CN108243756B - Micro-grafting method for grafting and rooting outside apple tissue culture seedling bottle - Google Patents
Micro-grafting method for grafting and rooting outside apple tissue culture seedling bottle Download PDFInfo
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Abstract
The invention provides a micro-grafting method for ex-bottle grafting rooting of tissue culture seedlings of apples, which comprises subculture of tissue culture seedlings of apples, rooting preparation, rooting of apple seedlings, management of rooting period, grafting of apple seedlings and management of grafting culture period. The invention simulates the grafting process of apple branches, leads the scion to be fully combined with the stock, takes root in a relatively closed matrix with proper humidity, then gradually removes the plastic film, carries out domestication of the grafted seedlings and directly enters the matrix cultivation stage. The method realizes the out-of-bottle grafting of the tissue culture apple seedlings, ensures high stability of the grafted seedlings, saves a large amount of manpower and material resources, simplifies production links and reduces production cost. The invention has simple technical method, low production cost and high success rate, and can embody direct economic benefit in production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a micro-grafting method for grafting and rooting outside an apple tissue culture seedling bottle.
Background
The tissue culture micro-grafting technology is created by Murashige, and is a technology for combining tissue culture and grafting rootstocks and scion tissue culture seedlings of different varieties under the condition of tissue culture. Because of the advantages of less required test materials, land conservation, convenient research and the like, the method is widely applied to the research and production of various fruit trees. However, the tissue culture seedlings are repeatedly transferred and cultured in special culture media under aseptic conditions, so that the production cost is high, the operation is difficult, the speed is relatively slow, and the large-scale and industrial production of the tissue culture grafted seedlings is restricted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a micro-grafting method for grafting and rooting outside a bottle of tissue culture seedlings of apples. The invention simulates the grafting process of mature fruit trees, leads the scion to be fully combined with the stock, takes root and grafts in a closed matrix with high humidity, then gradually removes the plastic film and directly enters a matrix cultivation stage.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a micro-grafting method for grafting and rooting outside an apple tissue culture seedling bottle, which comprises the following steps:
1) subculturing the tissue culture seedlings of the apples: cutting stem of tissue culture seedling of fructus Mali Pumilae into 0.5-1cm under aseptic condition, radially inserting into subculture medium, and subculturing;
2) rooting preparation: mixing the rooting substrate with water, and filling the rooting substrate with the humidity adjusted to 60% into a rooting container, wherein the thickness of the substrate is the same as the height of the rooting container;
3) apple seedling rooting: selecting the apple tissue culture seedling which grows vigorously by subculture, removing callus and bottom leaves, inserting stems of the apple tissue culture seedling into the rooting matrix, spraying water after the insertion is finished, and sealing a container;
4) and (3) management of a rooting period: regularly checking the humidity of the matrix, keeping the humidity at 50% -70%, and performing rooting culture;
5) apple seedling grafting: selecting rooted apple seedlings as stocks, cutting off leaves at the top ends, reserving stems with the root upwards length of 2.5cm-3cm, radially cutting the stems from the cut, cutting scions into wedges, inserting the wedges into the stocks, and winding and fixing the scions by using aluminum foil paper; spraying water on the surface, and sealing;
6) and (3) management in a grafting culture period: and regularly checking the matrix, keeping the humidity at 50% -60%, and grafting and culturing to obtain the grafted and rooted apple tissue culture seedling.
Further: the subculture medium in the step 1) consists of 6-BA with the final concentration of 0.5mg/L, IAA with the final concentration of 0.2mg/L, MS medium, coagulant, sucrose and water, and the pH value of the medium is adjusted to 5.8.
Further: the coagulants are agar, and the final concentration of the coagulants in the culture medium is 7.5 g/L.
Further: the condition of the subculture in the step 1) is 24 ℃, the photoperiod is 16/8h, and the subculture lasts 40 days.
Further: the rooting substrate in the step 2) is a mixture of turf, vermiculite and perlite in a ratio of 6:2: 2.
Further: the rooting culture in the step 4) is carried out under the conditions that the photoperiod is 12h/12h of illumination/darkness, the temperature is 22 ℃, the light intensity is 2000lx, and the culture lasts for 40 days.
Further: the grafting mode in the step 5) is as follows: cutting 0.4-0.5cm from the cut in radial direction; selecting the scions which grow vigorously and are consistent, reserving stems and partial leaves with the top ends of 1.5-2cm, cutting the base parts of the scions into wedges, inserting the tail ends of the scions into the rootstocks, and binding the interface parts by using aluminum foil paper.
Further: the conditions of grafting culture in the step 6) are 24 ℃, the photoperiod is 16/8h, the culture is carried out for 45 days, the illumination intensity is 2000lx, and the culture is carried out for 45 days.
Compared with the prior art, the invention has the advantages that: the invention simulates the grafting process of mature fruit trees, leads the scion to be fully combined with the stock, takes root and grafts in a closed matrix with high humidity, then gradually removes the plastic film and directly enters a matrix cultivation stage. The method realizes the out-of-bottle grafting of the tissue culture apple seedlings, ensures high stability of the grafted seedlings, saves a large amount of manpower and material resources, simplifies production links and reduces production cost. The technical method is simple, low in production cost and high in success rate, and represents direct economic benefit in production.
Drawings
FIG. 1 is a first diagram of apple seedling growth after completion of the grafting operation in examples 1-3, wherein the left side of FIG. 1 is apple seedling after completion of the grafting operation in example 2; the middle is apple seedlings after grafting culture for 45 days in example 3; on the right side are apple seedlings that were completed by grafting after 40 days of rooting culture in example 1.
FIG. 2 is a second graph of apple seedling growth after completion of the grafting operation in examples 1-3, wherein the left side of FIG. 2 is the grafted apple rooted seedling after 45 days of rooting grafting culture in example 2; the middle part is the grafted apple rooted seedling after rooting culture for 45 days in the embodiment 3; the grafted rooted apple seedlings after 45 days of grafting culture in example 1 are shown on the right.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The method for rooting and grafting the tissue culture apple seedlings outside the bottle comprises six steps of subculture of the tissue culture apple seedlings, rooting preparation, rooting of the apple seedlings, management of a rooting period, grafting of the apple seedlings and management of a grafting culture period, and specifically comprises the following steps:
1. subculturing the tissue culture seedlings of the apples: under aseptic condition, cutting stem of the cultured apple tissue culture seedling for 30-35 days into 0.5-1cm, radially inserting into subculture medium, and subculturing for 40 days at 24 deg.C under light cycle of 16/8 h;
the subculture medium consists of 6-BA with the final concentration of 0.5mg/L, IAA with the final concentration of 0.2mg/L, MS medium, coagulant, sucrose and water, and the pH value of the medium is adjusted to 5.8. The coagulants are agar, and the final concentration of the coagulants in the culture medium is 7.5 g/L.
2. Rooting preparation: selecting a flat-bottom tray container with the height of 6cm, placing domesticated rooting containers (4cm multiplied by 4cm, 6 multiplied by 12, including 72 in total) in the flat-bottom tray container, mixing rooting matrix (the rooting matrix is a mixture formed by grass carbon, vermiculite and perlite in a ratio of 6:2: 2) with water, adjusting the humidity of the mixture to 60%, and filling the wet rooting matrix into the rooting container, wherein the thickness of the matrix is the same as the height of the rooting container.
3. Apple seedling rooting: selecting subcultured vigorous apple tissue culture seedlings, removing callus and bottom leaves, inserting stems into the rooting matrix, pressing the surrounding matrix, spraying water after the whole disc is inserted, and covering a sealed container to keep the humidity of the container at 65%.
4. And (3) management of a rooting period: the substrate is checked regularly, the humidity is kept at 50% -70%, and rooting culture is carried out for 40 days. The conditions of rooting culture are that the photoperiod is 12h/12h of light/dark, the temperature is 22 ℃, and the light intensity is 2000 lx.
5. Apple seedling grafting: selecting rooted apple seedlings (Malus hupehensis Rehd) as stocks, cutting off top leaves, reserving stems with the root part of 2.5-3cm upwards, and radially cutting off 0.4-0.5cm from the cut; selecting Gala with vigorous and consistent growth as a scion, reserving a stem with the top end of 1.5-2cm and partial leaves, cutting the base part of the stem into a wedge shape, inserting the tail end of the scion into a stock, and binding the interface part by using aluminum foil paper. Spraying water on the surface, and sealing with a cover.
6. And (3) management in a grafting culture period: the substrate is inspected regularly, maintaining a humidity of 50-70%. The grafting culture conditions are 24 ℃, the light cycle is 16/8h, the culture is carried out for 45 days, and the illumination intensity is 2000 lx.
Example 2
The method for simultaneously grafting and rooting outside the apple tissue culture seedling bottle comprises four steps of subculture of the apple tissue culture seedling, preparation of grafting and rooting, grafting and rooting of the apple seedling and management of the grafting and rooting period, and specifically comprises the following steps:
1. subculturing the tissue culture seedlings of the apples: under the aseptic condition, cutting the stem of the tissue culture seedling of the apple which is cultured for 30-35 days into 0.5-1cm, radially inserting the stem into a subculture medium, and subculturing for 40 days;
2. grafting and rooting preparation: selecting a flat-bottom tray container with the height of 6cm, placing domesticated rooting containers (4cm multiplied by 4cm, 6 multiplied by 12, including 72 in total) in the flat-bottom tray container, mixing rooting matrix (same as example 1) with water, filling the wet rooting matrix into the rooting container, wherein the thickness of the matrix is the same as the height of the rooting container.
3. Grafting and rooting of apple seedlings: selecting rooted apple seedlings (Malus hupehensis Rehd) as stocks, cutting off top leaves, reserving stems with the root part of 2.5-3cm upwards, and radially cutting off 0.4-0.5cm from the cut; selecting Gala with vigorous and consistent growth as a scion, reserving a stem with the top end of 1.5-2cm and partial leaves, cutting the base part of the stem into a wedge shape, inserting the tail end of the scion into a stock, and binding the interface part by using aluminum foil paper. Spraying water on the surface, and sealing with a cover.
4. And (3) management of a grafting rooting culture period: the substrate is periodically checked, the humidity is kept at 50% -70%, and the culture is carried out for 45 days.
Example 3
The method for grafting the apple tissue culture seedling outside the bottle before rooting comprises six steps of subculture of the apple tissue culture seedling, grafting of the apple seedling, management of a grafting culture period, rooting preparation of the grafted seedling, rooting of the grafted seedling and management of the rooting culture period, and specifically comprises the following steps:
1. subculturing the tissue culture seedlings of the apples: under the aseptic condition, cutting the stem of the tissue culture seedling of the apple which is cultured for 30-35 days into 0.5-1cm, radially inserting the stem into a subculture medium, and subculturing for 40 days;
2. apple seedling grafting: selecting rooted apple seedlings (Malus hupehensis Rehd) as stocks, cutting off top leaves, reserving stems with the root upwards of 2cm, and radially cutting off 0.4-0.5cm from the cut; selecting Gala with vigorous and consistent growth as a scion, reserving a stem with the top end of 1.5cm and partial leaves, cutting the base part of the stem into a wedge shape, inserting the tail end of the scion into a stock, and binding the interface part by using aluminum foil paper.
3. And (3) management in a grafting culture period: the grafted seedling is cultured in the grafting culture medium. The grafting culture medium comprises MS, BA1.0mg/L, NAA0.05mg/L, a coagulant and sucrose, wherein the coagulant is agar, the final concentration of the coagulant in the culture medium is 7.5g/L, and the concentration of the sucrose is 30 g/L. The grafting culture conditions are 24 ℃, the light cycle is 16/8h, the culture is carried out for 45 days, and the illumination intensity is 2000 lx.
4. Rooting preparation of grafted seedlings: selecting a flat-bottom tray container with the height of 6cm, placing domesticated rooting containers (4cm × 4cm, 6 × 12, including 72) in the flat-bottom tray container, mixing a rooting matrix (Denmark PINDSRUP) with water, and filling the wet rooting matrix into the rooting container, wherein the thickness of the matrix is the same as the height of the rooting container;
5. rooting of the grafted seedling: selecting a grafting apple seedling with vigorous growth, removing the callus and partial leaves at the bottom, inserting the stem into a substrate rooting substrate (same as example 1), pressing the surrounding substrate, spraying water after the whole disc is inserted, and covering a sealing container to keep a certain humidity.
6. And (3) management of a rooting period: the matrix was checked periodically to prevent it from drying out. Rooting and culturing for 40 days. The MS culture medium components are as follows:
| macroelements | Final concentration in the culture medium (g.L)-1) |
| NH4NO3 | 1.65 |
| KH2PO4 | 1.9 |
| MgSO4.7H2O | 0.170.37 |
| Trace elements | Concentration in culture Medium (mg. L)-1) |
| FeSO4.7H2O | 27.8 |
| Na2EDTA | 37.3 |
| MnSO4.4H2O | 22.3 |
| ZnSO4.4H2O | 8.6 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| Na2MoO4.2H2O | 0.25 |
| CuSO4.5H2O | 0.025 |
| CoCl2.6H2O | 0.025 |
| Organic component | Concentration in culture Medium (mg. L)-1) |
| Glycine | 2.0 |
| Thiamine hydrochloride | 0.4 |
| Pyridoxine hydrochloride | 0.5 |
| Nicotinic acid | 0.5 |
| Muscle | 100 |
The experimental results are as follows:
table 1: examples 1-3 Effect of different treatments on survival Rate, root Length and root count of apple grafted seedlings
| Number of processes | Number of repetitions | Survival rate/% | Root length/cm | Root number of root | |
| Example 1 | 50 | 3 | 73.3 | 5.34±1.34 | 5.73±2.08 |
| Example 2 | 50 | 3 | 13.8 | 2.34±0.42 | 2.47±1.93 |
| Example 3 | 50 | 3 | 76.5 | 4.87±0.83 | 5.28±1.86 |
Table 2: examples 1-3 Effect of different treatments on root weight, leaf weight and cycle of apple graft seedlings
| Number of processes | Number of repetitions | Root weight/g | Leaf weight/g | |
| Example 1 | 50 | 3 | 17.37±5.32 | 29.15±4.06 |
| Example 2 | 50 | 3 | 6.82±2.61 | 18.43±1.58 |
| Example 3 | 50 | 3 | 11.96±2.74 | 20.87±1.65 |
Note: the period refers to the time from the subculture of the apple tissue culture seedling to the survival of the grafting.
Table 3: examples 1-3 Effect of different treatments on photosynthesis Rate and chlorophyll SPAD of apple grafts
| Number of processes | Number of repetitions | Photosynthetic Rate/μmol. m | Chlorophyll SPAD | |
| Example 1 | 50 | 3 | 13.28±2.47 | 43.79±3.57 |
| Example 2 | 50 | 3 | 7.02±1.73 | 31.04±2.86 |
| Example 3 | 50 | 3 | 8.21±1.94 | 30.85±2.93 |
Table 4: influence of different culture media on survival rate, root length and rooting number of apple grafted seedlings
| Culture medium | Number of processes | Number of repetitions | Survival rate/% | Root length/cm | Root number of root |
| Sand | 50 | 3 | 37.3 | 1.63±1.05 | 2.75±2.19 |
| Clay | 50 | 3 | 15.6 | 0.19±0.03 | 1.21±0.74 |
| Seedling raising substrate | 50 | 3 | 86.2 | 5.64±1.58 | 5.37±3.61 |
As shown in table 1-table 4 and fig. 1-2, the survival rate of the first rooting and then grafting method in example 1 is higher than that of the other two methods, the stress resistance of the rooted apple seedlings is stronger, the apple seedlings are easy to adapt to the change of rooting and grafting, and the first rooting and then grafting method is superior in both the average rooting length and the average rooting number of single plants. In example 2, the survival rate is low in the mode of grafting and rooting and the operation is minimum in the aspects of average rooting length, average root number per plant, root weight, leaf weight, photosynthetic rate and the like, apple seedlings are moved into a matrix from a culture medium, most of the apple seedlings are firstly withered by rootstocks, and therefore the scions are insufficient in nutrition and water supply and then die. The survival rate of the mode of rooting after grafting in the example 3 is not much different from that of the example 1, but the mode is weaker than that of the example 1 in the aspects of average rooting length, average root number of single plants, root weight, leaf weight and the like, and the grafting operation in the example 3 is carried out under aseptic conditions, so that the difficulty is higher than that of the grafting operation in the example 1, and the efficiency is low. Different culture media have different influences on apple grafted seedlings, wherein clay is most recommended to be adopted, and the survival rate, the root length and the rooting number of the cultured apple grafted seedlings are all the lowest levels. In contrast, the seedling substrate is the culture medium most favorable for the growth of apple grafted seedlings. Therefore, the method of taking the seedling substrate as the culture medium for rooting and grafting is more suitable for agricultural production.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (1)
1. A micro-grafting method for grafting and rooting outside an apple tissue culture seedling bottle comprises the steps of subculture of an apple tissue culture seedling, rooting preparation, rooting of the apple seedling, management of a rooting period, grafting of the apple seedling and management of a grafting culture period, and is characterized in that the micro-grafting method for grafting and rooting outside the apple tissue culture seedling bottle specifically comprises the following steps:
(1) subculturing the tissue culture seedlings of the apples: under aseptic condition, cutting stem of apple tissue culture seedling cultured for 30-35 days into 0.5-1cm, radially inserting into subculture medium, and subculturing for 40 days at 24 deg.C under light cycle of 16/8 h;
the subculture medium consists of 6-BA with the final concentration of 0.5mg/L, IAA with the final concentration of 0.2mg/L, MS culture medium, coagulant, cane sugar and water, and the pH value of the culture medium is adjusted to 5.8; the coagulator is agar, and the final concentration of the coagulator in the culture medium in which the coagulator is arranged is 7.5 g/L;
(2) rooting preparation: selecting a flat-bottom tray container, wherein the height of the flat-bottom tray container is 6cm, 72 domesticated rooting containers of 4cm multiplied by 4cm are placed in the flat-bottom tray container, grass carbon, vermiculite, perlite and water are mixed, the humidity of the rooting substrate is adjusted to 60%, the wet rooting substrate is filled in the rooting container, and the thickness of the substrate is the same as the height of the rooting container;
(3) apple seedling rooting: selecting subcultured vigorous apple tissue culture seedlings, removing callus and bottom leaves, inserting stems of the vigorous apple tissue culture seedlings into the rooting matrix, pressing the surrounding matrix, spraying water after the whole disc is inserted, and covering a sealed container to keep the humidity of the vigorous apple tissue culture seedlings at 65%;
(4) and (3) management of a rooting period: checking the matrix regularly, keeping the humidity at 50% -70%, and performing rooting culture for 40 days; the conditions of rooting culture are that the photoperiod is 12h/12h of light/dark, the temperature is 22 ℃, and the light intensity is 2000 lx;
(5) apple seedling grafting: selecting Malus hupehensis Rehd rooted apple seedlings as stocks, cutting off top leaves, reserving stems with the root part of 2.5-3cm long upwards, and cutting off the stems from the cut part by 0.4-0.5cm in radial direction; selecting Gala with vigorous and consistent growth as a scion, reserving a stem with the top end of 1.5-2cm and partial leaves, cutting the base part of the stem into a wedge shape, inserting the tail end of the scion into a stock, binding the interface part tightly by using aluminum foil paper, spraying water on the surface, and covering and sealing;
(6) and (3) management in a grafting culture period: periodically checking the matrix, keeping humidity at 50% -70%, culturing for 45 days at 24 deg.C under light cycle of 16/8h, and illuminating at 2000 lx.
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| CN109429757A (en) * | 2018-11-15 | 2019-03-08 | 山西省农业科学院生物技术研究中心 | A kind of indoor grafting of tender branch method of apple homozygous genotype strain test tube seedling |
| CN114402824A (en) * | 2022-01-27 | 2022-04-29 | 贵州省农业科学院 | Micro-grafting method for tea trees |
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