CN115380721A - Efficient cotton transgenic plant grafting technology - Google Patents
Efficient cotton transgenic plant grafting technology Download PDFInfo
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- CN115380721A CN115380721A CN202211227050.4A CN202211227050A CN115380721A CN 115380721 A CN115380721 A CN 115380721A CN 202211227050 A CN202211227050 A CN 202211227050A CN 115380721 A CN115380721 A CN 115380721A
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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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/50—Cotton
Abstract
The invention discloses an efficient cotton transgenic plant grafting technology, which specifically comprises the following steps: s1, preparing materials; s2, preparing rootstocks; transplanting the strong cotton rhizome plant cultured in the greenhouse into a plant tissue culture room; removing the upper part of the cotyledon; cutting the stem vertically to a depth of 2-3 cm; s3, preparing scions; selecting a transgenic cotton plant to be transplanted; only one unfolded leaf is reserved, and the rest leaves are removed; cutting the bottom of the transgenic cotton plant into a deep V shape; s4, grafting; vertically inserting the scion into the base of the V-shaped stock until the scion is stable; the growth environment was monitored daily until approximately two new leaves grew out of the scion and the grafts were transferred to the greenhouse; finally, the seeds are transferred into a large planting pot. According to the efficient cotton transgenic plant grafting technology, the transgenic cotton plant generated by somatic embryogenesis is regenerated by adopting the grafting technology, the survival rate of transplanting the transgenic cotton is remarkably improved by adopting the grafting technology, and the efficient cotton transgenic plant grafting technology is simple to operate and high in survival rate.
Description
Technical Field
The invention relates to the technical field of cotton cultivation, in particular to an efficient cotton transgenic plant grafting technology.
Background
The key step of improving crops by using a genetic engineering technology is to perform tissue culture on transgenic plants from a greenhouse to transplant the transgenic plants into fields, the seedlings obtained by the tissue culture are generally called test-tube seedlings or tissue culture seedlings, the test-tube seedlings grow in a constant-temperature, high-humidity, low-light and sterile closed environment for a long time, and have large difference with the external environment.
In the traditional method, a regenerated plant is transplanted under a greenhouse condition, multiple complex steps such as rooting, hardening, sterilizing and transplanting are usually involved, the culture environment and the transplanting environment are greatly different, the test tube seedling has poor capacity of adapting to the external environment, the survival rate of the test tube seedling directly transplanted into cotton is very low, the rooting and transplanting of the regenerated plant of cotton are difficult, the transplanting survival rate of the regenerated plant of cotton is low, more than 50% of plants die in the transplanting process, therefore, the low recovery rate of the transgenic cotton plant is an important bottleneck of the application of the transgenic cotton, and the survival rate can be improved by grafting and transplanting of robust plants.
Aiming at the problems, the innovative design is carried out on the basis of the original cotton transgenic plant planting technology.
Disclosure of Invention
The invention aims to provide an efficient cotton transgenic plant grafting technology to solve the problems that the cotton transgenic plant cultivation survival rate is low and the test popularization of the transgenic plant is not facilitated in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: an efficient grafting technology for cotton transgenic plants specifically comprises the following steps:
s1, preparing materials;
the preparation material comprises: transgenic cotton plants, robust cotton rhizomes, large and small planting pots, a soil mixture, an MS culture medium, a preservative film, a scalpel and a plastic bag;
s2, preparing rootstocks;
a. sterilizing the soil mixture at 120-130 deg.C for 30 min;
b filling the small planting pots with sterilized soil;
c. robust cotton rootstock plants are cultured in small plant pots in a greenhouse;
after 1.5-2 weeks, transferring the robust cotton rootstock plants into a plant tissue culture room;
e. removing the upper part of the cotyledon;
f. cutting the stem vertically to a depth of 2-3 cm;
s3, preparing scions;
a. selecting a transgenic cotton plant to be transplanted;
b. removing the transgenic cotton plant from the test tube;
c. only one unfolded leaf is reserved, and the rest leaves are removed;
d. cutting the bottom of the transgenic cotton plant into a deep V shape;
s4, grafting;
a. vertically inserting the scion into the base of the V-shaped stock until the scion is stable;
b. the graft part is wound and fixed by a preservative film;
c. covering the grafted plant by a plastic bag;
d. the grafted plants were grown under 16 hours of light/8 hours of dark cycle, 1500 lux of light, temperature set at 28 ± 2 ℃;
e. removing the twig growing on the rootstock node;
f. monitoring the soil daily, adding water and 1/2MS medium if necessary;
g.1 weeks later, removing the plastic bag or cup from the graft;
h. after about 2 weeks, the scion developed about two new leaves and the graft was transferred to the greenhouse;
i. after another 3 weeks, the plants were transferred to a large planting pot.
Preferably, the transgenic cotton plant is obtained by somatic embryo after agrobacterium-mediated transformation, and the robust cotton rhizome plant is a national cotton variety EZ9 cultivated by scientists of agricultural academy of sciences in north of huo.
Preferably, the large and small planting pots comprise small planting pots with the diameter of 15-20 cm and large planting pots with the diameter of about 30-35 cm, and are respectively used for planting and transplanting plants with different sizes in the early and later periods.
Preferably, the soil mixture is sandy or mucky soil pre-fertilized and the culture medium is Murashige and Skoog (MS) culture medium.
Preferably, the position of the stem cut vertically in the step S2 is the middle position of the stem, it is important to split the stem from the middle, so that the two sides can not be balanced, and the stem cutting depth is determined specifically until the stem vascular tissue meets the fixation of the scion.
Preferably, in the step S3, the bottom of the plant is cut into a deep V-shaped structure by a scalpel, and the cut surface of the V-shaped base of the cut is kept smooth so as to facilitate the firm butt joint and fixation of the scion and the rootstock.
Preferably, the size of the scion in the step S3 is not less than 0.3cm, the size of the scion is preferably 0.8-1.0cm, the survival rate of small scions with the size of 0.1-0.2cm is low, and the survival rate of large scions with the size of 0.8-1.0cm is as high as 95%.
Preferably, in the step S4, before two new leaves of the scion grow, the shoots growing on the nodes of the rootstock are kept removed, so as to avoid the scion from dying due to water shortage and nutrient shortage caused by more water and nutrient requirement of a plurality of leaves.
Preferably, the main purpose of covering the grafted plant in the step S4 is to maintain humidity, and the humidity is monitored in the previous week after grafting to maintain humidity at 70% -90%, so as to avoid the death of the scion caused by excessive water loss when no new vascular tissue is formed between the scion and the rootstock.
Compared with the prior art, the invention has the beneficial effects that: the efficient grafting technology for the cotton transgenic plants adopts the grafting technology to regenerate the transgenic cotton plants subjected to somatic embryogenesis, the recovery rate of the transgenic cotton plants is 65-92%, the survival rate of transplanted transgenic cotton is remarkably improved by the grafting technology, the grafting technology is simple to operate and high in survival rate, is suitable for basic research of the transgenic cotton plants, is also suitable for field planting and popularization, and is suitable for actual agricultural production.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The invention provides the following technical scheme:
example 1
An efficient grafting technology for cotton transgenic plants comprises the following steps:
s1, preparing materials;
the preparation material comprises the following steps: transgenic cotton plants, robust cotton rhizomes, large and small planting pots, soil mixtures, MS culture medium, preservative film, scalpel and plastic bag;
s2, preparing rootstocks;
a. sterilizing the soil mixture at 120 deg.C for 30 min;
b filling the small planting pot with sterilized soil;
EZ9 robust cotton rootstock plants are cultivated in small plant pots in the greenhouse;
after 1.5 weeks, robust cotton rootstock plants are moved into a plant tissue culture room;
e. removing the upper part of the cotyledon;
f. vertically cutting the stem to a depth of 2cm, wherein the vertical cutting position of the stem is the middle position of the stem;
s3, preparing scions;
a. selecting a transgenic cotton plant to be transplanted;
b. removing the transgenic cotton plant from the tube;
c. only one unfolded leaf is reserved, and the rest leaves are removed;
d. cutting the bottom of the transgenic cotton plant into a deep V shape, wherein the size of the scion is 0.9 +/-0.1 cm;
s4, grafting;
a. vertically inserting the scion into the base of the V-shaped stock until the scion is stable;
b. the graft part is wound and fixed by preservative film;
c. covering the grafted plant by a plastic bag;
d. the grafted plant is grown in a 16-hour light/8-hour dark cycle at a temperature of 28 +/-2 ℃ under 1500 lux light;
e. removing the twig growing on the rootstock node;
f. monitoring the soil daily, adding water and 1/2MS medium if necessary;
g.1 weeks later, remove the plastic bag or cup from the graft;
after h.2 weeks, approximately two new leaves grew out of the scion and the graft was transferred to the greenhouse;
i. after another 3 weeks, the plants were transferred to a large planting pot.
The transgenic cotton plant is obtained by somatic embryo after agrobacterium-mediated transformation, and the robust cotton root and stem plant is a national cotton variety EZ9 cultured by scientists of agricultural academy of sciences in Hubei province.
The large and small planting pots comprise a small planting pot with the diameter of 15 cm and a large planting pot with the diameter of about 30 cm, and are respectively used for planting and transplanting plants with different sizes in the front and the rear stages.
The soil mixture is sandy or clayey soil which is pre-applied with organic fertilizer, and the culture medium is Murashige and Skoog (MS) culture medium.
It is important to split the stem from the middle in the step S2, so that the two sides can not be balanced, and the stem cutting depth is determined by the stem vascular tissue until the scion is fixed.
In the step S3, the bottom of the plant is cut into a deep V-shaped structure by a scalpel, and the cut surface of the V-shaped base of the cut is kept smooth so as to be convenient for the butt joint and the fixation of the scion and the stock.
The survival rate of the big scions with the scions of 0.8-1.0cm in the step S3 is as high as 90%.
And S4, before two new leaves grow out from the scion, the shoots growing on the rootstock nodes are kept removed, and the scion is prevented from dying due to water shortage and insufficient nutrition because a plurality of leaves need more water and nutrition.
The main purpose of covering the grafted plant in the step S4 is to keep humidity, the humidity is monitored to keep the humidity at 75% in the previous week after grafting, the phenomenon that the scion dies due to excessive water loss when new vascular tissues are not formed between the scion and the rootstock is avoided, the previous week after grafting is very critical, the humidity is carefully monitored, and the low humidity often causes low survival rate of the graft.
Example 2
An efficient grafting technology for cotton transgenic plants specifically comprises the following steps:
s1, preparing materials;
the preparation material comprises: transgenic cotton plants, robust cotton rhizomes, large and small planting pots, a soil mixture, an MS culture medium, a preservative film, a scalpel and a plastic bag;
s2, preparing rootstocks;
a. sterilizing the soil mixture at 130 ℃ for 30 minutes;
b filling the small planting pots with sterilized soil;
EZ9 robust cotton rootstock plants are cultivated in small plant pots in the greenhouse;
d.2 weeks later, the robust cotton rootstock plants are moved into a plant tissue culture room;
e. removing the upper part of the cotyledon;
f. vertically cutting the stem to a depth of 3cm, wherein the vertical cutting position of the stem is the middle position of the stem;
s3, preparing scions;
a. selecting a transgenic cotton plant to be transplanted;
b. removing the transgenic cotton plant from the tube;
c. only one unfolded leaf is reserved, and the rest leaves are removed;
d. cutting the bottom of the transgenic cotton plant into a deep V shape, wherein the size of the scion is 0.9 +/-0.1 cm;
s4, grafting;
a. vertically inserting the scion into the base of the V-shaped stock until the scion is stable;
b. the graft part is wound and fixed by preservative film;
c. covering the grafted plant by a plastic bag;
d. the grafted plant is grown in a 16-hour light/8-hour dark cycle at a temperature of 28 +/-2 ℃ under 1500 lux light;
e. removing the twig growing on the rootstock node;
f. monitoring the soil daily, adding water and 1/2MS medium if necessary;
g.1 weeks later, removing the plastic bag or cup from the graft;
after h.2 weeks, approximately two new leaves grew out of the scion and the graft was transferred to the greenhouse;
i. after another 3 weeks, the plants were transferred to a large planting pot.
The transgenic cotton plant is obtained by somatic embryo after agrobacterium-mediated transformation, and the robust cotton root and stem plant is a national cotton variety EZ9 cultured by scientists of agricultural academy of sciences in Hubei province.
The large and small planting pots comprise small planting pots with the diameter of 20 cm and large planting pots with the diameter of about 35 cm, and are respectively used for planting and transplanting plants with different sizes in the front and the rear stages.
The soil mixture is sandy or clayey soil which is pre-applied with organic fertilizer, and the culture medium is Murashige and Skoog (MS) culture medium.
It is important to split the stem from the middle in the step S2, so that the two sides can be kept balanced, and the stem cutting depth is determined specifically until the stem vascular tissue meets the fixation of the scion.
In the step S3, the bottom of the plant needs to be cut into a deep V-shaped structure through a scalpel, and the cut surface of the V-shaped base of the cut is kept smooth so as to facilitate the butt joint and the firm fixation of the scion and the rootstock.
The survival rate of the big scions with the scions of 0.8-1.0cm in the step S3 is as high as 95 percent.
And S4, before two new leaves grow out from the scion, the shoots growing on the rootstock nodes are kept removed, and the scion is prevented from dying due to water shortage and insufficient nutrition because a plurality of leaves need more water and nutrition.
The main purpose of covering the grafted plant in the step S4 is to keep the humidity, the humidity is monitored to keep 85% in the previous week after grafting, the phenomenon that the scion dies due to excessive water loss when new vascular tissues are not formed between the scion and the rootstock is avoided, the previous week after grafting is very critical, the humidity is carefully monitored, and the low humidity often causes low survival rate of the graft.
Example 3
An efficient grafting technology for cotton transgenic plants specifically comprises the following steps:
s1, preparing materials;
the preparation material comprises: transgenic cotton plants, robust cotton rhizomes, large and small planting pots, soil mixtures, MS culture medium, preservative film, scalpel and plastic bag;
s2, preparing rootstocks;
a. sterilizing the soil mixture at 121 ℃ for 30 minutes;
b filling the small planting pots with sterilized soil;
EZ9 robust cotton rootstock plants are cultivated in small plant pots in the greenhouse;
d.2 weeks later, the robust cotton rootstock plants are moved into a plant tissue culture room;
e. removing the upper part of the cotyledon;
f. the stem is vertically cut to a depth of 2.5cm, and the vertical cut position of the stem is the middle position of the stem;
s3, preparing scions;
a. selecting a transgenic cotton plant to be transplanted;
b. removing the transgenic cotton plant from the tube;
c. only one unfolded leaf is reserved, and the rest leaves are removed;
d. cutting the bottom of the transgenic cotton plant into a deep V shape, wherein the size of the scion is 0.9 +/-0.1 cm;
s4, grafting;
a. vertically inserting the scion into the base of the V-shaped stock until the scion is stable;
b. the graft part is wound and fixed by preservative film;
c. covering the grafted plant by a plastic bag;
d. the grafted plants were grown under 16 hours of light/8 hours of dark cycle, 1500 lux of light, temperature set at 28 ± 2 ℃;
e. removing the tender branches growing on the rootstock nodes;
f. monitoring the soil daily, adding water and 1/2MS medium if necessary;
g.1 weeks later, removing the plastic bag or cup from the graft;
after h.2 weeks, approximately two new leaves grew out of the scion and the graft was transferred to the greenhouse;
i. after another 3 weeks, the plants were transferred to a large planting pot.
The transgenic cotton plant is obtained by somatic embryo after agrobacterium-mediated transformation, and the robust cotton rootstock plant is a national cotton variety EZ9 cultivated by scientists of agricultural academy of sciences of Hubei province.
The large and small planting pots comprise a small planting pot with the diameter of 15 cm and a large planting pot with the diameter of about 35 cm, and are respectively used for planting and transplanting plants with different sizes in the front and the rear stages.
The soil mixture is sandy or clayey soil which is pre-applied with organic fertilizer, and the culture medium is Murashige and Skoog (MS) culture medium.
In the step S2, it is important to split the stem from the middle, so that the two sides can be kept balanced, and the stem cutting depth is determined until the stem vascular tissue meets the fixation of the scion.
In the step S3, the bottom of the plant needs to be cut into a deep V-shaped structure through a scalpel, and the cut surface of the V-shaped base of the cut is kept smooth so as to facilitate the butt joint and the firm fixation of the scion and the rootstock.
The survival rate of the big scions with the scions of 0.8-1.0cm in the step S3 is as high as 94%.
And S4, before two new leaves grow out from the scion, the tender branches growing out from the rootstock nodes are kept removed, and the scion is prevented from dying due to water shortage and insufficient nutrition because a plurality of leaves need more water and nutrition.
The main purpose of covering the grafted plant in the step S4 is to keep humidity, the humidity is monitored in the previous week after grafting to keep the humidity at 80% +/-5%, the phenomenon that the scion dies due to excessive water loss when new vascular tissues are not formed between the scion and the stock is avoided, the previous week after grafting is very critical, the humidity is carefully monitored, and the low humidity often causes low survival rate of the graft.
By adopting the grafting technology to regenerate the transgenic cotton plant generated by somatic embryogenesis in the embodiment, the recovery rate of the transgenic cotton plant is 65-92%, the survival rate of transplanting the transgenic cotton is obviously improved by the grafting technology as shown in the following table, and the survival rate of the healthy regenerated plant can be as high as 94%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. An efficient cotton transgenic plant grafting technology is characterized in that: the method specifically comprises the following steps:
s1, preparing materials;
the preparation material comprises: transgenic cotton plants, robust cotton rhizomes, large and small planting pots, soil mixtures, MS culture medium, preservative film, scalpel and plastic bag;
s2, preparing rootstocks;
a. sterilizing the soil mixture at 120-130 deg.C for 30 min;
b filling the small planting pots with sterilized soil;
c. robust cotton rootstock plants are cultured in small plant pots in a greenhouse;
after 1.5-2 weeks, moving robust cotton rootstock plants into a plant tissue culture room;
e. removing the upper part of the cotyledon;
f. cutting the stem vertically to a depth of 2-3 cm;
s3, preparing scions;
a. selecting a transgenic cotton plant to be transplanted;
b. removing the transgenic cotton plant from the test tube;
c. only one unfolded leaf is reserved, and the rest leaves are removed;
d. cutting the bottom of the transgenic cotton plant into a deep V shape;
s4, grafting;
a. vertically inserting the scion into the base of the V-shaped stock until the scion is stable;
b. the graft part is wound and fixed by a preservative film;
c. covering the grafted plant by a plastic bag;
d. the grafted plant is grown in a 16-hour light/8-hour dark cycle at a temperature of 28 +/-2 ℃ under 1500 lux light;
e. removing the tender branches growing on the rootstock nodes;
f. monitoring the soil daily, adding water and 1/2MS medium if necessary;
g.1 weeks later, removing the plastic bag or cup from the graft;
h. after about 2 weeks, the scion developed about two new leaves and the graft was transferred to the greenhouse;
i. after another 3 weeks, the plants were transferred to a large planting pot.
2. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: the transgenic cotton plant is obtained by somatic embryo after agrobacterium-mediated transformation, and the robust cotton root and stem plant is a national cotton variety EZ9 cultured by scientists of agricultural academy of sciences in Hubei province.
3. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: the large and small planting pots comprise small planting pots with the diameter of 15-20 cm and large planting pots with the diameter of about 30-35 cm, and are respectively used for planting and transplanting plants with different sizes in the early and later periods.
4. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: the soil mixture is sandy or mucky soil which is pre-applied with organic fertilizer, and the culture medium is Murashige and Skoog (MS) culture medium.
5. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: and in the step S2, the vertical cut position of the stem is the middle position of the stem so as to keep balance of two sides, and the stem cutting depth is determined specifically until the stem vascular tissue meets the fixation of the scion.
6. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: in the step S3, the bottom of the plant needs to be cut into a deep V-shaped structure through a scalpel, and the cut surface of the V-shaped base of the cut is kept smooth so as to facilitate the butt joint and the fixation of the scion and the rootstock to be firm.
7. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: the size of the scion in the step S3 is not less than 0.3cm, and the size of the scion is preferably 0.8-1.0cm.
8. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: and in the step S4, before two new leaves of the scion grow, the shoots growing on the rootstock nodes are kept removed, so that the scion is prevented from dying due to water shortage and insufficient nutrition because a plurality of leaves need more water and nutrition.
9. The efficient grafting technique for cotton transgenic plants according to claim 1, characterized in that: the main purpose of covering the grafted plant in the step S4 is to keep humidity, the humidity is monitored in the previous week after grafting to keep the humidity at 70% -90%, and the phenomenon that the scion is dead due to excessive water loss when no new vascular tissue is formed between the scion and the stock is avoided.
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