Olea europaea crossbreeding method
By using pollen premix powder and conductive gel combined with ultrasonic treatment in olive hybridization breeding, the problems of low pollen activity and poor fertilization efficiency were solved, achieving efficient integration of resistance traits in olive varieties and shortening the breeding cycle.
Patent Information
- Application Number
- CN202511621215.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2025-12-19
AI Technical Summary
In olive hybridization breeding, pollen activity is low, fertilization efficiency is poor, and resistance traits are difficult to integrate. Traditional methods are time-consuming and consume a lot of manpower and resources, making it difficult to quickly obtain highly resistant varieties.
Pollen premix powder (a combination of pollen, boric acid, sucrose, GA3, and IAA) was used to activate the pollen germination signaling pathway. Conductive gel was used to form an ion channel network, which was combined with ultrasonic treatment to improve pollen tube growth rate and fertilization efficiency. A sealing agent was applied to prevent contamination, and superior male and female parents were selected.
It significantly improves fertilization efficiency and the probability of hybrid seed emergence, shortens the cultivation cycle of high-quality hybrid varieties, and rapidly obtains new olive varieties with high resistance.
Smart Images
Figure CN121153589A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant breeding technology, and in particular relates to a method for hybrid breeding of olive trees. Background Technology
[0002] Olive trees, often called the "Queen of Vegetable Oils" and internationally renowned as "fragrant soft gold," are used to produce edible olive oil, and their pulp can be made into various dried fruits. They are a world-famous woody oilseed and fruit-bearing tree species. However, most olive varieties require relatively dry growing environments and neutral to slightly alkaline soil. Chongqing has a subtropical monsoon climate with high humidity, significantly different from its native habitat. Early olive varieties, such as 'Fuo' and 'Pixiaoli,' were selected through selective breeding, but due to a lack of continued improvement efforts, the breeding program has stalled. Suitable varieties for local conditions remain relatively few. Hybrid breeding can combine the characteristics of different olive varieties to obtain olive varieties with higher resistance. However, due to the long growth cycle of olives, traditional hybrid breeding has many drawbacks. For example, it requires multiple generations of selection, usually taking more than ten years to obtain a stable variety, making it difficult to quickly respond to industry needs. The success rate of artificial hybridization is low, and large-scale screening is required to obtain the target traits (such as high oil content and stress resistance), which consumes a lot of human and material resources. The segregation of traits in hybrid offspring is complex, making it difficult to select superior plants and delaying the promotion of improved varieties. Therefore, there is an urgent need to study a fast, effective and stable hybrid breeding method to obtain olive varieties with high resistance and adaptability to the environment. Summary of the Invention
[0003] In view of this, the purpose of this invention is to provide a method for hybrid breeding of olive trees, which solves the problems of low pollen activity, poor fertilization efficiency, and difficulty in integrating resistance traits in olive hybridization.
[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for olive hybridization breeding, comprising the following steps: 1) Collect the pollen from the male parent, prepare pollen premix powder, and store at 0~6℃; 2) After coating the surface of the stigma of the mother plant with conductive gel, pollination is carried out using pollen premix powder, and then the pollinated stigma is ultrasonically treated. 3) After ultrasonic treatment, apply a column head sealant to the column head to complete artificial pollination; The pollen premix powder comprises the following components in the following mass ratio: Pollen: Boric acid: Sucrose: GA3: IAA = 80~120: 0.1~0.2: 10~15: 0.001~0.003: 0.0005~0.002; The conductive gel comprises the following components in the following mass ratio: Gellan gum: Trehalose: Boric acid: Chitosan quaternary ammonium salt: Methyl jasmonic acid: Water = 0.5~2:10~15:0.5~0.8:0.0005~0.0015:0.3~0.6:80~85.
[0005] Preferably, the parent olive variety includes Les Star.
[0006] Preferred paternal olive varieties that provide pollen include lentils.
[0007] Preferably, the sealing agent comprises components in the following mass ratio: Beeswax: Vaseline: Talc = 60~80: 20~30: 3~8.
[0008] Preferably, the degree of deacetylation of the chitosan quaternary ammonium salt is ≥90%.
[0009] Preferably, the conductive gel coating thickness is 1~3 mm.
[0010] Preferably, the ultrasonic treatment frequency is 20~40kHz, the ultrasonic intensity is 50~150W, the ultrasonic time is 5~15s, and the ultrasonic probe is 8~12cm away from the column head.
[0011] This invention provides the application of the hybridization breeding method in the cultivation of highly resistant olives.
[0012] Preferably, the resistance includes resistance to salt and alkali, resistance to moisture, resistance to peacock spot disease, leaf spot disease and / or fruit flies.
[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for hybrid breeding of olive trees. By limiting the composition of the premixed pollen, a permeable protective layer is constructed using boric acid and sucrose. GA3 (gibberellin) and IAA (auxin) synergistically activate the pollen germination signaling pathway at critical concentrations, ensuring sperm motility. A conductive gel is coated on the stigma to form an ion channel network, which, combined with a 20-40kHz ultrasonic field, generates a directional acoustic flow effect, increasing pollen tube growth rate by 2.1 times and precisely guiding it to the embryo sac, thereby improving fertilization efficiency. Then, a sealing agent is applied to the stigma after fertilization to prevent contamination by pollen from other olive varieties, increasing the probability of hybrid seed emergence. Finally, self-infertile female parents with good resistance and male parents with excellent traits are selected to further increase the probability of highly resistant hybrid olive seeds. This hybridization method significantly shortens the cultivation cycle of high-quality hybrid varieties, enabling the acquisition of highly resistant new varieties in a shorter time. Attached Figure Description
[0014] Figure 1 Image showing pollen maturation; Figure 2Image of the mother plant's flower buds; Figure 3 Image of the mother plant's flower buds; Figure 4 Image showing the application of conductive gel; Figure 5 This is an illustration of the bagging effect. Detailed Implementation
[0015] This invention provides a method for olive hybridization breeding, comprising the following steps: 1) Collect the pollen from the male parent, prepare pollen premix powder, and store at 0~6℃; 2) After coating the surface of the stigma of the mother plant with conductive gel, pollination is carried out using pollen premix powder, and then the pollinated stigma is ultrasonically treated. 3) After ultrasonic treatment, apply a column head sealant to the column head to complete artificial pollination; In this invention, the pollen premix powder comprises components in the following mass ratio: Pollen:boric acid:sucrose:GA3:IAA = 80~120:0.1~0.2:10~15:0.001~0.003:0.0005~0.002, preferably pollen:boric acid:sucrose:GA3:IAA = 90~110:0.12~0.18:11~14:0.0015~0.0025:0.0007~0.0015, and even more preferably pollen:boric acid:sucrose:GA3:IAA = 100:0.15:12:0.002:0.001.
[0016] In this invention, the parent olive variety providing the pollen preferably includes lentil, and the storage temperature of the pollen premix is 0~6℃, preferably 1~5℃, and more preferably 4℃.
[0017] In this invention, the parent olive variety preferably includes *Lagerstroemia indica*, and the conductive gel is preferably applied when there is mucus on the stigma of the parent plant. The conductive gel comprises the following components in the following mass ratio: Gellan gum: trehalose: boric acid: chitosan quaternary ammonium salt: methyl jasmonic acid: water = 0.5~2:10~15:0.5~0.8:0.0005~0.0015:0.3~0.6:80~85, preferably gellan gum: trehalose: boric acid: chitosan quaternary ammonium salt: methyl jasmonic acid: water = 0.7~1.5:11~14:0.55~0.7:0.0007~0.0012:0.4~0.55:81~84, further preferably gellan gum: trehalose: boric acid: chitosan quaternary ammonium salt: methyl jasmonic acid: water = 1:12:0.6:0.001:0.5:82.
[0018] The degree of deacetylation of the chitosan quaternary ammonium salt is preferably ≥90%.
[0019] In this invention, the thickness of the conductive gel coating is preferably 1-3 mm, more preferably 1.5-2.5 mm, and even more preferably 2 mm.
[0020] In this invention, after applying the conductive gel, pollination is performed using a premixed pollen powder, with a preferred pollination amount of 300-600 g / hm. 2 Further preferred is 400~500g / hm 2 A further preferred value is 450g / hm 2 .
[0021] In this invention, after pollination, the parent plant is ultrasonically treated. Preferably, the ultrasonic treatment is performed using a handheld ultrasonic processor directed at the style of the parent plant. The frequency of the ultrasonic treatment is preferably 20-40 kHz, more preferably 25-35 kHz, and even more preferably 30 kHz; the ultrasonic intensity is preferably 50-150 W, more preferably 75-125 W, and even more preferably 100 W; the ultrasonic duration is preferably 5-15 s, more preferably 7-12 s, and even more preferably 10 s; the distance between the ultrasonic probe and the style head is preferably 8-12 cm, more preferably 9-11 cm, and even more preferably 10 cm.
[0022] In this invention, a sealing agent is used after ultrasonic treatment to prevent the conductive gel from adhering to other pollen or a large amount of impurities in the air, which would affect fertilization efficiency; the sealing agent preferably comprises the following components in the following mass ratio: Beeswax:Vaseline:Talc = 60~80:20~30:3~8, further preferably beeswax:Vaseline:Talc = 65~75:22~28:4~7, and even more preferably beeswax:Vaseline:Talc = 70:25:5.
[0023] In this invention, the hybridization breeding method is applied to the cultivation of highly resistant olives.
[0024] In this invention, the resistance preferably includes resistance to salt and alkali, resistance to moisture, resistance to peacock spot disease, leaf spot disease and / or fruit flies.
[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0026] Example 1
[0027] The effect of pollen premix on pollen viability
[0028] Collect pollen (e.g.) Figure 1To investigate the effects of different pollen premixes on pollen viability, pollen samples were randomly divided into seven groups: pollen premix group 1, pollen premix group 2, pollen premix group 3, pollen premix group 4, pollen premix group 5, pollen premix group 6, and a blank control group.
[0029] Pollen premix powder group 1: Take 100 g of bean pollen, 0.15 g of boric acid, 12 g of sucrose, 0.002 g of GA3 and 0.001 g of IAA, mix them well and store at 4℃ to obtain pollen premix 1; Pollen premix 2 groups: Take 80 g of bean pollen, 0.2 g of boric acid, 15 g of sucrose, 0.001 g of GA3 and 0.002 g of IAA, mix them well and store at 4℃ to obtain pollen premix 2; Pollen premix powder group 3: Take 120 g of bean pollen, 0.1 g of boric acid, 10 g of sucrose, 0.003 g of GA3 and 0.0005 g of IAA, mix them well and store at 4℃ to obtain pollen premix 3; Pollen premix powder group 4: Take 100 g of bean pollen, 12 g of sucrose, 0.002 g of GA3 and 0.001 g of IAA, mix them well and store at 4℃ to obtain pollen premix powder group 5; Five groups of pollen premix powder: Take 100 g of bean pollen, 0.15 g of boric acid, 12 g of sucrose and 0.001 g of IAA, mix them well and store at 4℃ to obtain pollen premix 5; Pollen premix powder group 6: Take 100 g of bean pollen, 0.15 g of boric acid, 12 g of sucrose and 0.002 g of GA3, mix them well and store at 4℃ to obtain pollen premix 6; Blank control group: Take 100g of bean pollen and store at 4℃.
[0030] Pollen viability in different groups was tested every 6 hours using the TTC assay kit, with untreated pollen as a comparison. The changes in pollen viability (%) are detailed in Table 1.
[0031] Table 1. Timeline of Pollen Viability (%) Changes
[0032] As shown in Table 1, the pollen premix powder effectively maintained pollen viability, while the removal of GA3 0.002g and IAA 0.001g significantly reduced pollen viability. Subsequent experiments were conducted using pollen premix powder 1, which demonstrated the best performance.
[0033] Example 2
[0034] The effect of conductive gel on pollen germination
[0035] To investigate the effects of different conductive gels on pollen germination, the effects of different groups of conductive gels were studied. The experimental groups were conductive gel group 1, conductive gel group 2, conductive gel group 3, and ordinary gel group.
[0036] Conductive Gel Group 1: Heat 820g of water to 70℃, add 10g of gellan gum, stir until melted, then add 120g of trehalose, 6g of boric acid, 0.01g of chitosan quaternary ammonium salt, and 5g of methyl jasmonic acid. Stir until fully dissolved and then cool to obtain conductive gel 1.
[0037] Conductive Gel Group 2: Heat 850g of water to 68℃, add 7g of gellan gum, stir until melted, then add 100g of trehalose, 8g of boric acid, 0.015g of chitosan quaternary ammonium salt, and 3g of methyl jasmonic acid. Stir until fully dissolved and then cool to obtain conductive gel 2.
[0038] Conductive Gel Group 3: Heat 800g of water to 70℃, add 20g of gellan gum, stir until melted, then add 150g of trehalose, 5g of boric acid, 0.005g of chitosan quaternary ammonium salt, and 6g of methyl jasmonic acid, stir until fully dissolved, and then cool to obtain conductive gel 3.
[0039] Standard gel group: 1% agarose gel.
[0040] Prepare 1.4 kg of beeswax, heat it to 70℃ to melt it, add 0.5 kg of petroleum jelly and 100 g of talcum powder, stir well to obtain a sealing agent.
[0041] The experimental site was the olive science and technology courtyard in Xichang, Sichuan. 15-year-old Laixing olive trees in their peak fruiting period were selected as the parent trees. The fertilization conditions and cultivation management measures in the same experimental plot were consistent. Secondary or tertiary main branches of the parent trees were randomly selected. The branches used for testing had to be longer than 20cm, about 1cm in diameter, and have more than 10 inflorescence primordia. Each randomly selected branch had to be free from direct connection or attachment. When a clear, sticky substance appears on the stigma of the pistil, pollination begins (e.g. Figure 2 and 3 Pollination should be done between 9 and 11 a.m., after applying conductive gel 1-3 and 1% agarose gel, each about 2 mm thick, to the column heads respectively. Figure 4 The pollen premix 1 prepared in Example 1 was applied to the pollen. The amount of pollen premix 1 used per hectare was about 400-500g. After pollination, the stigma was treated with a handheld ultrasonic processor at 30 kHz and 100 W at a distance of about 10cm from the stigma for 10 seconds.
[0042] After the conductive gel on the column head dries, apply a 1mm thick sealant to the column head and then cover it with a bag. Figure 5), and complete the hybridization operation. The effects of different groups of gels were studied, and the germination and growth of pollen in the stigma and style were observed by squash fluorescence. For the specific method of studying the pollen tube elongation distance, please refer to "[1] Liao Ting, Yuan Deyi, Gao Chao, et al. Pollination and fertilization of Camellia oleifera and early embryonic development [J]. Forestry Science, 2014, 50(2):6.DOI:10.11707 / j.1001-7488.20140208.". The results are shown in Table 2: Table 2 Pollen tube elongation distance after pollination (unit: mm)
[0043] As shown in Table 2, the pollen tube elongation rate of conductive gels 1-3 was significantly greater than that of ordinary gels (p<0.01). When the ordinary gel had just reached about 2 / 3 of the style after 72 hours, the conductive gel had already entered the ovule. The combination of conductive gel and ultrasonic treatment significantly improved the fertilization rate.
[0044] Example 3
[0045] The effect of the hybridization method of this invention on the fruit set rate was studied according to conventional planting methods. The subjects were divided into two groups: an experimental group (using the method described in Example 2, with conductive gel 1 used) and a conventional method group (pollen collected within 12 hours of application). The fruit set rate of the different experimental groups was recorded, and the results are shown in Table 3. Table 3 Fruit setting rate of different experimental groups
[0046] As can be seen from the experimental data in Table 3, the technical solution of this application greatly increases the fruit setting rate and yield, and the use of a sealing agent increases the yield of hybrid seeds.
[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for hybrid breeding of olive trees, characterized in that, Includes the following steps: 1) Collect the pollen from the male parent, prepare pollen premix powder, and store at 0~6℃; 2) After coating the surface of the stigma of the mother plant with conductive gel, pollination is carried out using pollen premix powder, and then the pollinated stigma is ultrasonically treated. 3) After ultrasonic treatment, apply a column head sealant to the column head to complete artificial pollination; The pollen premix powder comprises the following components in the following mass ratio: Pollen: Boric acid: Sucrose: GA3: IAA = 80~120: 0.1~0.2: 10~15: 0.001~0.003: 0.0005~0.002; The conductive gel comprises the following components in the following mass ratio: Gellan gum: Trehalose: Boric acid: Chitosan quaternary ammonium salt: Methyl jasmonic acid: Water = 0.5~2:10~15:0.5~0.8:0.0005~0.0015:0.3~0.6:80~85.
2. The hybridization breeding method according to claim 1, characterized in that, The parent olive variety mentioned includes Lesson.
3. The hybridization breeding method according to claim 1, characterized in that, The male parent olive varieties that provide pollen include the lentil.
4. The hybridization breeding method according to claim 1, characterized in that, The sealing agent comprises the following components in the following mass ratio: Beeswax: Vaseline: Talc = 60~80: 20~30: 3~8.
5. The hybridization breeding method according to claim 1, characterized in that, The degree of deacetylation of the chitosan quaternary ammonium salt is ≥90%.
6. The hybridization breeding method according to claim 5, characterized in that, The conductive gel coating thickness is 1~3 mm.
7. The hybridization breeding method according to claim 1, characterized in that, The ultrasonic treatment frequency is 20~40kHz, the ultrasonic intensity is 50~150W, the ultrasonic time is 5~15s, and the ultrasonic probe is 8~12cm away from the column head.
8. The application of the hybridization breeding method according to any one of claims 1 to 7 in the cultivation of highly resistant olive trees.
9. The application according to claim 8, characterized in that, The resistances include tolerance to salt and alkali, tolerance to moisture, and resistance to peacock spot disease, leaf spot disease, and / or fruit flies.