CN113796219A - Watermelon tissue culture seedling out-of-tube grafting and transplanting method - Google Patents
Watermelon tissue culture seedling out-of-tube grafting and transplanting method Download PDFInfo
- Publication number
- CN113796219A CN113796219A CN202111038083.XA CN202111038083A CN113796219A CN 113796219 A CN113796219 A CN 113796219A CN 202111038083 A CN202111038083 A CN 202111038083A CN 113796219 A CN113796219 A CN 113796219A
- Authority
- CN
- China
- Prior art keywords
- tissue culture
- grafting
- seedling
- watermelon
- seedlings
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
-
- 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
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses an external grafting and transplanting method of a watermelon tissue culture seedling test tube, belonging to the technical field of watermelon grafting. The invention discloses an out-of-tube grafting and transplanting method for watermelon tissue culture seedlings, which comprises stock preparation, scion preparation, tissue culture seedling hardening, grafting and grafting seedling transplanting. The tissue culture seedling can be used for grafting after about 1cm, and does not need to root, so that the time is saved; the method is easy to implement, simple and convenient to operate and high in survival rate, and the problem of low transplanting survival rate of the tissue culture regenerated plants of the watermelons is solved; provides a new approach for the application of grafting technology and a scientific research system for the research of grafting related theories.
Description
Technical Field
The invention relates to the technical field of watermelon grafting, in particular to an external grafting and transplanting method of a watermelon tissue culture seedling test tube.
Background
The watermelon is the fifth fruit in the world, and the cultivation area, total output and per-capita consumption of the watermelon in China all reside in the first place in the world. The research of biological engineering techniques of insect resistance, disease resistance, quality improvement and the like of watermelons needs to establish a regeneration system for rapid and efficient propagation of watermelons, cultivate a large amount of tissue culture seedlings, and induce rooting to obtain complete test tube plants. However, the transplanted tissue culture seedlings of the watermelons are not easy to survive, and have poor growth vigor, low survival rate and lower flowering and fruit setting rate.
The micro-grafting is a technology for grafting scions and stocks through tissue culture, is a combination of plant tissue culture and grafting technology, and comprises two types of grafting in vitro and grafting out of vitro. Micro-grafting has been widely applied to the aspects of rapid detection of plant viruses, propagation and preservation of precious breeding materials, removal of plant viruses, rapid propagation of industrial seedlings and the like. The micro grafting technology outside the test tube is to graft the non-toxic tissue cultured seedling as scion and the strong seedling formed by seed germination as stock in natural environment. Compared with the conventional tissue culture seedling transplanting method, the method saves the rooting culture of the tissue culture seedling and saves the time; the stock developed by the seeds can provide sufficient nutrient sources for the scions, resist diseases, promote the rapid growth of the scions, adapt to the transplanting environment quickly and have high survival rate. Although the micro-grafting technology is applied to various crops such as tomatoes, peanuts, apples and the like, no report on the application of the watermelon tissue culture seedling on the watermelon exists so far because the tissue of the watermelon tissue culture seedling is tender, the operation technical requirement is high, and the culture condition is strict.
Therefore, the technical personnel in the field need to solve the problem of providing the watermelon tissue culture seedling out-of-tube grafting and transplanting method, improving the transplanting survival rate of the tissue culture seedling and improving the growth condition of the tissue culture seedling.
Disclosure of Invention
In view of the above, the invention provides an out-of-tube grafting and transplanting method for watermelon tissue culture seedlings, which takes watermelon tissue culture seedlings as scions and wild watermelons developed from seeds as stocks and adopts a grafting method for grafting, and the method is easy to implement, simple and convenient to operate, high in survival rate and wide in application range.
In order to achieve the purpose, the invention adopts the following technical scheme:
a watermelon tissue culture seedling out-of-tube grafting and transplanting method comprises the following specific steps:
(1) stock preparation: the method comprises the following steps of (1) carrying out surface disinfection on wild watermelon seeds, sowing the seeds in a nutrition pot or a plug tray, carrying out intensive seedling culture, flattening two cotyledons, and taking the seeds as a stock when true leaves are just exposed;
(2) preparing scions: sterilizing seeds of watermelon (both cultivar and wild watermelon), performing tissue culture and rapid propagation, and performing thickening culture on tissue culture seedlings with the length of 1.0-2.0cm to serve as scions;
(3) hardening tissue culture seedlings: placing the tissue culture seedlings subjected to thickening culture in the step (2) into a greenhouse, and exposing the tissue culture seedlings in the greenhouse for hardening seedlings;
(4) grafting: cutting off a growing point and another cotyledon of the rootstock from one side of the cotyledon of the rootstock in the step (1) by a blade at an angle of 45 degrees by adopting a grafting method; taking out the domesticated tissue culture seedlings in the step (3), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; aligning the stock with the section of the scion, attaching the stock and the scion together, and fixing the stock and the scion tightly by using a grafting clamp;
(5) culturing a grafted body: culturing in a sunlight greenhouse shed after grafting, starting to grow new leaves after 10-12 days, and performing conventional management to keep the grafted seedlings to grow normally;
(6) transplanting grafted seedlings: and (3) hardening the surviving grafted seedlings in a sunlight greenhouse for 6-7 days after 20-25 days of grafting, transplanting, and managing according to a conventional cultivation mode.
Further, the step (2) of disinfecting the watermelon seeds comprises the following steps: taking full watermelon seeds, soaking the seeds in purified water at 35 ℃ for 5-6h, and slightly peeling off shells; soaking in 70% ethanol for l min, and washing with sterile water for 3 times; soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and drying with sterile filter paper.
Further, the tissue culture rapid propagation in the step (2) is carried out at the temperature of 24-26 ℃, the illumination intensity of 2500-.
Further, the specific process of performing thickening culture on the tissue culture seedling in the step (2) is as follows: 10-15 days before the micro-grafting in the step (4), opening the cover of the tissue culture bottle for the tissue culture seedling growing to 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L once aiming at the leaf blade of the tissue culture seedling, then covering the cover of the tissue culture bottle for continuous culture, and increasing the illumination intensity to 5000-.
Further, the specific process of the tissue culture seedling hardening-off in the step (3) is as follows: placing the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; and (3) acclimatizing the tissue culture seedling to natural illumination and day-night variation of temperature and humidity, and carrying out micro-grafting after 6-7 days.
Further, the specific steps of the graft culture in the step (5) are as follows: placing the grafted watermelon tissue culture seedlings obtained in the step (4) in a small shed of a sunlight greenhouse, spraying a bactericide (800 times of 50% carbendazim solution) in the small shed of the sunlight greenhouse for disinfection, covering a film tightly, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilating after 7 days of grafting, and basically healing the grafted seedling interface after 10-12 days of grafting and entering normal growth.
The invention provides a method for preserving a special breeding intermediate material of watermelons. The direct planting survival rate of tissue seedlings such as somatic cell regeneration plants, transgenic plants, stem tip culture plants and the like is low, the application of distant hybridization, biotechnology and cell engineering in the aspects of plant genetic improvement and the like is restricted, and the grafting and transplanting method can improve the condition.
According to the technical scheme, compared with the prior art, the invention discloses and provides the method for grafting and transplanting the watermelon tissue culture seedlings outside the test tube, which has the following beneficial effects:
(1) the tissue culture seedling can be grafted outside the test tube after about 1cm, and does not need to take root, thereby saving time and omitting the process of regenerating the root system of the tissue culture seedling;
(2) the grafting method is simple, the contact area is large, the survival rate after transplanting in the field is high, the survival rate of tissue culture seedling transplanting is greatly improved, and labor and time are saved;
(3) the method has wide application, can preserve special breeding intermediate materials of the watermelon, can also carry out germplasm resource innovation on the watermelon, and can also carry out seedling production;
(4) provides a new approach for the application of grafting technology and a scientific research system for the research of grafting related basic theories.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A watermelon tissue culture seedling out-of-tube grafting and transplanting method comprises the following specific steps:
(1) cleaning the vessels (plug tray and nutrition bowl) for seedling and cultivation, sterilizing the substrate (grass peat and vermiculite), and fumigating the greenhouse with bactericide and insecticide.
(2) 4 wild watermelon stocks were selected for testing and 3 and 6 varieties of cucurbita pepo were used as controls (table 1), and the seeds were sterilized in 1.5% NaClO solution for 20min each with agitation once.
(3) After disinfection, the mixture is washed with clean water and kneaded until the water becomes colorless and transparent.
(4) Soaking the disinfected seeds in warm water at 50-60 ℃ for 6 hours, wrapping the seeds with gauze or a wet towel, accelerating germination in a constant temperature box at 27 ℃, and germinating for 24-30 hours.
(5) Picking out the seeds with uniform germination potential for sowing: and filling a seedling raising hole disc with a substrate (peat: vermiculite: 1), watering thoroughly, sowing single seeds, covering with a mulching film, preserving heat and moisture, controlling the temperature to be 24-30 ℃ in the daytime and the temperature to be about 18 ℃ at night after seedling emergence, and preventing vain growth. Two cotyledons are flat, and the cotyledons can be used as stocks for grafting when the true leaves are just exposed.
(6) Preparing scions: soaking filled 'Zaojia 8424' watermelon seeds in purified water at 35 ℃ for 5-6h, then slightly peeling off shells, soaking in 70% alcohol for l min, washing with sterile water for 3 times, then soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and then absorbing water on the surfaces of the seeds by sterile filter paper. Then, the tissue culture is carried out under the conditions of 24-26 ℃, illumination intensity of 2500-.
(7) 10-15 days before grafting, opening the cover of the tissue culture bottle for the tissue culture seedling with the length of 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L once aiming at the leaf blade of the tissue culture seedling, then covering the cover of the tissue culture bottle for continuous culture, and increasing the illumination intensity by one time, namely the illumination intensity is 5000-6000 lux, and performing thickening culture to serve as a scion.
(8) Hardening tissue culture seedlings: placing the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; the tissue culture seedling is acclimatized to natural illumination and day and night change of temperature and humidity, and micro-grafting is carried out after 6-7 days.
(9) Grafting: adopting a grafting method, firstly cutting off a growing point and another leaf from one side of a rootstock cotyledon by a blade at an angle of 45 degrees; taking out the domesticated tissue culture seedlings in the step (8), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; the stock is aligned with the section of the scion and is attached together, and the stock is fixed tightly by a grafting clamp, so that the grafted watermelon tissue culture seedling is obtained.
(10) Culturing a grafted body: placing the grafted watermelon tissue culture seedlings in a sunlight greenhouse shed, spraying 800 times of 50% carbendazim solution in the sunlight greenhouse shed for disinfection, covering the film tightly, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilation can be carried out after 7 days of grafting, and grafting seedling joints are basically healed after 10-12 days of grafting, and the seedlings grow normally. And after 20-25 days, counting the survival rate. The results are shown in Table 1.
TABLE 1 influence of different stocks on the survival rate of watermelon tissue culture seedlings grafted outside test tubes
The results in table 1 show that the compatibility and survival rate of wild watermelons are higher than those of pumpkin and bottle gourd stocks, and in 4 wild watermelon varieties, the stem thickness of the stocks is related to the size of seeds, the seed grain is large, the stem thickness is large, the seed grain is small, the stem thickness is small, and when the stocks are grafted with varieties with small stem thickness of the stocks, the ratio of the joint area is large, the survival rate is high, so that the wild watermelon stocks with small seed grains are suitable to be selected.
(11) After about 25 days of grafting, the surviving grafted seedlings are hardened in a sunlight greenhouse for 6 to 7 days, can be transplanted and are managed according to a conventional cultivation mode.
Example 2
A watermelon tissue culture seedling out-of-tube grafting and transplanting method comprises the following specific steps:
(1) cleaning the vessels (plug tray and nutrition bowl) for seedling and cultivation, sterilizing the substrate (grass peat and vermiculite, and fumigating the greenhouse with bactericide and insecticide).
(2) Wild watermelon T1 was selected as a stock and sterilized in 1.5% NaClO solution for 20min with agitation once.
(3) After disinfection, the mixture is washed with clean water and kneaded until the water becomes colorless and transparent.
(4) Soaking the sterilized seeds in warm water of 50-60 deg.C for 6h, wrapping with gauze or wet towel, and germinating in a thermotank of 27 deg.C for 24-30 h.
(5) Picking out the seeds with uniform germination potential for sowing: filling a seedling raising hole disc with a substrate (peat: vermiculite: 1), watering thoroughly, sowing single seeds, covering with a mulching film, keeping the temperature and moisture, controlling the temperature to be 24-30 ℃ in the daytime and 18 ℃ at night after seedling emergence, and preventing vain growth. Two cotyledons are flat, and the cotyledons can be used as stocks for grafting when the true leaves are just exposed.
(6) Preparing scions: soaking filled 'Zaojia 8424' watermelon seeds in purified water at 35 ℃ for 5-6h, then slightly peeling off shells, soaking in 70% alcohol for l min, washing with sterile water for 3 times, then soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and then absorbing water on the surfaces of the seeds by sterile filter paper. Then, the tissue culture is carried out under the conditions of 24-26 ℃, illumination intensity of 2500-.
(7) 10-15 days before grafting, opening the cover of a tissue culture bottle for tissue culture seedlings growing to 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L (wherein, spraying 200, 300 and 500mg/L of mepiquat chloride is not sprayed as a contrast) once aiming at the leaves of the tissue culture seedlings, then covering the cover of the tissue culture bottle for continuous culture, and doubling the illumination intensity, namely, the illumination intensity is 4000 lux, for thickening culture to be used as scions.
(8) Hardening off the tissue culture seedlings, namely putting the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; the tissue culture seedling is acclimatized to natural illumination and day and night change of temperature and humidity, and micro-grafting is carried out after 6-7 days.
(9) Grafting: adopting a grafting method, firstly cutting off a growing point and another leaf from one side of a rootstock cotyledon by a blade at an angle of 45 degrees; taking out the domesticated tissue culture seedlings in the step (8), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; the stock is aligned with the section of the scion, is attached together and is fixed tightly by a grafting clamp. And obtaining the grafted watermelon tissue culture seedling.
(10) Culturing a grafted body: placing the grafted watermelon tissue culture seedling in a sunlight greenhouse shed, spraying 800 times of 50% carbendazim solution in the sunlight greenhouse shed for disinfection, covering the film tightly, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilation can be carried out 7 days after grafting, and grafting seedling joints are basically healed and grow normally 10-12 days later. And after 20-25 days, counting the survival rate. The results are shown in Table 2.
TABLE 2 influence of mepiquat chloride of different concentrations on the stem thickness of tissue culture seedling of watermelon and the survival rate of grafting
The results in Table 2 show that the grafting survival rate is highest by adopting the mepiquat chloride with the concentration of 400 mg/L.
(11) After about 25 days of grafting, the surviving grafted seedlings are hardened in a sunlight greenhouse for 6 to 7 days, can be transplanted and are managed according to a conventional cultivation mode.
Example 3
A watermelon tissue culture seedling out-of-tube grafting and transplanting method comprises the following specific steps:
(1) cleaning the vessels (plug tray and nutrition bowl) for seedling and cultivation, sterilizing the substrates (grass peat and vermiculite), and fumigating the greenhouse with bactericide and insecticide;
(2) selecting wild watermelon T1 as a stock, and sterilizing in 1.5% NaClO solution for 20min, wherein the stock is stirred once;
(3) after disinfection, washing with clear water and kneading until the water becomes colorless and transparent;
(4) soaking the sterilized seeds in warm water at 50-60 deg.C for 6h, wrapping with gauze or wet towel, and germinating in a thermotank at 27 deg.C for 24-30 h;
(5) picking out the seeds with uniform germination potential for sowing: filling a seedling raising hole disc with a substrate (peat: vermiculite: 1), watering thoroughly, sowing single seeds, covering with a mulching film, keeping the temperature and moisture, controlling the temperature to be 24-30 ℃ in the daytime and 18 ℃ at night after seedling emergence, and preventing vain growth. Two cotyledons are flat, and can be used for grafting when the true leaves are just exposed.
(6) Preparing scions: soaking filled 'Zaojia 8424' watermelon seeds in purified water at 35 ℃ for 5-6h, then slightly peeling off shells, soaking in 70% alcohol for l min, washing with sterile water for 3 times, then soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and then absorbing water on the surfaces of the seeds by sterile filter paper. Then, the tissue culture and rapid propagation are carried out under the conditions of 24-26 ℃, illumination intensity of 2500-.
(7) 10-15 days before grafting, opening the cover of a tissue culture bottle for tissue culture seedlings growing to 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L once aiming at the leaves of the tissue culture seedlings, then covering the cover of the tissue culture bottle for continuous culture, and increasing the illumination intensity by one time, namely the illumination intensity is 5000-plus 6000 lux, and performing thickening culture to serve as scions.
(8) Hardening off the tissue culture seedlings, namely putting the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; and (3) hardening the tissue culture seedlings for 6 days (wherein hardening seedlings are 0, 3 and 9 as controls) after the tissue culture seedlings adapt to natural illumination and day-night variation of temperature and humidity, and then grafting.
(9) Grafting: adopting a grafting method, firstly cutting off a growing point and another leaf from one side of a rootstock cotyledon by a blade at an angle of 45 degrees; taking out the domesticated tissue culture seedlings in the step (8), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; the stock is aligned with the section of the scion and is attached together, and the stock is fixed tightly by a grafting clamp, so that the grafted watermelon tissue culture seedling is obtained.
(10) Culturing a grafted body: placing the grafted watermelon tissue culture seedling in a sunlight greenhouse shed, spraying 800 times of 50% carbendazim solution in the sunlight greenhouse shed for disinfection, covering the film tightly, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilation can be carried out 7 days after grafting, and grafting seedling joints are basically healed and grow normally 10-12 days later. After 20-25 days, the survival rate was counted, and the results are shown in Table 3.
TABLE 3 influence of days for hardening tissue culture seedlings on survival rate of grafting in vitro
The results in Table 3 show that the grafting survival rate is highest after the seedling is hardened for 6 to 7 days by adopting the method.
(11) After about 25 days of grafting, the surviving grafted seedlings are hardened in a sunlight greenhouse for 6 to 7 days, can be transplanted and are managed according to a conventional cultivation mode.
Example 4
A watermelon tissue culture seedling out-of-tube grafting and transplanting method comprises the following specific steps:
(1) cleaning the vessels (plug tray and nutrition bowl) for seedling and cultivation, sterilizing the substrate (grass peat and vermiculite), and fumigating the greenhouse with bactericide and insecticide.
(2) Wild watermelon T1 was selected as a stock and sterilized in 1.5% NaClO solution for 20min with agitation once.
(3) After disinfection, the mixture is washed with clean water and kneaded until the water becomes colorless and transparent.
(4) Soaking the disinfected seeds in warm water at 50-60 ℃ for 6 hours, wrapping the seeds with gauze or a wet towel, accelerating germination in a constant temperature box at 27 ℃, and germinating for 24-30 hours.
(5) Picking out the seeds with uniform germination potential for sowing: and filling a seedling raising hole disc with a substrate (peat: vermiculite: 1), watering thoroughly, sowing single seeds, covering with a mulching film, preserving heat and moisture, controlling the temperature to be 24-30 ℃ in the daytime and the temperature to be about 18 ℃ at night after seedling emergence, and preventing vain growth. Two cotyledons are flat, and can be used for grafting when the true leaves are just exposed.
(6) Preparing scions: soaking filled 'Zaojia 8424' watermelon seeds in purified water at 35 ℃ for 5-6h, then slightly peeling off shells, soaking in 70% alcohol for l min, washing with sterile water for 3 times, then soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and then absorbing water on the surfaces of the seeds by sterile filter paper. Then, the tissue culture is carried out under the conditions of 24-26 ℃, illumination intensity of 2500-.
(7) 10-15 days before grafting, opening the cover of a tissue culture bottle for tissue culture seedlings growing to 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L once aiming at the leaves of the tissue culture seedlings, then covering the cover of the tissue culture bottle for continuous culture, and increasing the illumination intensity by one time, namely the illumination intensity is 5000-plus 6000 lux, and performing thickening culture to serve as scions.
(8) Hardening tissue culture seedlings: placing the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; the tissue culture seedling is acclimatized to natural illumination and day and night change of temperature and humidity, and is grafted after 6 days of seedling acclimatization.
(9) Grafting: the method of grafting (in which grafting and cleft grafting are controls) is used.
Pasting and connecting: firstly, cutting off a growing point and another leaf from one side of a rootstock cotyledon by a blade at an angle of 45 degrees; taking out the domesticated tissue culture seedlings in the step (8), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; the stock is aligned with the section of the scion and is attached together, and the stock is fixed tightly by a grafting clamp, so that the grafted watermelon tissue culture seedling is obtained.
Cleft grafting: firstly cutting off main leaves and a central growing point of the stock, only leaving two cotyledons, splitting the two cotyledons from the middle of the two cotyledons, cutting the bottom of the scion into a wedge shape, inserting the wedge shape into the cut of the stock, and finally clamping the wedge shape by a plastic clamp.
Top plug-in method: when grafting, the stock needs to be larger than the scion, a first true leaf and a growing point of the stock are firstly cut off, then a grafting needle with the thickness close to that of a hypocotyl of the scion is inserted at one side of a cotyledon of the stock, a stem of the scion is cut into a corresponding inclined plane, and finally the inserted scion is inserted on the stock and is clamped by a plastic clamp.
(10) Culturing the grafted body, placing the grafted watermelon tissue culture seedling in a sunlight greenhouse shed, spraying 800 times of 50% carbendazim solution in the sunlight greenhouse shed for disinfection, covering the film tightly, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilation can be carried out 7 days after grafting, and grafting seedling joints are basically healed and grow normally 10-12 days later. After 20-25 days, the survival rate was counted, and the results are shown in Table 4.
TABLE 4 influence of different grafting methods on the survival rate of watermelon tissue culture seedlings grafted outside the test tube
The results in Table 4 show that the method for grafting the watermelon tissue culture seedlings has the highest survival rate, is probably related to the larger joint surface of grafting and is more suitable for grafting the watermelon tissue culture seedlings in vitro.
(11) And (3) hardening the survived grafted seedlings in a sunlight greenhouse for 6-7 days after about 25 days of grafting, and managing the seedlings in a movable greenhouse or a greenhouse according to a conventional cultivation mode.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A watermelon tissue culture seedling out-of-tube grafting and transplanting method is characterized by comprising the following specific steps:
(1) stock preparation: the method comprises the following steps of (1) carrying out surface disinfection on wild watermelon seeds, sowing the seeds in a nutrition pot or a plug tray, carrying out intensive seedling culture, flattening two cotyledons, and taking the seeds as a stock when true leaves are just exposed;
(2) preparing scions: sterilizing watermelon seeds, carrying out tissue culture and rapid propagation, and carrying out thickening culture on tissue culture seedlings with the length of 1.0-2.0cm to serve as scions;
(3) hardening tissue culture seedlings: placing the tissue culture seedlings subjected to thickening culture in the step (2) into a greenhouse, and exposing the tissue culture seedlings in the greenhouse for hardening seedlings;
(4) grafting: cutting off a growing point and another cotyledon of the rootstock from one side of the cotyledon of the rootstock in the step (1) by a blade at an angle of 45 degrees by adopting a grafting method; taking out the domesticated tissue culture seedlings in the step (3), and obliquely cutting stems into corresponding inclined planes by using a grafting knife; aligning the stock with the section of the scion, attaching the stock and the scion together, and fixing the stock and the scion tightly by using a grafting clamp;
(5) culturing a grafted body: culturing in a sunlight greenhouse shed after grafting, starting to grow new leaves after 10-12 days, and performing conventional management to keep the grafted seedlings to grow normally;
(6) transplanting grafted seedlings: and (3) hardening the surviving grafted seedlings in a sunlight greenhouse for 6-7 days after 20-25 days of grafting, transplanting, and managing according to a conventional cultivation mode.
2. The method for the out-of-tube grafting and transplanting of the watermelon tissue culture seedling according to claim 1, wherein the step (2) of disinfecting the watermelon seed comprises the following steps: taking full watermelon seeds, soaking the seeds in purified water at 35 ℃ for 5-6h, and slightly peeling off shells; soaking lmin in 70% alcohol, and washing with sterile water for 3 times; soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 3 times, and drying with sterile filter paper.
3. The method for grafting and transplanting the watermelon tissue culture seedling outside the test tube as claimed in claim 2, wherein the tissue culture rapid propagation in the step (2) is performed under the conditions of 24-26 ℃, illumination intensity of 2500-.
4. The method for grafting and transplanting the watermelon tissue culture seedling outside the test tube according to claim 3, wherein the specific process of thickening and culturing the tissue culture seedling in the step (2) is as follows: 10-15 days before the micro-grafting in the step (4), opening the cover of the tissue culture bottle for the tissue culture seedling growing to 1.0-2.0cm, spraying mepiquat chloride with the concentration of 400mg/L once aiming at the leaf blade of the tissue culture seedling, then covering the cover of the tissue culture bottle for continuous culture, and increasing the illumination intensity to 5000-.
5. The method for transplanting the watermelon tissue culture seedling out of the test tube for grafting according to claim 4, wherein the specific process of hardening the tissue culture seedling in the step (3) is as follows: placing the tissue culture seedlings after the thickening culture into a greenhouse, opening a cover of a tissue culture bottle, and enabling the tissue culture seedlings to be in the conditions of natural illumination and the temperature and humidity of the greenhouse, wherein the daytime temperature is 23-28 ℃, the nighttime temperature is 18-20 ℃, and the humidity is 55-65%; and (3) acclimatizing the tissue culture seedling to natural illumination and day-night variation of temperature and humidity, and carrying out micro-grafting after 6-7 days.
6. The method for the out-of-tube grafting and transplanting of the watermelon tissue culture seedling according to claim 5, wherein the specific steps of the grafting body culture in the step (5) are as follows: placing the grafted watermelon tissue culture seedlings obtained in the step (4) in a small shed of a sunlight greenhouse, spraying a bactericide in the small shed of the sunlight greenhouse for disinfection, tightly covering a film, covering a sunshade net, and keeping the temperature at 25-28 ℃ and the humidity at more than 95%; removing shading objects in the morning and at the evening after grafting for 5 days, gradually increasing transparency according to seedling conditions, prolonging light transmission time, and keeping shading after 10 days; ventilating after 7 days of grafting, and basically healing the grafted seedling interface after 10-12 days of grafting and entering normal growth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111038083.XA CN113796219A (en) | 2021-09-06 | 2021-09-06 | Watermelon tissue culture seedling out-of-tube grafting and transplanting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111038083.XA CN113796219A (en) | 2021-09-06 | 2021-09-06 | Watermelon tissue culture seedling out-of-tube grafting and transplanting method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113796219A true CN113796219A (en) | 2021-12-17 |
Family
ID=78894765
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111038083.XA Pending CN113796219A (en) | 2021-09-06 | 2021-09-06 | Watermelon tissue culture seedling out-of-tube grafting and transplanting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113796219A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103782811A (en) * | 2014-02-26 | 2014-05-14 | 武汉市农业科学研究所 | Watermelon tissue culture seedling test tube micro-grafting method |
| CN103782686A (en) * | 2014-02-26 | 2014-05-14 | 武汉市农业科学研究所 | Watermelon tissue culture seedling grafting method |
| CN106358770A (en) * | 2016-08-31 | 2017-02-01 | 宿州市埇桥区九里梦种植专业合作社 | Method for raising watermelon seedlings by grafting |
| CN107509536A (en) * | 2017-10-18 | 2017-12-26 | 广州市农业科学研究院 | A kind of outer micro-grafting method of test tube of balsam pear tissue-cultured seedling |
| CN110178564A (en) * | 2019-07-04 | 2019-08-30 | 周志疆 | A kind of method of Tissue-cultured Tomato Plants micrografting |
-
2021
- 2021-09-06 CN CN202111038083.XA patent/CN113796219A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103782811A (en) * | 2014-02-26 | 2014-05-14 | 武汉市农业科学研究所 | Watermelon tissue culture seedling test tube micro-grafting method |
| CN103782686A (en) * | 2014-02-26 | 2014-05-14 | 武汉市农业科学研究所 | Watermelon tissue culture seedling grafting method |
| CN106358770A (en) * | 2016-08-31 | 2017-02-01 | 宿州市埇桥区九里梦种植专业合作社 | Method for raising watermelon seedlings by grafting |
| CN107509536A (en) * | 2017-10-18 | 2017-12-26 | 广州市农业科学研究院 | A kind of outer micro-grafting method of test tube of balsam pear tissue-cultured seedling |
| CN110178564A (en) * | 2019-07-04 | 2019-08-30 | 周志疆 | A kind of method of Tissue-cultured Tomato Plants micrografting |
Non-Patent Citations (2)
| Title |
|---|
| 王振龙主编: "《植物组织培养》", 30 June 2007, 北京：中国农业大学出版社 * |
| 陈云香编著: "《西瓜施肥新技术》", 30 April 1996, 北京：中国农业出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102119660B (en) | Method for rooting culture,seedling adaptation and transplantation of alpine rose tissue-cultured seedling in greenhouse | |
| CN108157180B (en) | Open type factory rapid propagation method for potato virus-free seedlings | |
| CN112586346B (en) | Tree eggplant, and cultivation method, rapid propagation method and application thereof | |
| CN110192524A (en) | A method of using base of leaf and peduncle kryptoblast as the in vitro fast breeding of the zingiberaceous plant of explant | |
| CN107926715A (en) | A kind of eggplant or/and the engrafting and cultivating method of capsicum or/and tomato | |
| JPH10191776A (en) | Vegetative propagation method of camphor-wood by cuttage | |
| CN107197746B (en) | Breeding method of cunninghamia lanceolata field excellent resources | |
| CN109042330A (en) | A kind of method for tissue culture of spindle tree | |
| CN111034617B (en) | Method for breeding tea seedlings by culturing young embryo tissues of Yunnan large-leaf tea trees | |
| KR100868523B1 (en) | Mass propagation method through in vitro aseptic germination of Calanthe spp. | |
| CN109863997A (en) | A kind of method for tissue culture covering mulberry seedling | |
| CN108450332A (en) | A kind of Chinese toon stem section method for tissue culture | |
| KR101064947B1 (en) | The mass producing method of regenerated plant from the leaf segment of calanthe discolor | |
| CN108849500A (en) | The culture medium and method of a kind of rescue of lotus embryo and the development of offspring's fast-growth | |
| CN111656978B (en) | Cutting propagation method of distant hybrid summer wintersweet | |
| CN114568307A (en) | Method for rapidly breeding seedlings by utilizing alpinia katsumadai stem tips | |
| JPS6258934A (en) | Mass propagation of potato by tissue culture | |
| CN113973673A (en) | Method for propagating amorphophallus bulbifer seedlings | |
| CN113796219A (en) | Watermelon tissue culture seedling out-of-tube grafting and transplanting method | |
| CN112293252A (en) | Artificial efficient clonal propagation method of dendrobium santalinum | |
| CN112930899A (en) | Grafting seedling method for efficiently utilizing melon stocks | |
| JPS6371121A (en) | Mass production of yam brood bud by tissue culture | |
| CN112616671A (en) | Method for obtaining industrial hemp single plant tissue culture seedlings | |
| CN111448985A (en) | Tissue culture method of rosa tenuifolia | |
| KR100322351B1 (en) | A method for producing seedlings of Acanthopanax koreanum NAKAI by tissue culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211217 |
|
| RJ01 | Rejection of invention patent application after publication |