Artificial pollination method and sexual breeding method for radix ophiopogonis

Abstract

The invention provides an artificial pollination method and a sexual breeding method for radix ophiopogonis, and belongs to the technical field of traditional Chinese medicine breeding. The invention provides an artificial pollination method of radix ophiopogonis and a sexual reproduction breeding method of radix ophiopogonis based on the artificial pollination method of radix ophiopogonis, which comprises the following steps: firstly, selecting blooming plants of different varieties (strains), which are more than biennial, as male parents and female parents; secondly, selecting non-flowering II-stage male parents, bagging, selecting I-stage female parent flower buds, removing peeled petals, removing stamens, and bagging; and then, after the female parent flower buds without stamens bloom, uncovering the bags, pollinating the blooming female parent with the pollen of the blooming male parent ophiopogon japonicus, and directionally breeding the hybrid seeds of the new variety of ophiopogon japonicus with high yield, good resistance and excellent quality through an artificial hybridization technology. Hybrid seedlings are cultivated through the seeds, the heterosis of different dwarf lilyturf varieties (lines) is kept by utilizing the tillering reproductive capacity of the dwarf lilyturf, and the method can be widely popularized and applied in production.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine breeding, and particularly relates to an artificial pollination method and a sexual breeding method for Ophiopogon japonicus. Background Art

[0002] Ophiopogon japonicus is the dried root of Ophiopogon japonicus (Lf) Ker-Gawl., a plant of the Liliaceae family. It is a commonly used bulk root medicinal material. It was first recorded in Shennong's Herbal Classic and listed as a top grade. It is a variety included in the Chinese Pharmacopoeia. It has the effects of nourishing yin and moistening the lungs, clearing the heart and removing troubles, and benefiting the stomach and producing body fluids. It is mainly used to treat dry cough due to lung dryness, vomiting blood, hemoptysis, pulmonary tuber, lung abscess, asthenia, fever, thirst, damage to body fluids due to fever, dry throat and dry mouth, and constipation. At present, most of the Ophiopogon japonicus tubers circulating in the Chinese herbal medicine market are cultivated products, mainly from Sichuan and Zhejiang. The Ophiopogon japonicus produced in Zhejiang is called Hangzhou Ophiopogon japonicus, which is concentrated in Cixi, Yuyao and Xiaoshan in the Hangzhou Bay area of ​​southeastern Zhejiang. The Ophiopogon japonicus produced in Sichuan is called Sichuan Ophiopogon japonicus, which is mainly cultivated in Mianyang and Santai in the Fujiang River Basin.

[0003] In current cultivation, Ophiopogon japonicus is propagated asexually by division, and has long been self-propagated and self-sown, resulting in a single variety. At the same time, the existing varieties are degenerating, such as the reduction of disease resistance and changes in root characteristics, which have led to a decline in the quality and yield of Sichuan Ophiopogon japonicus, restricting the development of the Ophiopogon japonicus industry. The existing varieties are bred with yield and resistance as the main breeding goals, and are mostly bred using conventional breeding techniques. The lack of quality breeding concepts makes it difficult to reflect the quality characteristics of traditional Chinese medicine and adapt to the diversified needs of the market. There are currently three new varieties of Ophiopogon japonicus, Sichuan Ophiopogon japonicus No. 1, Sichuan Ophiopogon japonicus No. 2 and Fumai No. 1, all of which are obtained through purification and rejuvenation based on local varieties using systematic breeding technology. The breeding is not predictable, and breeding techniques are in urgent need of innovation. Summary of the invention

[0004] The invention provides an artificial pollination method and a sexual breeding method for Ophiopogon japonicus, which utilize artificially controlled pollination to purposefully aggregate the excellent traits of existing Ophiopogon japonicus germplasm, effectively solve the problem of Ophiopogon japonicus hybrid breeding, and achieve the purpose of artificial pollination breeding of Ophiopogon japonicus hybrid species.

[0005] The present invention provides an artificial pollination method for Ophiopogon japonicus, comprising the following steps: (1) emasculating and bagging the female plant's flower buds when they are swollen but not yet opened;

[0006] (2) On the day of flowering but before the anthers open, pick the male parent flower, collect and dry the pollen;

[0007] (3) Evenly pollinate the dried and preserved pollen onto the stigma of the fully opened flower of the bagged mother plant.

[0008] In a preferred embodiment of the present invention, the emasculation in step (1) includes selecting the middle and lower flowers of each inflorescence for emasculation one day before or on the day when the flowers of the female plant Ophiopogon japonicus are to bloom; during the emasculation, all stamens are removed to avoid damaging the stigma, ovary and petals.

[0009] In a preferred embodiment of the present invention, the emasculation also includes removing excess inflorescences.

[0010] In a preferred embodiment of the present invention, step (2) comprises refrigerating and drying the picked male parent flowers in a clean indoor environment, collecting pollen after most anthers have cracked, and placing the collected pollen in a cryopreservation tube for drying and storage;

[0011] The dry preservation comprises placing the cryopreservation tube containing the pollen in a desiccator for drying, and placing the desiccator in a 4° C. environment for storage.

[0012] In a preferred embodiment of the present invention, in step (3), the dried pollen is taken out 1 hour before the pollination, gradually restored to room temperature, and then artificial pollination is performed.

[0013] In a preferred embodiment of the present invention, the pollination time in step (3) is after 11:00.

[0014] In a preferred embodiment of the present invention, the pollination includes direct pollination and pollination using the suspension after the pollen is mixed with a culture solution to form a suspension;

[0015] The culture solution includes components with the following concentrations: 75g / L-100g / L sucrose, 10mg / L-50mg / L H3BO3, 50mg / L-100mg / L Ca(NO3)2·4H2O and 150mg / L-300mg / L KNO3, with a pH of 7-8.

[0016] The present invention also provides application of the artificial pollination method in sexual reproduction and breeding of Ophiopogon japonicus.

[0017] The present invention provides a method for sexual reproduction and breeding of ophiopogon japonicus, comprising the following steps: selecting a flowering ophiopogon japonicus variety or strain of two years or more as a parent, using the above-mentioned artificial pollination method to pollinate the stigma of a female parent with pollen taken from the male parent, collecting seeds after culturing until seed formation, and testing and storing the seeds;

[0018] After sowing, raising seedlings and planting qualified seeds, the characteristics and quality of the hybrids are evaluated, and tillering propagation is carried out for individual plants that meet the expected characteristics.

[0019] In a preferred embodiment of the present invention, the pollination time is from early to mid-May to mid-June.

[0020] Beneficial effects: The present invention provides an artificial pollination method for Ophiopogon japonicus, and a sexual reproduction and breeding method for Ophiopogon japonicus created based on the artificial pollination method for Ophiopogon japonicus. First, two-year-old or older flowering plants of different varieties (lines) are selected as male and female parents; secondly, the male parent of stage II is selected for bagging, and the flower buds of the female parent of stage I are selected, the petals are peeled off, the stamens are removed, and the bagging is performed; then, 2 to 3 days after the stamens are removed from the flower buds of the female parent, the bagging is opened, and the flowering female parent is pollinated with pollen of the flowering male parent Ophiopogon japonicus every day, and hybrid seeds of a new variety of Ophiopogon japonicus with high yield, good resistance and excellent quality are selectively bred through artificial hybridization technology. Hybrid seedlings are cultivated through seeds, and the heterosis between different Ophiopogon japonicus varieties (lines) is maintained by utilizing the tillering reproduction ability of Ophiopogon japonicus, and can be widely promoted and applied in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The flower morphology of Ophiopogon japonicus in stages I, II and III;

[0022] Figure 2 This is a diagram of pollen germination and stigma activity at different times. From left to right and from top to bottom, it represents 07:00, 09:00, 11:00, 13:00, 15:00, 17:00 and 19:00;

[0023] Figure 3 This is a picture of the fruit setting of hybrid Ophiopogon japonicus. DETAILED DESCRIPTION

[0024] The present invention provides an artificial pollination method for Ophiopogon japonicus, comprising the following steps: (1) emasculating and bagging the female plant's flower buds when they are swollen but not yet opened;

[0025] (2) On the day of flowering but before the anthers open, pick the male parent flower, collect and dry the pollen;

[0026] (3) Evenly pollinate the dried and preserved pollen onto the stigma of the fully opened flower of the bagged mother plant.

[0027] In the present invention, the flowers of the Ophiopogon japonicus are characterized by being numerous and small, and the flowering period of a single flower of the Ophiopogon japonicus is 1 day, and the petals are closed and persistent after flowering. The flowers of the Ophiopogon japonicus are divided into stages, such as Figure 1 As shown, the buds of Ophiopogon japonicus are swollen, the petals are not open or slightly open, and they are slightly hard when pinched (different from the opened flowers that are soft and open when pinched). The flowers at this time are in stage I; on the day of flowering, the flowers are half open and the anthers are not split, which is called stage II; after 15:00 on the day of flowering, the petals unfold and the stigma is most receptive, which is stage III.

[0028] The present invention emasculates the female parent flower. On the day before or on the day when the female parent Ophiopogon japonicus flower is about to bloom, about 5 flowers with proper development in the middle and lower parts and in the first stage are selected from each inflorescence. The harder ones are unopened buds when gently pinched by hand, and the soft ones are flowers that have been opened. The lower part of the flower is pinched with the index finger and thumb of the left hand, and the tip of the pointed tweezers is inserted into the center of the flower. The flower is slightly opened with force to expose the stamens, and then the stamens are gently clamped and pulled out with tweezers. The direction of force during pulling out is consistent with the direction of flower growth. All six stamens are pulled out to avoid damaging the stigma and ovary, and petals are not damaged. The petals are guaranteed to be in a normal state, and the redundant inflorescence is cut off. The inflorescence is covered with a sulfuric acid paper bag, and the bag mouth is sealed with a paper clip for standby use. The emasculation is completed.

[0029] The present invention picks flowers from male parent plants, and then uses the flowers to collect pollen. In the present invention, the stamens of Ophiopogon japonicus generally split and spread pollen at 9:30-13:00. During this time, healthy male parent flowers in flower phase II are selected and picked, brought back indoors, placed in a clean uncovered culture dish, placed in a 1-4°C environment for cold storage and drying, taken out after most anthers split, pollen is shaken off, and pollen is collected in a clean cryopreservation tube, the bottle cap is tightly closed, a label is attached to the outside of the tube, indicating the collection time, place, and provenance, and then the centrifuge tube containing the pollen is placed in a small desiccator and placed in a 4°C refrigerator for storage and standby use.

[0030] The present invention uses pollen collected from male plants to artificially pollinate the emasculated female plants. Before the artificial pollination, for example, the pollen is taken out 1 hour before artificial hybridization and gradually restored to room temperature, and then directly pollinated. The pollen can also be mixed with a culture solution, mixed into a suspension, and then pollinated. The culture solution includes the following components at the following concentrations: 75g / L to 100g / L sucrose, 10mg / L to 50mg / L H3BO3, 50mg / L to 100mg / L Ca(NO3)2·4H2O and 150mg / L to 300mg / L KNO3, pH 7 to 8. In one embodiment, the composition of the culture solution is: 50g / L sucrose, 10mg / L H3BO3, 100mg / L Ca(NO3)2·4H2O and 300mg / L KNO3.

[0031] Whether the present invention is direct pollination or suspension pollination, generally a brush or cotton swab is used to dip the pollen or suspension, and then the pollen is evenly applied to the stigma of the emasculated female parent flower in the III stage, and the flowers are pollinated one by one, bagged, and hung with tags for marking, indicating the names of the male and female parents and the pollination date. The flowers that are not successfully pollinated fall off on the 2nd to 3rd day, and the ovary begins to swell 4 to 5 days after the successful pollination, and the petals remain, and the bag is removed for registration. The pollination time of the present invention is generally after 11:00, and 11:00-13:00 on sunny days. During this period, the germination rate of Ophiopogon japonicus pollen is high, and the receptivity of the pistil is relatively strong; after 15:00, the stigma has strong receptivity and can be maintained until the flower is closed.

[0032] The present invention also provides application of the artificial pollination method in sexual reproduction and breeding of Ophiopogon japonicus.

[0033] The present invention can construct artificial hybridization technology through artificial pollination method, and selectively breed hybrid seeds of new varieties of Ophiopogon japonicus with high yield, good resistance and excellent quality. Hybrid seedlings are cultivated from seeds, and the heterosis between different Ophiopogon japonicus varieties (lines) is maintained by utilizing the tillering reproduction ability of Ophiopogon japonicus, and can be widely promoted and applied in production.

[0034] The present invention provides a method for sexual reproduction and breeding of ophiopogon japonicus, comprising the following steps: selecting a flowering ophiopogon japonicus variety or strain of two years or more as a parent, using the above-mentioned artificial pollination method to pollinate the stigma of a female parent with pollen taken from the male parent, collecting seeds after culturing until seed formation, and testing and storing the seeds;

[0035] After sowing, raising seedlings and planting qualified seeds, the characteristics and quality of the hybrids are evaluated, and tillering propagation is carried out for individual plants that meet the expected characteristics.

[0036] In the present invention, two-year-old or older flowering plants of different varieties (lines) are used as male parents (♂) or female parents (♀) for field artificial hybridization, and the hybridization time is from early to mid-May to mid-June during the flowering period.

[0037] After the artificial pollination, the present invention carries out reasonable management of hybrid plants, timely fertilization and watering, and prevention of diseases and insect pests. The field management method refers to the Sichuan Province (Mianyang City) local standard "Geographical Indication Product Fucheng Ophiopogon Part 4: Planting Technology Regulations". The present invention picks seeds in the second year after artificial pollination, such as around Qingming Festival in the second year, picks seeds, tests and stores them; then sows, raises seedlings, and plants qualified seeds respectively, observes the growth of hybrid offspring under normal cultivation and management conditions, and then selects plants according to breeding goals, and evaluates yield and quality; finally, the preferred single plants that meet the target traits are propagated by tillering to maintain hybrid vigor for further application.

[0038] In order to further illustrate the present invention, an artificial pollination method and a sexual breeding method for Ophiopogon japonicus provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Investigate the morphological characteristics and flowering period of single flower of Ophiopogon japonicus

[0041] Survey method: Select 10 spikes of plants with consistent growth and strong growth, mark them with signs, observe them once a day until the end of the flowering period, randomly select 10 single flowers from each inflorescence, and record the state of the flowers, the opening process, and the changes in the stamens and pistils during each observation. Randomly select 30 ophiopogon scapes, measure the length of the scape, the length of the part with flowers on the scape, and the number of flowers on each scape, randomly select 30 flowers to directly measure the length and width of the inner and outer perianth pieces, and randomly select 30 flowers to measure the flower diameter and the length of the anther style.

[0042] The scape is significantly shorter than the leaves, 19.82±2.54cm long; 26.45±4.66 flowers are borne on it, the flowering part of the scape is 5.54±0.57cm long, the pedicel is 3.21±0.41mm long, and the joint is located above or near the middle; the flowers are solitary or in pairs in the axils of the bracts. The bracts are lanceolate, with acuminate tips; the tepals are lanceolate, the outer tepals are 4.47±0.27mm long, and the inner tepals are 4.09±0.38mm long, white or lavender; the anthers are triangular-lanceolate, 2.93±0.27mm long; the style is 3.32±0.26mm long, relatively thick, wide at the base, and gradually narrows upwards. The scape blooms for about 20 days, starting from the lower and middle lower parts of the scape, and each scape blooms 0-5 flowers per day. After flowering, the ovary begins to swell in 4-5 days, and the petals remain. Most flowers fall off after blooming, which may be due to external forces or unsuccessful fertilization. The flowering period of a single flower of Ophiopogon japonicus is 1 day.

[0043] 2. Investigate pollen germination and stigma activity at different times

[0044] The results are as follows Figure 2 As shown in the figure, Ophiopogon japonicus opens at around 09:00, and the pollen is already active at 7:00 before flowering, with a pollen germination rate of 13.10%. The pollen has relatively high activity from 11:00 to 17:00, with a germination rate of more than 20%. The stigma receptivity on the day of flowering is low from 7:00 to 9:00, high from 11:00 to 13:00, and strong after 15:00, which can be maintained until the flower closes.

[0045] Table 1 Pollen viability and stigma receptivity during the development of Ophiopogon japonicus

[0046]

[0047]

[0048] In Table 1, “-” indicates no grantability, “+ / -” indicates partial grantability, “+” indicates low grantability, “++” indicates higher grantability, and “+++” indicates the strongest grantability.

[0049] 3. Investigate the fruit setting rate of artificial pollination

[0050] The artificial pollination fruit setting rate of Ophiopogon japonicus germplasm was counted for two consecutive years. Each combination of Ophiopogon japonicus germplasm materials included wild purple flower material No. 48, Sichuan Ophiopogon japonicus was Sichuan Ophiopogon japonicus No. 1 Sichuan Pharmaceutical 2010003, and Zhejiang Ophiopogon japonicus was a conventional variety. Figure 3 As shown, the pollination situation under natural conditions: the fruit setting rate of Sichuan Ophiopogon No. 1 without emasculation, bagging, and free pollination is 21.94%, and the highest fruit setting rate is 48.57% in the intraspecific hybridization combination of ♂48×♀ Sichuan Ophiopogon No. 1. When Sichuan and Zhejiang Ophiopogon are hybridized, the fruit setting rate is low when Sichuan Ophiopogon is the male parent, only 5.26%, and the fruit setting rate is improved when Zhejiang Ophiopogon is the male parent, which is 14.06%.

[0051] 4. Investigate the fruit setting rate of direct pollination and suspension pollination

[0052] Material No. 48 was selected as the male parent and Sichuan Ophiopogon No. 1 was selected as the female parent for direct pollination and suspension culture pollination. 50 flowers were treated in each process, and the fruit setting rate was examined after one week. The experimental results showed that the direct pollination method was simple and convenient, with a fruit setting rate of 48.57%. The suspension culture pollination steps were cumbersome, and the fruit setting rate was significantly improved to 57.14%. At the same time, it can solve the problem of different varieties flowering periods. It is expected that new varieties of Ophiopogon with high yield and good quality can be bred from the hybrid plants of Ophiopogon.

[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for artificial pollination of Ophiopogon japonicus, characterized in that: The following steps are involved: (1) When the female plant's flower buds are swollen but not yet open, the buds are emasculated and bagged; (2) On the day of flowering but before the anthers open, pick the male parent flower, collect and dry the pollen; (3) Evenly pollinate the dried and preserved pollen onto the stigma of the fully opened flower of the bagged mother plant.

2. The artificial pollination method according to claim 1, characterized in that: The detasseling in step (1) includes selecting the middle and lower flowers of each inflorescence for detasseling one day before or on the day when the flowers of the female plant Ophiopogon japonicus are to bloom; when detasseling, all stamens are removed to avoid damaging the stigma, ovary and petals.

3. The artificial pollination method according to claim 2, characterized in that: The detasseling also includes removing excess inflorescences.

4. The artificial pollination method according to claim 1, characterized in that: Step (2) comprises refrigerating and drying the picked male parent flowers in a clean indoor environment, collecting pollen after most anthers have cracked, and placing the collected pollen in a cryopreservation tube for drying and storage; The dry preservation comprises placing the cryopreservation tube containing the pollen in a desiccator for drying, and placing the desiccator in a 4° C. environment for storage.

5. The artificial pollination method according to claim 1, characterized in that: Step (3) takes out the dried pollen 1 hour before the pollination, gradually returns to room temperature, and then performs artificial pollination.

6. The artificial pollination method according to claim 1 or 5, characterized in that: The pollination time in step (3) is after 11:

00.

7. The artificial pollination method according to claim 6, characterized in that: The pollination includes direct pollination and pollination using the suspension after the pollen is mixed with the culture solution to form a suspension; The culture solution includes components with the following concentrations: 75g / L-100g / L sucrose, 10mg / L-50mg / L H3BO3, 50mg / L-100mg / L Ca(NO3)2·4H2O and 150mg / L-300mg / L KNO3, with a pH of 7-8.

8. Use of the artificial pollination method according to any one of claims 1 to 7 in sexual reproduction and breeding of Ophiopogon japonicus.

9. A method for sexual reproduction and breeding of Ophiopogon japonicus, characterized in that: The following steps are involved: Select a flowering ophiopogon japonicus variety or strain that is two years old or older as a parent, use the artificial pollination method described in any one of claims 1 to 7 to pollinate the stigma of the female parent with pollen taken from the male parent, collect seeds after culturing until seed formation, and test and store the seeds; After sowing, raising seedlings and planting qualified seeds, the characteristics and quality of the hybrids are evaluated, and tillering propagation is carried out for individual plants that meet the expected characteristics.

10. The sexual reproduction breeding method according to claim 9, characterized in that: The pollination time is from early to mid-May to mid-June.

Images