CN115136805A - Double-stock micro-grafting method for grapes - Google Patents
Double-stock micro-grafting method for grapes 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
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
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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
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
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Abstract
The invention belongs to the technical field of grafting, and particularly relates to a double-stock micro-grafting method for grapes, which comprises the following steps: (1) cultivating tissue culture seedlings: after the grape explants are disinfected, inoculating the grape explants on a culture medium A for culture until complete plants grow out, and obtaining scion seedlings and stock seedlings; (2) grafting: taking one internode of each scion seedling and each stock seedling as a scion and a stock, grafting the scion and the two stocks in a cleft grafting mode, jointing the cuts of the scions with the cuts of the two stocks, and fixing the joints to obtain grafted seedlings; (3) grafting seedling culture: and inoculating the grafted seedling to a culture medium B for culture until a callus is formed at the interface, the rootstock takes root, and the scion grows to grow a new tip. The scion and the rootstock are healed into a complete plant, can normally grow and develop, and provides a new way for the propagation of the grape.
Description
Technical Field
The invention relates to the technical field of grafting, in particular to a double-stock micro-grafting method for grapes.
Background
The grape is one of the favorite fruits of people, has a very important position in the gardening industry, and the propagation method of grape grafting seedlings is green branch grafting and hard branch grafting at present. China gives priority to the green branch grafting method. When the green branch is grafted, generally when the young shoots of the stock grow to 50cm in the current year, 20-30 cm are cut and reserved, and the cleft grafting method is adopted for grafting.
The grafting can enhance the growth vigor of plants, prolong the harvesting period, improve the yield and the quality, prolong the storage time after harvesting, enhance the tolerance to low temperature or high temperature, enhance the resistance to salt and heavy metal ions, enhance the drought resistance or waterlogging resistance, improve the water utilization efficiency, defend diseases, improve the resistance of root-knot nematodes, control weeds, cultivate new plant varieties and the like. After the grafting operation is finished, a series of reactions such as nutrient transportation, signal substance exchange, cell recognition and proliferation, tissue reconstruction and the like occur between the rootstock and the scion in the healing process of the grafting interface. The selection of the stock and the scion is a key factor for ensuring the survival rate of the grafted seedling, and the difference of the affinity of the stock and the scion has important influence on the healing and the growth of new plants.
The traditional single-rootstock grafting technology can effectively improve the cold resistance, the root aphid resistance and the saline-alkali resistance of grape plants, but has limited resistance range for most rootstock varieties, higher resistance to individual adversities and poor effect on other adversities. The double-rootstock grafting is a cultivation technology for grafting one scion by two (seed) rootstocks, has the possibility of integrating the characteristics of the two (seed) rootstocks to a certain extent, and further has positive influence on enhancing the stress resistance of grafted fruits and improving the fruit quality.
The tissue culture micro-grafting technology was established in 1972 and is a vegetative propagation technology combining tissue culture and grafting. Usually, the method is divided into in-vitro grafting and out-of-vitro grafting. Grafting in a test tube is to obtain the stock and the scion through tissue culture, wherein the grafting operation is carried out under the aseptic condition, and then the stock and the scion are continuously cultured in the test tube; the scion grafted outside the test tube is subjected to tissue culture to obtain a nontoxic tissue culture seedling or a field seedling subjected to disinfection treatment, the stock is a robust seedling formed by germinating seeds, and grafting is carried out in a natural environment, but the micro-grafting propagation of grapes is not reported at present.
In the existing grape double-rootstock green branch grafting method, two rootstocks are cultured firstly, after roots and sprouts, the two rootstocks are close together, and grafting is performed after shearing, and because micro-grafting is performed in a test tube, the two rootstocks are difficult to ensure that the cutting positions are parallel and level, and grafting ports are not at the same height, the grafting can not be completed, so that the green branch grafting method is not suitable for micro-grafting.
Disclosure of Invention
In order to solve the technical problems, the invention provides a double-rootstock micro-grafting method for grapes, provides a new way for propagation of the grapes, and provides a practical basis for solving the problems of insufficient stress resistance and single resistance in the existing grape grafting.
A double-rootstock micro-grafting method for grapes comprises the following steps:
(1) Cultivating tissue culture seedlings: after the grape explants are disinfected, inoculating the grape explants on a culture medium A for culture until root systems grow below and complete plants with at least three stem sections grow above the grape explants, and obtaining scion seedlings and stock seedlings;
(2) Grafting: taking one internode of each scion seedling and each stock seedling as a scion and a stock, grafting the scion and the two stocks in a cleft grafting mode, jointing the cut of the scion and the cuts of the two stocks, and fixing the joint to obtain a grafted seedling;
(3) Culturing grafted seedlings: and (4) inoculating the grafted seedling to a culture medium B for culturing until callus is formed at the joint, the rootstock takes roots, and the scion grows to form a new shoot.
Preferably, the grape explant is a stem section with a single bud, which is cut from a tender branch or a tender branch of a hardwood water culture.
Preferably, the cabernet sauvignon tissue culture seedling is used as the scion, and the 5BB and Beada tissue culture seedling is used as the rootstock.
Preferably, the culture medium A in the step (1) is 1/2MS, and the hormones in the culture medium A are 0.2-0.5mg/LIBA and 0.2-0.5 mg/L6-BA.
Preferably, the culture conditions in the step (1) are: the temperature is 26 ℃, the illumination is 2500lux-3000lux, the illumination is 16h every day, and the darkness is 8 h.
Preferably, the grafting specifically comprises: cutting off a scion seedling and two stock seedlings on the surface close to a culture medium, namely the lower end of a tissue culture seedling, respectively taking a internode without bud leaves, cutting one stock along the middle position of the morphological upper end of the stock, cutting the other stock at the same position by the same length, cutting the scion at the middle position of the morphological lower end by the same length, then leaning the cuts of the two stock together, respectively inserting two cuts of the scion into the cuts of the two stock, and finally integrally wrapping and fixing the two connectors.
Preferably, the length of the rootstock cut is 5-8mm, and the direction is parallel to the stem section.
Preferably, the culture medium B in the step (3) is 3/4MS, and the hormones in the culture medium B are 0.5-1.0mg/L IBA and 0.5-1.0 mg/L6-BA.
Preferably, the culture conditions in step (3) are: the temperature is 26 ℃, the illumination is 2500lux-3000lux, the illumination is 16 hours per day, and the illumination is 8 hours dark.
Compared with the prior art, the invention has the beneficial effects that:
1. after the sterile tissue culture seedling is obtained, the thicker part of the lower part of the sterile tissue culture seedling is taken as a scion and a stock, then double-stock micro grafting is carried out in a cleft grafting mode, and finally a grafting port is fixed by binding tin foil paper, so that the grafting port can be tightly combined, and the scion and the stock are healed into a complete plant through the micro grafting, so that the growth and development can be normally carried out, and a new way is provided for the propagation of grapes.
2. Compared with the green branch grafting and the hard branch grafting, the micro-grafting has many advantages, is carried out in a laboratory, occupies small space, is not limited by seasons and time, and is convenient for scientific research; the grafting affinity of different combinations of the stock and the scion is researched more quickly; rapidly cultivating virus-free plants; detecting plant viruses and removing viruses and the like;
3. the stress resistance regulator can be used for enhancing the single stress resistance of grape plants, can also add different stress resistance to the grape plants, reduces the production cost, reduces the manpower consumption and improves the capacity of the plants for absorbing nutrition.
Drawings
FIG. 1 is a schematic diagram illustrating the cultivation of tissue culture seedlings according to the present invention;
FIG. 2 is a schematic view of the scion and the stock after cultivation according to the present invention;
FIG. 3 is a schematic view of the scion and the stock which are well treated before grafting;
FIG. 4 is a schematic diagram of the grafting of the double stocks of the present invention;
FIG. 5 is a schematic diagram of the completion of grafting by the grafting method of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
Example 1
(1) Tissue culture seedling cultivation
Cutting off tender branches of grape tender branches or hard branches subjected to water culture, cutting off all leaves, washing with running water for 2h, cutting into stem segments with single buds in an ultra-clean workbench by using a scalpel, and placing into a sterilized tissue culture bottle for surface disinfection.
During disinfection, the surface of the tea is disinfected for 30s by 70 percent alcohol, and the tea is washed for 3 times by sterile water; then, the mixture is disinfected with 10% sodium hypochlorite for 15min and washed with sterile water for 3 times. Absorbing water on the surface of the explant by using sterile filter paper, and shearing off the head and tail parts of the stem sections subjected to sterile browning by using sterile scissors for later use.
Sterilizing forceps, scissors and scalpel in the inoculating loop sterilizer, cooling, and inoculating. Cutting the branches into single-bud stem sections by using sterilized scissors, clamping the single-bud stem sections by using forceps, inoculating the single-bud stem sections on a 1/2MS +0.2mg/L IBA +0.2 mg/L6-BA culture medium in a sterile tissue culture bottle, covering a bottle cover, and marking the name, date and the like of materials.
Placing the inoculated material on a culture frame of a tissue culture room, and culturing under a light/dark period of 16/8h to obtain a scion seedling and a stock seedling, wherein the scion seedling and the stock seedling are shown in figure 1;
(2) Grafting
Scion seedlings and stock seedlings are cut in a super clean workbench, and are shown in figure 2; cutting a scion seedling and two stocks at the lower end close to the surface of a culture medium, namely the lower end of a tissue culture seedling, wherein a relatively thick internode is cut without bud leaves, the morphological upper end and the morphological lower end are distinguished, one stock is cut by a sterile scalpel at the middle position along the morphological upper end of the stock for 5mm, the direction of the scalpel is parallel to a stem segment and forms a straight line with the stem segment, the other stock is cut at the same position for the same length, so that the cut looks like a V-shape, and the scion is cut by the scalpel at the middle position of the morphological lower end for the same length, so that the cut looks like an inverted V-shape, as shown in figure 3; then, the two stocks are leaned together to form a VV shape, two cuts of the scion are respectively inserted into the cuts of the two stocks, namely, the 'V' is grafted to the VV, and finally, the two connectors are integrally wrapped by sterilized tin foil paper to obtain grafted seedlings, wherein the grafted seedlings are shown in figure 4;
(3) Post-grafting management
The grafted seedlings are inoculated on a 6-BA culture medium of 3/4MS +0.5mg/L IBA +0.5mg/L, as shown in figure 5, the temperature and the illumination time of a tissue culture room are controlled, and the temperature of the tissue culture room is controlled to be 26 ℃, the illumination intensity is 2500lux, the illumination intensity is 16h every day, and the illumination time is 8 h.
Example 2
The difference between the example 2 and the example 1 is that (1) the components of the culture medium in the tissue culture seedling cultivation are 1/2MS +0.5mg/L IBA +0.5 mg/L6-BA, and (3) the components of the culture medium in the management after grafting are 3/4MS +1.0mg/L IBA +1.0 mg/L6-BA, and the other steps and conditions are the same as the example 1.
Example 3
The difference between the embodiment 3 and the embodiment 1 lies in that (1) the components of the culture medium in the tissue culture seedling cultivation are 1/2MS +0.3mg/L IBA +0.3 mg/L6-BA, and (3) the components of the culture medium in the management after grafting are 3/4MS +0.8mg/L IBA +0.8 mg/L6-BA, and the other steps and conditions are the same as the embodiment 1.
Example 4
Example 4 differs from example 1 in that the length of the "V" cut is 8mm, and the other steps and conditions are the same as in example 1.
To verify the effects of the micro-grafting of the present invention on low temperature stress, drought stress and low temperature drought co-stress, the following experiments were performed:
the 5BB and Beada tissue culture seedlings are used as double stocks, and the Cabernet Sauvignon tissue culture seedlings are used as scions to be grafted according to the method. The grafted seedling is subjected to three experiments of low-temperature stress, drought stress and low-temperature drought co-stress, 10 double-stock plants with good growth vigor are selected in each experiment, meanwhile, 5BB and Bedad tissue culture seedlings are respectively used as single stocks, and cabernet sauvignon tissue culture seedlings are used as scions and are grafted according to a conventional single-stock grafting mode to serve as a control.
1.1 Low temperature stress experiment
10 grape grafted seedlings of different types are taken, after hardening, the plants are put into a constant-temperature incubator with the temperature of 0 ℃ and the relative humidity of 80% for culturing for 48 hours, mature leaves of the grape seedlings are taken for analyzing the relative conductivity and the content of malondialdehyde, and the influence of the grafting mode on the cold resistance of the grape grafted seedlings is shown in table 1.
1.2 drought stress experiments
10 grafted seedlings of different types of grapes are taken, and drought stress treatment is carried out from 0d to 7d after hardening. And 7d, taking mature leaves of different types of grafted seedlings to analyze the relative conductivity and the malondialdehyde content, wherein the influence of the grafting mode on the drought resistance of the grape grafted seedlings is shown in a table 2.
1.3 Low temperature drought Co-stress experiment
10 grafted seedlings of different types of grapes are taken, and drought stress treatment is carried out from 0d to 7d after hardening. Culturing in a constant-temperature incubator at 25 ℃ and relative humidity of 80% until the temperature reaches 0 ℃ after 6d, continuously culturing for 48h under the condition of relative humidity of 80%, taking mature leaves of different types of grafted seedlings to analyze relative conductivity and malondialdehyde content, wherein the influence of the grafting mode on the low-temperature and drought co-stress resistance of the grape grafted seedlings is shown in Table 3.
2. Method for measuring each index
2.1 index of freezing injury
Evaluation and quantity statistics are carried out according to the following 5 grades according to the injury degree:
stage 0: the leaves are normal and not damaged;
level 1: only a few leaf margins had mild shriveling wilting;
and 2, stage: less than half of the leaves die due to wilting, but the main stem is not dried;
and 3, level: more than half of the leaves die due to wilting;
4, level: all plants die;
according to the formula F (%) = ∑ (freezing damage value x number of corresponding freezing damage plants)/(freezing damage maximum value x total number of investigated plants) × 100%
2.2 relative conductivity measurement
Punching 10 small discs with a puncher with the diameter of 5mm on the blades, then placing the small discs in a 20mL centrifuge tube filled with 10mL deionized water, vacuumizing to enable the small discs to be completely immersed in liquid, covering a centrifuge tube cover, sealing with a sealing film, placing at a dark place for 24 hours at room temperature, measuring the conductivity with a daily use conductivity meter (DDS-307 type), and measuring the conductivity with an R 1 And (4) showing. Heating in 100 deg.C boiling water bath for 20min, cooling to room temperature, measuring conductivity, and measuring with R 2 Is represented by R 0 Relative conductivity was calculated as deionized water conductivity using the following formula to characterize the relative permeability of the plasma membrane.
Relative conductivity (%) = (R) 1 -R 0 )/(R 2 -R 0 )×100％
2.3 assay of malondialdehyde
Placing 0.8g of grape seedling leaf in mortar precooled by liquid nitrogen, adding a small amount of quartz sand, placing on ice bath, grinding into homogenate, adding 8.0mL of precooled 50mmol/L (pH7.8) PBS buffer (containing 2% polyvinylpyrrolidone PVP, PVP is added at present), and heating to 4 deg.C at 10000 r.min -1 Centrifuging for 20min, collecting 1mL of supernatant, adding 2mL of thiobarbituric acid into 1mL of phosphate buffer solution with pH value of 7.8 as blank reference, shaking, developing in boiling water bath for 15min, cooling, performing color comparison at wavelength of 450nm, 532nm and 600nm, and recording absorbance.
Calculation of the malondialdehyde concentration C MDA ＝6.45×(D 532 -D 600 )-0.56×D 450
MDA content [ mu mol g -1 FW]＝C MDA (μmol·L -1 ) X volume of extractive solution (mL)/fresh weight of plant tissue (g)
3. Results of the experiment
TABLE 1 Effect of grafting methods on Cold resistance of grape grafts
In a certain low temperature range, the generation and elimination of free radicals in the plant body are in a balanced state, so that membrane lipid peroxidation is avoided, but under the condition of low temperature cold injury, active oxygen free radicals are increased and can cause membrane lipid peroxidation when the active oxygen free radicals exceed a threshold value, malonaldehyde is one of the most important products of membrane lipid peroxidation, malonaldehyde is accumulated, the membrane leaks, the permeability is increased, electrolyte is leaked out, the relative conductivity of cytoplasm is increased to cause damage to a cell membrane system, and as can be seen from table 1, the freezing injury index of a grape grafted seedling obtained by the grafting method with 5BB + Betada as double stocks is 36.2 +/-1.7, the relative conductivity is 42.7 +/-1.6, and the content of malonaldehyde is 6.9 +/-0.2 mu mol g -1 FW, compared with the grape grafted seedling obtained by the grafting method of 5BB single rootstock and Beada single rootstock, the freezing injury index, the relative conductivity and the malondialdehyde content of the grape grafted seedling are superior to those of the single rootstock.
TABLE 2 Effect of grafting mode on drought resistance of grape grafted seedlings
The malondialdehyde and the relative conductivity are physiological indexes reflecting the damage degree of the plant under the stress of adverse circumstances, so that the action mechanisms of the malondialdehyde and the relative conductivity are the same no matter under the adverse circumstances such as salt, high temperature, low temperature, drought and the like, and as can be seen from table 2, the relative conductivity of the grape grafted seedling obtained by the grafting method with 5BB + Betada as the double rootstock is 49.2 +/-1.1, and the content of the malondialdehyde is 7.8 +/-0.3 mu mol g -1 Compared with grape grafted seedlings obtained by grafting methods of 5BB single stocks and Beada single stocks, FW has better relative conductivity and malondialdehyde content than single stocks.
Table 3 results of the Effect of grafting methods on Low temperature and drought Co-stress resistance of grape grafted seedlings
As can be seen from Table 3, the relative conductivity of the grape grafted seedling obtained by the grafting method using 5BB + Betada as the double rootstock is 53.6 +/-2.6, and the malonaldehyde content is 10.7 +/-0.2 mu mol.g -1 Compared with grape grafted seedlings obtained by grafting methods of 5BB single stocks and Beada single stocks, FW has better relative conductivity and malondialdehyde content than single stocks.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A double-rootstock micro-grafting method for grapes is characterized by comprising the following steps:
(1) Cultivating tissue culture seedlings: after the grape explants are disinfected, inoculating the grape explants on a culture medium A for culture until root systems grow below and complete plants with at least three stem sections grow above, and obtaining scion seedlings and rootstock seedlings;
(2) Grafting: taking one internode of each scion seedling and each stock seedling as a scion and a stock, grafting the scion and the two stocks in a cleft grafting mode, jointing the cuts of the scions with the cuts of the two stocks, and fixing the joints to obtain grafted seedlings;
(3) And (3) grafting seedling culture: and (4) inoculating the grafted seedling to a culture medium B for culturing until callus is formed at the joint, the rootstock takes roots, and the scion grows to form a new shoot.
2. The method for double-rootstock micro-grafting of the grapes according to claim 1, wherein the grape explants are stem sections with single buds which are cut from grape tender branches or tender branches of hard branch water culture.
3. The method for micro-grafting a grape double-rootstock according to claim 1, wherein a Cabernet Sauvignon tissue culture seedling is used as the scion, and a 5BB and Beada tissue culture seedling is used as the rootstock.
4. The method for double-rootstock micro-grafting of grapes according to claim 1, wherein the culture medium A in the step (1) is 1/2MS, and hormones contained in the culture medium A are IBA of 0.2-0.5mg/L and 6-BA of 0.2-0.5 mg/L.
5. The method for double-rootstock micro-grafting of grapes according to claim 4, wherein the culture conditions in the step (1) are as follows: the temperature is 26 ℃, the illumination is 2500lux-3000lux, the illumination is 16h every day, and the darkness is 8 h.
6. The grape double-rootstock micro-grafting method according to claim 1, wherein the grafting is specifically as follows: cutting off scion seedlings and two stock seedlings on the surface close to a culture medium, namely the lower end of a tissue culture seedling, respectively taking a internode without bud leaves, cutting one stock along the middle position of the morphological upper end of the stock, cutting the other stock at the same position by the same length, cutting the scion at the middle position of the morphological lower end by the same length, then leaning the cuts of the two stock together, respectively inserting two cuts of the scion into the cuts of the two stock, and finally integrally wrapping and fixing the two connectors.
7. The method for double-rootstock micro-grafting of grapes according to claim 6, wherein the length of the rootstock cut is 5-8mm, and the direction is parallel to the stem section.
8. The method for the double-rootstock micro-grafting of grapes according to claim 1, wherein the culture medium B in the step (3) is 3/4MS, and the hormones contained in the culture medium B are 0.5-1.0mg/LIBA and 0.5-1.0 mg/L6-BA.
9. The method for double-rootstock micro-grafting of grapes according to claim 8, wherein the culture conditions in the step (3) are as follows: the temperature is 26 ℃, the illumination is 2500lux-3000lux, the illumination is 16h every day, and the darkness is 8 h.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN88102063A (en) * | 1987-04-08 | 1988-10-26 | 葡萄香槟开发集团 | The method of producing wooden or half Woody plant nursery stock is to obtain nursery stock particularly grape seedlings and the nursery stock that obtained |
| JPH10262452A (en) * | 1997-03-28 | 1998-10-06 | Nippon Derumonte Kk | Single stem training graft nursery plant of tomato |
| CN103918485A (en) * | 2014-04-22 | 2014-07-16 | 四川农业大学 | Multi-stock grafting method for watermelon plant |
| CN108848994A (en) * | 2018-04-18 | 2018-11-23 | 宁夏林业研究院股份有限公司 | A kind of method of suitable grape micrografting seedling Liquid Culture |
| CN113115654A (en) * | 2019-12-30 | 2021-07-16 | 西北农林科技大学 | Grape double-stock green branch grafting method |
-
2022
- 2022-08-09 CN CN202210950394.1A patent/CN115136805A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN88102063A (en) * | 1987-04-08 | 1988-10-26 | 葡萄香槟开发集团 | The method of producing wooden or half Woody plant nursery stock is to obtain nursery stock particularly grape seedlings and the nursery stock that obtained |
| JPH10262452A (en) * | 1997-03-28 | 1998-10-06 | Nippon Derumonte Kk | Single stem training graft nursery plant of tomato |
| CN103918485A (en) * | 2014-04-22 | 2014-07-16 | 四川农业大学 | Multi-stock grafting method for watermelon plant |
| CN108848994A (en) * | 2018-04-18 | 2018-11-23 | 宁夏林业研究院股份有限公司 | A kind of method of suitable grape micrografting seedling Liquid Culture |
| CN113115654A (en) * | 2019-12-30 | 2021-07-16 | 西北农林科技大学 | Grape double-stock green branch grafting method |
Non-Patent Citations (1)
| Title |
|---|
| 李雪雪等: "响应面法优化葡萄微嫁接组培快繁体系", 《湖北农业科学》, vol. 58, no. 9, pages 69 - 74 * |
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