Plant embryo in-vitro rescue method

Abstract

The invention relates to the technical field of plant sexual reproduction, in particular to a plant embryo in-vitro rescue method which comprises the following steps: selecting a plant female parent needing embryo rescue; preparing a treating fluid containing a plant hormone regulator; fixing a continuous release device on a branch below the silique or legume of the maternal plant; the treatment liquid is applied to the continuous release device, so that active ingredients in the treatment liquid are absorbed by the branches and transported to the siliques or pods. The externally applied plant hormone regulator can be upwards transported to ovules through the phloem and the xylem and directly acts on the hybrid embryo with development stagnation. Hormonal balance in the embryo is remodeled through cooperative regulation and control of hormones, and a stagnation signal is reversed into a development signal, so that the normal development process of the embryo is rescued and completed in situ without in-vitro operation, and the method is simple to operate, high in success rate, time-saving and labor-saving, and convenient to popularize and apply.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant sexual reproduction, in particular to a method for in vitro rescue of plant embryos. BACKGROUND

[0002] Distant hybridization can introduce excellent gene resources in wild species into cultivated varieties, which is an effective way for germplasm innovation and new variety breeding. However, due to reproductive isolation in distant hybridization, the hybrid embryos develop abnormally or stagnate, and it is difficult to obtain hybrid species.

[0003] In recent years, with the continuous improvement of in vitro embryo rescue technology, hybrid species can be obtained to some extent. However, the in vitro embryo rescue technology needs to separate the hybrid embryo from the fruit pod first and then culture it in plant tissue culture medium. The separation process is easy to cause embryo damage, and the culture period is long, which leads to very low embryo rescue efficiency. In addition, the in vitro embryo rescue technology has complex operation process and high difficulty, which cannot meet the needs of agricultural production, and needs to consume more material resources, financial resources and time.

[0004] Therefore, it is of great significance to develop an in vitro plant embryo rescue technology with high efficiency, simple operation, low difficulty and low cost. SUMMARY

[0005] The present application aims to overcome the problems of operation difficulty, long period, low rescue efficiency, time-consuming and labor-consuming in the existing in vitro embryo rescue technology, and provides a method for in vitro rescue of plant embryos. The method provides a sustained release device fixed under the branch below the fruit pod and creates a slight wound, and the active ingredients in the treatment solution are released continuously and slowly, and are transported by vascular tissue. The exogenous plant hormone regulator can be transported upward to the ovule through phloem and xylem, and directly act on the development-stagnant hybrid embryo, thereby rescuing and completing the normal development process of the embryo in situ without in vitro operation. The method is simple to operate, has high success rate, saves time and labor, and is convenient for popularization and application.

[0006] The present application provides a method for in vitro rescue of plant embryos, comprising the following steps:

[0007] Selecting a plant female parent in need of embryo rescue;

[0008] Configuring a treatment solution containing a plant hormone regulator;

[0009] Fixing a sustained release device on the branch below the silique or pod of the female parent plant;

[0010] The treatment liquid is applied to the sustained release device, and active ingredients in the treatment liquid are absorbed and transported to the silique or pod via the branch.

[0011] The application provides a method for in-vitro rescue of plant embryos, which comprises the following steps: selecting a plant female parent in need of embryo rescue; configuring a treatment liquid containing a plant hormone regulator; fixing a sustained release device on a branch below a silique or pod of the female parent plant; and applying the treatment liquid to the sustained release device, so that active ingredients in the treatment liquid are absorbed and transported to the silique or pod via the branch. The exogenously applied plant hormone regulator is transported upward to the ovule via phloem and xylem, and directly acts on the development-stagnant hybrid embryo. The synergistic regulation of the hormones reshapes the hormone balance inside the embryo, reverses the stagnation signal into a development signal, thereby in-situ rescuing and completing the normal development process of the embryo without off-body operation. The method is simple in operation, high in success rate, time-saving and labor-saving, and is convenient for popularization and application.

[0012] Further, the plant female parent is a plant with development-stagnant or abnormal embryos after distant hybridization.

[0013] Further, the plant hormone regulator comprises a growth promoter and / or a growth inhibitor antagonist.

[0014] Further, the growth promoter comprises auxin and / or gibberellin; and the growth inhibitor antagonist is an abscisic acid synthesis inhibitor.

[0015] Further, the abscisic acid synthesis inhibitor is sodium tungstate (Na2WO4).

[0016] IAA promotes cell wall relaxation and cell elongation by activating the expression of cell wall acid expansin genes and regulating the activity of proton pumps on the cell membrane; GA3 promotes cell division and embryo tissue differentiation by activating key factors in the gibberellin signaling pathway. At the same time, Na2WO4, an abscisic acid synthesis inhibitor, effectively blocks the biosynthesis of abscisic acid in the embryo and surrounding tissues by inhibiting the activity of key enzymes. As a positive regulator of embryo dormancy and abortion, the significant reduction of the concentration of ABA directly alleviates the programmed development block caused by reproductive isolation.

[0017] Further, the sustained release device comprises a carrier with a cavity and an adsorbent material arranged in the cavity; the carrier is provided with an opening for the adsorbent material to communicate with the external environment; and the device is attached to the branch by a fixing member.

[0018] Further, the carrier is a rubber tube, and / or the adsorbing material is a cotton sheet. Preferably, the length of the rubber tube is 2.5-3 cm, and the inner diameter is 0.2-0.5 cm; a small hole is arranged in the middle of the rubber tube, and the diameter of the small hole is 0.1-0.2 cm.

[0019] Further, before fixing the device, a wound surface is first made on the branch at a position corresponding to the opening of the device.

[0020] Further, the application time of the treatment liquid is between the first day after hybrid pollination and the embryo development to the globular embryo stage.

[0021] Further, the treatment liquid is applied to the sustained release device in a periodic replenishment manner.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application provides a method for in vitro rescue of plant embryos, comprising selecting a plant female parent in need of embryo rescue; configuring a treatment liquid containing a plant hormone regulator; fixing a sustained release device on a branch below a silique or legume of the female parent plant; and applying the treatment liquid into the sustained release device, so that the effective components in the treatment liquid are absorbed and transported to the silique or legume via the branch. The exogenously applied plant hormone regulator can be transported upward to the ovule via the phloem and xylem, and directly acts on the developing hybrid embryo. The synergistic regulation of the hormones reshapes the hormone balance inside the embryo, reverses the stop signal to a development signal, thereby in situ rescuing and completing the normal development process of the embryo without the need for ex vivo operation. The method is simple to operate, has a high success rate, saves time and labor, and is convenient for popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The plant embryo in vitro rescue sustained release device comprises a carrier and an adsorbing material arranged in the carrier. Figure 1 Figure A in the drawings is a sustained release device for embryo in vitro rescue. Figure 1 Figure B in the drawings is a cotton sheet with slow release solution characteristics. Figure 1 Figure C in the drawings is a rubber tube. Figure 1 Figure D in the drawings is a fixing clamp.

[0025] Figure 2 Figure is a placement diagram of the sustained release device for embryo rescue after hybridization of Chinese cabbage and cabbage. Figure 2 The red arrow in the drawings indicates the placement position of the embryo rescue device.

[0026] Figure 3 Figure is the growth of ovules after hybridization and pollination of Suzhouqing and cabbage for 18 days under different hormone / inhibitor treatments.

[0027] Figure 4 Ovule growth of Suzhou green and cabbage 18 days after cross-pollination under different hormone / inhibitor treatments. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0029] Unless otherwise specified, the use of terms such as "upper," "lower," "left," "right," "center," "inner," and "outer" to indicate orientation or positional relationships in the description of specific embodiments of the present invention is based on the orientation or positional relationships shown in the accompanying drawings, or the orientation or positional relationship in which the product / equipment / device is typically placed during use. These terms are merely for the purpose of facilitating the description of the present invention or simplifying the description in specific embodiments, enabling those skilled in the art to quickly understand the solution, and do not indicate or imply that a particular device / component / element must have a specific orientation, or be constructed and operated in a specific positional relationship. Therefore, they should not be construed as limitations on the present invention.

[0030] Furthermore, the use of terms such as "horizontal," "vertical," "suspended," and "parallel" does not imply that the corresponding device / component / element must be absolutely horizontal, vertical, suspended, or parallel, but rather that it can be slightly tilted or have a deviation. For example, "horizontal" merely means that its direction is more horizontal relative to "vertical," not that the structure must be completely horizontal, but that it can be slightly tilted. Alternatively, it can be simplified to mean that the corresponding device / component / element, when set in a "horizontal," "vertical," "suspended," or "parallel" direction, can have an error / deviation of ±10% relative to the corresponding direction, more preferably within ±8%, more preferably within ±6%, more preferably within ±5%, and more preferably within ±4%. As long as the corresponding device / component / element is within the error / deviation range, it can still achieve its function in the present invention.

[0031] Furthermore, the use of terms such as "first," "second," and "third" in terminology is merely for distinguishing descriptions of identical or similar components and should not be interpreted as emphasizing or implying the relative importance of a particular component.

[0032] Furthermore, in the description of the embodiments of the present invention, "several", "more than", and "a number of" represent at least two. The number can be any number, such as 2, 3, 4, 5, 6, 7, 8, or 9, and can even exceed nine.

[0033] Furthermore, in the description of the technical solution of this invention, unless otherwise explicitly specified / limited / restricted, the terms "set up," "install," "connect," "link," "provided with," "laid out," and "arranged" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to common connection methods in the art, such as welding, riveting, bolting, and threaded connections. Such connections can be mechanical, electrical, or communication connections; they can be direct connections or indirect connections through an intermediate medium; and they can refer to the internal communication between two components.

[0034] In vitro embryo rescue technology requires separating the hybridized embryos from the pods and culturing them in plant tissue culture media. This separation process is highly susceptible to embryo damage, and the long culture period results in extremely low embryo rescue efficiency. Furthermore, in vitro embryo rescue technology is complex and difficult to implement, failing to meet the needs of agricultural production, and requires significant material, financial, and time resources.

[0035] The first aspect of this embodiment provides a method for in vitro rescue of plant embryos, including the following steps:

[0036] Select the plant parent plants for which embryo rescue is required;

[0037] Prepare a treatment solution containing plant hormone regulators;

[0038] A continuous release device is fixed on a branch below the siliques or pods of the mother plant.

[0039] The treatment liquid is applied to the continuous release device, so that the active ingredients in the treatment liquid are absorbed through the branches and transported to the siliques or pods.

[0040] This method provides a slow-release device fixed to the branch below the pod, creating a minor wound. The active ingredients in the treatment solution are continuously and slowly released and targeted via vascular tissue. Exogenously applied plant hormone regulators can be transported upwards through the phloem and xylem to the ovule, directly acting on the developmentally arrested hybrid embryo. This allows for the in-situ rescue and completion of normal embryonic development without the need for ex vivo manipulation. The method is simple to operate, has a high success rate, saves time and labor, and is easy to promote and apply.

[0041] In some embodiments, the parent plant is a plant whose embryonic development has stopped or is abnormal after distant hybridization.

[0042] In some embodiments, the plant hormone regulator includes growth promoters and / or growth inhibitor antagonists.

[0043] In some embodiments, the growth promoter comprises auxin and / or gibberellin (GA3); the growth inhibitor antagonist is an abscisic acid synthesis inhibitor. In some embodiments, the auxin is indole-3-acetic acid (IAA), and the concentration of IAA is 6-8 mg / L. In some embodiments, the concentration of gibberellin is 2-5 mg / L.

[0044] In some embodiments, the abscisic acid synthesis inhibitor is sodium tungstate (Na2WO4). Preferably, the concentration of sodium tungstate is 1000-3000 mg / L.

[0045] IAA promotes cell wall relaxation and cell elongation by activating the expression of acidic dilatation protein genes in the cell wall and regulating the activity of proton pumps on the cell membrane. GA3 synergistically promotes cell division and embryonic tissue differentiation by activating key factors in the gibberellin signaling pathway. The ABA synthesis inhibitor Na2WO4 effectively blocks the biosynthesis of abscisic acid in the embryo and surrounding tissues by inhibiting the activity of key enzymes. As a positive regulator of embryonic dormancy and abortion, the significant reduction in ABA concentration directly alleviates programmed developmental arrest caused by reproductive isolation.

[0046] Furthermore, the continuous release device includes a carrier with a cavity and an adsorbent material disposed within the cavity; the carrier has an opening that allows the adsorbent material to communicate with the external environment; the device is attached to the branch by a fixing member.

[0047] In some embodiments, the continuous release device includes a carrier having a cavity and an adsorbent material disposed within the cavity; the carrier has an opening that allows the adsorbent material to communicate with the external environment; the device is attached to the branch by a fixing member.

[0048] In some embodiments, the carrier is a rubber tube, and / or the absorbent material is a cotton sheet. Preferably, the rubber tube has a length of 2.5cm-3cm and an inner diameter of 0.2cm-0.5cm; a small hole with a diameter of 0.1cm-0.2cm is provided in the middle of the rubber tube.

[0049] In some embodiments, before fixing the device, a wound surface is made on the branch at a position corresponding to the opening of the device.

[0050] In some embodiments, the treatment solution is applied between the first day after hybridization pollination and the embryo's development to the spherical embryo stage.

[0051] In some embodiments, the treatment solution is applied to the continuous release device in a periodic replenishment manner. In some embodiments, the dosage of the plant hormone regulator is 100-300 μL each time, injected once every 7-15 days.

[0052] To better understand the technical solutions of the above embodiments, the following more detailed specific embodiments are provided for further explanation.

[0053] Example 1

[0054] This embodiment includes an in vitro embryo rescue device, which contains two plant hormones (IAA and GA3) and a hormone synthesis inhibitor (ABA hormone synthesis inhibitor Na2WO4).

[0055] 1. Select Chinese cabbage and kale with a distant hybridization background as the female and male parents, respectively. Hybridize when the female and male parents are in full bloom (with 5-6 lateral branches). First, carefully peel open the flower buds of the Chinese cabbage (female parent) with tweezers and remove its stamens and petals. Then, evenly apply the pollen of the kale (male parent) to the stigma of the Chinese cabbage and mark it. This marks the first day after hybridization.

[0056] 2. Hormone / inhibitor combinations were set up, with the experimental group receiving the hormone / inhibitor combination AG according to the principles of single and mixed administration. The experimental group received the in vitro embryo rescue device on the first day after hybridization. The control group did not receive any embryo rescue device after hybridization.

[0057] A: IAA 7.36 mg / L + 4 mg / L;

[0058] B: IAA 7.36mg / L+4mg / L+100mg / LNa2WO4;

[0059] C: IAA 7.36mg / L+4mg / L+200mg / LNa2WO4;

[0060] D: IAA 7.36mg / L+4mg / L+2000mg / L Na2WO4;

[0061] E: 100 mg / L Na2WO4;

[0062] F: 200 mg / L Na2WO4;

[0063] G: 2000mg / L Na2WO4.

[0064] 3. Install embryo rescue devices: such as Figure 1 and Figure 2As shown, select two rubber tubes, 2.5-3 cm long and 0.3 cm in inner diameter. Cut a small hole (0.1 cm in diameter) at the middle end of each tube. Place shredded cotton (with slow-evaporating properties) inside the hole. Secure the rubber tube containing the cotton to the branch below the hybridized pod using a clamp. Make a minor wound on the branch with a sterile blade to facilitate better absorption of the hormone / inhibitor. Inject the pre-prepared hormone solution AG into the cotton inside the device using a 1 ml syringe. Each hormone combination can be injected into the rescue device on the branch on the day of hybridization, and 7 and 8 days after hybridization.

[0065] like Figure 3 As shown, the ovule growth of Suzhou green and cabbage 18 days after cross-pollination under different hormone / inhibitor treatments is illustrated. The study found that appropriate concentrations of IAA, GA and ABA inhibitors can significantly increase the proportion of normal embryos in interspecific hybridization.

[0066] like Figure 4 As shown, the ovule growth of Suzhou green and cabbage 18 days after cross-pollination under different hormone / inhibitor treatments is presented. The study found that an appropriate concentration of ABA inhibitor can significantly increase the proportion of normal embryos in interspecific hybridization.

[0067] Therefore, this invention provides a method for in vitro rescue of plant embryos, comprising: selecting a female plant for embryo rescue; preparing a treatment solution containing a plant hormone regulator; fixing a continuous release device on a branch below the silique or pod of the female plant; and applying the treatment solution to the continuous release device, so that the active ingredients in the treatment solution are absorbed through the branch and transported to the silique or pod. The exogenously applied plant hormone regulator can be transported upward through the phloem and xylem to the ovule, directly acting on the developmentally arrested hybrid embryo. The synergistic regulation of hormones reshapes the hormonal balance within the embryo, reversing the stagnation signal into a developmental signal, thereby rescuing and completing the normal development process of the embryo in situ without the need for in vitro manipulation. This method is simple to operate, has a high success rate, saves time and effort, and achieves unexpected results.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro rescue of plant embryos, characterized in that, Includes the following steps: Select the plant parent plants for which embryo rescue is required; Prepare a treatment solution containing plant hormone regulators; A continuous release device is fixed on a branch below the siliques or pods of the mother plant. The treatment liquid is applied to the continuous release device, so that the active ingredients in the treatment liquid are absorbed via the branches and transported to the siliques or pods.

2. The method for in vitro rescue of plant embryos according to claim 1, characterized in that, The parent plant is a plant whose embryonic development has stopped or is abnormal after distant hybridization.

3. The method for in vitro rescue of plant embryos according to claim 1, characterized in that, The plant hormone regulators include growth promoters and / or growth inhibitor antagonists.

4. The method for in vitro rescue of plant embryos according to claim 3, characterized in that, The growth promoters include auxins and / or gibberellins; the growth inhibitor antagonists are abscisic acid synthesis inhibitors.

5. The method for in vitro rescue of plant embryos according to claim 4, characterized in that, The abscisic acid synthesis inhibitor is sodium tungstate (Na2WO4).

6. The method for in vitro rescue of plant embryos according to claim 1, characterized in that, The continuous release device includes a carrier with a cavity and an adsorbent material disposed within the cavity; the carrier has an opening that allows the adsorbent material to communicate with the external environment; the device is attached to the branch by a fixing member.

7. The method for in vitro rescue of plant embryos according to claim 6, characterized in that, The carrier is a rubber tube, and / or the adsorbent material is a cotton sheet.

8. The method for in vitro rescue of plant embryos according to claim 6, characterized in that, Before fixing the device, a wound surface is made on the branch at a position corresponding to the opening of the device.

9. The method for in vitro rescue of plant embryos according to claim 1, characterized in that, The treatment solution is applied between the first day after hybridization pollination and the embryo's development to the spherical embryo stage.

10. The method for in vitro rescue of plant embryos according to claim 1, characterized in that, The treatment fluid is applied to the continuous release device in a periodic replenishment manner.

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