Quinoa female first-ripening female parent-based flowering phase synergy and compound pollination two-line hybridization method

Abstract

The invention discloses a flowering phase synergy and compound pollination two-line hybridization method based on quinoa female first-ripening female parents, and belongs to the technical field of crop breeding and hybrid seed production. According to the method, quinoa plants with the female first-ripening characteristic serve as female parents, and the pollen scattering period of the male parents and the pollen receiving window period of the female parents are overlapped through cooperative adjustment of the flowering periods of the male parents; when florets of the female parent are in a full blooming state, stigma branches are exposed, transparent mucus appears on the surfaces of the stigma branches, and corresponding anther does not crack, it is judged that the female parent is in an optimal pollination window period; hybrid seeds are obtained by combining wind-medium supplementary pollination, male parent pollen collection and short-time preservation and supplementary pollination of a pollinator in the period. The female parent has self-pollination and fruiting capabilities, so that the invention belongs to a quinoa two-line hybridization method.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding and hybrid seed production technology, specifically involving a method for carrying out two-line hybridization using quinoa as the female promatonic parent, and more particularly involving a method for obtaining hybrid seeds based on female promatonic phenotypic identification, pollination window determination, synergistic regulation of male parent flowering period, natural wind-assisted pollination, male parent pollen collection and short-term preservation, pollinator-assisted supplementary pollination, and pollination. Background Technology

[0002] Quinoa has high nutritional value, wide ecological adaptability and good industrial development prospects. Hybrid breeding is an important technical approach to combine high yield, high quality and stress resistance and create new germplasm.

[0003] However, quinoa florets are small and inflorescence structures are complex, which often leads to problems in conventional artificial hybridization, such as difficulty in identifying pollination windows, difficulty in emasculation, high labor intensity, easy damage to floral organs, unstable seed setting rate, and low efficiency in population combination construction.

[0004] Among the existing publicly available methods, there are hybridization techniques that utilize the protisthemorrhagic characteristic of quinoa. For example, the method in publication number CN108901832A utilizes the characteristic of the female parent maturing before the stamens to achieve artificial pollination hybridization without emasculation. Such methods are of positive significance in simplifying the emasculation process, but in the context of mass seed production, they may still face problems such as insufficient continuity of male parent pollination, inadequate wind pollination coverage, and low efficiency of relying entirely on artificial re-pollination per flower or per ear.

[0005] Through long-term breeding practice, the inventors discovered a type of quinoa material with stable or relatively stable protandry characteristics. This type of material is not a male-sterile line; rather, after the florets open, the pistils become pollinating before the stamens, and it can still produce seeds through self-pollination without cross-pollination. Therefore, this type of material is suitable as a maternal parent in two-line hybridization.

[0006] Further research indicates that the promatogenic female parent suitable for the application of this invention typically possesses one or more of the following characteristics: First, the florets are fully open when they enter the pollination stage; second, the perianth segments are missing, degenerate, small, or do not obscure the pollination structure at the tip of the pistil, facilitating identification and pollination; third, two or three exposed filamentous pollination structures are usually visible at the tip of the pistil, botanically corresponding to stigma branches or stigma lobes, functionally similar to the pollination sites of corn silks; fourth, the appearance of transparent mucus on the surface of the stigma branches indicates pistil maturity, while the dehiscence of the anthers, opening of the pollen sacs, or entry into a pollen-shedding state indicates stamen maturity; fifth, the flowering process is relatively consistent among inflorescences on the same plant or among plants in the same batch; sixth, in some embodiments, the female parent plant is shorter than or no taller than the male parent plant, which is more conducive to pollen migration and assisted pollination.

[0007] Furthermore, after pollen falls onto the surface of the stigma branches, it germinates to form pollen tubes. The pollen tubes grow downward along the style and enter the ovary and ovules to complete fertilization. Therefore, the stigma branches are not only an important morphological marker for determining whether the parent plant has entered the pollination stage, but also the main pollination site when artificial pollination and wind-assisted pollination are carried out.

[0008] Therefore, it is still necessary to provide a systematic two-line hybridization method that focuses on the female-maturing maternal parent of quinoa, takes into account flowering period coordination, population pollination efficiency, natural wind pollination assistance, and artificial supplementary pollination. Summary of the Invention

[0009] The purpose of this invention is to provide a two-line hybridization method based on quinoa female pre-mature parent with coordinated flowering and compound pollination, in order to solve the problems of difficulty in determining the pollination window, low efficiency of relying entirely on individual flower artificial pollination, insufficient pollination coverage of the population, and unstable seed setting rate in existing quinoa hybridization. It is especially suitable for large-scale two-line hybridization seed production and hybridization combination construction of quinoa populations.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] S1. Selection of maternal parent: Select quinoa plants, strains or materials with female prematurity as maternal parents; the florets of the maternal parent are fully open during the pollination stage, with missing, degenerated, or small perianth segments or minimal obstruction of the stigma branches, which facilitates observation of the pollination structure at the pistil tip and pollination operations; the maternal parent is not a male-sterile line, but has the ability to self-pollinate and produce fruit.

[0012] S2. Male parent configuration and flowering period coordination: Select male parent varieties, lines or materials according to the target hybridization combination, and make the pollen shedding period of the male parent overlap or partially overlap with the pollination window of the female parent through at least one method selected from synchronous sowing, staggered sowing, planting density adjustment, water and fertilizer adjustment, temperature and light environment control or a combination thereof.

[0013] S3. Determination of the pollination window of the female parent: Continuously observe the inflorescence or floret of the female parent. When the stigma branch at the tip of the pistil (functionally similar to the pollination part of corn silk) is exposed and transparent mucus appears on its surface, the pistil is determined to be pollinating active. When the anther of the corresponding floret has not yet dehisced, the pollen sac has not opened, or has not yet entered the stage of large-scale pollen shedding, the stamen is determined to be immature. The period when the pistil matures significantly earlier than the stamen is determined as the pollination window period.

[0014] S4. Natural wind-assisted pollination: During the overlapping flowering periods of the male and female parents, the pollen from the male parent is promoted to migrate to the inflorescence of the female parent through reasonable spatial layout, row orientation, planting ratio configuration, utilization of natural wind power or artificial airflow assistance, so as to form assisted pollination.

[0015] S5. Collection and short-term preservation of male parent pollen: Fresh pollen is collected at the beginning, peak, or interval of pollen shedding of the male parent; the pollen is preferably used immediately, or used after short-term preservation under conditions that maintain pollen activity.

[0016] S6. Pollinator-assisted supplementary pollination: During the pollination window of the female parent, a pollinator is used to apply the collected male parent pollen to the female parent inflorescence or floret to supplement or enhance pollination; the pollinator is one or more of the following: brushing type, blowing type, spot pollination type, and micro-spreading type device.

[0017] S7. Isolation, Marking and Harvesting: After pollination, isolate and mark the female parent inflorescences, individual plants or plots, and harvest hybrid seeds by single ear, single plant or plot after maturity.

[0018] Preferably, the "fully open state" refers to a state in which the florets are open to a large extent, the stigma branches are clearly visible, and the pollination contact interface is fully exposed.

[0019] Preferably, the flowering process of the female promatonic parent plants is highly consistent among the inflorescences of the same plant or among plants in the same batch, so as to improve the efficiency of pollination window identification and pollination operation.

[0020] Preferably, in some embodiments, the height of the female plant is lower than or no higher than that of the male plant, and the male plant is located on the upwind side of the prevailing wind, so as to facilitate pollen migration and artificial pollination.

[0021] Preferably, the short-term storage conditions for the pollen are 4-10℃, relative humidity 50-70%, and storage time not exceeding 24 hours.

[0022] Preferably, the pollinator is used for supplementary pollination within 0-48 hours after the appearance of stigma mucus, and more preferably the first supplementary pollination is completed within 24 hours.

[0023] The present invention has at least the following beneficial effects:

[0024] (1) Based on the characteristics of the female promatized mother plant, such as the florets being fully open, the perianth segments obscuring the stigma branches less, and the stigma branches having transparent mucus on their surface, the pollination window can be identified more accurately.

[0025] (2) The female pistil matures first and the stamen matures later, which can reduce or avoid complicated emasculation operations and reduce damage to the flower parts;

[0026] (3) By coordinating the flowering period of the male parent and wind-assisted pollination, the pollination coverage of the population can be improved;

[0027] (4) By collecting and storing the male parent's pollen for a short period of time and supplementing the pollination with a pollinator, pollination can be strengthened during the critical window period, thereby improving the stability of hybrid seed acquisition;

[0028] (5) The female parent retains the ability to self-pollinate and set fruit. This invention does not rely on cytoplasmic male sterile lines and maintainer lines, and is suitable for constructing a two-line hybrid seed production technology system for quinoa. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the method flow of the present invention;

[0030] Figure 2 A schematic diagram of the florets of quinoa promatifer when it enters the pollination stage;

[0031] Figure 3 A schematic diagram for determining pistil maturity;

[0032] Figure 4 This is a diagram illustrating how to determine if stamens are immature.

[0033] Figure 5 A schematic diagram illustrating the synergy of parental flowering periods and spatial configuration;

[0034] Figure 6 A schematic diagram of composite pollination using wind-assisted pollination and supplementary pollinator pollination. Detailed Implementation

[0035] Example 1: Morphological identification and screening of promatogenic female parent plants

[0036] Using over 2000 quinoa germplasm resources as the observation subjects, phenotypic trait surveys and flowering period observations were conducted for several consecutive years. The materials included conventional germplasm resources, intermediate breeding materials, segregated progeny materials, and EMS-induced mutagenesis materials. Inflorescence morphology, floret opening status, stigma branching exposure, stigma surface pollination activity, anther dehiscence, and the order of male and female maturation were continuously recorded and compared from the budding stage to the flowering stage. From this, maternal materials with protruder female characteristics were selected.

[0037] Preferably, observations are made once a day from 8:00-10:00 AM and 3:00-5:00 PM, starting from the initial flowering period, and the flowering consistency between different inflorescences on the same plant and between plants in the same batch is recorded.

[0038] The screening results showed that when a certain type of material entered the pollination stage, the florets were fully open, with missing, degenerated, or small perianth segments or minimal obstruction of the stigma branches, making it easier to observe the pollination structure at the tip of the pistil. At this time, transparent mucus was visible on the surface of the stigma branches, while the anthers had not yet dehisced or released a large amount of pollen, exhibiting obvious characteristics of promatization.

[0039] Materials with the above characteristics and good flowering uniformity were selected as candidate parent materials, and individual plants were labeled and continuously observed.

[0040] Example 2: Determination of Pollination Window

[0041] The parent candidate materials in Example 1 were observed daily starting from the initial flowering stage.

[0042] When the florets are fully open, the stigma branches are clearly exposed, and transparent mucus appears on their surface, the pistil is considered to be pollinating. If the anthers have not yet dehisced, the pollen sacs have not opened, or only a few florets have begun to shed pollen, then this period is determined to be the cross-pollination window.

[0043] If the anthers have already dehisced extensively and are in a state of heavy pollen shedding, they are no longer considered as preferred cross-pollination windows.

[0044] Example 3: Coordination of male parent flowering period and spatial configuration

[0045] Select male parent materials according to the target hybridization combination, and make the pollen shedding period of the male parent overlap with the pollination window of the female parent or form a continuous pollen supply zone by synchronous sowing, staggered sowing, adjustment of planting density, water and fertilizer adjustment or environmental adjustment.

[0046] In one embodiment, the male parent can be sown earlier to ensure that the male parent has started to supply pollen when the female parent enters the pollination window; in another embodiment, a continuous pollen belt can be formed by staggered sowing to extend the pollination time.

[0047] In field plots, parent plants can be configured in a ratio of 1:3 to 1:6 (father:mother). The father plant is preferably located on the windward side of the prevailing wind. In some implementations, the height of the father plant is higher than that of the mother plant, and the height of the mother plant is lower than or not higher than that of the father plant, in order to facilitate wind-assisted pollination and manual pollination.

[0048] Example 4: Collection and short-term preservation of paternal pollen

[0049] Collect fresh pollen from the male parent during the early or peak pollination period, preferably using it directly for re-pollination on the day of collection.

[0050] For short-term storage, pollen can be temporarily stored in a relatively clean, low-temperature and suitable humidity environment. The preferred storage conditions are 4-10℃, relative humidity 50-70%, and storage time not exceeding 24 hours.

[0051] The purpose of the aforementioned short-term preservation is to balance the time difference between the pollen shedding period of the male parent and the pollination window of the female parent, rather than to store pollen for a long period of time.

[0052] Example 5: Combined pollination using wind-assisted pollination and pollinator supplementation

[0053] In areas where the flowering periods of the parent plants overlap, natural wind, reasonable row orientation, and spatial layout are first used to promote the migration of pollen from the parent plants to the inflorescences of the parent plants, so as to form basic pollination coverage.

[0054] Meanwhile, after the female parent enters the pollination window, a pollinator is used to apply the collected male parent pollen to the female parent inflorescence or florets, forming a supplementary or enhanced pollination to natural wind-assisted pollination.

[0055] The pollinator can be a brush-type, blowing-type, spot-type, or micro-spreading type device; preferably, the re-pollination should be completed within 0-48 hours after the appearance of transparent mucus on the surface of the stigma branches, and preferably the first re-pollination should be carried out within 24 hours.

[0056] Example 6: Isolation, Harvesting, and Two-Line Hybridization System Description

[0057] After pollination, the pollinated inflorescences, individual plants, or plots are marked, isolated, and managed. Once the seeds mature, they are harvested as individual ears, individual plants, or in sections to obtain hybrid seeds.

[0058] The female promatonic parent is not a cytoplasmic male sterile line and can still obtain seeds through self-pollination without cross-pollination. Therefore, this invention does not rely on maintainer lines and restorer lines, but uses the time difference between the male and female maturation of the female promatonic parent to achieve cross-pollination, which belongs to the two-line hybridization method.

[0059] This system retains the self-propagation ability of the maternal parent and can introduce pollen from the paternal parent during the target window period, making it suitable for the construction of quinoa breeding combinations and large-scale seed production.

[0060] Example 7: Verification of Hybridization Authenticity

[0061] White-seeded quinoa material ProA was selected as the female parent, and black-seeded quinoa material was selected as the male parent. Hybridization was performed using the synergistic flowering and compound pollination method described in this invention. Continuous self-pollination observations showed that the female parent ProA exhibited stable genetic expression, and its self-pollinated offspring completely retained the characteristics of the female parent.

[0062] After planting the resulting hybrid offspring to form F1 plants, it was observed that all the seeds produced by the F1 plants at maturity were black; at the same time, the height of the F1 plants was close to that of the male parent and significantly taller than that of the female parent, and the leaf and stem colors were more similar to those of the male parent.

[0063] Since the offspring exhibit distinct paternal introgression characteristics in seed color and vegetative traits compared to the self-crossed offspring of the maternal parent, the resulting offspring can be confirmed as true hybrids. If necessary, molecular marker sampling tests can be used to further verify the authenticity of the hybridization.

[0064] Table 1. Record of the effects of different pollination treatments

[0065]

Claims

1. A two-line hybridization method based on quinoa promatized female parent with synchronized flowering and compound pollination, characterized in that, The process includes the following steps: selecting quinoa materials with pre-mature female characteristics as the female parent; adjusting the overlap or partial overlap between the pollen shedding period of the male parent and the pollination window of the female parent; during the period when the female parent's pistils are mature but the stamens are immature, using natural wind or artificial airflow to promote the migration of male parent pollen to the female parent inflorescence to form auxiliary pollination, and after collecting male parent pollen, using a pollinator to supplement pollination of the female parent inflorescence or florets; after pollination, isolating and marking the female parent inflorescences, individual plants or plots, and harvesting hybrid seeds after maturity; wherein, the florets of the female parent are in a fully open state during the pollination stage, and the stigma branches at the tip of the pistil are exposed and have pollination activity, and the female parent has the ability to self-pollinate and set fruit.

2. The method according to claim 1, characterized in that, When the parent plant enters the pollination window, the florets are in a fully open state.

3. The method according to claim 1 or 2, characterized in that, The tepals of the female parent florets are missing, degenerate, small, or do not obstruct the stigma branches as much as possible to facilitate identification and pollination.

4. The method according to any one of claims 1 to 3, characterized in that, The criteria for determining whether a pistil is pollinating include exposed stigma branches and the presence of transparent mucus on its surface.

5. The method according to any one of claims 1 to 4, characterized in that, The criteria for determining immature stamens include the anthers not being dehiscent, the pollen sacs not being opened, or not having entered the pollen shedding stage.

6. The method according to any one of claims 1 to 5, characterized in that, The methods for regulating the flowering period of the male parent include one or more of the following: synchronous sowing, staggered sowing, staggered sowing to form a continuous pollen supply zone, planting density regulation, water and fertilizer regulation, and temperature and light environment control.

7. The method according to any one of claims 1 to 6, characterized in that, The parent plants are configured in a ratio of 1:3 to 1:6, with the parent plant located on the upwind side of the prevailing wind, and in some embodiments, the parent plant is taller than the parent plant.

8. The method according to any one of claims 1 to 7, characterized in that, The pollen from the male parent is collected at the early or peak of pollen shedding and used immediately or after short-term storage at 4-10℃ and relative humidity of 50-70% for no more than 24 hours.

9. The method according to any one of claims 1 to 8, characterized in that, The pollinator is one or more of the following: brushing, blowing, spot application, and micro-spreading devices, and the pollinator is used to supplement the pollination within 0-48 hours after the appearance of transparent mucus on the surface of the stigma branches.

10. The method according to any one of claims 1 to 9, characterized in that, The flowering process of the female promatonic parent plants is consistent among the inflorescences of the same plant or among plants in the same batch, thereby improving the efficiency of pollination window identification and group pollination operation.

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