Induction method of olive tetraploid

Abstract

The invention discloses an olive tetraploid induction method, and belongs to the field of olive breeding. The induction method provided by the invention comprises the step of treating growing points among cotyledons of the olive sterile tissue culture seedlings by using an induction solution, wherein the induction solution is a mixed solution which is prepared by using a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent and contains 0.1%-0.5% of colchicine and 0.02%-0.04% of oryzalin. According to the method, the production of the olive tetraploid can be effectively induced, and the method has relatively low material death rate and relatively high material ploidy variation rate.

Description

Technical Field

[0001] The invention belongs to the technical field of olive breeding, and in particular relates to an olive tetraploid induction method. Background Art

[0002] Olive (Olea europaea L.) is an evergreen tree of the genus Oleaceae and an important oil crop. Olive oil cold-pressed from fresh olive fruit is a natural high-quality edible vegetable oil, rich in nutrients and functional active ingredients. It is a good product for both medicine and food, known as "liquid gold", and has extremely high economic value. However, a large amount of pomace is produced during the production and processing of olive oil. According to statistics, 35 to 40 kg of pomace will be produced for every 100 kg of fresh fruit, and its main components are olive peel, pits and residual pulp. At present, the processing technology of olive fruit mainly includes two-phase separation process and three-phase separation process, but no matter which process can achieve 100% oil extraction rate, the oil content of pomace is still 5% to 8%. Although chemical reagents can be used to extract residual pomace oil, due to cost and technical limitations, the extracted pomace oil cannot be eaten and can only be used in the industrial field, resulting in great waste.

[0003] Polyploidy is widely present in higher plants and is considered to be one of the important mechanisms for plants to adapt to the environment and form species. In addition to being able to directly and quickly produce new varieties, it is also an important germplasm resource. Studies have shown that polyploidy improves the protein synthesis capacity and efficiency of plants, changes their morphological and physiological characteristics such as morphology, growth and development, stress resistance, and fertility, and lays the foundation for the selection and development of new plant varieties and the development and utilization of germplasm resources. Artificially induced polyploids generally have the characteristics of vigorous growth, large fruits, few or no seeds, high yield, strong adaptability and stress resistance, and can be fixed through asexual reproduction. Excellent traits can be used for a long time in production. Through the cultivation of polyploid olives, obtaining olives with large fruits, small cores, or even no cores will inevitably bring a revolutionary breakthrough to the olive industry. At the same time, polyploidy leads to changes in crop metabolism and growth and development progress, which will also affect the composition and content of the contents of polyploid crop fruits, and improve the nutritional quality of fruits and olive oil. However, although the artificial induction technology of fruit tree polyploidy has matured, the research on the induction of olive polyploidy is still lagging behind. 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 prior art in the field. Summary of the invention

[0005] In view of one or more problems existing in the prior art, the present invention provides a method for inducing tetraploid olive, which comprises treating the growth points between cotyledons of sterile tissue culture seedlings of olive 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 with 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 culture seedlings of olive 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 seedlings 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 tetraploid, which, based on the above-mentioned induction method, further comprises hardening the tissue culture seedlings after the tetraploid induction, and transplanting them into soil after hardening.

[0013] In another aspect, the present invention provides an induction solution suitable for inducing olive tetraploid, 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 tetraploidy to the greatest extent.

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

[0018] (3) In the tetraploid breeding 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 of flow cytometry were obtained.

[0020] Figure 2 The phenotypic differences between tetraploid individuals in the treatment group; A is the diploid wild-type control of the same period, and the phenotypes of B, C, and D are short, multi-branched, and tall, respectively.

[0021] Figure 3 The leaves of tetraploid individuals are shown in Figure 1. A is a diploid wild-type control of the same period, and the rest (BE) are tetraploids. 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 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 "including" 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 specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0024] The methods for obtaining various biological materials described in the examples are only provided as a way of obtaining them in an experiment to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions 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] The olive fruits were selected to be large, full, and free of pests and diseases, and the sampling location was the nursery of the Academy of Forestry.

[0029] 2. Obtaining seeds

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

[0031] 3. Establishment of sterile system

[0032] The seed shell was cracked open to remove the seed kernel, and after disinfection (the disinfection method was: the seeds were rinsed with running water for 2-3 hours, moved to the clean bench, washed with 75% alcohol for 45 seconds, washed with sterile water once, and then treated with 6% sodium hypochlorite solution for 10 minutes. After the treatment, the seeds were rinsed with sterile water for 5-6 times), and gently cut along the seed ridge with a scalpel in the clean bench, and the healthy embryo was carefully removed and transplanted into the culture medium (the formula was Murashige & Skoog Basal Medium (MS) + 0.05mg / L GA + 25g mannitol + 3.5g plant gel, pH = 6.0). After 1 week, the cotyledons turned green and the radicles began to elongate. After about 2 weeks, the cotyledons unfolded, and the polyploid induction treatment could be started.

[0033] 4. Tetraploid induction

[0034] Using the aseptic tissue culture seedlings of olive with cotyledons just opened as the material, the aseptic induction liquid was dripped at the growth point between the cotyledons by the drip method, and the olive was induced to form homologous polyploids for 24 hours. The induction liquid used 0.2% dimethyl sulfoxide (DMSO) aqueous solution as the solvent, and a mixed solution of 0.3% colchicine and 0.03% oryzalin was prepared. When dripping, a 1mL sterile syringe was used to carefully drop the droplets between the two cotyledons. If the droplets were found to be dry, they should be replenished in time. After the treatment, the treated area was washed with distilled water.

[0035] 5. Polyploidy detection

[0036] The DNA content of the polyploid induced population of olive was detected by flow cytometry (CyFlowSpace). When the plants grow up, the third and fourth pairs of new leaves with vigorous growth from the top downward are taken for flow cytometry detection. Be careful to avoid collecting the first and second pairs of leaves at the top to prevent the plants from stopping growing due to stress; at the same time, old leaves should not be collected, as such leaves are hard in texture and have more secondary products, which can easily cause adverse effects on flow cytometry detection. Place two new leaves in a culture dish, take 500μL of pre-cooled mGb lysis solution (purchased from Shanghai Shangbao Biotechnology Co., Ltd.) and add it to the dish, and quickly chop the leaves with a sharp blade. Note that throughout the process, the entire material should be immersed in the lysis solution to better free the cell nucleus. Add 1mL of lysis solution to the chopped leaves. After incubating on ice for 5 minutes, avoid impurities, aspirate the liquid in the dish, filter it into a centrifuge tube using a 300-mesh filter, and collect the cell nucleus at low temperature and low speed centrifugation (4℃, 1000rpm, 5min). Then carefully remove the supernatant, add 100 μL of pre-cooled mGb dissociation solution and 150 μL of PI staining solution, mix well, and resuspend on ice in the dark for 15 minutes. Then transfer the sample to the loading tube and collect 5000 to 10000 particles. All detections were performed at the same gain. Figure 1 The results of flow cytometry detection are shown, where panel a is the original 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, peak P2 is the wild-type control sample (diploid), and peak P4 is sample No. 29 of the treatment group. As shown by the horizontal axis, peak P4 is about twice the peak of P2, and the peak is concentrated without any mixed peaks. Since the gain is the same during detection, the peak channel position can reflect the relative size of the sample genome, that is, it corresponds to different ploidy relationships.

[0037] 6. Hardening the seedlings

[0038] Unscrew the lid of the tissue culture bottle, add an appropriate amount of tap water to the bottle to submerge the culture medium 1-2cm deep, place it in an artificial climate chamber, adapt for 1-2 weeks, and add water from time to time to keep it moist. Then move it into the greenhouse. Carefully pull out the seedlings, wash the culture medium attached to the roots with clean water, plant them in moist soil, cover them with a plastic cover to keep them moist for about a week, and then uncover them. The conditions of the artificial climate chamber are: 16h light, 8h dark, 24℃, and 50% humidity. The soil matrix is ​​pre-disinfected with 0.3-0.5% potassium permanganate solution to prevent the seedlings from mold and rot. The matrix composition is humus: vermiculite: perlite = 3:1:1.

[0039] 7. Trait statistics and analysis

[0040] About one year after the seedlings were transplanted into the soil, the plant height, ground diameter, leaf length, leaf width and leaf thickness of the tetraploid and diploid control groups of the same batch of olives were measured. The stem was measured 2 cm above the soil surface, and the fourth and fifth pairs of leaves with the top facing downward were selected to measure the leaf length, leaf width and leaf thickness using a vernier caliper.

[0041] Table 1 below shows the intra-group statistical results of plant height and ground diameter of tetraploid and diploid control groups, and Table 2 below shows the independent sample test results of plant height and ground diameter of tetraploid and diploid control groups. Table 3 below shows the intra-group statistical results of leaf length, leaf width and leaf thickness of tetraploid and diploid control groups, and Table 4 below shows the independent sample test results of leaf length, leaf width and leaf thickness of 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 leaf conditions of polyploid individuals are shown, among which A is a diploid wild type control of the same period, and the rest are polyploids. All leaves were taken from the fourth and fifth pairs of leaves from the top downward.

[0042] Depend on Figure 2 As shown in Table 1-2, tetraploid plants have variations 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 between tetraploid plants, the average plant height and average ground diameter of tetraploid plants are lower than those of diploid controls, but neither is significant. Figure 3 As shown in Table 3-4, the leaf thickness of tetraploid plants is significantly different from that of diploid plants, and the surface of tetraploid leaves is rougher and the leaf color is darker green. Similarly, due to the large individual differences between tetraploid plants, the average leaf length and leaf width of the two do not constitute a significant difference. This shows 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 is in accordance with that of Example 1, except that different induction solutions are used for polyploid induction. Specifically, the induction solution used in Example 2 is a mixed solution of colchicine with a concentration of 0.1% and oryzalin with a concentration of 0.02% using a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent; the induction solution used in Example 3 is a mixed solution of colchicine with a concentration of 0.5% and oryzalin with a concentration of 0.04% using a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent.

[0053] Comparative Example 1

[0054] Comparative Example 1 was performed in accordance with the procedures of Example 1, 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 are performed according to the operation of Example 1, except that different induction solutions are used for polyploid induction. Specifically, the induction solution used in Comparative Example 2 is a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a colchicine concentration of 0.3% is prepared; the induction solution used in Comparative Example 3 is a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a colchicine concentration of 0.3% and a dimethoate concentration of 0.03% are prepared; the induction solution used in Comparative Example 4 is a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a colchicine concentration of 0.6% and a oryzalin concentration of 0.05% are prepared; the induction solution used in Comparative Example 5 is a 0.2% dimethyl sulfoxide (DMSO) aqueous solution as a solvent, and a colchicine concentration of 0.05% and a oryzalin concentration of 0.01% are prepared.

[0057] Table 5 below shows the results of Examples 1-3 and Comparative Examples 1-5 for the material mortality rate, material ploidy variation rate and tetraploid yield for polyploid induction of olive. It can be seen that the mortality rate, ploidy variation rate and tetraploid yield of the induced material 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 description is only a preferred embodiment of the present invention and is 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing tetraploid olive, characterized in that: The induction method comprises treating the growth points between cotyledons of olive sterile 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 with 0.2% dimethyl sulfoxide aqueous solution as solvent.

2. The induction method according to claim 1, characterized in that: The induction solution is a mixed solution containing 0.3% to 0.5% colchicine and 0.03% to 0.04% oryzalin, prepared by using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

3. The induction method according to claim 1 or 2, characterized in that: The method of using the inducing liquid to treat the growth points between the cotyledons of the olive sterile tissue culture seedlings is to use a drip method to treat the growth points between the cotyledons, wherein the drip method is operated in the following manner: using a sterile syringe to drop the inducing liquid between two cotyledons, and after the droplets dry up, the droplets need to be replenished in time; after the treatment is completed, the treated area is washed with distilled water.

4. The induction method according to any one of claims 1 to 3, characterized in that: The treatment time is 22-26 hours.

5. The induction method according to any one of claims 1 to 4, 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 mannitol + 3-4 g plant gel, pH = 6.

0.

6. The induction method according to any one of claims 1 to 5, characterized in that: The tetraploid yield of the induction method is above 16%.

7. The induction method according to claim 6, characterized in that: The tetraploid yield of the induction method is above 24%.

8. A method for cultivating olive tetraploid, characterized in that: The cultivation method includes, on the basis of the induction method described in any one of claims 1 to 7, further comprising hardening the tissue culture seedlings after the tetraploid induction, and transplanting them into the soil after hardening.

9. An induction solution suitable for inducing tetraploidy in olive, characterized in that: The induction solution is a mixed solution containing 0.1% to 0.5% colchicine and 0.02% to 0.04% oryzalin, prepared by using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

10. The induction solution according to claim 9, characterized in that: The induction solution is a mixed solution containing 0.3% to 0.5% colchicine and 0.03% to 0.04% oryzalin, prepared by using 0.2% dimethyl sulfoxide aqueous solution as a solvent.

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