A method for quickly identifying the parthenocarpic ability of Fortunella hindsii strains

Abstract

The present invention discloses a method for rapidly identifying the parthenocarpic ability of Fortunella hindsii strains. By selecting flowers at the large bud stage with fully developed floral organs and ready to bloom during the full bloom period of Fortunella hindsii, removing the stigmas, and observing the development of the ovaries 14 - 21 days after the treatment through multiple steps, the identification of Fortunella hindsii parthenocarpic strains can be completed in the early stage of fruit development. This method omits cumbersome tasks such as bagging and separating individually, can identify Fortunella hindsii strains with parthenocarpic ability in the early stage of fruit development, greatly reduces the identification difficulty, improves the identification efficiency, and can be applied to the screening of related Fortunella hindsii varieties. At the same time, it provides effective methods and ideas for the identification of other citrus varieties and the research of seedless fruits, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of crop breeding, and particularly relates to a method for rapidly identifying the parthenocarpic ability of Fortunella hindsii strains. Background Art

[0002] According to the genetic characteristics of plants themselves, the flowering and fruiting of plants mostly go through a series of processes such as pollination and fertilization, and finally develop into fruits. In 1890, Sturtevant proposed that there is a phenomenon of seedless fruits in nature. In 1902, Noll used the term parthenocarpy to represent the production of seedless fruits without pollination or under other stimuli (Gustafson, 1942). At present, it is generally believed that parthenocarpy generally refers to the phenomenon that the ovary produces seedless fruits without pollination or without fertilization even after pollination. Parthenocarpy can be divided into two categories according to whether external stimuli are required: natural parthenocarpy and stimulative parthenocarpy. No matter which type of parthenocarpy, it ultimately does not produce seeds and forms seedless fruits.

[0003] Parthenocarpy is a very important economic and agronomic trait in horticultural crops, and it has many advantages: (1) The produced seedless fruits are convenient for eating and processing and are deeply loved by consumers; (2) Varieties with parthenocarpic ability reduce the dependence on pollinator trees, lower production costs, simplify growth conditions, and can still flower and fruit especially in greenhouses or under adverse climatic conditions. Identifying and comprehensively mastering the parthenocarpic ability of different materials is an important condition for cultivating high-quality parthenocarpic varieties.

[0004] Citrus belongs to the Rutaceae family and the Citrus genus, and is the world's largest category of fruits. Citrus generally has parthenocarpic ability, but due to its long juvenile period, highly heterozygous genome, and the presence of nucellar embryos, the relevant research process is slow. At present, there are few systematic studies on the parthenocarpic ability of Citrus germplasm resources, and there is a lack of a simple method for evaluating parthenocarpic ability. Fortunella hindsii, also known as Fortunella hindsii var. chintienensis, Fortunella margarita, etc., belongs to the Fortunella genus of the subfamily Aurantioideae of the Rutaceae family, and is a wild woody plant. The plant is short, almost a shrub or small tree, and can be used as an excellent dwarfing rootstock for Ponkan, Banana Orange, and Pomelo. China is the origin of wild Fortunella hindsii, and its wild resources are distributed in mountainous areas such as Fujian, Guangdong, and Zhejiang. Fortunella hindsii has the characteristics of early flowering and fruiting of seedlings, dwarfing, and strong resistance. After sowing by seeds, it can flower and fruit in one to two years, and there is a situation of flowering multiple times a year. Cao Hongbo, Zhu Chenqiao, etc. used Fortunella hindsii as a genetic transformation material and successfully obtained transgenic plants, showing the great potential of Fortunella hindsii as a citrus model plant.

[0005] Previous studies often screened parthenocarpic lines by emasculating and bagging a large number of citrus fruits and observing whether the fruits were seedless at maturity. Zhou X et al. investigated 113 germplasms of pomelo, orange, mandarin and their related species. By emasculating and bagging, the parthenocarpy index at fruit maturity = [parthenocarpic fruit set rate (%) + proportion of parthenocarpic fruit mass (%)] / 2 was used to estimate the autonomous parthenocarpic ability of different germplasms. However, this method is labor-intensive and time-consuming. Moreover, the flowers of Fortunella hindsii are extremely small and easily fall off, and it is difficult to set fruit under bagging conditions, which greatly increases the screening difficulty. Therefore, it is necessary to develop a method for quickly identifying the parthenocarpic ability of Fortunella hindsii lines to effectively screen natural parthenocarpic Fortunella hindsii materials.

[0006] References:

[0007] Cao Hongbo. Cytological and metabolic studies on transgenic regulation of carotenoid accumulation in citrus [D]. Huazhong Agricultural University, 2012.

[0008] Deng Xiuxin. Progress in the improvement of world citrus varieties [J]. Acta Horticulturae Sinica, 2005. (06): 1140-1146.

[0009] Zhou X, Wakana A, Kim J H, Sakai K, Kajiwara K, Mizunoe Y. Parthenocarpy in Citrus accessions with special focus on relatives of Kunenbo (C. nobilis Lour. var. kunep Tanaka). Scientia Horticulturae, 2018a. 232: 29–39. Summary of the Invention

[0010] In order to solve the defects and deficiencies in the above-mentioned prior art, the present invention provides a method for quickly identifying the parthenocarpic ability of Fortunella hindsii lines.

[0011] In order to achieve the object of the present invention, the technical solution adopted by the present invention is as follows:

[0012] In some embodiments, a method for quickly identifying the parthenocarpic ability of Fortunella hindsii lines includes the following steps:

[0013] (1) Select plant materials of Fortunella hindsii parents and hybrid offspring, and perform stigma removal treatment; (2) Count the fruit set rate of the plant materials 7-21 days after the treatment in step (1), where the fruit set rate = actual number of ovaries of the fruit tree / total number of treated flowers × 100%, and screen the plant materials according to the fruit set rate.

[0014] Further, observe the early ovary development of the plant materials screened in step (2). Those with ovaries that can grow and develop normally on the branches and show obvious swelling can set fruits. Select the materials that can set fruits. If at least one fruit in the fruits of the materials is light green in color, rounder and smaller compared to the fruits pollinated under the same conditions, it can be determined that they have parthenocarpic ability.

[0015] In some embodiments, a method for quickly identifying the parthenocarpic ability of Fortunella hindsii plant lines includes the following steps:

[0016] (1) During the full-bloom period of Fortunella hindsii, select large bud-stage flowers with fully developed floral organs and ready to bloom. Carefully peel off the petals, select flower buds with anthers not yet dehisced, remove the style and stigma, and make marks.

[0017] (2) Observe the ovary state 14 - 21 days after the treatment in step (1). Those with ovaries that can grow and develop normally on the branches and show obvious swelling can set fruits. Select the materials that can set fruits. If at least one fruit in the fruits of the materials is light green in color, rounder and smaller compared to the fruits pollinated under the same conditions, it can be determined that they have parthenocarpic ability.

[0018] Preferably, the number of styles and stigmas removed is at least 20 for each plant material treated.

[0019] Preferably, the Fortunella hindsii is a multi-year-old adult tree.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention first proposes a method for quickly and simply identifying the parthenocarpic ability of Fortunella hindsii. This method does not require bagging or separate isolation, and can identify parthenocarpic materials at the early stage of fruit development. It can be applied to the screening of related Fortunella hindsii varieties. At the same time, it provides an effective method and idea for the identification work of other citrus varieties and the research on seedless fruits, and lays a good foundation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Referring to the accompanying drawings, the disclosure of the present invention will become more understandable. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention.

[0023] Figure 1-1 is a structural diagram of Fortunella hindsii flowers;

[0024] Figure 1-2 is a schematic diagram of removing the stigma of Fortunella hindsii;

[0025] Figure 2-1 is a schematic diagram of the early development of parthenocarpic ovaries;

[0026] Figure 2-2 Schematic diagram of parthenocarpy and early ovary development of control plants (-N unpollinated, -P pollinated);

[0027] Figure 2-3 Schematic diagram of dynamic development of pollinated and unpollinated ovaries of PN01 (scale bars are all 1 cm);

[0028] Figure 3-1 Cross-sectional views of early pollinated (left) and unpollinated (right) fruits of PN01;

[0029] Figure 3-2 Cross-sectional views of pollinated (left) and unpollinated (right) fruits of PN01 at the mature stage. Detailed implementation manners

[0030] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Embodiment 1

[0032] In the germplasm resource nursery of Huazhong Agricultural University, perennial adult Fortunella hindsii population materials with good growth and no obvious pests and diseases were selected. The source and quantity information of a total of 65 parent and offspring materials are shown in Table 1.

[0033] Among them, the single-embryo resource PN of Fortunella hindsii was collected from Puning County, Guangdong Province and grafted onto Poncirus trifoliata var. sabonii rootstock, with strong growth and a large number of flowers. PN01, PN02, and PN03 were derived from different mother trees, and each had three grafted offspring as pollination mother trees.

[0034] The single-embryo resource LT02 was collected from Yongding County, Fujian Province, and LT02-4 was a self-crossed offspring seedling.

[0035] The multi-embryo resource DB02 of Fortunella hindsii was derived from Dingnan County, Jiangxi Province.

[0036] The research group previously constructed a hybrid segregation population with PN / LT as the female parent and DB02 as the male parent, and selected some materials from it as the research objects of this experiment.

[0037] Table 1 Source and quantity information of 65 materials

[0038]

[0039] During the full-bloom stage of Fortunella hindsii, on a sunny and moderately warm day, select large-bud stage flowers with fully developed floral organs and about to bloom. Carefully peel back the petals with forceps, being careful not to dislodge them. Select flower buds with anthers that have not yet shed pollen, remove the stigmas of the styles to prevent cross-pollination, and make good marks.

[0040] Count the fruit-setting rate of the ovaries every 7 days. The fruit-setting rate of the ovaries refers to the percentage of the number of ovaries remaining on the tree after removing the stigmas of the styles among the total number of treated flowers, that is, fruit-setting rate = actual number of ovaries on the fruit tree / total number of treated flowers × 100%. The early fruit-setting rates of the ovaries of the parent plants are shown in Table 2-1, those of the 1-progeny are shown in Table 2-2, those of the 2-progeny are shown in Table 2-3, those of the 3-progeny are shown in Table 2-4, and those of the 4-progeny are shown in Table 2-5.

[0041] Table 2-1 Statistical table of early fruit-setting rate of ovaries of parent plants

[0042] Serial number Number 7D 14D 21D 1 PN01 83.33％ 50.00％ 27.78％ 2 PN02 82.35％ 64.71％ 41.18％ 3 PN03 93.75％ 37.50％ 12.50％ 4 LT02-4 72.00％ 0.00％ 0.00％ 5 DB02 70.37％ 48.15％ 0.00％

[0043] Table 2-2 Statistical table of early fruit-setting rate of ovaries of 1-progeny

[0044] Serial number Number 7D 14D 21D 1 1-18 50.00％ 0.00％ 0.00％ 2 1-3 19.23％ 0.00％ 0.00％ 3 1-26 0.00％ 0.00％ 0.00％ 4 1-10 39.13％ 8.70％ 0.00％ 5 1-36 50.00％ 0.00％ 0.00％ 6 1-11 0.00％ 0.00％ 0.00％ 7 1-33 15.79％ 0.00％ 0.00％ 8 1-7 52.17％ 4.35％ 0.00％ 9 1-19 70.83％ 12.50％ 0.00％ 10 1-14 0.00％ 0.00％ 0.00％ 11 1-21 0.00％ 0.00％ 0.00％ 12 1-38 40.00％ 15.00％ 0.00％ 13 1-44 40.00％ 3.33％ 0.00％ 14 1-15 73.91％ 4.35％ 0.00％ 15 1-29 0.00％ 0.00％ 0.00％

[0045] Table 2-3 Statistical table of early fruit-setting rate of ovaries of 2-progeny

[0046]

[0047]

[0048] Table 2-4 Statistical table of early fruit-setting rate of ovaries of 3-progeny

[0049] Serial number Number 7D 14D 21D 1 3-89 0.00％ 0.00％ 0.00％ 2 3-61 38.10％ 0.00％ 0.00％ 3 3-261 0.00％ 0.00％ 0.00％ 4 3-160 45.45％ 36.36％ 0.00％ 5 3-250 69.57％ 4.35％ 0.00％ 6 3-500 25.93％ 0.00％ 0.00％ 7 3-287 0.00％ 0.00％ 0.00％ 8 3-129 45.00％ 0.00％ 0.00％ 9 3-294 13.64％ 0.00％ 0.00％ 10 3-299 0.00％ 0.00％ 0.00％ 11 3-225 44.00％ 8.00％ 0.00％ 12 3-95 0.00％ 0.00％ 0.00％ 13 3-82 40.00％ 0.00％ 0.00％ 14 3-287 54.55％ 0.00％ 0.00％ 15 3-205 0.00％ 0.00％ 0.00％

[0050] Table 2-5 Statistical table of early fruit-setting rate of ovaries of 4-progeny

[0051]

[0052]

[0053] The results show that most plants can set fruits within 7 days after treatment, with the fruit-setting rate ranging from 14% to 94%. Among them, the fruit-setting rates of PN01, PN02, and PN03 are higher than 80%. 14 days after treatment, a large number of fruits dropped, and the ovaries of nearly 66% of the plants all fell off. The fruit-setting rates of the remaining plants were 4% - 65%. And 21 days after flowering, only the ovaries of the three materials PN01, PN02, and PN03 could grow and develop normally.

[0054] Observe the dynamic development of the early ovaries of the three materials PN01, PN02, and PN03, asFigure 2-1 As shown, on the 14th day after the stigma removal treatment, the unpollinated ovaries could all grow normally and showed obvious swelling, that is, they could set fruits. The fruits produced were similar in size, round in shape, and light green in color.

[0055] During the whole development process, the unpollinated fruits of the same plant material of PN01 were always smaller than the pollinated fruits. In addition, there were also obvious differences in color. The color of the unpollinated fruits was lighter green and they matured earlier. After being cut when mature, the fruits were completely seedless; while the color of the pollinated fruits was darker and they matured later, as Figure 2-3 、 3-1 、shown in Figure 3-2.

[0056] Taking LT02-4 as a control without parthenocarpy ability, as Figure 2-2 shown, under the same conditions of stigma removal and without pollination and fertilization, the ovaries of LT02-4 did not show obvious swelling in the early stage and gradually withered and fell off at 14-21 days along with the development of the plant; while after pollination, the fruits of LT02-4 swelled and developed normally and were oval in shape.

[0057] In summary, PN01, PN02 and PN03 still set fruits 14-21 days after the stigma removal treatment, could grow and develop normally, and showed obvious swelling of the ovaries. At least one of the fruits was light green in color, round in shape and smaller. Therefore, they have the ability of parthenocarpy.

[0058] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, they can also make several substitutions or modifications to these described embodiments, and these substitution or modification methods should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for rapidly identifying the parthenocarpic ability of a fructus kumquat strain, characterized in that: The steps include: (1) When the kumquat is in full bloom, select flowers in the large bud stage with fully developed flower organs and buds about to bloom, carefully peel off the petals, select buds whose anthers have not yet shed pollen, remove the stigma, and mark them; (2) Observe the state of the ovary 21 days after the treatment in step (1). If the ovary can grow and develop normally on the branch and is obviously swollen, it is considered that the material can bear fruit. Select the material that can bear fruit. (3) Among the fruits of the material screened in step (2), at least one fruit is light green in color and is round and smaller than the fruits pollinated under the same conditions, which indicates that the material has parthenocarpic ability.

2. The method for rapidly identifying the parthenocarpy ability of axillaris strain according to claim 1, characterized in that: The number of stigmas to be removed is at least 20 for each plant.

3. A method for rapidly identifying the parthenocarpy ability of axillaris strain according to claim 1 or 2, characterized in that: The kumquats are all perennial trees.

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