) and preparing an extravase rooting facility: putting the substrate into a container to flatten the surface of the substrate;
3) preparing a rooting material: cutting off callus at the base of the ginger subculture seedling;
4) and (3) planting the tissue culture seedlings: planting the cut single ginger plantlets into a container filled with a matrix, wherein the bottom of the container is provided with a water suction hole, the bottom of the container is provided with a tray, and a transparent covering is covered above the container to ensure that the ginger plantlets are in a moisture-preserving environment;
5) and managing the rooting period: after culturing for 12-15 days, growing roots at the base of the plantlet; lifting the covering; adding 1-2cm of water into the tray when the substrate is dry, and transplanting the substrate to a field after wet culture for 15-20 days;
the method has the advantages that the temperature is 20-30 ℃, the humidity is 70-90%, and the light intensity is 25-30 mu mol.m during the culture process -2 ·s -1 Photoperiod 14-16 h.d -1 。
2. The method of claim 1, wherein: and (3) standard of single plantlet: the plant height is 3-5cm, and the stem thickness is more than or equal to 0.2 cm.
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