Artificial hybridization method for cultivating new yellow peony varieties by inter-subgroup induced pollination

Abstract

The invention discloses an artificial hybridization method for cultivating a new yellow peony variety through guided pollination between subgroups. The method utilizes the flowering period inheritance of a fleshy flower disc subgroup, freezes and stores pollen due to its strong desiccation resistance to solve the problem of different flowering periods between subgroups, removes part of petals to promote ovary development, and treats guided pollen through ultraviolet irradiation to carry out artificial hybridization breeding between subgroups, fully taps the genetic material of yellow germplasm resources in the fleshy flower disc subgroup, solves the problem that the flowering periods of existing cultivated varieties are similar and yellow varieties are scarce in existing cultivated varieties, creatively completes the germplasm innovation of the new yellow peony variety, and has significant social and economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of peony breeding, in particular to an artificial hybridization method for cultivating a new yellow peony variety through inter-subgroup induced pollination. Background Art

[0002] Peony belongs to the Paeoniaceae family, Paeonia genus, Paeonia section ( Sect. Moutan DC. ), the group is divided into leathery disc subgroup and fleshy disc subgroup. The discs of each species in the leathery disc subgroup are leathery and cup-shaped, almost completely covering the carpels. There are five species in total, namely Yangshan peony, ovate peony, purple-spotted peony, Sichuan peony and dwarf peony; the discs of various species in the fleshy disc subgroup are fleshy and triangular, only covering the base of the carpels. There are four species in total, namely yellow peony, large-flowered yellow peony, purple peony and narrow-leaved peony. Among them, yellow peony, narrow-leaved peony and purple peony are also called Yunnan peony.

[0003] The wild species of peony are all native to China. After introduction and breeding, they spread to all parts of the world. After more than 1,000 years of natural selection and artificial cultivation, there are now more than 1,200 cultivated varieties. In the evolutionary history of peony, due to the influence of population distribution, geographical environment and human factors, the fleshy disc subgroup within the peony group has basically had no relationship with the leathery disc subgroup for a long time. Except for a very few varieties, most of the existing ornamental peony varieties are formed by hybridization evolution within the leathery disc subgroup, and the base of the fleshy disc subgroup is not involved. Peony has nine major flower colors. There are many red, purple, pink and white varieties, and fewer black, blue and green varieties. In particular, yellow varieties are very rare. Currently, only the American Sea Yellow, Golden Island and Japanese Yellow Crown are pure yellow in garden ornamental applications. The fleshy disc subgroup peony has the characteristics of late flowering, long group flowering period, rare yellow and orange, etc., and is a very good genetic resource. Artificial hybridization of yellow peony, large-flowered yellow peony and leathery disc varieties in the fleshy disc subgroup is a new way to breed new yellow peony varieties. However, there are problems in artificial hybridization between peony subgroups, such as inconsistent flowering period, stigma not recognizing heterologous pollen, and selective pollen growth disorder. How to use the yellow peony germplasm resources of the fleshy disc subgroup, carry out artificial hybridization between subgroups, expand the gene range of yellow peony genetic material, create new yellow peony germplasm, solve the problem that the flowering period of existing cultivated varieties is similar and the yellow varieties are scarce in existing cultivated varieties, and breed new yellow peony varieties has become an urgent issue to be solved. Summary of the invention

[0004] The present invention provides an artificial hybridization method for cultivating a new yellow peony variety by guided pollination between subgroups to solve the above technical problems. The method utilizes the genetic diversity of flowering period and flower color of fleshy disc subgroup germplasm resources, and overcomes the mismatch of flowering period of parent materials between subgroups by culturing, drying and freezing and storing target pollen; removes petals of pollinated flowers during the flower bud color-transmitting period to stimulate the development of ovary and improve the degree of maturity; and uses ultraviolet irradiation to form guided pollen, artificial hybridization guided pollination and other technologies to carry out hybridization breeding between subgroups, so as to overcome the difficulties of mismatch of parent flowering period, unrecognized stigma and pollen growth disorder in hybridization between peony subgroups, deeply explore the genetic diversity of yellow germplasm resources in the fleshy disc subgroup, and cultivate a new yellow peony variety.

[0005] The technical solution adopted by the present invention is: an artificial hybridization method for cultivating a new variety of yellow peony by inter-subgroup induced pollination, comprising the following steps:

[0006] Step 1: Select suitable parent material

[0007] The female parent is a rose-shaped, chrysanthemum-shaped, or crown-shaped white or yellow peony cultivar in the leathery disc subgroup, with normal stamens, partially or completely developed into petals, and pistils in normal form or slightly changed, with a certain fruiting ability and the ability to produce a small amount of seeds; the male parent is a yellow peony or a large-flowered yellow peony in the fleshy disc subgroup;

[0008] Step 2: Collect and cultivate male pollen

[0009] In the spring of the first year, select healthy male plants, select fully developed flower buds at the initial flowering stage, open the petals by hand when the petals are not open and the stamens are not mature, use tweezers to collect the stamens from the filaments, and put them into a marked and sealable container; spread the collected pollen on sulfuric acid paper, put it in a constant temperature incubator to cultivate it, so that the pollen is fully mature, and different varieties should be stored separately; put the mature pollen in a blast drying oven to dry, and wait for it to be used when the water content of the pollen drops to 10%;

[0010] Step 3: Freeze and store male pollen

[0011] The dried pollen is sieved through a 30-mesh sieve to remove filaments and debris, and the sieved pollen is weighed and divided into cryopreservation tubes, which are sealed after adding desiccant, marked, and stored at ultra-low temperature;

[0012] Step 4: Treat the pollen with ultraviolet light

[0013] In the spring of the second year, at the beginning of the peony flowering period, mature pollen of early-flowering peony varieties in the leathery disc subgroup was collected to make guided pollen, which was then air-dried or placed in a drying oven for drying, treated with ultraviolet light, and cultured on a 10% sucrose + 0.1% boric acid medium. The pollen could not germinate at all, indicating that the irradiation treatment was successful.

[0014] Step 5: Male parent pollen viability determination

[0015] 48 hours before artificial hybridization pollination, take out the cryopreservation tube of the male parent pollen, thaw it quickly, dip the pollen and culture it on 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is greater than 20%, it proves that the pollen has germination ability after frozen storage and can be used.

[0016] Step 6: Mix the stored male parent pollen with the ultraviolet-treated Mongolian pollen in a mixing ratio of 1:1;

[0017] Step 7: Processing of the female parent flower buds

[0018] The mother plant is a rose-type, chrysanthemum-type, crown-type white or yellow peony variety in the leathery disc subgroup. When the buds are broken, the petals are slightly loose, and the stamens are immature, all the stamen petals and stamens are removed, and only the outer petals are retained and sealed in a bag in time;

[0019] Step 8: Observe the stigma and pollinate it 5 hours after it starts to secrete mucus; during pollination, apply the mixed pollen to the stigma, repeat the application on the stigma once on the second day, and quickly put it in an isolation bag and seal it after pollination;

[0020] Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

[0021] The male parent pollen collected in step 2 is cultured in a constant temperature incubator at 23°C for 24 hours; the mature pollen is placed in a 25°C forced air drying oven for drying, and is set aside when the moisture content of the pollen drops to 10%;

[0022] In step three, the male parent pollen is stored in ultra-low temperature. The pollen is packaged and stored in cryogenic tubes, sealed after adding a silica gel desiccant, placed in a -50°C refrigerator for 2 hours, and then placed in a -196°C liquid nitrogen tank. Check regularly during one year, and add liquid nitrogen in time when the residual liquid nitrogen amount is 1 / 3 of the total amount.

[0023] In step 4, the Mongolian pollen is irradiated with ultraviolet light using a 30W ultraviolet lamp at a distance of 0.5 meters for 60 hours.

[0024] In step 5, the male parent pollen is quickly thawed after ultra-low temperature freezing storage by placing the cryotube in running water for 2 hours and placing it in a 4-5°C refrigerator for use.

[0025] The pollination time in step eight is between 9 and 11 am.

[0026] Among them, in step three, the pollen is ultra-low temperature stored by ultra-low temperature storage, which means that the desiccation resistance of peony pollen is fully utilized to store the biological material in an environment of -80°C to -196°C. Ultra-low temperature can greatly slow down the metabolism and aging process of pollen, maintain the stability of organisms, and inhibit the physiological metabolic intensity to the maximum extent. The life metabolic activities are almost stopped, which can ensure the relatively stable vitality of pollen for a long time and effectively extend the storage time of pollen. The appropriate temperature can achieve a very good storage effect. The pollen after storage has a strong germination power, which can effectively preserve the pollen of wild peonies and effectively solve the problem of different flowering periods among peony subgroups.

[0027] Among them, step 4 uses ultraviolet irradiation to treat the guided pollen, which can improve the microphysiological environment of the stigma, confuse the recognition effect of the stigma, provide specific recognition substances for the male parent pollen, and reduce the obstruction of the stigma-specific secretions to the heterologous pollen.

[0028] Among them, in step 7, the female parent selects rose-type, chrysanthemum-type, crown-type white or yellow peony varieties in the leathery disc subgroup, removes the stamen petal and all stamens when the buds are broken and the petals are slightly loose, and only retains the outer petals; this is because the petals grow and develop rapidly at this stage, and the competition pressure on the ovary and stigma is the greatest, and the buds develop to a moderate size at this time, which is easy to operate without hurting the stigma, and promotes the ovary to develop more completely; if it is too early, the buds are small and difficult to operate, and the proportion of wounds formed is too large, and improper operation will cause the growth and development of the buds to stagnate. The outer petals are retained to protect the carpels and stigma, and they are not directly exposed to the external environment, so as not to affect the later growth and development of the ovary and cause accidental damage to the carpels and stigma.

[0029] Wherein the pollination time in step eight is at 9-11 o'clock in the morning every day, because this stage is the best time for pollination in peony hybrid breeding work, and is the vigorous time of stigma secretion mucus in one day. Pollination is delayed to 5h after stigma starts to secrete mucus, because experiments have shown that delayed pollination can utilize the identification of guided pollen to effectively overcome the recognition mechanism of stigma, improve the germination ability of target pollen, and preferred pollen is easily stimulated by mucus to germinate and form pollen tube, enter ovary, and complete fertilization process. The 2nd day needs to repeat pollination 1 time, in order to improve the success rate of guided pollination. Rapidly putting isolation bag after pollination is to prevent wound from getting sick, avoids the entry of external miscellaneous pollen.

[0030] The beneficial effects of the present invention are:

[0031] The invention utilizes yellow peony germplasm resources in the fleshy disc subgroup unique to my country, overcomes the problem of inconsistent flowering periods of parent materials between subgroups through pollen cultivation and frozen storage; removes part of the petals when the petals are fluffy during the bud stage to the early flowering stage, which is easy to operate and does not damage the stigma, thereby promoting more complete development of the ovary; treats the pollen by ultraviolet irradiation, and can significantly improve the microphysiological environment of the stigma during guided pollination, confuse the recognition effect of the stigma, and simultaneously provide specific recognition substances for the pollen of the male parent, reduce the obstruction of the specific secretion of the stigma to the heterologous pollen, and other technologies to carry out hybrid breeding between subgroups, fully explore the genetic materials of the yellow germplasm resources in the fleshy disc subgroup, cultivate new yellow peony varieties, solve the problem that the flowering periods of existing cultivated variety groups are similar and the yellow varieties in the existing cultivated varieties are scarce, creatively complete the germplasm innovation of the new yellow peony varieties, and have significant social and economic benefits. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The artificial hybridization method of the present invention is used to carry out inter-subgroup induced pollination to cultivate a new yellow peony variety, and peonies with strong growth potential, luxuriant branches and leaves, and strong branches and trunks are selected as test materials. The female parent is a rose-type, chrysanthemum-type, or crown-type white or yellow peony cultivar in the leathery disc subgroup, and the male parent is a yellow peony or a large-flowered yellow peony in the fleshy disc subgroup for hybridization. The specific implementation methods of the present invention are further described in detail below in conjunction with examples.

[0034] Example 1

[0035] An artificial hybrid breeding method for cultivating late-flowering peonies by inter-subgroup induced pollination comprises the following steps:

[0036] Step 1: Select suitable parent material

[0037] The female parent is the Jingyu peony, a cultivar in the Zhongyuan peony cultivar group of the leathery disc subgroup. It blooms very early, has a crown-shaped flower, is pure white in color, has no purple spots on the petals, has a high flowering rate, and has strong branching ability. Only a few stamens are normal, partially or fully developed into petals, and the pistils are normal or slightly changed, and can produce a small amount of seeds; the male parent is the large-flowered yellow peony in the fleshy disc subgroup, with pure color and strong growth potential.

[0038] Step 2: Collect and cultivate male pollen

[0039] In the spring of the first year, healthy large-flowered yellow peony plants were selected, and fully developed buds were selected during the initial flowering period. When the petals were not open and the stamens were immature, the petals were opened by hand, and the stamens were collected from the filaments with tweezers and placed in a marked and closable container; the collected pollen was spread on sulfuric acid paper and placed in a 23°C constant temperature incubator for 24 hours to fully mature the pollen, and different varieties should be stored separately; the mature pollen was placed in a 25°C blast drying oven for drying, and the pollen was set aside when the water content dropped to 10%;

[0040] Step 3: Freeze and store male pollen

[0041] The dried pollen was sieved through a 30-mesh sieve to remove filaments and debris. The sieved pollen was weighed and divided into cryopreservation tubes. One silica gel desiccant was added and sealed. The tubes were marked and placed in a -50℃ refrigerator for 2 hours. The tubes were then placed in a -196℃ liquid nitrogen tank. The tubes were checked regularly throughout the year. When the residual amount of liquid nitrogen was 1 / 3 of the total amount, liquid nitrogen was added in time.

[0042] Step 4: Treat the pollen with ultraviolet light

[0043] In the early flowering period of the peony in spring of the second year, mature pollen of the early-flowering variety White Fengdan in the leathery disc subgroup was collected to make guided pollen, which was dried in the air or placed in a drying oven. It was irradiated with a 30W ultraviolet lamp at 0.5 meters for 60 hours and cultured on a 10% sucrose + 0.1% boric acid medium. The pollen did not germinate at all, proving that the irradiation treatment was successful.

[0044] Step 5: Male parent pollen viability determination

[0045] 48 hours before artificial hybridization pollination, take out the cryopreserved tubes in the liquid nitrogen tank, place them in running water for 2 hours, put them in a 4-5℃ refrigerator, dip the pollen and culture them on a 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is >20%, it proves that the pollen has germination ability after frozen storage and can be used;

[0046] Step 6: Mix the stored male parent pollen with the ultraviolet-treated Mongolian pollen in a mixing ratio of 1:1;

[0047] Step 7: Processing of the female parent flower buds

[0048] The mother plant is the Jingyu peony variety in the Zhongyuan cultivar group. When the petals are fluffy at the bud break stage, all the stamen petals and stamens are removed, and only the outer petals are retained and sealed in a bag in time;

[0049] Step 8: Observe the stigma and pollinate 5 hours after it starts to secrete mucus. The pollination time is between 9 and 11 am. During pollination, apply mixed pollen to the stigma. Repeat the application of the stigma once on the second day. After pollination, quickly put on the isolation bag.

[0050] Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

[0051] Example 2

[0052] An artificial hybrid breeding method for cultivating late-flowering peonies by inter-subgroup induced pollination comprises the following steps:

[0053] Step 1: Select suitable parent material

[0054] The female parent is the cultivar Yaohuang peony in the Zhongyuan peony cultivar group, which is known as the "king of flowers". It is yellow and creamy yellow, with a crown-shaped flower shape, which is very elegant and gorgeous. However, its stamens are partially or completely developed into petals, and the pistils are slightly or partially changed, and basically no seeds are produced. Our patented technology has enabled it to produce a small amount of normal seeds; the male parent is the yellow peony in the fleshy disc subgroup, with pure color and a small number of petals;

[0055] Step 2: Collect and cultivate male pollen

[0056] In the spring of the first year, healthy yellow peony plants were selected, and fully developed buds were selected during the initial flowering period. When the petals were not opened and the stamens were immature, the petals were opened by hand, and the stamens were collected from the filaments with tweezers and placed in a marked and closable container; the collected pollen was spread on sulfuric acid paper and placed in a 23°C constant temperature incubator for 24 hours to fully mature the pollen, and different varieties should be stored separately; the mature pollen was placed in a 25°C blast drying oven for drying, and the pollen was set aside when the water content dropped to 10%;

[0057] Step 3: Freeze and store male pollen

[0058] The dried pollen was sieved through a 30-mesh sieve to remove filaments and debris. The sieved pollen was weighed and divided into cryopreservation tubes. One silica gel desiccant was added and sealed. The tubes were marked and placed in a -50℃ refrigerator for 2 hours. The tubes were then placed in a -196℃ liquid nitrogen tank. The tubes were checked regularly throughout the year. When the residual amount of liquid nitrogen was 1 / 3 of the total amount, liquid nitrogen was added in time.

[0059] Step 4: Treat the pollen with ultraviolet light

[0060] In the spring of the second year, mature pollen from the early-flowering variety Peony White Fengdan in the leathery disc subgroup was collected as guide pollen. After being air-dried or dried in a drying oven, it was irradiated with a 30W ultraviolet lamp at 0.5 meters for 60 hours and cultured on a 10% sucrose + 0.1% boric acid medium. The pollen did not germinate at all, proving that the irradiation treatment was successful.

[0061] Step 5: Male parent pollen viability determination

[0062] 48 hours before artificial hybridization pollination, take out the cryopreserved tubes in the liquid nitrogen tank, place them in running water for 2 hours, put them in a 4-5℃ refrigerator, take out the pollen and culture it on a 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is >20%, it proves that the pollen has germination ability after frozen storage and can be used;

[0063] Step 6: Mix the stored male parent pollen with the ultraviolet-treated Mongolian pollen in a mixing ratio of 1:1;

[0064] Step 7: Processing of the female parent flower buds

[0065] The mother plant is selected from the Yaohuang peony variety in the Zhongyuan cultivar group. When the petals are fluffy during the bud break stage, all the stamen petals and stamens are removed, leaving only the outer 1-2 rounds of large petals and then bagging and sealing in time;

[0066] Step 8: Observe the stigma and pollinate 5 hours after it starts to secrete mucus. The pollination time is between 9 and 11 am. During pollination, apply mixed pollen to the stigma. Repeat the application of the stigma once on the second day. After pollination, quickly put on the isolation bag.

[0067] Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

[0068] Example 3

[0069] An artificial hybrid breeding method for cultivating late-flowering peonies by inter-subgroup induced pollination comprises the following steps:

[0070] Step 1: Select suitable parent material

[0071] The female parent is the cultivar Chuehuanghuang Mudan in the Zhongyuan Peony cultivar group, with light yellow flowers, hard petals, upright plant shape, normal stamens or a small number of petals, normal pistils or slightly smaller pistils, and can produce seeds; the male parent is the yellow peony in the fleshy disc subgroup;

[0072] Step 2: Collecting pollen from the male parent

[0073] In the spring of the first year, healthy yellow peony plants were selected, and fully developed buds were selected during the initial flowering period. When the petals were not opened and the stamens were immature, the petals were opened by hand, and the stamens were collected from the filaments with tweezers and placed in a marked and closable container; the collected pollen was spread on sulfuric acid paper and placed in a 23°C constant temperature incubator for 24 hours to fully mature the pollen, and different varieties should be stored separately; the mature pollen was placed in a 25°C blast drying oven for drying, and the pollen was set aside when the water content dropped to 10%;

[0074] Step 3: Freeze and store male pollen

[0075] The dried pollen was sieved through a 30-mesh sieve to remove filaments and debris. The sieved pollen was weighed and divided into cryopreservation tubes. One silica gel desiccant was added and sealed. The tubes were marked and placed in a -50℃ refrigerator for 2 hours. The tubes were then placed in a -196℃ liquid nitrogen tank. The tubes were checked regularly throughout the year. When the residual amount of liquid nitrogen was 1 / 3 of the total amount, liquid nitrogen was added in time.

[0076] Step 4: Treat the pollen with ultraviolet light

[0077] In the spring of the second year, mature pollen from the early-flowering variety Peony White Fengdan in the leathery disc subgroup was collected as guide pollen. After being air-dried or dried in a drying oven, it was irradiated with a 30W ultraviolet lamp at 0.5 meters for 60 hours and cultured on a 10% sucrose + 0.1% boric acid medium. The pollen did not germinate at all, proving that the irradiation treatment was successful.

[0078] Step 5: Male parent pollen viability determination

[0079] 48 hours before artificial hybridization pollination, take out the cryopreserved tubes in the liquid nitrogen tank, place them in running water for 2 hours, put them in a 4-5℃ refrigerator, dip the pollen and culture them on a 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is >20%, it proves that the pollen has germination ability after frozen storage and can be used;

[0080] Step 6: Mix the stored male parent pollen with the ultraviolet-treated Mongolian pollen in a mixing ratio of 1:1;

[0081] Step 7: Processing of the female parent flower buds

[0082] The mother plant is selected from the goose yellow peony variety in the leathery disc subgroup of the original cultivar group. When the petals are fluffy at the bud break stage, all the stamens and stamens are removed, and only the outer petals are retained and sealed in a bag in time.

[0083] Step 8: Observe the stigma and pollinate 5 hours after it starts to secrete mucus. The pollination time is between 9 and 11 am. During pollination, apply mixed pollen to the stigma. Repeat the application of the stigma once on the second day. After pollination, quickly put on the isolation bag.

[0084] Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

[0085] Example 4

[0086] An artificial hybrid breeding method for cultivating late-flowering peonies by inter-subgroup induced pollination comprises the following steps:

[0087] Step 1: Select suitable parent material

[0088] The female parent is the Baiyupan peony, a cultivar in the Zhongyuan peony cultivar group, with lotus-shaped flowers, pure white color, wide and long petals, long pedicels, large number of flowers, vigorous growth, normal stamens or a small number of petals, and normal or slightly changed pistils, which can produce seeds; the male parent is the large-flowered yellow peony in the fleshy disk subgroup;

[0089] Step 2: Collecting pollen from the male parent

[0090] In the spring of the first year, healthy large-flowered yellow peony plants were selected, and fully developed buds were selected during the initial flowering period. When the petals were not open and the stamens were immature, the petals were opened by hand, and the stamens were collected from the filaments with tweezers and placed in a marked and closable container; the collected pollen was spread on sulfuric acid paper and placed in a 23°C constant temperature incubator for 24 hours to fully mature the pollen, and different varieties should be stored separately; the mature pollen was placed in a 25°C blast drying oven for drying, and the pollen was set aside when the water content dropped to 10%;

[0091] Step 3: Freeze and store male pollen

[0092] The dried pollen was sieved through a 30-mesh sieve to remove filaments and debris. The sieved pollen was weighed and divided into cryopreservation tubes. One silica gel desiccant was added and sealed. The tubes were marked and placed in a -50℃ refrigerator for 2 hours. The tubes were then placed in a -196℃ liquid nitrogen tank. The tubes were checked regularly throughout the year. When the residual amount of liquid nitrogen was 1 / 3 of the total amount, liquid nitrogen was added in time.

[0093] Step 4: Treat the pollen with ultraviolet light

[0094] In the spring of the second year, mature pollen from the early-flowering variety Peony White Fengdan in the leathery disc subgroup was collected as guide pollen. After being air-dried or dried in a drying oven, it was irradiated with a 30W ultraviolet lamp at 0.5 meters for 60 hours and cultured on a 10% sucrose + 0.1% boric acid medium. The pollen did not germinate at all, proving that the irradiation treatment was successful.

[0095] Step 5: Male parent pollen viability determination

[0096] 48 hours before artificial hybridization pollination, take out the cryopreserved tubes in the liquid nitrogen tank, place them in running water for 2 hours, put them in a 4-5℃ refrigerator, dip the pollen and culture them on a 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is >20%, it proves that the pollen has germination ability after frozen storage and can be used;

[0097] Step 6: Mix the stored male parent pollen with the ultraviolet-treated Mongolian pollen in a mixing ratio of 1:1;

[0098] Step 7: Processing of the female parent flower buds

[0099] The mother plant is the Baiyupan peony variety in the Zhongyuan cultivar group. When the petals are fluffy during the bud break stage, all the stamen petals and stamens are removed, and only the outer petals are retained and sealed in a bag in time;

[0100] Step 8: Observe the stigma and pollinate 5 hours after it starts to secrete mucus. The pollination time is between 9 and 11 am. During pollination, apply mixed pollen to the stigma. Repeat the application of the stigma once on the second day. After pollination, quickly put on the isolation bag.

[0101] Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

Claims

1. An artificial hybridization method for cultivating a new variety of yellow peony by inter-subgroup induced pollination, characterized in that: The following steps are involved: Step 1: Select suitable parent material The female parent is Jingyu peony in the leathery disc subgroup, and the male parent is large-flowered yellow peony in the fleshy disc subgroup; or the female parent is Yaohuang peony in the leathery disc subgroup, and the male parent is yellow peony in the fleshy disc subgroup; or the female parent is Chuehuang peony in the leathery disc subgroup, and the male parent is yellow peony in the fleshy disc subgroup; or the female parent is Baiyupan peony in the leathery disc subgroup, and the male parent is large-flowered yellow peony in the fleshy disc subgroup; Step 2: Collect and cultivate male pollen In the spring of the first year, healthy male plants were selected. When the buds were in their early flowering stage, fully developed buds were selected. When the petals were not open and the stamens were immature, the petals were opened by hand, and the stamens were collected from the filaments with tweezers and placed in a marked and closable container. The collected pollen was spread on sulfuric acid paper and placed in a 23°C constant temperature incubator for 24 hours to fully mature the pollen. Different varieties should be stored separately. The mature pollen was placed in a 25°C forced air drying oven for drying and was set aside when the water content of the pollen dropped to 10%. Step 3: Freeze and store male pollen The dried pollen was passed through a 30-mesh sieve to remove filaments and debris. The sieved pollen was weighed and divided into cryopreservation tubes. One silica gel desiccant was added and sealed. The tubes were marked and placed in a -50℃ refrigerator for 2 hours. The tubes were then placed in a -196℃ liquid nitrogen tank for ultra-low temperature freezing. The tubes were checked regularly throughout the year. When the residual amount of liquid nitrogen was 1 / 3 of the total amount, liquid nitrogen was added in time. Step 4: Treat the pollen with ultraviolet light In the spring of the second year, at the beginning of the flowering period of the peony, mature pollen of the early-flowering peony varieties in the leathery disc subgroup was collected to prepare the guided pollen, which was then air-dried or placed in a drying oven for drying, and irradiated with ultraviolet light at a distance of 0.5 meters using a 30W ultraviolet lamp for 60 hours. The pollen was cultured on a 10% sucrose + 0.1% boric acid medium, and the pollen could not germinate at all, indicating that the irradiation treatment was successful. Step 5: Male parent pollen viability determination 48 hours before artificial hybridization pollination, take out the cryopreservation tube of the male parent's pollen, put it in running water for 2 hours, thaw it quickly, put it in a 4-5℃ refrigerator for later use, dip the pollen and culture it on 10% sucrose + 0.1% boric acid medium. If the pollen germination rate is greater than 20%, it proves that the pollen has germination ability after frozen storage and can be used; Step 6: Mix the stored male parent pollen with the UV-treated Mongolian pollen in a mixing ratio of 1:1; Step 7: Processing of the female parent flower buds The mother plant is Jingyu peony, Yaohuang peony, young goose yellow peony or Baiyupan peony. When the buds are broken, the petals are slightly loose, and the stamens are immature, all the stamens are removed, and only the outer petals are retained and sealed in time. Step 8: Observe the stigma and pollinate it 5 hours after it starts to secrete mucus; during pollination, apply the mixed pollen to the stigma, repeat the application on the stigma once on the second day, and quickly put it in an isolation bag and seal it after pollination; Step 9. Open the breeding isolation bag 8-10 days after pollination and observe the growth and development of the ovary. If the ovary is obviously enlarged, it proves that fertilization has been successful and the ovules have developed. Continue to strengthen the water and fertilizer management of the mother plant until seed harvesting and sowing.

2. The artificial hybridization method for cultivating a new variety of yellow peony by inter-subgroup induced pollination according to claim 1, characterized in that: The pollination time in step eight is between 9 and 11 am.