CN108271682B - Directional hybridization seed production method for stevia rebaudiana - Google Patents
Directional hybridization seed production method for stevia rebaudiana Download PDFInfo
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Abstract
The invention discloses a method for producing stevia rebaudiana by directional hybridization, belonging to the field of seed production of commercial crops. The method comprises the steps of isolation planting of male and female parent plants, collection and preservation of pollen, preparation of pollination liquid, liquid spraying or liquid instillation pollination and collection of stevia rebaudiana seeds. According to the directional hybridization seed production method of stevia rebaudiana provided by the invention, the hybridization seed setting rate can be improved, the purity of hybrid seeds can be guaranteed, and the seed yield can be improved.
Description
Technical Field
The invention belongs to the field of seed production of commercial crops, and particularly relates to a directional hybridization seed production method of stevia rebaudiana.
Background
Stevia rebaudiana belongs to self-incompatible plants, and most varieties are self-pollinated and are not fruity, and seeds are produced by hybridization.
The existing hybrid seed production method of stevia rebaudiana comprises the steps of planting cuttage propagation seedlings or seed direct seeding seedlings of various parent strains in the same field, and carrying out pollination and mutual hybridization by using media such as bees to prepare hybrid seeds of the stevia rebaudiana. These methods have the following disadvantages: the male parent and the female parent have high requirement on flower-period encounter, low hybrid seed setting rate, low seed purity, great male parent plant waste and high seed production cost.
In addition, in the existing stevia rebaudiana hybrid seed production method, different parents are planted in the same land, and because the stevia rebaudiana hybrid seed production method is a natural hybrid, the parents and the plants can mutually accept the pollen of the other side to form seeds through the pollination effect of media such as bees and the like. Because of the habits of light stevia rebaudiana seeds, having crown hair, being easy to fly around with the wind after being mature and the like, the incorporation of seeds formed by backcrossing with male parent plants with insufficient agronomic characters as female parents is difficult to eliminate when the seeds are harvested, so that the seeds have low purity and high mixing degree.
Meanwhile, because the flowering phase of the same plant of the stevia rebaudiana lasts about one month more, the difference of the maturation phase among the seeds is large, and the stevia rebaudiana seeds are easy to scatter around along with the wind after maturation. The conventional stevia rebaudiana seed collecting method is to harvest seeds of female parent plants at one time and shed the seeds after drying the female parent plants in the sun or manually shake the female parent plants in a field at certain time intervals to harvest the seeds when most of the seeds are mature. The seeds obtained by the former seed harvesting method have higher purity, and the defects of low seed harvesting rate, inconsistent maturity, low germination rate, difficult later-stage selection and the like; the seed obtained by the latter harvesting method has better maturity consistency and high seed yield, but the obtained seed has low purity, high mixing degree, labor and time consumption, and is easy to damage plants, so that the harvested seeds at the later stage contain immature seeds falling from injured branches. Therefore, the two harvesting modes finally cause the defects of low purity, low germination rate, high production cost, low late field planting efficiency and the like of the hybrid seeds in different degrees.
In addition, in the preparation of other artificial pollination solutions for crops which have been published at present, the required components are complex, for example, the formula of the pollination solution published in patent 201610497946.2 contains ten compounds such as sucrose, glucose, boric acid, guar gum, soybean lecithin, dodecyl hydroxypropyl phosphate, betaine, potassium nitrate, ammonium chloride and choline chloride, and the increase of the components causes that general farmers are difficult to purchase and accurately prepare in sufficient quantity, thereby affecting the pollination success rate and the effective popularization of the technology.
Disclosure of Invention
The invention aims to: aiming at the problems in the stevia rebaudiana hybrid seed production, the invention provides a simple and effective artificial pollination liquid formula and a stevia rebaudiana hybrid seed modern production process.
The purpose of the invention can be realized by the following technical scheme:
a method for directional hybridization seed production of stevia rebaudiana by pollination liquid comprises the following steps:
1) carrying out isolated planting on male parent plants and female parent plants;
2) collecting and preserving pollen: collecting pollen in the flowering period of the male parent, and storing the obtained pollen at a low temperature;
3) preparation of pollination liquid: weighing the following substances in parts by weight: d-sodium erythorbate, polyethylene glycol 4000 and deionized water which is 10-20 times of the total mass of the D-sodium erythorbate and the polyethylene glycol 4000 are uniformly stirred to obtain a pollen suspension buffer solution; adding pollen into the suspension buffer solution to prepare a pollen suspension buffer solution to obtain pollination solution;
4) liquid spraying or liquid dripping pollination: when the female parent plant of the stevia rebaudiana is in the flowering phase, taking the pollination liquid obtained in the step 3) every day or at intervals of 1-3 days, and spraying the pollination liquid on the stigmas of the newly opened female parent plant;
5) collecting stevia rebaudiana seeds: after pollination, mature seeds are collected by using a gauze bag for covering plants one by one or by laying a gauze 0-50 cm away from the ground in rows.
The technical scheme of the invention is as follows: the isolation planting method of the male parent plant and the female parent plant for pollen collection comprises but is not limited to area isolation, effective isolation by means of a gauze screen for constructing a net room and the like, the isolation planting of the male parent plant aims to conveniently realize acquisition of high-purity pollen with consistent genotype by using pollen collection organisms such as Chinese bees, bumblebees and the like, and the isolation planting of the female parent plant aims to facilitate automatic operation, save pollen and improve pollination efficiency during subsequent spraying or instillation pollination.
The technical scheme of the invention is as follows: the pollen collecting method comprises the steps of manually separating pollen and collecting the pollen by using bees.
The technical scheme of the invention is as follows: the collection period of the florets used for manually separating the pollen comprises, but is not limited to the period when the height of the florets and the total bracts is equal, the tops of the florets are white and the florets are about to be opened, and any period when the pollen has germination vigor before and after the florets are opened.
The technical scheme of the invention is as follows: the period of collecting the pollen by the bee is any period when the pollen has germination vigor after the florets are opened.
The technical scheme of the invention is as follows: the pollen collection time is 6 in the morning: 00-6 in the afternoon: 00;
the technical scheme of the invention is as follows: the bee includes but not limited to various bee organisms with stevia rebaudiana powder collection capability, such as Chinese bee, bumble bee, and the like.
The technical scheme of the invention is as follows: the pollen collected by the bees is automatically shed when the bees are in the nest by using tools including but not limited to a pollen shedding plate and the like.
The technical scheme of the invention is as follows: the low-temperature preservation temperature of the pollen is-252 ℃ to 4 ℃.
The technical scheme of the invention is as follows: the mass ratio of the D-sodium erythorbate to the polyethylene glycol 4000 is 0.01-10: 0.05 to 50.
The technical scheme of the invention is as follows: the pollen concentration in the pollen suspension buffer solution is 20-100 particles/microliter.
The technical scheme of the invention is as follows: the liquid instillation or liquid spraying pollination period is carried out every day or 1-3 days after the stevia rebaudiana female parent plant enters the flowering period (small flowers are opened).
The technical scheme of the invention is as follows: the pollination time by liquid instillation or liquid spraying is 6: 00-6 in the afternoon: 00;
the technical scheme of the invention is as follows: the stevia rebaudiana seed collecting method is characterized in that mature seeds are collected in a mode that gauze bags are used for bagging plant by plant or gauze is laid in a line spacing place by 0-50 cm and the like, the aperture of each gauze bag or gauze is 0.1-2.0mm, and the seeds falling on the gauze can be harvested by adopting machinery including but not limited to a dust collector and the like.
The technical scheme of the invention is as follows: the seeds can be harvested once at certain time intervals in the seed harvesting period, and can also be harvested once after all the seeds are mature and all the seeds are vibrated to fall on a gauze or a mesh bag by adopting a vibration plant method.
According to the stevia rebaudiana directional hybrid seed production method, a platform can be provided for pollen directional collection based on assistance of manpower, robots, mechanical arms, bees and the like, pollen collection convenience is improved, directional collection of target pollen is realized, and pollen purity is guaranteed.
According to the stevia rebaudiana directional hybrid seed production method, the leaves of male parent plants can be harvested in time after pollen is collected, waste of the leaves of the male parent plants in the production of stevia rebaudiana hybrid seeds is effectively avoided, and thus the production cost of the stevia rebaudiana hybrid seeds is reduced.
According to the directional hybridization seed production method of stevia rebaudiana, the activity of excellent male parent pollen is guaranteed, the waste of high-quality pollen resources is reduced, and the application of the excellent pollen resources in hybridization breeding is promoted;
according to the method for directional hybrid seed production of stevia rebaudiana provided by the invention, the production of the stevia rebaudiana hybrid seed is completely free from the control of the florescence consistency of parents, the free combination between the parents is realized, and the technical guarantee is provided for the production of more excellent combined hybrid seeds;
according to the directional hybridization seed production method of stevia rebaudiana, the complex components required by pollen buffer solution preparation in the traditional method are reduced, and the pollination success rate and the popularization of the technology are improved;
according to the method for directional hybridization seed production of stevia rebaudiana provided by the invention, the influence of high-temperature and high-humidity adverse environment in the flowering phase on the pollen activity in the traditional method is reduced, and the stability and controllability of the production of the stevia rebaudiana hybrid seed are improved;
according to the directional hybridization seed production method of stevia rebaudiana, the probability of directionally receiving pollen of a male parent by florets of female parent plants is improved, and the hybridization seed setting rate of the florets of the female parent plants is improved;
according to the directional hybridization seed production method of stevia rebaudiana provided by the invention, the mixing of seeds from male parent plants is thoroughly eliminated, the purity of hybrid seeds is ensured, and the planting benefit of farmers and the processing benefit of subsequent extraction and processing enterprises are ensured;
according to the method for directional hybrid seed production of stevia rebaudiana provided by the invention, conditions are created for harvesting stevia rebaudiana seeds used in combination with machinery at a later stage, loss caused by scattering of early mature seeds everywhere and labor expenditure in harvesting the stevia rebaudiana hybrid seeds are reduced, and the production benefit of the stevia rebaudiana hybrid seeds is improved;
according to the directional hybridization seed production method of the stevia rebaudiana, the harvest frequency can be reduced on the premise of ensuring the harvest quantity of the seeds, the labor expenditure is reduced, and the production benefit is improved;
according to the directional hybridization seed production method of stevia rebaudiana provided by the invention, one-time harvest after all seeds are completely mature is realized, the consistency of the maturity of the seeds is ensured, and the germination rate of the seeds is improved.
According to the directional hybridization seed production method of the stevia rebaudiana, the following problems in the seed production of the stevia rebaudiana hybrid seeds are mainly solved:
1) the invention adopts the isolated planting of the male parent, thereby providing a platform for the high-efficiency and high-purity collection of pollen resources.
2) The invention adopts the low-temperature preservation of the pollen, thereby ensuring the pollen activity and promoting the effective application of excellent pollen germplasm resources.
3) The invention adopts isolated planting of female parent, which provides conditions for automatic operation in late stage pollination liquid spraying or liquid drop hybridization and improves the hybridization utilization efficiency of pollen.
4) The invention provides a simple pollen buffer solution preparation method, thereby improving the generalization of the technology and ensuring the pollination success rate.
5) Because the invention adopts manual work, robot or mechanical arm to assist multiple directional pollination, the probability that the female parent florets receive the pollen of the male parent is improved, and the hybridization seed setting rate is improved.
6) The invention adopts the isolated planting of the female parent, thereby avoiding the mixing of seeds formed by taking male parent plants as the female parent and effectively ensuring the purity of hybrid seeds.
7) Because the invention adopts the isolated planting of the female parent, conditions are created for harvesting the hybrid stevia rebaudiana seeds by machinery in the later period.
8) Because the invention adopts the isolated planting of the female parent, conditions are created for collecting mature seeds in the later period by uniformly laying a gauze 0-50 cm away from the ground in rows and the like.
9) Because the invention adopts the modes of laying gauze among lines and collecting mature seeds, the invention creates conditions for harvesting stevia rebaudiana seeds by machinery in the later period.
In conclusion, due to the adoption of the stevia rebaudiana directional hybridization seed production scheme, the method has the beneficial effects that:
the invention breaks through the limitation of the seed production mode of the traditional stevia rebaudiana hybrid seeds, provides a directional hybrid seed production method, and provides a new direction for the production process of the plant hybrid seeds which comprise stevia rebaudiana, are self-fruitless, have inconsistent seed maturity, light seeds, have crown hairs and are easy to scatter around along with wind after maturation; the utilization efficiency of pollen of the male parent can be improved, and the problem that the production of hybrid seeds is limited by the consistency of the florescence of the male parent and the female parent and the influence of high-temperature and high-humidity environment on the activity of the pollen and the like is solved; the purity, the maturity consistency, the germination rate and the like of the hybrid seeds can be guaranteed; the problems of high artificial dependence, unstable seed quality and the like in the production of stevia hybrid seeds can be solved; the production cost can be reduced, and the seed production benefit of seed production enterprises can be improved; high-quality seeds can be produced, and the planting benefit of downstream farmers and the processing benefit of extraction processing enterprises are guaranteed; the application of related scientific achievements and agricultural machinery in the production of stevia hybrid seeds can be promoted better; can better play the role of the stevia products in improving the health of human bodies and is beneficial to the sustainable and healthy development of the stevia industry.
The advantages of the modern stevia hybrid seed production process are mainly embodied in the following aspects:
1) by isolated planting of male parent plants, a platform is provided for pollen directional collection based on assistance of manpower, robots, mechanical arms, bees and the like, pollen collection convenience is improved, directional collection of target pollen is realized, and pollen purity is guaranteed;
2) by the isolated planting of the male parent plant, the leaves of the male parent plant can be harvested in time after the collection of the florets and the pollen of the male parent plant is finished, so that the waste of the leaves of the male parent plant in the production of the stevia hybrid seeds is effectively avoided, and the production cost of the stevia hybrid seeds is reduced;
3) by storing the pollen at low temperature for a long time, the activity of the excellent male parent pollen is ensured, the waste of high-quality pollen resources is reduced, and the application of the excellent pollen resources in hybrid seed production is promoted;
4) through long-term low-temperature preservation of pollen, the production of stevia hybrid seeds is thoroughly free from the restriction of the florescence consistency of parents, the free combination between the parents is realized, and the technical guarantee is provided for the production of more excellent combined hybrid seeds;
5) by storing the pollen at low temperature for a long time, the influence of high-temperature, high-humidity and adverse environment in the flowering phase on the pollen activity in the traditional method is reduced, and the stability and controllability of the stevia hybrid seed production are improved;
6) the simplified preparation method of the pollen buffer solution is provided, the technical risk caused by multiple components is reduced, and the technical stability and the popularization are improved;
7) the controllable directional repeated pollination assisted by manpower, robots and mechanical arms improves the probability that the florets of the female parent plant directionally receive the pollen of the male parent, and improves the hybridization seed setting rate;
8) through the physical isolation planting of the female parent plant, the mixing of seeds from the male parent plant is thoroughly eliminated, the purity of hybrid seeds is effectively guaranteed, the planting benefit of a field at the later stage is improved, and the planting benefit of farmers and the processing benefit of a subsequent extraction processing enterprise are guaranteed;
9) mature seeds are collected by the modes of paving gauze screens among rows and the like, conditions are created for the harvest of stevia rebaudiana seeds used in combination with machinery at the later stage, the labor expenditure in the harvest of stevia rebaudiana hybrid seeds and the loss caused by the scattering of the early mature seeds are reduced, and the production benefit of the stevia rebaudiana hybrid seeds is improved;
10) because the modes of covering the gauze bags one by one or paving the gauze in lines and the like are utilized, the harvesting frequency can be reduced on the premise of ensuring the harvesting quantity, the labor expenditure in the harvest of the stevia rebaudiana hybrid seeds is reduced, and the production benefit of the stevia rebaudiana hybrid seeds is improved;
11) due to the fact that the gauze bags are sleeved one by one or gauze is laid among lines, all seeds are harvested at one time after being completely mature, the uniformity of the maturity of the seeds is guaranteed, and the germination rate of the seeds is improved.
Detailed Description
The invention is further illustrated by the following examples, without limiting the scope of the invention:
example 1
A method for directional hybridization seed production of stevia rebaudiana comprises the following steps:
the used male parent material is Nannong 1066 cutting propagation seedling, and the female parent material is Cannon No. 1 cutting propagation seedling.
(1) Collecting florets: after the south nong 1066 plant enters flowering stage, 10: before 00, picking up and fixing unrooted florets with liquid nitrogen, and storing at-80 ℃ for later use;
(2) preparation of pollen suspension buffer: weighing the following substances, 10 parts of D-sodium erythorbate, 50 parts of polyethylene glycol 4000 and deionized water which is 10 times of the total mass of the two substances to obtain a pollen suspension buffer solution for later use.
(3) Preparation of pollination liquid: taking the preserved florets obtained in the step (1), freezing, grinding and crushing, adding a pollen suspension buffer solution according to the weight ratio of 1:50, homogenizing under an ice bath condition, filtering by four layers of gauze, passing through a filter screen with the aperture of 0.1mm, standing for 30min, carefully removing a supernatant, and adjusting the pollen concentration to 50 per microliter by using the pollen suspension buffer solution to obtain a pollination solution;
(4) artificial pollination: after the sweet nong No. 1 plant enters the florescence, dripping the pollen pollination liquid prepared in the step (3) onto the small stylobate, wherein the dripping time is selected from 6: 00-10 in the morning: 00 and 3 in the afternoon: 00-6: 00, pollinating once a day from the initial flowering stage until all the small flowers are opened.
(5) Bagging: before and after artificial pollination, nylon mesh bags with the aperture of 0.8 mm are used for bagging isolation, and the maturing rate is investigated after the seeds are mature.
Comparative examples 1 to 1
The difference from example 1 is that the artificial pollination method is to pollinate the full bloom florets with pollen pollination liquid by a micro sprayer, and the rest operation is the same as example 1.
Comparative examples 1 to 2
The difference from example 1 is that the pollen suspension buffer contains only sodium D-erythorbate.
Comparative examples 1 to 3
The difference from example 1 is that the pollen suspension buffer contains only polyethylene glycol 4000.
Comparative examples 1 to 4
The difference from example 1 is that the pollen suspension buffer contains 20 parts of D-sodium erythorbate and 50 parts of polyethylene glycol 4000
Comparative examples 1 to 5
The difference from example 1 is that the pollen suspension buffer contains: 20 parts of cane sugar, 10 parts of glucose, 4 parts of boric acid, 7 parts of guar gum, 2 parts of soybean lecithin, 1.5 parts of dodecyl hydroxypropyl phosphate betaine, 1 part of potassium nitrate, 1 part of ammonium chloride and 0.5 part of choline chloride
And (5) naturally pollinating a control group.
The following table shows the plant seed setting rate obtained by each method, and the results show that the directional hybridization seed production method of stevia rebaudiana can greatly improve the hybridization seed setting rate.
Example 2
A method for directional hybridization seed production of stevia rebaudiana comprises the following steps:
the used male parent material is Nannong 1066 cutting propagation seedling, and the female parent material is Cannon No. 1 cutting propagation seedling.
(1) Collecting florets: after the south nong 1066 plant enters flowering stage, 10: before 00, picking up and fixing unrooted florets with liquid nitrogen, and storing at-120 ℃ for later use;
(2) preparation of pollen suspension buffer: weighing the following substances, 1 part of D-sodium erythorbate, 50 parts of polyethylene glycol 4000 and deionized water which is 15 times of the total mass of the two substances to obtain a pollen suspension buffer solution for later use.
(3) Preparation of pollination liquid: taking the preserved florets obtained in the step (1), freezing, grinding and crushing, adding a pollen suspension buffer solution according to the weight ratio of 1:50, homogenizing under an ice bath condition, filtering by four layers of gauze, passing through a filter screen with the aperture of 0.1mm, standing for 30min, carefully removing a supernatant, and adjusting the pollen concentration to 60 per microliter by using the pollen suspension buffer solution to obtain a pollination solution;
(4) artificial pollination: after the sweet nong No. 1 plant enters the florescence, dripping the pollen pollination liquid prepared in the step (3) onto the small stylobate, wherein the dripping time is selected from 6: 00-10 in the morning: 00 and 3 in the afternoon: 00-6: 00, pollinating once a day from the initial flowering stage until all the small flowers are opened.
(5) Bagging: before and after artificial pollination, nylon mesh bags with the aperture of 0.8 mm are used for bagging isolation, and the maturing rate is investigated after the seeds are mature.
Comparative example 2-1
The difference from the example is that the artificial pollination method is to pollinate the full bloom florets by pollen pollination liquid by a micro sprayer, and the rest operation is the same as the example 2.
Comparative examples 2 to 2
The difference from example 2 is that the pollen suspension buffer contains only sodium D-erythorbate.
Comparative examples 2 to 3
The difference from example 2 is that the pollen suspension buffer contains only polyethylene glycol 4000.
Comparative examples 2 to 4
The difference from example 2 is that the pollen suspension buffer contains 20 parts of D-sodium erythorbate and 50 parts of polyethylene glycol 4000
Comparative examples 2 to 5
The difference from example 1 is that the pollen suspension buffer contains: 20 parts of cane sugar, 10 parts of glucose, 4 parts of boric acid, 7 parts of guar gum, 2 parts of soybean lecithin, 1.5 parts of dodecyl hydroxypropyl phosphate betaine, 1 part of potassium nitrate, 1 part of ammonium chloride and 0.5 part of choline chloride
And (5) naturally pollinating a control group.
The following table shows the plant seed setting rate obtained by each method, and the results show that the directional hybridization seed production method of stevia rebaudiana can greatly improve the hybridization seed setting rate.
Example 3
A method for directional hybridization seed production of stevia rebaudiana comprises the following steps:
the used male parent material is Nannong 1066 cutting propagation seedling, and the female parent material is Cannon No. 1 cutting propagation seedling.
(1) Collecting florets: after the south nong 1066 plant enters flowering stage, 10: before 00, picking up and fixing unrooted florets with liquid nitrogen, and storing at-200 ℃ for later use;
(2) preparation of pollen suspension buffer: weighing the following substances, 10 parts of D-sodium erythorbate, 1 part of polyethylene glycol 4000 and deionized water which is 20 times of the total mass of the two substances to obtain a pollen suspension buffer solution for later use.
(3) Preparation of pollination liquid: taking the preserved florets obtained in the step (1), freezing, grinding and crushing, adding a pollen suspension buffer solution according to the weight ratio of 1:50, homogenizing under an ice bath condition, filtering by four layers of gauze, passing through a filter screen with the aperture of 0.1mm, standing for 30min, carefully removing a supernatant, and adjusting the pollen concentration to 80 per microliter by using the pollen suspension buffer solution to obtain a pollination solution;
(4) artificial pollination: after the sweet nong No. 1 plant enters the florescence, dripping the pollen pollination liquid prepared in the step (3) onto the small stylobate, wherein the dripping time is selected from 6: 00-10 in the morning: 00 and 3 in the afternoon: 00-6: 00, pollinating once a day from the initial flowering stage until all the small flowers are opened.
(5) Bagging: before and after artificial pollination, nylon mesh bags with the aperture of 0.8 mm are used for bagging isolation, and the maturing rate is investigated after the seeds are mature.
Comparative example 3-1 is different from the example in that the artificial pollination method is to pollinate full-bloom florets with pollen pollination liquid by a microsprayer, and the rest of the operation is the same as that of example 3.
Comparative examples 3 to 2
The difference from example 2 is that the pollen suspension buffer contains only sodium D-erythorbate and the rest of the procedure is the same as example 2.
Comparative examples 3 to 3
The difference from example 2 is that the pollen suspension buffer contains only PEG 4000, and the rest of the procedure is the same as in example.
Comparative examples 3 to 4
The difference from example 3 is that 20 parts of sodium D-isoascorbate and 50 parts of polyethylene glycol 4000 are contained in the pollen suspension buffer, and the rest of the procedure is the same as in example.
Comparative examples 3 to 5
The difference from example 1 is that the pollen suspension buffer contains: the procedure of 20 parts of sucrose, 10 parts of glucose, 4 parts of boric acid, 7 parts of guar gum, 2 parts of soybean lecithin, 1.5 parts of dodecyl hydroxypropyl phosphate betaine, 1 part of potassium nitrate, 1 part of ammonium chloride and 0.5 part of choline chloride was repeated.
And (5) naturally pollinating a control group.
The following table shows the plant seed setting rate obtained by each method, and the results show that the directional hybridization seed production method of stevia rebaudiana can greatly improve the hybridization seed setting rate.
Claims (3)
1. A method for directional hybridization seed production of stevia rebaudiana by pollination liquid is characterized in that: the method comprises the following steps:
1) carrying out isolated planting on male parent plants and female parent plants;
2) collecting and preserving pollen: collecting pollen in the flowering period of the male parent, and storing the obtained pollen at a low temperature;
3) preparation of pollination liquid: weighing the following substances in parts by weight: d-sodium erythorbate, polyethylene glycol 4000 and deionized water which is 10-20 times of the total mass of the D-sodium erythorbate and the polyethylene glycol 4000 are uniformly stirred to obtain a pollen suspension buffer solution; adding pollen into the suspension buffer solution to prepare a pollen suspension buffer solution to obtain pollination solution;
4) liquid spraying or liquid dripping pollination: when the female parent plant of the stevia rebaudiana is in the flowering phase, taking the pollination liquid obtained in the step 3) every day or at intervals of 1-3 days, and spraying the pollination liquid on the stigmas of the newly opened female parent plant;
5) collecting stevia rebaudiana seeds: after pollination, collecting mature seeds by using a gauze bag to sleeve plants one by one or by laying a gauze 0-50 cm away from the ground in a row mode;
wherein: the mass ratio of the D-sodium erythorbate to the polyethylene glycol 4000 is 1: 5. 1:50 or 10: 1.
2. the method of directional hybridization breeding of stevia rebaudiana as claimed in claim 1, wherein: the low-temperature preservation temperature is-252 ℃ to 4 ℃.
3. The method of directional hybridization breeding of stevia rebaudiana as claimed in claim 1, wherein: the pollen concentration is 20-100 particles/microliter pollen suspension buffer solution.
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| CN102318549B (en) * | 2011-08-16 | 2012-07-25 | 南京农业大学 | Method for saving cost and improving efficiency for liquid pollination of pear tree |
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