CN111758406A - Grafting preservation method for kiwi fruit in-vitro resources - Google Patents
Grafting preservation method for kiwi fruit in-vitro resources Download PDFInfo
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- CN111758406A CN111758406A CN202010595371.4A CN202010595371A CN111758406A CN 111758406 A CN111758406 A CN 111758406A CN 202010595371 A CN202010595371 A CN 202010595371A CN 111758406 A CN111758406 A CN 111758406A
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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
- 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
- A01H4/001—Culture apparatus for tissue culture
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- 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
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Abstract
The invention discloses a grafting preservation method of kiwi fruit in vitro resources, and relates to the technical field of preservation and breeding of in vitro resources. The method comprises the following steps: A. obtaining a scion in-vitro sterile material; B. strong seedling cultivation of the scions; C. preparing and treating rootstocks; D. grafting operation; E. and (5) management after grafting. Compared with the prior art, the invention has the following advantages and positive effects: the method has the advantages that production can be carried out all the year round, the growth environment condition is artificially controlled, and a new way is provided for the timely preservation and breeding of kiwi fruit germplasm resources; secondly, the operation is simple and easy, the survival rate is high, the application range is wide, and various crop breeding materials can be stored; the preparation of the scions is carried out by water culture sprouting, so that the method is convenient and quick, the time is saved, and the efficiency is improved; fourthly, directly grafting the main stem of the excised material as a scion to the branch of the adult stock species by adopting a micro-grafting operation means, wherein the survival rate can reach more than 80 percent; and fifthly, the observation of the phenological stages such as flowering and fruiting characters can be accelerated by adopting a micro-grafting operation means.
Description
Technical Field
The invention relates to the technical field of preservation and breeding of in vitro resources, in particular to a grafting preservation method of in vitro resources of kiwi fruits.
Background
Kiwi fruit[Actinidia chinensis Planch]Is a large-scale deciduous woody vine plant of Actinidiaceae, Actinidia. The kiwi fruit is soft in texture and sour and sweet in taste. The kiwi fruit praised as "fruit king" is sour, sweet and tasty, has rich nutrition, and is a nourishing fruit for the old, children and weak and many patients. It contains rich vitamin C, vitamin A, vitamin E, potassium, magnesium and cellulose, and in the first 26 kinds of fruits with the largest consumption in the world, the Chinese gooseberry is most abundant and comprehensive. The Vc, Mg and trace elements in the kiwi fruit have the highest content. In the kiwi fruit germplasm resource conservation research, the traditional cuttage and grafting can not meet the increasing resource conservation demand, the tissue culture in-vitro preservation breeding has the gradually highlighted advantages in recent years, but the problems of difficult preservation, difficult rooting and low transplanting survival rate of breeding intermediate materials exist in the breeding related to the tissue culture, the field introduction material has great difficulty in the survival rate of transplanting of the materials preserved by tissue culture due to the land property and the low introduction and cultivation survival rate, and seasonal factors influence the introduction of kiwi fruits and the various problems of grafting, cuttage cultivation survival rate and the like of specific kiwi fruit materials.
Disclosure of Invention
The invention aims to provide a method for grafting and preserving in-vitro kiwi fruit resources, which solves the problems of difficult preservation and difficult rooting of breeding intermediate materials related to tissue culture, the problems of land property and low introduction and cultivation survival rate of field introduction resources preserved in-vitro, and the problems of influence of seasonal factors on introduction and grafting and cuttage cultivation survival rate of specific kiwi fruit materials.
In order to achieve the purpose, the invention adopts the following technical scheme:
specifically, the method comprises the following steps:
A. obtaining a scion in-vitro sterile material: culturing kiwi fruit in water in room, cutting into 20-30cm long sections, sealing the upper end of the hard branch with paraffin wax, soaking the lower end of the hard branch 5-8cm in an open bottle containing 0.1-0.2% carbendazim and 30-50mg/L naphthylacetic acid solution, replacing the aqueous solution for 2-3 days, irradiating with 3500-4000Lux light at 18-25 deg.C for 7-15 days, sterilizing the young buds growing for 3-5 days, removing leaves of the young buds, cutting young stem sections, inoculating to a quick culture medium, culturing under 8h/d light, 2500Lux light intensity, 3000Lux light at 22-26 deg.C, and axillary bud propagation at 15-20 days, rapidly propagating to obtain multiple buds at the base, 45d is a subculture period;
B. strong seedling cultivation of scions: after 2-3 subcultures, cutting a single robust tender bud or a small amount of multiple shoots, transferring the single robust tender bud or the small amount of multiple shoots to a strong seedling culture medium, wherein the culture condition is 12h/d illumination, the illumination intensity is 3000-;
C. preparation and treatment of rootstock: the rootstock is a hole tray seeding seedling in the same year, transplanting and pot changing culture are carried out after germination, after stumping and trimming in winter, the rootstock sprouts in the spring of the second year, and robust twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm are selected as the rootstock;
D. grafting operation: preparing in vitro scions, grafting and selecting to be carried out in the morning at the ambient temperature of 15-35 ℃, cutting a plastic cylinder into 0.5-1.0cm segments, sterilizing at 121 ℃ for 20min for later use, selecting robust rootstock twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm, cutting off blades at the position 2-4cm above the base of the twigs along the base, cutting off the top end of the twigs along the position 3-4cm upwards from the base of the twigs, quickly sleeving the plastic cylinder on the base of the twigs, sleeving the plastic cylinder on the base, exposing the polished rod part with the blades removed, simultaneously opening a tissue culture bottle cap of the in vitro scions, cutting a target species stem segment with the length of 1-2cm and the stem diameter of 0.1-0.2cm, cutting the base into a wedge shape after removing the blades of the base, quickly transversely cutting the blades downwards at the center of the rootstock twigs with the depth of 0.8-1.0cm, quickly inserting the scions into the rootstock, after the grafting is finished, the plastic cylinder is pulled upwards to the grafting opening of the scion and the stock and fixed, finally a self-sealing bag of 3cm multiplied by 5cm is taken to cover the grafting position in an inverted mode, the self-sealing bag is sealed for moisture preservation, and a gap of 0.5-1.0cm is leaked from the tender tip of the stock;
E. and (3) management after grafting: after grafting, conventionally managing the stock pot seedlings, controlling the environmental temperature at 15-35 ℃, shading and cooling when the environmental temperature is higher than 35 ℃, entering a facility greenhouse for management when the environmental temperature is lower than 10 ℃, observing the states of the stocks and the scions every day, timely erasing sprouts at the base parts of the stocks, pulling open a seal of a self-sealing bag opening after one week, and removing the self-sealing bag after the grafting opening is completely healed after 2-3 weeks.
The innovation of the invention is to provide a grafting preservation method of kiwi fruit in vitro resources. Excellent kiwi in-vitro germplasm resource tissue culture bottle seedlings successfully preserved in a tissue culture room are difficult to root, the transplanting survival rate is low, the transplanting management cost of the tissue culture seedlings is high, the wild species resource introduction environment is poor in adaptability, and the like.
Compared with the prior art, the invention has the following advantages and positive effects:
the method has the advantages that production can be carried out all the year round, the growth environment condition is artificially controlled, and a new way is provided for the timely preservation and breeding of kiwi fruit germplasm resources;
secondly, the operation is simple and easy, the survival rate is high, the application range is wide, and various crop breeding materials can be stored;
the preparation of the scions is carried out by water culture sprouting, so that the method is convenient and quick, the time is saved, and the efficiency is improved;
fourthly, directly grafting the main stem of the excised material as a scion to the branch of the adult stock species by adopting a micro-grafting operation means, wherein the survival rate can reach more than 80 percent;
and fifthly, the observation of the phenological stages such as flowering and fruiting characters can be accelerated by adopting a micro-grafting operation means.
Detailed Description
Method and device
1. The culture medium in step A
Cutting a stem section with 0.5-1.0cm of water culture sprout, sterilizing the stem section, placing the stem section on a sprout induction culture medium for induction culture, wherein the culture medium comprises MS + 6-BA 1.0-3.0mg/L + IBA 0.1-1.0mg/L +30% sucrose +0.8% agar, MS + TDA1.0-3.0mg/L + NAA0.1-0.5mg/L +30% sucrose +0.8% agar, and the pH value is 5.5-6.0.
2. The medium in step B
The strong seedling culture medium is MS + 6-BA 0.1-1.0mg/L + IBA 0.01-0.05mg/L +30% sucrose +0.8% agar for culture and MS + TDZ0.1-1.0mg/L + NAA0.01-0.05mg/L +30% sucrose +0.8% agar, and its pH value is 5.5-6.0.
3. Step E of valve bag
The self-sealing bag is a bone-blowing self-sealing bag made of PO film, and can achieve 75-90% of light transmittance for light with the wavelength of 400-800 nm.
Second, example
Example 1
A. Obtaining a scion in-vitro sterile material: culturing hard kiwi fruit to be cultivated in water indoors, cutting the hard kiwi fruit into segments with the length of 20-30cm, sealing the upper end cut of the hard kiwi fruit with paraffin wax, soaking the lower end of the hard kiwi fruit with the length of 5-8cm in an open bottle filled with an aqueous solution containing 0.1-0.2% carbendazim and 30-50mg/L naphthylacetic acid, replacing the aqueous solution for 2-3d, irradiating 3500 and 4000Lux with light, controlling the temperature at 18-25 ℃, germinating for 7-9d, sterilizing young axillary buds growing for 3-5d after germination on a clean bench, removing the leaves of the young buds, cutting the stem segments with the length of 0.5-1.0cm, inoculating the stem segments with the length of 0.1 mg/L IBA, 30% sucrose and 0.8% agar, the pH value is 5.5-6.0, the culture condition is 8h/d illumination, the illumination intensity is 2500lux, the temperature is 22-26 ℃, axillary buds of the stem segments are quickly propagated and sprout after 15-20d, a plurality of buds are regenerated at the base part, and 45d is a subculture period;
B. strong seedling cultivation of in vitro scions: after 3 subcultures, cutting strong single tender shoots or a small amount of multiple shoots, transferring the cut strong single tender shoots or the small amount of multiple shoots to a strong seedling culture medium MS + 6-BA 0.2 mg/L + IBA 0.02mg/L +30% sucrose +0.8% agar for culture at the pH of 5.5-6.0 under the condition of 12h/d illumination, the illumination intensity of 3000-. After 25 days, the main stem of the bud grows and is thick. After strong seedling culture is carried out for 50-60 days, the diameter of the main stem of the in vitro material reaches 0.1-0.2cm, and the in vitro material can be used as a scion for grafting, and the scion is in the optimal state;
C. preparation and treatment of rootstock: the rootstock is a hole tray seeding seedling in the same year, transplanting and pot changing culture are carried out after germination, after stumping and trimming in winter, the rootstock sprouts in the spring of the second year, and robust tender branches with tender tip length of 4-6cm and stem diameter of 0.1-0.3cm are selected as the rootstock;
D. grafting operation: preparing in vitro scions, grafting and selecting to be carried out in the morning of the ambient temperature of 15-35 ℃, cutting a plastic cylinder into 0.5-1.0cm segments, sterilizing at 121 ℃ for 20min for later use, selecting robust rootstock twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm, cutting off blades at the position 2-4cm above the base of the twigs along the base, cutting off the top end of the twigs along the position 3-4cm upwards from the base of the twigs, quickly sleeving the plastic cylinder on the base of the twigs, sleeving the plastic cylinder on the base, exposing the polished rod part with the blades removed, simultaneously opening a tissue culture bottle cap of the in vitro scions, cutting a target species stem segment with the length of 1-2cm and the stem diameter of 0.1-0.2cm, cutting the base into a wedge shape after removing the base blades, quickly transversely cutting the blade downwards at the center of the slightly tender cut to a depth of 0.8-1.0cm, quickly inserting the scions into the rootstock cut, and one end of the cambium of the scion and the stock is aligned, and after the alignment is finished, the plastic cylinder is pulled upwards to the grafting opening of the scion and the stock and is fixed. And finally, taking a PO film of 3cm multiplied by 5cm to blow a bone self-sealing bag to cover the grafting part in an inverted manner, sealing the self-sealing bag to preserve moisture, wherein a gap of 0.5-1.0cm is leaked from the tender tip of the stock, and the self-sealing bag has a light transmittance of 90% to the light energy with the wavelength of 800nm of 400-.
E. And (3) management after grafting: after grafting, conventionally managing the stock pot seedlings, controlling the environmental temperature to be 15-35 ℃, shading when the environmental temperature is higher than 35 ℃, and managing in a greenhouse when the environmental temperature is lower than 10 ℃. Observing the states of the rootstock and the scion every day, timely erasing the sprouts at the base of the rootstock, pulling a self-sealing bag opening after one week, removing the self-sealing bag after the grafting opening is completely healed after 2-3 weeks, performing conventional management, wherein after 2 months, the grafted body grows well, and the survival rate reaches 85%.
Example 2
A. Obtaining a scion in-vitro sterile material: culturing hard branches of Chinese gooseberry resource to be cultivated in water indoors, cutting the hard branches into segments with the length of 20-30cm, sealing the cuts at the morphological upper ends of the hard branches with paraffin wax, soaking the morphological lower ends of the hard branches with the length of 5-8cm in an open bottle filled with aqueous solution containing 0.1-0.2% carbendazim and 30-50mg/L naphthylacetic acid, replacing the aqueous solution once for 2-3d, giving illumination of 3500 and 4000Lux, controlling the temperature at 18-25 ℃, germinating for 12-15d, taking young axillary buds growing for 3-5d after germination, sterilizing on a clean bench, removing the redundant leaves at the bases of the young buds, cutting 0.5-0.8cm of terminal buds, inoculating the terminal buds on a rapid propagation culture medium for cultivation, selecting MS + TDZ1.0mg/L + NAA0.5mg/L +30% sucrose +0.8% agar, pH5.5-6.0, and culturing under the condition of 8h/d illumination, the illumination intensity is 3000lux, and the temperature is 22-26 ℃. After 30 days, the base part of the axillary bud regenerates and buds, and 45 days is a subculture period;
B. strong seedling cultivation of in vitro scions: after 3 times of subculture, cutting a single strong tender bud or a small amount of multiple buds, transferring the single strong tender bud or the small amount of multiple buds to a strong seedling culture medium MS + TDZ0.5mg/L + NAA0.02mg/L +30% sucrose +0.8% agar for culture at the pH of 5.5-6.0 under the condition of 12h/d illumination, the illumination intensity of 3500lux and the temperature of 22-26 ℃. After 25 days, the main stem of the bud grows and is thick, the diameter of the main stem of the in vitro material reaches 0.1-0.2cm after the strong seedling is cultured for 60 days, and the in vitro material can be used as a scion for grafting, and the scion is in the optimal scion state.
C. Preparation and treatment of rootstock: the rootstock is a hole tray seeding seedling in the same year, transplanting and pot changing culture are carried out after germination, after stumping and trimming in winter, the rootstock sprouts in the spring of the second year, and robust tender branches with tender tip length of 4-6cm and stem diameter of 0.1-0.3cm are selected as the rootstock;
D. grafting operation: preparing in vitro scions, grafting and selecting to be carried out in the morning of the ambient temperature of 15-35 ℃, cutting a plastic cylinder into 0.5-1.0cm segments, sterilizing at 121 ℃ for 20min for later use, selecting robust rootstock twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm, cutting off blades at the position 2-4cm above the base of the twigs along the base, cutting off the top end of the twigs along the position 3-4cm upwards from the base of the twigs, quickly sleeving the plastic cylinder on the base of the twigs, sleeving the plastic cylinder on the base, exposing a polished rod part with the blades removed, simultaneously opening a tissue culture bottle cap of the in vitro scions, cutting a target species stem segment with the length of 1-2cm and the stem diameter of 0.1-0.2cm, cutting the base into a wedge shape after removing the base blades, quickly transversely cutting the base downwards at the center of the tender rootstock cut with the depth of 0.8-1.0cm, quickly inserting the scions into the rootstock cut, one end of the cambium of the scion and the stock is aligned, after the alignment, the plastic cylinder is pulled upwards to the grafting port of the scion and the stock and fixed, finally, a 3cm multiplied by 5cm PO film is taken to blow a bone self-sealing bag to cover the grafting part in an inverted manner, the self-sealing bag is sealed for moisture preservation, the sealing of the self-sealing bag is only carried out by leaking a gap of 0.5-1.0cm from the tender tip of the stock, and the self-sealing bag can reach 90% of light transmittance to the light energy with the wavelength of 800nm and 400 plus materials;
E. and (3) management after grafting: after grafting, conventionally managing the stock pot seedlings, controlling the environmental temperature to be 15-35 ℃, shading when the environmental temperature is higher than 35 ℃, and managing in a greenhouse when the environmental temperature is lower than 10 ℃. Observing the states of the rootstock and the scion every day, timely erasing the sprouts at the base of the rootstock, pulling a self-sealing bag opening after one week, removing the self-sealing bag after the grafting opening is completely healed after 2-3 weeks, and after 2 months, the grafted body grows well and the statistical survival rate reaches 80%.
Claims (4)
1. The grafting preservation method of the kiwi fruit in-vitro resource is characterized by comprising the following steps:
A. obtaining a scion in-vitro sterile material: culturing kiwi fruit in water in room, cutting into 20-30cm long sections, sealing the upper end of the hard branch with paraffin wax, soaking the lower end of the hard branch 5-8cm in an open bottle containing 0.1-0.2% carbendazim and 30-50mg/L naphthylacetic acid solution, replacing the aqueous solution for 2-3 days, irradiating with 3500-4000Lux light at 18-25 deg.C for 7-15 days, sterilizing the young buds growing for 3-5 days, removing leaves of the young buds, cutting young stem sections, inoculating to a quick culture medium, culturing under 8h/d light, 2500Lux light intensity, 3000Lux light at 22-26 deg.C, and axillary bud propagation at 15-20 days, rapidly propagating to obtain multiple buds at the base, 45d is a subculture period;
B. strong seedling cultivation of scions: after 2-3 subcultures, cutting a single robust tender bud or a small amount of multiple shoots, transferring the single robust tender bud or the small amount of multiple shoots to a strong seedling culture medium, wherein the culture condition is 12h/d illumination, the illumination intensity is 3000-;
C. preparation and treatment of rootstock: the rootstock is a hole tray seeding seedling in the same year, transplanting and pot changing culture are carried out after germination, after stumping and trimming in winter, the rootstock sprouts in the spring of the second year, and robust twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm are selected as the rootstock;
D. grafting operation: preparing in vitro scions, grafting and selecting to be carried out in the morning at the ambient temperature of 15-35 ℃, cutting a plastic cylinder into 0.5-1.0cm segments, sterilizing at 121 ℃ for 20min for later use, selecting robust rootstock twigs with the length of 4-6cm and the stem diameter of 0.1-0.3cm, cutting off blades at the position 2-4cm above the base of the twigs along the base, cutting off the top end of the twigs along the position 3-4cm upwards from the base of the twigs, quickly sleeving the plastic cylinder on the base of the twigs, sleeving the plastic cylinder on the base, exposing the polished rod part with the blades removed, simultaneously opening a tissue culture bottle cap of the in vitro scions, cutting a target species stem segment with the length of 1-2cm and the stem diameter of 0.1-0.2cm, cutting the base into a wedge shape after removing the blades of the base, quickly transversely cutting the blades downwards at the center of the rootstock twigs with the depth of 0.8-1.0cm, quickly inserting the scions into the rootstock, after the grafting is finished, the plastic cylinder is pulled upwards to the grafting opening of the scion and the stock and fixed, finally a self-sealing bag of 3cm multiplied by 5cm is taken to cover the grafting position in an inverted mode, the self-sealing bag is sealed for moisture preservation, and a gap of 0.5-1.0cm is leaked from the tender tip of the stock;
E. and (3) management after grafting: after grafting, conventionally managing the stock pot seedlings, controlling the environmental temperature at 15-35 ℃, shading and cooling when the environmental temperature is higher than 35 ℃, entering a facility greenhouse for management when the environmental temperature is lower than 10 ℃, observing the states of the stocks and the scions every day, timely erasing sprouts at the base parts of the stocks, pulling open a seal of a self-sealing bag opening after one week, and removing the self-sealing bag after the grafting opening is completely healed after 2-3 weeks.
2. The grafting preservation method of kiwi isolated resource according to claim 1, wherein the culture medium in step a:
cutting a stem section with 0.5-1.0cm of water culture sprout, sterilizing the stem section, placing the stem section on a sprout induction culture medium for induction culture, wherein the culture medium comprises MS + 6-BA 1.0-3.0mg/L + IBA 0.1-1.0mg/L +30% sucrose +0.8% agar, MS + TDA1.0-3.0mg/L + NAA0.1-0.5mg/L +30% sucrose +0.8% agar, and the pH value is 5.5-6.0.
3. The grafting preservation method of kiwi isolated resource according to claim 1, wherein the culture medium in step B:
the strong seedling culture medium is MS + 6-BA 0.1-1.0mg/L + IBA 0.01-0.05mg/L +30% sucrose +0.8% agar for culture and MS + TDZ0.1-1.0mg/L + NAA0.01-0.05mg/L +30% sucrose +0.8% agar, and its pH value is 5.5-6.0.
4. The grafting preservation method of kiwi isolated resource according to claim 1, wherein kiwi isolated resource is obtained by grafting kiwi isolated resource onto kiwi isolated resource
The valve bag in the step E:
the self-sealing bag is a bone-blowing self-sealing bag made of PO film, and can achieve 75-90% of light transmittance for light with the wavelength of 400-800 nm.
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