A method for inducing olive tetraploid

Abstract

The present invention discloses a method for inducing tetraploid olives, belonging to the field of olive breeding. The induction method provided by the present invention comprises treating the growth points between the cotyledons of sterile tissue-cultured olive seedlings with an induction solution, wherein the induction solution is a mixed solution containing 0.1% to 0.5% colchicine and 0.02% to 0.04% oryzalin, prepared using a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent. The method of the present invention can effectively induce the production of tetraploid olives, while achieving a relatively low material mortality rate and a high material ploidy variation rate.

Description

Technical Field

[0001] The invention belongs to the technical field of olive breeding, and particularly relates to a method for inducing olive tetraploid. Background Art

[0002] Olive (Olea europaea L.), an evergreen tree in the genus Oleaceae, is a major oilseed crop. Olive oil, cold-pressed from fresh olive fruit, is a natural, high-quality edible vegetable oil rich in nutrients and functionally active ingredients. It is a highly valued food and medicine, known as "liquid gold." However, the production and processing of olive oil produces a significant amount of pomace. According to statistics, every 100 kg of fresh olive fruit produces 35-40 kg of pomace, primarily consisting of the olive peel, pit, and residual pulp. Currently, the main olive fruit processing technologies include two-phase separation and three-phase separation, but neither process achieves 100% oil extraction, with the pomace oil content remaining between 5% and 8%. While chemical reagents can be used to extract residual pomace oil, cost and technical limitations mean the extracted pomace oil cannot be consumed and can only be used industrially, resulting in significant waste.

[0003] Polyploidy is widespread in higher plants and is considered a key mechanism for plant adaptation and speciation. In addition to enabling the rapid and direct generation of new varieties, it also represents a valuable germplasm resource. Research has shown that polyploidy enhances a plant's protein synthesis capacity and efficiency, leading to changes in morphological and physiological characteristics such as morphology, growth and development, stress tolerance, and fertility. This has laid the foundation for the selection and utilization of new plant varieties and germplasm resources. Artificially induced polyploids generally exhibit vigorous growth, large fruit, few or no seeds, high yield, strong adaptability, and stress tolerance. These desirable traits can be fixed through asexual reproduction, enabling long-term production utilization. The development of polyploid olives with large fruit, small seeds, or even seedless seeds is poised to revolutionize the olive industry. Furthermore, the alterations in crop metabolism and growth and development caused by polyploidy can affect the composition and content of polyploid fruit, potentially improving the nutritional quality of the fruit and olive oil. However, while the technology for artificially inducing polyploidy in fruit trees has matured, research on polyploidy induction in olives remains underdeveloped. How to apply polyploidy induction technology to olive trees to obtain high-yield and high-efficiency plants is a technical problem that needs to be solved urgently.

[0004] The contents of the background technology section are merely the technologies known to the inventors and do not necessarily represent the existing technologies in this field. Summary of the Invention

[0005] In response to one or more problems existing in the prior art, the present invention provides, on one hand, a method for inducing olive tetraploids, which comprises treating the growth points between the cotyledons of sterile olive tissue culture seedlings with an induction solution, wherein the induction solution is a mixed solution containing 0.1% to 0.5% colchicine and 0.02% to 0.04% oryzalin, prepared using a 0.2% dimethyl sulfoxide aqueous solution as a solvent.

[0006] In some embodiments, the induction solution is a mixed solution containing 0.3% to 0.5% colchicine and 0.03% to 0.04% oryzalin, prepared using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

[0007] In some embodiments, the use of induction liquid to treat the growth points between the cotyledons of the sterile tissue cultured olive seedlings is to use a drip method to treat the growth points between the cotyledons, wherein the drip method is operated as follows: using a sterile syringe to drop drops of the induction liquid between two cotyledons, and after the droplets dry up, they need to be replenished in time; after the treatment is completed, the treated area is washed with distilled water.

[0008] In some embodiments, the treatment time is 22-26 hours.

[0009] In some embodiments, the olive sterile tissue culture plantlets are obtained by transplanting the embryos of olive seeds into a culture medium and culturing for 12-16 days, wherein the culture medium has a formula of Murashige & Skoog Basal Medium (MS) + 0.04-0.06 mg / L GA + 20-30 g mannitol + 3-4 g plant gel, pH = 6.0.

[0010] In some embodiments, the tetraploid yield of the induction method is greater than 16%.

[0011] In some embodiments, the tetraploid yield of the induction method is greater than 24%.

[0012] On the other hand, the present invention provides a method for cultivating olive tetraploids, which, in addition to the above-mentioned induction method, further comprises hardening the tissue culture seedlings after the tetraploids are induced, and transplanting them into soil after hardening.

[0013] In another aspect, the present invention provides an induction solution suitable for inducing olive tetraploids, which is a mixed solution containing 0.1% to 0.5% colchicine and 0.02% to 0.04% oryzalin, prepared using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

[0014] In some embodiments, the induction solution is a mixed solution containing 0.3% to 0.5% colchicine and 0.03% to 0.04% oryzalin, prepared using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

[0015] The present invention has the following beneficial effects:

[0016] (1) In the tetraploid induction method provided by the present invention, colchicine + oryzalin are used as inducers, which can effectively induce tetraploidy and at the same time ensure the survival rate of the induced tetraploids to the greatest extent.

[0017] (2) The tetraploid induction method provided by the present invention adopts the dripping method, which has the advantages of low tetraploid plant mortality and high efficiency compared with the immersion method.

[0018] (3) In the tetraploid cultivation method provided by the present invention, the advantages of directly planting embryos compared to traditional germination are: high survival rate, stable environment, and droplets are not easy to evaporate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results are detected by flow cytometry.

[0020] Figure 2 The phenotypic differences among tetraploid individuals in the treatment groups are shown in Figure 2. A is a diploid wild-type control of the same period, and the phenotypes of B, C, and D are dwarf, multi-branched, and tall, respectively.

[0021] Figure 3 The following are the leaves of tetraploid individuals; A is a diploid wild-type control from the same period, and the others (BE) are tetraploid. All leaves were taken from the fourth and fifth pairs of leaves from the top. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are 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 making creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "containing" will be understood to include the stated elements or components, without excluding other elements or other components.

[0023] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0024] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of the biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0025] In this specification, the terms "tetraploid" and "polyploid" are used interchangeably.

[0026] Example 1: Induction and cultivation of olive tetraploid

[0027] 1. Material selection

[0028] Olive fruits with large, plump grains and no diseases or insect pests were selected, and the source of the materials was the nursery of the Academy of Forestry.

[0029] 2. Obtaining seeds

[0030] Peel off the pulp, remove the seeds, wash and drain, and set aside after the seeds are completely dry.

[0031] 3. Establishment of sterile system

[0032] The seed shell is cracked open to remove the kernel. After disinfection (disinfection method: rinse the seeds with running water for 2-3 hours, transfer to a clean bench, rinse with 75% alcohol for 45 seconds, rinse once with sterile water, then treat with 6% sodium hypochlorite solution for 10 minutes. After treatment, rinse the seeds 5-6 times with sterile water), gently incise along the seed ridge with a scalpel in a clean bench. Carefully remove the healthy embryo and transplant it into a culture medium (Murashige & Skoog Basal Medium (MS) + 0.05mg / L GA + 25g mannitol + 3.5g phytagel, pH = 6.0). After one week, the cotyledons turn green and the radicle begins to elongate. Approximately two weeks after the cotyledons expand, polyploid induction can begin.

[0033] 4. Tetraploid induction

[0034] Using sterile tissue cultured olive seedlings with newly opened cotyledons, a sterile induction solution was dripped onto the growing points between the cotyledons for 24 hours to induce autopolyploid formation in the olive. The induction solution consisted of a 0.2% dimethyl sulfoxide (DMSO) aqueous solution containing 0.3% colchicine and 0.03% oryzalin. The solution was carefully placed between the two cotyledons using a 1 mL sterile syringe. If the solution dried up, it was replenished promptly. After treatment, the treated area was rinsed with distilled water.

[0035] 5. Polyploidy detection

[0036] DNA content in the induced polyploid population of olive trees was measured using flow cytometry (CyFlowSpace). After the plants matured, the third and fourth pairs of actively growing leaves from the top were collected for flow cytometry analysis. Care was taken to avoid collecting the first and second pairs of leaves from the top to prevent plant growth from cessation due to stress. Older leaves should also be avoided, as these are harder and contain more secondary products, which can adversely affect flow cytometric analysis. Two new leaves were placed in a Petri dish. 500 μL of pre-chilled mGb lysis buffer (purchased from Shanghai Shangbao Biotechnology Co., Ltd.) was added to the dish. The leaves were quickly minced with a sharp blade. Throughout the process, the entire material should be submerged in the lysis buffer to facilitate the isolation of cell nuclei. An additional 1 mL of lysis buffer was added to the minced leaves. After incubation on ice for 5 minutes, the liquid in the dish was aspirated to avoid impurities and filtered through a 300-mesh filter into a centrifuge tube. The nuclei were collected by low-speed centrifugation (4°C, 1000 rpm, 5 minutes). The supernatant was then carefully aspirated, and 100 μL of pre-chilled mGb dissociation buffer and 150 μL of PI staining solution were added. Mix thoroughly, and resuspend on ice for 15 minutes in the dark. The sample was then transferred to a loading tube, and 5,000 to 10,000 particles were collected. All measurements were performed at the same gain. Figure 1 The results of flow cytometry detection are shown, where panel a is the raw data of flow cytometry detection of the wild-type control sample (diploid) and one of the polyploid samples (No. 29, tetraploid); panel b intuitively shows the relative relationship between the DNA content of the two. In the figure, the P2 peak is the wild-type control sample (diploid), and the P4 peak is sample No. 29 in the treatment group. As shown by its horizontal axis, the P4 peak is approximately twice the P2 peak, and the peak is concentrated and there are no other peaks. Since the gain is the same during detection, the peak channel position can reflect the relative size of the sample genome, that is, corresponding to different ploidy relationships.

[0037] 6. Hardening the seedlings

[0038] Unscrew the cap of the tissue culture flask and add an appropriate amount of tap water to cover the culture medium to a depth of 1-2 cm. Place in a climatic chamber and acclimate for 1-2 weeks, adding water occasionally to maintain moisture. Then, transfer to a greenhouse. Carefully remove the seedlings, rinse the culture medium from the roots with clean water, and plant them in moist soil. Cover with a plastic cover to retain moisture for about a week before removing. The climatic chamber conditions are: 16 hours of light, 8 hours of darkness, 24°C, and 50% humidity. Pre-disinfect the soil substrate with a 0.3-0.5% potassium permanganate solution to prevent mold and rot. The substrate composition is a 3:1:1 ratio of humus: vermiculite:perlite.

[0039] 7. Trait statistics and analysis

[0040] Approximately one year after seedlings were transplanted into soil, plant height, ground diameter, leaf length, leaf width, and leaf thickness were measured for tetraploid olive seedlings and diploid olive seedlings from the same planting. Measurements were taken from the stem 2 cm above the soil surface, and leaf length, width, and thickness were measured using a vernier caliper on the fourth and fifth pairs of leaves, facing downward.

[0041] Table 1 below shows the intra-group statistical results of plant height and base diameter of the tetraploid and diploid control groups, and Table 2 below shows the independent sample test results of plant height and base diameter of the tetraploid and diploid control groups. Table 3 below shows the intra-group statistical results of leaf length, leaf width, and leaf thickness of the tetraploid and diploid control groups, and Table 4 below shows the independent sample test results of leaf length, leaf width, and leaf thickness of the tetraploid and diploid control groups. Figure 2 The phenotypic differences between polyploid individuals in the treatment groups are shown, where A is a diploid wild-type control of the same period, and the phenotypes of B, C, and D are short, multi-branched, and tall, respectively; Figure 3 The following table shows the leaves of polyploid individuals, where A is a diploid wild-type control at the same stage, and the others are polyploids. All leaves were taken from the fourth and fifth pairs of leaves from the top.

[0042] Depend on Figure 2 As shown in Table 1-2, tetraploid plants have different types of variation, such as tall, short, and multi-branched ( Figure 2 B is short, C is multi-branched, and D is tall). Due to the large differences among tetraploid plants, the average height and average ground diameter of tetraploid plants are lower than those of diploid controls, but neither is significant. Figure 3 As shown in Tables 3-4, the leaf thickness of tetraploid plants is significantly different from that of diploid plants. Furthermore, the leaf surface of tetraploid plants is rougher and the leaf color is darker green. Similarly, due to the large individual variability among tetraploid plants, the average leaf length and width of the two plants do not differ significantly. This indicates that leaf thickness is an important trait for distinguishing tetraploids from diploids.

[0043] Table 1: Intra-group statistical results of plant height and ground diameter

[0044]

[0045] Table 2: Independent sample test results for plant height and ground diameter

[0046]

[0047] Table 3: Intra-group statistical results of leaf length, leaf width and leaf thickness

[0048]

[0049] Table 4: Independent sample test results for leaf length, leaf width and leaf thickness

[0050]

[0051] Example 2-3

[0052] The operation of Example 2-3 was carried out in accordance with Example 1, except that different induction solutions were used for polyploid induction. Specifically, the induction solution used in Example 2 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a mixed solution of colchicine at a concentration of 0.1% and oryzalin at a concentration of 0.02% was prepared; the induction solution used in Example 3 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a mixed solution of colchicine at a concentration of 0.5% and oryzalin at a concentration of 0.04%.

[0053] Comparative Example 1

[0054] Comparative Example 1 The operation of Example 1 was followed, except that the induction solution was used to soak the sterile olive seeds for 24 h during polyploid induction.

[0055] Comparative Examples 2-5

[0056] Comparative Examples 2-5 were performed according to the procedures of Example 1, except that different induction solutions were used for polyploid induction. Specifically, the induction solution used in Comparative Example 2 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a colchicine concentration of 0.3% was prepared; the induction solution used in Comparative Example 3 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a mixed solution of colchicine at a concentration of 0.3% and pendimethalin at a concentration of 0.03% was prepared; the induction solution used in Comparative Example 4 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a mixed solution of colchicine at a concentration of 0.6% and oryzalin at a concentration of 0.05% was prepared; the induction solution used in Comparative Example 5 was a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a mixed solution of colchicine at a concentration of 0.05% and oryzalin at a concentration of 0.01% was prepared.

[0057] Table 5 below shows the results of Examples 1-3 and Comparative Examples 1-5 for the induction of polyploidy in olive trees, including the mortality rate, ploidy variation rate, and tetraploid yield. It can be seen that the mortality rate, ploidy variation rate, and tetraploid yield of the induced materials are related to the composition, content, and induction method of the induction solution.

[0058] Specifically, as the concentrations of colchicine and oryzalin increased in Examples 2, 1, and 3, the induction rate increased to a certain extent, but the mortality rate also increased accordingly; the mortality rate of the material in Comparative Example 2 was similar to that of the material in Example 1, and the induction rate was relatively low; the mortality rate of the material in Comparative Example 1 was low, the mortality rate of the material in Comparative Example 3 was high, the mortality rate of Comparative Example 4 was the highest, and the mortality rate of Comparative Example 5 was the lowest, but the induction efficiency was extremely low; the ploidy variation rate and tetraploid yield of the material in Example 1 were significantly higher than those in Comparative Examples 1-5, and Comparative Example 1 could hardly be induced based on the data and had no practical application value.

[0059] Table 5: Induction results of Examples 1-3 and Comparative Examples 1-5 for olive materials

[0060] deal with Material mortality rate Material ploidy variation rate Tetraploid yield Example 1 27.66％ 44.68％ 24.46％ Example 2 15.79％ 10.53％ 2.11％ Example 3 55.56％ 22.23％ 16.67％ Comparative Example 1 16.37％ 0 0 Comparative Example 2 28.00％ 23.58％ 7.53％ Comparative Example 3 49.09％ 8.93％ 1.82％ Comparative Example 4 72.00％ 2.67％ 0 Comparative Example 5 13.33％ 6.67％ 1.33％

[0061] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for inducing tetraploid olive, characterized in that: The induction method comprises treating the growth point between the cotyledons of the sterile tissue cultured olive seedlings with an induction solution, wherein the induction solution is a mixed solution composed of 0.3% to 0.5% colchicine and 0.03% to 0.04% oryzalin, prepared using a 0.2% dimethyl sulfoxide aqueous solution as a solvent; The method of using the inducing liquid to treat the growth points between the cotyledons of the sterile tissue cultured olive seedlings is to use a dripping method to treat the growth points between the cotyledons, wherein the dripping method is operated as follows: using a sterile syringe to drop a drop of the inducing liquid between two cotyledons, and after the drop dries up, it is necessary to replenish the drop in time; after the treatment is completed, the treated area is washed with distilled water.

2. The induction method according to claim 1, characterized in that The treatment time is 22-26 h.

3. The induction method according to claim 1 or 2, characterized in that The method for obtaining the olive sterile tissue culture seedlings is to transplant the embryos of olive seeds into a culture medium and culture them for 12-16 days, wherein the formula of the culture medium is: Murashige & Skoog Basal Medium (MS) + 0.04-0.06 mg / L GA + 20-30 g / L mannitol + 3-4 g / L plant gel, pH = 6.

0.

4. The induction method according to claim 1 or 2, characterized in that The tetraploid yield of the induction method reaches more than 16%.

5. The induction method according to claim 1 or 2, characterized in that: The tetraploid yield of the induction method reaches more than 24%.

6. A method for cultivating olive tetraploids, characterized in that: The cultivation method includes, in addition to the induction method according to any one of claims 1 to 5, further comprising hardening the tissue culture seedlings after tetraploid induction and transplanting them into soil after hardening.

Images