CN104719163A - Method for increasing acclimatization planting percent of blueberry tissue culture test-tube seedlings - Google Patents

Abstract

The invention discloses a method for improving the seedling rate of blueberry tissue culture test-tube seedlings, which comprises the steps of adding basic medium to the cultivation of strong seedlings in tomato bottles, the cultivation of strong seedlings in miniature stems of robust tissue cultured indoors and high-density rooting outside of bottles, and the cultivation of micro Hardening and transplanting of tissue culture rooted seedlings. The added tomato culture medium stabilizes its low-pH growth environment, and the seedlings cultivated are more robust than the seedlings of the prior art; the outdoor rooting is changed to indoor rooting outside the bottle, and the operation method is simple and stable, and the rooting stability reduces the difficulty of management; the matrix material adopts perlite , light in weight, easy to carry, extremely efficient in rooting and transplanting, and easy to obtain materials. The blueberry micro-rooted seedlings obtained by this technical scheme have an effective seedling quantity of 2.5 to 3 times that of the original technology (adopting complete seedlings), and the number of root systems of each seedling reaches 10. The seedlings are strong and strong, and the rooting rate reaches more than 95%. Seedling raising cost when blueberry group cultured seedlings rooted and transplanted.

Description

A method for improving the seedling rate of blueberry tissue culture test-tube plantlets

technical field

The invention belongs to the technical field of tissue culture and breeding of plant seedlings, and in particular relates to a method for improving the seedling rate of blueberry tissue culture test tube seedlings.

Background technique

Blueberry is known as "the king of antioxidants, the fruit of color contact lenses", and it has more and more health care value for modern urbanites who live a fast-paced life. my country's blueberry industry has begun to take shape after nearly 15 years of rapid development, but the area and output are far from meeting the needs of the domestic market. At present, the production of domestic blueberry seedlings mainly relies on tissue culture technology, and its technical difficulties are concentrated in the links of strong seedlings in the bottle, rooting outside the bottle, hardening and transplanting. The "three lows" problems (hereinafter referred to as "three lows" problems) such as low reproduction coefficient, low rooting rate, and low seedling transplanting operation efficiency (hereinafter referred to as "three lows") generally exist, which greatly increases the cost of cultivating blueberry seedlings and the price of seedlings, thereby restricting blueberry production. industry development.

Contents of the invention

The invention aims at integrating and innovating a method for improving the seedling rate of blueberry tissue culture test-tube seedling hardening and systematically solving the "three lows" problems existing in the prior art.

The technical solution adopted in the present invention is, a method for improving the seedling rate of blueberry tissue culture test-tube plantlet hardening, carrying out primary culture and subculture proliferation cultivation under aseptic conditions, and obtaining aseptic subculture seedlings after culturing On top of that, do the following:

Step 1, adopt the WPM medium that adds tomato to subculture seedling to carry out strong seedling culture, obtain robust bottle seedling;

Step 2, carry out the high-density rooting culture of the miniature stem section outside the indoor bottle to the strong bottle seedlings, and prepare the miniature rooting seedlings;

Step 3, carry out outdoor seedling hardening and transplanting management to the micro-rooted seedlings, and cultivate standard tissue culture container seedlings.

The present invention is also characterized in that,

In step 1, adopt the WPM substratum that adds tomato to secondary seedling to carry out strong seedling culture and specifically implement according to the following steps:

Step 1.1. Inoculate the subcultured seedlings on the blueberry subculture medium. The formula of the blueberry subculture medium is WPM+ZTR 1mg/L+tomato 5g/L. There is no need to adjust the pH value, and transfer once. Among them, blueberry subculture The base is the WPM medium added with tomatoes;

Step 1.2, adjust the formula of blueberry subculture medium to WPM+ZTR 0.5mg/L+tomato 10g/L, no need to adjust the pH value, and obtain a sufficient number of robust bottle seedlings after multiple transfers. It is the WPM medium added with tomatoes.

For the secondary seedlings, use the WPM medium added with tomatoes to carry out strong seedling cultivation operations. Cut the stem segment with 1 to 2 single buds and inoculate the medium. tree.

In the step 2, carry out the high-density rooting culture of the miniature stem section outside the indoor bottle to the strong bottle seedling and specifically implement according to the following steps:

Step 2.1, cutting and processing of blueberry strong seedling stems: open the bottle cap in the air, use tweezers to clamp out the test tube seedlings, cut off the part with the medium at the base, and then cut into stems with 2 buds, use Collect it in a clean container, soak it in the hormone solution for 3 seconds and remove it;

Step 2.2, cutting of the stem section: the rooting matrix is placed in a plastic lunch box, and the blueberry miniature rooting stem section prepared in step 2.1 is placed in the plastic lunch box filled with the rooting matrix, about 100 plants per box, and the direct cutting is changed to perforated cuttings;

Step 2.3, sealing and rooting: Immediately seal it with a plastic wrap, place it at a temperature of 21° C. to 28° C. and light conditions of 2000 to 3000 lux and cultivate it for 21 days.

The preparation and sterilizing method of rooting nutrient solution in the step 2 are: the WPM substratum of preparation 1L adds water and is diluted to 5L, and the solution after dilution adjusts the pH value to 4.5, sterilizes (changing autoclave sterilization is boiling sterilization, hereinafter the same) . Cooling after sterilization to obtain the rooting nutrient solution, standby; the preparation method of the hormone solution is: take IBA or NAA 1g, dissolve with a small amount of 1N NaOH solution, settle to 1L, adjust the pH value of the diluted solution to 5.0, sterilized and cooled for later use; the preparation method of the rooting matrix is: according to the weight ratio of perlite and rooting nutrient solution of 1:5, the two are mixed to obtain the rooting matrix.

The seedling hardening and transplanting of step 3 blueberry tissue culture micro-rooted seedlings are specifically implemented according to the following steps:

Step 3.1, Seedling hardening: Under the environmental conditions of temperature 20-25°C, light 2000lux, and relative humidity 70-90%, cut a hole in the plastic wrap of the rooting seedling lunch box in step 2.3, harden the seedlings for 1-2 days, and then remove it completely Plastic wrap, water and continue to harden seedlings for 1 to 2 days;

Step 3.2, transplanting: After the seedling hardening is completed, collect the blueberry micro-rooted seedlings in a plastic box container. When transplanting, use tweezers to pick up the rooted blueberry micro-rooted seedlings and put them directly into the hole tray containing the transplanting matrix, and place them with In the seedbed of the sprinkler irrigation system, spray water from time to time to keep it moist;

Step 3.3, potting: when the new shoots of the plug seedlings grow 4-5 leaves, transplant them into the pots with the substrate. Among them, the plastic pots have a diameter of 8-10 cm, and then move them into the nursery for normal management.

The specific preparation method of the transplanting substrate is as follows: Weigh 70 parts of peat, 10 parts of perlite, 10 parts of yellow heart soil, 8 parts of rice husk, and 2 parts of controlled-release fertilizer according to the mass percentage, mix evenly, soak and disinfect, and pour water thoroughly before transplanting. .

The beneficial effects of the present invention are: the methods of strong seedlings in the bottle, high-density concentrated rooting outside the indoor bottle, outdoor hardened seedling transplanting and other methods adopted by the technical solution have improved the seedling rate and work efficiency, and the system solves the problem of blueberry industrialized group cultivation of seedlings. The "three lows" problem. Compared with existing technologies of the same kind, the advantages are: 1. step 1 subculture medium adds tomato organic matter to carry out strong seedling cultivation, which improves the robustness of test-tube seedlings, is more conducive to rooting and enhances its resistance to adverse environments; blueberry When growing naturally, they belong to plants that prefer strong acidic soil conditions. Since tomatoes are rich in organic acids, the buffering capacity of the medium added with tomatoes to maintain a low pH value is greatly enhanced, which reduces the adverse effects of the pH rise of the unadded medium in the later period, and can also Save the trouble of adjusting the pH every time the medium is prepared. ②The indoor high-density rooting technology outside the bottle used in step 2 changes the sterilization method and implements indoor operation, which is simple and convenient; in terms of environmental control, the indoor controllability is strong, and the management is convenient and intensive, thus ensuring the stability of its rooting; ③The outdoor hardening and transplanting of micro-rooted seedlings in step 3, compared with the non-rooted seedlings of the same technology, requires both adaptation to the environment and rooting when domesticated. Its adaptability to environmental changes is greatly enhanced, and the rooting rate reaches 95%, which is higher than the domestic average. Higher than more than 10 percentage points; 4. step 3 rooting section only has 2 buds, only this stage just improves more than 2 times than the seedling reproduction coefficient of prior art (5～6 buds). ⑤Perlite is used as a support for rooting, the material is easy to obtain and light in weight, and it is easy to operate when transplanting. Each person can transplant 0.8 to 10,000 plants per day. The transplanting efficiency is twice that of the existing domestic technology, which significantly reduces labor costs. .

Description of drawings

Fig. 1 is microscopic photos of rooting of miniature seedlings, wherein, a is a microscopic photo of rooting of miniature seedlings; b is a microscopic photo of rooting of miniature seedlings; c is a picture of survival after rooting; d is a picture of large-scale rooting indoors.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The invention provides a method for improving the seedling rate of blueberry tissue culture test-tube seedlings, which is specifically implemented according to the following steps: take semi-lignified or non-lignified young shoots of blueberries for aseptic inoculation, and perform primary culture and subculture multiplication culture. On the basis of obtaining the progeny seedlings, carry out the following operations:

Step 1, the strong seedling cultivation of the subcultured seedlings; the blueberry aseptic seedlings that have entered the rapid multiplication stage are carried out for strong seedling cultivation. The cultivation of strong seedlings is divided into two stages: the medium of the first stage consists of: basic medium, hormones and organic additives. The basic medium is WPM medium, the added hormone is trans-zeatin nucleoside (ZTR), the concentration is 1mg/L, the organic additive is tomato, the concentration is 5g/L, no need to adjust the pH value. In the second stage, the hormone formula of the medium was adjusted to ZTR 0.5mg/L, and the concentration of organic additive tomato was adjusted to 10g/L, without adjusting the pH value. In the first stage, transfer once, and in the second stage, transfer multiple times, and the interval between each transfer is 1 month, until a sufficient number of strong bottle seedlings are cultivated. For each transfer operation, it is required to cut the stem segment with 2 single buds. The culture container is 250ml canned bottle or potato bottle, and the number of inoculated plants in each bottle is 10-15.

Step 2. Indoor and outdoor high-density rooting culture of strong bottle seedlings, prepare two kinds of rooting solutions: rooting nutrient solution (1/5WPM nutrient solution) and hormone solution (1mg/mL IBA solution). The preparation method of 1/5WPM nutrient solution is: prepare 1L of WPM medium, add water and dilute to 5L, adjust the pH value of the diluted solution to 4.5, boil and sterilize, then cool and set aside. The preparation method of the hormone solution is as follows: weigh 1g of IBA, dissolve it with a small amount of 1N NaOH solution, dilute to 1L, adjust the pH value of the diluted solution to 5.0, boil and sterilize, then cool down for later use.

Mix clean perlite and rooting nutrient solution evenly according to the weight ratio of 1:5, put it into a disposable transparent lunch box, and the volume is 1/3 of the volume of the lunch box, and then the rooting matrix is obtained. Take out the new shoots of blueberry aseptic seedlings with good growth directly from the culture bottle without ultra-clean workbench, cut off the part with the medium at the base, and then cut it into stems with 1 to 2 buds. Soak the stems in the hormone solution for 3 seconds and remove them. First, punch holes with a pencil-thick wooden stick. Put the soaked stems directly in the holes, and cut them into the rooting matrix. One plant per hole, a total of about 100 plants. Immediately seal it with plastic wrap, place it at a temperature of 25 ± 1°C, and cultivate it for 21 days under 2000-3000lux light environment conditions; obtain high-density miniature seedlings, as shown in Figure 1, wherein a is a microscope photo of miniature seedlings taking root; b The other is microscopic photos of rooting of miniature seedlings.

Step 3, the hardening and transplanting of the blueberry micro-rooted seedlings, at a temperature of 25±1° C., with a light of 2000 lux, and a relative humidity of 70-90%, for 3-5 days of transitional hardening. Transplanting after seedling hardening, specifically: weigh 70% of peat, 15% of perlite, 8% of yellow core soil, 8% of rice husk, and 2% of controlled-release fertilizer according to the mass percentage, mix evenly, fill 50 or 50% after soaking and disinfection 72-hole plug tray, water thoroughly before transplanting for later use. When transplanting, use tweezers to pick up the rooted blueberry micro-tissue culture seedlings and cut them in the hole tray, and place them in the seedbed with a sprinkler irrigation system. After about 20 to 30 days, the new shoots of the seedlings can be transplanted into the seedbed for normal management.

When the blueberry seedlings are cultured in a test tube to a sufficient number under sterile conditions, they must be transplanted outdoors for colonization. When transplanting, because the seedlings in the test tube have grown very fast and the plants are young, they must undergo a strict and slow transition of temperature and humidity before they can adapt to the environment. external environmental conditions. The degree of growth of seedlings and whether they take root will greatly affect the survival rate of domestication.

The natural growth of blueberries is suitable for strong acidic soil conditions (pH 4.2-5.5), and the cultivation of blueberry tissue culture seedlings also requires low pH conditions to maintain the absorption of iron, etc., so the medium requires relatively acidic medium conditions. When the culture medium is sterilized and during the cultivation process, the excessive change range of the pH value condition in the matrix will affect the proliferation and growth of blueberry seedlings. Tomatoes contain acidic organic matter such as citric acid, and their buffering ability to maintain low pH is greatly enhanced. The present invention maintains low pH conditions in the medium by gradually adding organic matter in tomatoes during the subculture process of blueberry tissue culture seedlings. It stabilizes the iron and other nutrients needed by blueberries in the matrix, and at the same time, the unknown organic nutrients in tomatoes provide active nutrients for the growth of blueberries, which improves the robustness of blueberry tissue culture seedlings, thereby increasing the rooting rate of blueberry tissue culture seedlings.

Using indoor rooting technology to improve the rooting rate and reproduction coefficient, using tomato, perlite and other materials are easy to obtain, the quality is stable, and the complete seedlings are used for domestication, which improves the resistance of the seedlings to adverse environments, and solves the problem of both rooting and To domesticate the double obstacles, thereby improving the transplanting survival rate and seedling rate. It completely solved the "three lows" and other problems encountered when the blueberry group cultured seedlings were rooted outdoors when dipping the rooting powder to cut the seedlings and hardened them. It is suitable for simple factory production.

The blueberry miniature rooting seedling that makes through this technical scheme, effective seedling quantity is 2.5～3 times of original technology (adopting complete seedling), and the root system quantity of every seedling reaches 10, and seedling stock is strong and strong, and rooting rate reaches more than 95%. The cuttings are perforated cuttings, and the rooting matrix support is perlite, the root system is not sticky and the matrix is light, the operation method for rooting and transplanting is simple, the efficiency is extremely high, and the materials are easy to obtain, which greatly reduces the cost of rooting and transplanting when cultivating blueberry seedlings.

Claims (7)

1. improve a method for blueberry tissue culture test-tube plantlet hardening planting percent, it is characterized in that, aseptically carry out Initial culture and shoot proliferation and cultivate, carry out following operation again cultivating on the basis obtaining aseptic Regenerated plant:

Step 1, Regenerated plant adopted and adds the WPM medium of tomato and carry out strong seedling culture, obtain healthy and strong bottle seedling;

Step 2, healthy and strong bottle seedling carried out to the outer miniature stem section high density culture of rootage of indoor bottle, prepare miniature seedling of taking root;

Step 3, outdoor hardening and transplanting and management are carried out to miniature seedling of taking root, cultivate standard group culture container seedling.

2. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 1, is characterized in that, adopts the WPM medium adding tomato to carry out strong seedling culture specifically implement according to following steps in described step 1 to Regenerated plant:

Step 1.1, be inoculated on blueberry subculture medium by Regenerated plant, blueberry squamous subculture based formulas is WPM+ZTR 1mg/L+ tomato 5g/L, without the need to adjust ph, transfers 1 time, and wherein, blueberry subculture medium is the WPM medium adding tomato;

Step 1.2, blueberry squamous subculture based formulas is adjusted to WPM+ZTR 0.5mg/L+ tomato 10g/L, without the need to adjust ph, after repeatedly switching, obtain the healthy and strong bottle seedling of sufficient amount, wherein, blueberry subculture medium is the WPM medium adding tomato.

3. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 2, it is characterized in that, the described WPM medium to Regenerated plant employing interpolation tomato cuts in the stem section inoculation medium of band 1 ~ 2 simple bud when carrying out strong seedling culture operation, culture vessel adopts 250ml Cans or potato bottle, every bottle graft kind quantity 10 ~ 15.

4. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 2, is characterized in that, carries out the outer miniature stem section high density culture of rootage of indoor bottle specifically implement according to following steps in described step 2 to healthy and strong bottle seedling:

The clip of step 2.1, blueberry stem in strong sprout section and process: open bottle cap in atmosphere, press from both sides out test-tube plantlet with tweezers, cut off the part of base portion with medium, then be cut into the stem section of band 2 buds, collect with clean container, soak 3s at once and pull out in hormone solution;

The cuttage of step 2.2, stem section: matrix of taking root is inserted in plastics cutlery box, is placed in splendid attire by the miniature stem section of taking root of blueberry that step 2.1 is obtained and takes root in the plastics cutlery box of matrix, about the strain of every box 100, changes cuttage for punching cuttage;

Step 2.3, sealing are taken root: immediately with preservative film sealing, being placed in temperature is 21 DEG C ~ 28 DEG C, and illumination condition is cultivate 21 days under 2000 ~ 3000lux photoenvironment condition.

5. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 4, it is characterized in that, the compound method of nutrient solution of taking root in described step 2 is: the WPM medium of preparation 1L is diluted with water to 5L, solution adjust ph after dilution is to 4.5, (changing autoclaving is boiling sterilization in sterilizing, lower same), cool after sterilizing and namely obtain taking root nutrient solution, for subsequent use; The compound method of described hormone solution is: take IBA or NAA 1g, dissolves, be settled to 1L by the NaOH solution of a small amount of 1N, by the solution adjust ph to 5.0 after dilution, cools for subsequent use after sterilizing; The compound method of described matrix of taking root is: according to pearlite interstitial substance and nutrient solution part by weight 1:5 of taking root, by both mixing i.e. obtained matrix of taking root.

6. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 5, is characterized in that, hardening and the transplanting of the miniature seedling of taking root of described step 3 blueberry tissue culture are specifically implemented according to following steps:

Step 3.1, hardening: temperature 20 ~ 25 DEG C, illumination 2000lux, under relative moisture 70 ~ 90% environmental condition, one openning scratched by step 2.3 seedling cutlery box preservative film of taking root, and hardening 1 ~ 2 day, throws off preservative film afterwards completely, waters and continue hardening 1 ~ 2 day;

Step 3.2, transplanting: collect the miniature seedling of taking root of blueberry in plastic casing container after hardening completely, directly put into the cave dish of splendid attire transplanting medium with the miniature seedling of taking root of blueberry that tweezers gripping is taken root during transplanting, be placed in the seedbed with sprinkling irrigation system, moisturizing of irregularly spraying water;

Step 3.3, upper basin: until Plug seedling young sprout grow 4-5 open blade time, carrying substrates transplants upper basin, and wherein, plastic basin bore 8-10cm, namely moves into nursery and carry out normal management.

7. the method for raising blueberry tissue culture test-tube plantlet hardening planting percent according to claim 6, it is characterized in that, the concrete preparation method of described transplanting medium is as follows: take 70 parts, peat according to mass percent, perlite 10 parts, yellow soil 10 parts, 8 parts, rice husk, Controlled Release Fertilizer 2 parts is puddled evenly, soaking disinfection, waters permeable for subsequent use before transplanting.