A cutting rapid propagation method for transgenic bottle gourd

Abstract

The present invention discloses a method for rapid propagation of transgenic gourds by cuttings. The method uses side branches of transgenic gourds as materials and adopts a cutting culture method for rapid propagation. The method propagates the transgenic gourds by combining hydroponic cuttings with soil-based cuttings. Side branches of transgenic gourds before the flowering period are selected and, after processing, are first hydroponically cultivated and then soil-cultivated. This method effectively improves the rooting rate of the gourds and shortens the seedling raising cycle. The method is not restricted by the environment or season, has a high survival rate, and offspring can stably maintain the excellent traits of the mother plant. The method also has a short propagation cycle and low costs.

Description

Technical Field

[0001] The present invention relates to the technical field of plant propagation, and mainly relates to a method for asexual propagation of plants, and specifically to a method for asexual propagation of cloned seedlings by cuttings of transgenic bottle gourd side branches. Background Art

[0002] Gourd [Lagenaria siceraria (Mol.) Standl.] (2n=2x=22) belongs to the Cucurbitaceae family and is a cultivated species of the genus Cucurbita. It is one of the specialty summer vegetables in southern my country. Gourd is thermophilic, light-loving, and heat-resistant. It is mainly planted in spring and autumn in southern China. It cannot flower and bear fruit normally when temperatures are too low. Currently, no research related to the functional genomics of gourd has been reported at home or abroad. Genetic transformation of gourd is very difficult, with a success rate of less than one in a thousand. Gourd-positive transgenic plants are extremely valuable research vehicles. The uncontrollable output cycle of transgenic experiments sometimes causes transgenic plants to encounter environmental conditions that are not suitable for flowering and bearing fruit. If left unchecked, the precious transgenic plants will not be able to be propagated or even survive, resulting in heavy losses for research. At the same time, in actual work, the transgenic gourd plant often encounters problems such as the small number of seeds, which necessitates a 3-5 month propagation cycle, or the transgenic gourd plant seed fertility is poor and cannot be sexually propagated, seriously hindering project progress. Therefore, it is very necessary to invent a convenient, efficient and highly consistent cutting propagation method to ensure the preservation and propagation of precious gourd transgenic positive plants and meet the needs of gourd molecular biology research.

[0003] At present, there are no reports of cutting propagation technology for gourd, especially transgenic gourd, at home and abroad. Conventional soil-based cutting technology is to directly insert the trimmed cutting seedlings into a solid culture medium for rooting culture, supplemented by nutrient solution to induce rooting of the cutting seedlings. However, transgenic gourd plants have the problem of weak plant growth potential, poor regeneration and differentiation ability, and are not suitable for conventional soil-based cutting technology. Hydroponic cutting uses water or nutrient solution as the culture medium, with the advantages of simple facilities, convenient management, clean and pollution-free, and low cost. At the same time, it can also be precisely controlled according to the plant's nutrient requirements, making it convenient to observe and understand the growth status of the plant. It is less affected by the natural environment and can achieve a seedling raising cycle covering the whole year. At present, there is an urgent need for a cutting propagation technology suitable for transgenic gourd. The combination of soil-based cutting and hydroponic cutting will effectively give play to the advantages of both and achieve the purpose of asexual propagation of transgenic gourd side branch cutting. Summary of the Invention

[0004] The present invention aims to address the current lack of methods for rapid propagation of gourds by cuttings in production and scientific research, and to provide an original and convenient method for obtaining a large number of transgenic gourd plant cuttings in a short period of time. The present invention is the first invention of a method for rapid propagation of transgenic gourd plants by cuttings, providing an important means for propagation of transgenic gourd plants in seed preservation and molecular biology research.

[0005] In one aspect, the present invention provides a method for rapid propagation of transgenic gourd by cuttings, comprising the following steps:

[0006] 1) Using a transgenic bottle gourd plant as a mother plant, selecting bottle gourd side branches 10-20 cm long as cuttings; cutting the cuttings from the stem nodes;

[0007] 2) completely immersing 1-2 stem nodes at the lower end of the cuttings obtained in step 1) in a nutrient solution; and hydroculturing the cuttings until the stem nodes turn white and adventitious roots grow;

[0008] 3) transplanting the cuttings obtained in step 2) to a soil base for cutting, burying the stem nodes that have grown adventitious roots in the substrate, exposing the leaves and leaf buds at the top, and watering with 200-300 ml of sterile water; cultivating in the soil for 7-10 days until the cuttings sprout a large number of 2-4 cm long adventitious roots;

[0009] 4) Transplanting the cuttings obtained in step 3) together with the substrate into nutrient soil.

[0010] In the present invention, the transgenic bottle gourd used for testing is code-named HD-1. During its production cycle, the environmental conditions are not suitable for flowering and fruiting, and it is difficult to propagate using conventional methods.

[0011] Preferably, the cutting angle at the stem node in step 1) is 40-50°. Furthermore, the cutting seedlings are cut quickly with a sharp blade at 40-50°, and the incision is required to be smooth.

[0012] Preferably, the leaves and tendrils at 1-2 nodes at the lower end of the cutting seedlings obtained in step 1) need to be removed.

[0013] More preferably, the cuttings obtained in step 1) need to retain 2-4 leaves and 1-2 leaf buds at the top.

[0014] Normally, a 10-20 cm long gourd side branch contains only 4-5 nodes. The upper nodes need to retain leaf buds, and only 1-2 nodes are used to differentiate adventitious roots at the lower end.

[0015] Preferably, the nutrient solution in step 2) is a rooting nutrient solution; each bead cutting seedling requires 400 ml of rooting nutrient solution. In some embodiments, multiple bead cutting seedlings can be hydroponically cultivated at the same time, and 5-10 bead cutting seedlings generally require 2-4 L.

[0016] Preferably, the hydroponic conditions in step 2) are: 12 hours of light per day, 3000 lx of light intensity, 26°C of temperature, and 60% of humidity; 12 hours of darkness per day, 20°C of temperature, and 40% of humidity; and the nutrient solution is replaced every 2 days.

[0017] Typically, the hydroponic method involves pouring 2-4 L of prepared rooting nutrient solution into a culture container, fixing the prepared cuttings on a foam board, and completely immersing the stem nodes in the nutrient solution.

[0018] Preferably, the substrate for soil-based cuttings in step 3) is vermiculite with a thickness of 3-4 cm; preferably, the vermiculite is sterilized in a high-pressure steam autoclave at 121°C.

[0019] Preferably, the soil culture conditions in step 3) are: daily light duration of 12 hours, light intensity of 4000 lx, temperature of 26°C, and humidity of 60%; daily dark duration of 12 hours, temperature of 20°C, and humidity of 40%; and watering with 200-300 ml of sterile water once a day.

[0020] Preferably, the rooting culture solution is an aqueous solution containing rooting powder with a concentration of 3 g / L; preferably, the rooting culture solution also contains 2 ml / L of dilution solution of benzyl ammonium chloride.

[0021] Preferably, the transgenic bottle gourd plant used as the mother plant in step 1) is a transgenic bottle gourd plant that has been soil-cultured for at least 30 days.

[0022] In some preferred embodiments, the most preferred method of the present invention comprises the following steps:

[0023] (1) Preparation of cuttings

[0024] The transgenic gourd seedlings produced in the laboratory were trained and then planted in a soil culture medium consisting of a mixture of nutrient soil and vermiculite (3:1). The planting conditions were: 16 hours of daylight, a temperature of 20-28°C, and a humidity of 40-60%.

[0025] (2) Design of cutting container

[0026] Use a blue opaque plastic tray as a cutting container, which is 60 cm long, 40 cm wide and 4 cm high. Take a foam board of the same size and evenly punch round holes with a diameter of 2 cm on it to cover the plastic tray. Sterilize it and set aside.

[0027] (3) Preparation of hydroponic nutrient solution: The hydroponic nutrient solution is based on sterilized deionized water, with 3g / L of rooting powder (common name: trace element water-soluble fertilizer) and 2ml / L of dilution of benzyl alcohol added. The rooting powder can be purchased from Taobao e-commerce, such as Shandong Ainong Trace Element Foliar Fertilizer.

[0028] (4) Cutting selection: As described in (1), transgenic bottle gourds that have been cultured for at least 30 days are used as mother plants, and side branches or main branches with 2-3 leaf buds, 4-5 leaves, and 3-4 stem nodes on the mother plants are selected as cuttings.

[0029] (5) Hydroponic cuttings: Pour 3L of prepared hydroponic nutrient solution into the culture container. Cut the selected cuttings at a 45° angle to the branch. The cuttings should be 10-20cm long, with 2-4 leaves and 1-2 leaf buds. Ensure that the leaf buds are exposed and not submerged in the nutrient solution. Then insert the cuttings into the small holes of the foam board, ensuring that 1-2 stem nodes are submerged in the nutrient solution, and then place them in the culture container. 5-10 cuttings can be placed in the same container.

[0030] (6) Hydroponic cutting management: 12 hours of light, 3000 lx of light intensity, 26°C, 60% humidity; 12 hours of darkness, 20°C, 40% humidity. Change the nutrient solution every 2 days. Adventitious roots will appear on the cuttings after 6-7 days of cultivation.

[0031] (7) Soil-based cuttings: When the stem nodes of the cuttings turn white and adventitious roots grow, they can be moved to the soil for cuttings. The stem nodes with adventitious roots should be buried deep in the substrate, exposing the leaves and leaf buds at the top, and watered with 200-300 ml of sterile water.

[0032] (8) Soil-based cutting management: light duration 12h, light intensity 4000lx, temperature 26℃, humidity 40%, dark duration 12h, temperature 20℃, humidity 40%, and sterile water irrigation once a day.

[0033] (9) Planting: After 7-10 days of soil-based cuttings, the cuttings will sprout a large number of 2-4 cm long adventitious roots. Transplant the cuttings together with the substrate into nutrient soil and manage them in the same way as conventional gourd cultivation.

[0034] The terminology of the present invention

[0035] Gourd

[0036] Bottle gourd [Lagenaria siceraria (Mol.) Standl.] (2n=2x=22) is a cultivated species of the genus Cucurbitaceae, and is one of the specialty summer vegetables in southern my country.

[0037] Culture container

[0038] The culture container can be a hydroponic container commonly used in the art. In some specific embodiments, an opaque cooked material tray can be selected as the culture container. Furthermore, a blue opaque plastic tray can be used. The size of the hydroponic container is selected according to actual needs. In some embodiments, the culture container is 60 cm long, 40 cm wide, and 4 cm high. Furthermore, in order to facilitate the positioning and cultivation of the cuttings, in some embodiments, a foam board of the same size can be evenly punched with circular holes with a diameter of 4 cm and covered on the plastic tray, and then sterilized for use.

[0039] hydroponic nutrient solution

[0040] To sterile deionized water, add 3g / L of rooting powder (common name: trace element water-soluble fertilizer) and 2ml / L of dilution of benzyl ammonium chloride. The rooting powder is purchased from Taobao, such as Shandong Ainong Trace Element Foliar Fertilizer. The main technical indicators are Mn+Zn+B+Mo ≥ 10%, mercury ≤ 5mg / kg, arsenic ≤ 10mg / kg, cadmium ≤ 10mg / kg, lead ≤ 50mg / kg, and chromium ≤ 50mg / kg.

[0041] Selection of cuttings

[0042] The gourd has many side branches and vigorous growth during its growth process, so the present invention selects the side branches of the gourd as cuttings, the side branches need to be selected before the flowering period, and the uppermost node of the selected side branches needs to have 1-2 leaf buds, the cutting length needs to include 3-4 stem sections (10-20 cm), the lower stem section has the highest rooting rate and the best plant growth, followed by the middle section, and the upper section has the worst rooting rate and growth, and is easy to enter the reproductive growth stage, and begins to bloom when the plant is still small, which affects later utilization and seed yield.

[0043] The advantages and beneficial effects of the present invention are as follows:

[0044] 1. the present invention is that the water culture of gourd genetically modified plant is attempted for the first time in combination with soil-based cutting and propagation technology, gourd breeding is very strict to environmental conditions and is only suitable for cultivation and breeding in spring and autumn, and transgenic gourd is due to the uncontrollability of the transgenic test output cycle, and after obtaining rare transgenic material, has to adopt the cutting clone propagation technology to preserve rare germplasm resources. The water culture of gourd genetically modified material is combined with soil-based cutting method, there is the advantage of available throughout the year, controllable environment, convenient management and time-saving and labor-saving, and the clone plant growth consistency of cuttage is better, can preferably keep the peculiar character of female parent, particularly can be used for molecular breeding research and seed propagation.

[0045] 2. The present invention uses a light-proof culture tray as a culture container to reduce the problem of phytoplankton growth in the nutrient solution due to long-term light exposure. It uses 75% alcohol for disinfection, effectively avoiding the phenomenon of mold and fungus growth of cuttings during hydroponics due to bacteria in the culture container, and also isolates the spread of soil-borne diseases. It also occupies little space and is suitable for wide promotion.

[0046] 3. the present invention uses soil culture seedling side shoot branch to carry out cuttage, and gourd can sprout more side branches during growth, and most side branches need to be pruned by pruning in conventional gourd breeding process, and the main reason is that plant nutrition supply is insufficient, and pruning and pruning make plant enter reproductive growth stage faster, and these pruned side branches have excellent phenotype and growth potential equally. Cuttings are all easily obtained throughout the year, and this method can be carried out cutting and expansion throughout the year, is not limited by season and temperature, and effective cutting time is extended.

[0047] 4. The nutrient solution prepared by the present invention is nutritionally balanced and reasonable, and the dosage and method of use of trace element fertilizers are adjusted to be suitable for inducing the differentiation and germination of adventitious roots of transgenic gourds in the nutrient solution. The cutting seedlings grow robustly and uniformly, are simple to operate, easy to manage, practical and efficient, eliminate diseases and insect pests, and save the labor costs of management measures such as irrigation, spraying pesticides, and deworming required for traditional soil cuttings.

[0048] 5. This invention combines hydroponic cuttings with soil-based cuttings to overcome the weak growth potential and poor differentiation and regeneration ability of transgenic bottle gourds. Hydroponic cuttings are first used to induce wound healing and adventitious rooting, followed by soil-based cuttings to strengthen the seedlings. This improves the survival rate of the cuttings and reduces their disease susceptibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0050] Figure 1 This is the operation flow in the embodiment. DETAILED DESCRIPTION

[0051] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0052] Unless otherwise specified, all reagents used in the examples of the present invention are commercially available and can be purchased through commercial channels. Chemicals and consumables used in the experiments, such as nutrient soil, vermiculite, plastic trays, rooting powder, and benomyl, were purchased from the market and met industrial production standards.

[0053] Example 1

[0054] (1) Preparation of cuttings

[0055] Transgenic bottle gourd HD-1 was used as the test material for the experiment. The planting medium was a mixed nutrient soil (nutrient soil: vermiculite ratio of 3:1). The planting conditions were: 12 hours of light, 20-30°C temperature, and 40-60% humidity.

[0056] (2) Design of cutting container

[0057] Use a blue opaque plastic tray as a cutting container, which is 60 cm long, 40 cm wide and 4 cm high. Take a foam board of the same size and evenly punch round holes with a diameter of 2 cm on it, then cover the plastic tray. Wash the container twice with deionized water, then wipe the surface of the container with 75% alcohol, and place it in a dry and ventilated place to dry before use.

[0058] (3) Preparation of hydroponic nutrient solution: The hydroponic nutrient solution uses sterilized deionized water as the solvent. 3g of rooting powder (common name: trace element water-soluble fertilizer) and 2ml of dilution of benzyl alcohol are added to each liter of deionized water. The rooting powder can be purchased from Taobao e-commerce, such as Shandong Ainong Trace Element Foliar Fertilizer.

[0059] (4) Cutting selection: The transgenic bottle gourd plants that have been cultured for at least 30 days are used as mother plants. The side branches before the flowering period of the mother plants are selected for cuttings. The top node of the selected side branches needs to have 1-2 leaf buds. The length of the cuttings needs to include 3-4 stem sections and be 10-20 cm.

[0060] (5) Hydroponic cuttings: Pour 3L of prepared hydroponic nutrient solution into the culture container. Cut the selected transgenic side branch cuttings at a 45° angle to the branch. The cuttings should be 10-20cm long, with 3-4 leaves and 1-2 leaf buds. Then insert them into the small holes of the foam board, ensuring that the leaf buds and leaves are exposed to full sunlight and not submerged in the nutrient solution. Ensure that 1-2 stem nodes are immersed in the nutrient solution and place them in the culture container.

[0061] (6) Hydroponic cutting management: 12 hours of light, 3000 lx of light intensity, 26°C temperature, 60% humidity; 12 hours of darkness, 20°C temperature, 40% humidity. Change the nutrient solution every 2 days. After 6-7 days of hydroponic culture, the nodes of the cuttings immersed in the nutrient solution will turn white, crack, and produce adventitious roots.

[0062] (7) Soil-based cuttings: When the stem nodes of the cuttings turn white and adventitious roots grow, they can be moved to the soil for cuttings. Use Yilan brand horticultural vermiculite sterilized by high-pressure steam as the cutting medium. Bury the stem nodes with adventitious roots deep in the medium, exposing the leaves and leaf buds at the top, and water with 200-300ml of sterile water.

[0063] (8) Soil-based cutting management: 12 hours of light, 4000 lx of light intensity, 26°C temperature, 40% humidity; 12 hours of darkness, 20°C temperature, 40% humidity. Water with 200 ml of sterile water daily. Transplant to conventional soil when adventitious roots reach 2-4 cm in length as observed through the bottom.

[0064] (9) Planting: After 7-10 days of soil-based cuttings, the cuttings will sprout a large number of 2-4 cm long adventitious roots. Transplant the cuttings together with the vermiculite substrate into HAWITA brand nutrient soil and manage them in the same way as conventional gourd cultivation.

[0065] in, Figure 1 Shown are the morphologies of cutting seedlings at various stages.

[0066] Multiple control experiments were conducted using the same procedures as in Example 1, with various modifications to the steps. Control group 1 was not hydroponically cultured; controls 2-5 were hydroponically cultured, but the concentration of rooting powder in the hydroponic nutrient solution in step 3) was modified, and a translucent plastic tray was used as the cutting container in step 2). The final adventitious root induction rate, plant susceptibility rate, and plant survival rate of the cuttings obtained in Example 1 and the control groups were statistically analyzed and are shown in Table 1.

[0067] Table 1

[0068] Adventitious root induction rate Plant susceptibility Plant survival rate Soil-based cuttings 15％ 70％ 10％ Hydroponic cuttings (7g / L) 50％ 50％ 30％ Hydroponic cuttings (5g / L) 60％ 45％ 50％ Hydroponic cuttings (3g / L) 80％ 40％ 70％ Hydroponic cuttings (1g / L) 60％ 60％ 55％ Hydroponic cuttings (3g / L) + opaque container 85％ 20％ 80％

[0069] This shows that the soil-water combined cutting technique of Example 1 is well suited for the cutting propagation of transgenic bottle gourd.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are possible within the skill of the art. Any modifications, equivalent substitutions, or improvements to the present invention are intended to fall within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of transgenic gourd by cuttings, characterized in that: The following steps are involved: 1) Using the transgenic bottle gourd plant as the mother plant, select bottle gourd side branches 10-20 cm long as cuttings; cut the cuttings from the stem nodes; 2) completely immersing 1-2 stem nodes at the lower end of the cuttings obtained in step 1) in a nutrient solution; and hydroculturing the cuttings until the stem nodes turn white and adventitious roots grow; 3) Transplanting the cuttings obtained in step 2) into a soil base for cuttings. Bury the stem nodes that have grown adventitious roots in the soil, leaving the top leaves and leaf buds exposed. Water with 200-300 ml of sterile water. Cultivate in the soil for 7-10 days until the cuttings sprout numerous 2-4 cm long adventitious roots. 4) transplanting the cuttings obtained in step 3) together with the substrate into nutrient soil; The nutrient solution is an aqueous solution containing rooting powder with a concentration of 3 g / L; the nutrient solution also contains 2 ml / L of dilution solution of benzyl ammonium chloride.

2. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: In step 1), the cutting angle at the stem node is 40-50°.

3. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: The leaves and tendrils at 1-2 nodes at the lower end of the cutting seedlings obtained in step 1) need to be removed.

4. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: The cutting seedlings obtained in step 1) need to retain 2-4 leaves and 1-2 leaf buds on the top.

5. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: In step 2), each cutting seedling requires 400 ml of rooting nutrient solution.

6. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: The hydroponic conditions in step 2) are as follows: 12 hours of light per day, 3000 lx of light intensity, 26° C., and 60% humidity; 12 hours of darkness per day, 20° C., and 40% humidity; and the nutrient solution is replaced every 2 days.

7. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 6, wherein: The culture container used for hydroponics in step 2) is a light-proof container.

8. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: In step 3), the substrate for soil-based cuttings is vermiculite with a thickness of 3-4 cm.

9. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 8, wherein: The vermiculite is sterilized in a high-pressure steam autoclave at 121°C.

10. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: The soil culture conditions in step 3) are as follows: daily light duration of 12 hours, light intensity of 4000 lx, temperature of 26°C, and humidity of 60%; daily dark duration of 12 hours, temperature of 20°C, and humidity of 40%; and watering with 200-300 ml of sterile water once a day.

11. The method for rapid propagation of transgenic bottle gourd by cuttings according to claim 1, wherein: The transgenic bottle gourd plant used as the mother plant in step 1) is a transgenic bottle gourd plant that has been soil-cultured for at least 30 days.

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