Method for rapidly inducing calluses and proliferating by utilizing swallow seeds

Abstract

The invention provides a method for rapidly inducing calluses and proliferating by utilizing swallow flower seeds. According to the method, the calluses are directly induced by the swallow flower seeds for the first time, the induction time is short, the calluses can be induced after the swallow flower seeds serve as explants and are disinfected for 30 days, and the induction rate reaches 58.48%. After the seeds are sterilized for 5 days, the sterility rate is 95.56%, the sterility rate in the callus induction period is 99.17%, and the sterility rate in the callus subculture period is 100%. The callus is high in growth speed and good in activity, the proliferation rate of a 2mm callus block mass is 81.54%, the browning rate of the callus during subculture is only 5.36%, and the callus can be proliferated for a long time. The method is easy to master, low in cost and high in efficiency, materials are easy to obtain, the method is not limited by seasons, a large number of calluses of the swallow flowers can be provided in a short time, and a foundation is laid for follow-up establishment of a swallow flower efficient improved variety propagation system and a genetic transformation system.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for rapidly inducing callus and proliferation using spathiphyllum spatholobi seeds. Background Art

[0002] Iris laevigata Fisch. is a perennial wetland plant of the genus Iris in the family Iridaceae. It is native to high-altitude wetlands in northeast and southwest my country and is widely distributed in Japan, North Korea, Russia and other regions. With its large blue-purple flowers (flowering period from May to June) and strong cold resistance and water and humidity resistance, Iris laevigata Fisch. has become an important ornamental plant for wetland ecological restoration and waterscape construction. However, its traditional propagation method has significant bottlenecks: seed propagation takes 3 years to bloom; as a large wetland plant, ramet propagation is inconvenient, the ramet coefficient is low and it is easily affected by root rot, which makes it difficult to meet the needs of large-scale production and breeding. Establishing a rapid propagation method is crucial for plant research, breeding, application and commercial production. Tissue culture is an effective means to quickly expand the propagation coefficient of Iris laevigata Fisch. It is also a necessary basis for genetic engineering breeding of Iris laevigata Fisch. However, there is currently little research on the rapid establishment of tissue culture systems for Iris laevigata Fisch. It is very necessary and urgent to develop tissue culture propagation methods for Iris laevigata Fisch.

[0003] Callus tissue carries all the genetic information of the whole plant, has the omnipotence of biosynthesis of secondary metabolites and plant regeneration, and is the basic material required for genetic transformation. However, iris is a monocot without cambium, and faces two major challenges in callus induction: First, the regeneration ability of Iridaceae is lower than that of Amaryllidaceae, Araceae and Liliaceae in the monocot class (Wei Xiaoyu et al. Research Progress on Tissue Culture and Rapid Propagation Technology of Iris Plants [J]. Molecular Plant Breeding, 2024, 22(19): 6500-6513.), and callus induction is difficult and time-consuming. Second, existing explants have temporal and spatial limitations. Flower stem nodes and young leaves are used as explants with high contamination rates and serious endophyte contamination, and the operation window period is short. Sterile hypocotyls, sterile leaf sheaths, and sterile root segments rely on the supply of sterile seedlings. In addition, existing explants have long callus induction cycles and low induction rates (Liu Yujia. Establishment of tissue culture system of Swallow's jasmine and preliminary exploration of genetic transformation system [D]. Northeast Forestry University, 2020. DOI: 10.27009 / d.cnki.gdblu.2020.000757.). Although some studies have obtained regenerated plants through in vitro culture of immature fruits (Zhao Hongbo, Zhang Chao, Fu Jianxin, et al. A method for rapid in vitro propagation of Swallow's jasmine using immature fruits [P]. Zhejiang Province: CN201510217132.4, 2016-09-07.), the acquisition of explants is limited by the unique fruit development stage.

[0004] In contrast, as explants, the seeds of the Chinese swallowtail have three unique advantages: first, they are available all year round. The seeds of the Chinese swallowtail are available all year round, and the problem of seed germination difficulties has been overcome (DB23 / T 3054-2021, Technical regulations for seed stratification treatment of Chinese swallowtail seeds [S]), so the materials used are not restricted by time and space; second, the success rate of disinfection is high. The seeds of the Chinese swallowtail are easy to remove the seed coat, and the seeds are hard and can withstand multiple washings and long-term disinfection at high concentrations; third, embryonic cells have stronger dedifferentiation potential and can quickly induce callus tissue. The present invention realizes the direct induction of callus tissue from Chinese yew seeds for the first time, with a short induction time. Callus tissue can be induced 30 days after disinfection, with an induction rate of 58.48%, a sterility rate of 99.17% during induction, and a sterility rate of 100% during subculture. A lower browning rate (5.36%) is obtained through an optimized method, and the callus tissue grows fast and has good activity. The proliferation rate of a 2 mm callus mass is 81.54%, which can be used for long-term proliferation and provide a large amount of callus tissue, laying a technical foundation for rapid propagation of Chinese yew tissue culture, gene editing breeding, and industrial production of secondary metabolites. Summary of the invention

[0005] The purpose of the present invention is to provide a method for quickly establishing callus induction and proliferation of jasmine seeds by using seeds as explants, through the steps of explant acquisition, explant disinfection, callus induction, callus proliferation and the like. This method can quickly obtain a large amount of callus with bright yellow color and good condition, which is conducive to subsequent redifferentiation to form adventitious buds and further cultivation into complete plants, thereby achieving rapid reproduction of jasmine and providing a basis for the subsequent establishment of a genetic transformation system of jasmine.

[0006] In order to realize the above technology, the technical solution adopted by the present invention is: a method for rapidly inducing callus and proliferation using seeds of spatholobi, comprising the following steps:

[0007] Step 1: Obtain explants. Select the seeds of the Chinese marigold that have been harvested and stratified for germination that year as explants. Remove the seed coats and rinse them for later use.

[0008] Step 2: Explant disinfection: Take the clean seeds treated in step 1, shake and clean the peeled seeds for 5 minutes with detergent water, rinse with running water for 30 minutes, and wash thoroughly. In a sterile table, shake and clean with 75% ethanol solution for 1 minute, rinse with sterile water 3 times, 1 minute each time, then put in 4% NaClO solution and shake and disinfect for 25 minutes, rinse with sterile water 5 times, 1 minute each time, and place the disinfected seeds on sterile filter paper.

[0009] Step 3: Callus induction: press the seeds obtained in step 2 into the callus induction medium. The culture dish is 90 mm in size, and 30 seeds are placed in each culture dish. After the culture dish is sealed, it is cultured in a dark environment at a temperature of 25±1°C. After 5 days, select the culture dish without fungal contamination, select the seeds without bacteria from it, inoculate it into the callus induction medium, and culture it in the dark for 25 days.

[0010] Step 4: Callus proliferation. The sterile seeds with robust callus obtained in step 3 are subcultured to callus proliferation medium. After 30 days, the callus is cut and placed on sterile filter paper. The browned tissue is removed and cut into small pieces with a diameter of 2 mm. The pieces are continued to be placed on callus proliferation medium and cultured at 26°C in the dark. The pieces are subcultured once every 30 days.

[0011] The callus induction medium described in step 3 is MS+1.0 mg / L 6-BA+0.4 mg / L NAA+1.0 mg / L 2,4-D+30 g / L sucrose+7.0 g / L agar, with a pH of 5.8-6.0.

[0012] The callus proliferation medium described in step 4 is MS+0.5 mg / L 6-BA+0.2 mg / L NAA+0.5 mg / L 2,4-D+30 g / L sucrose+7.0 g / L agar, with a pH of 5.8-6.0.

[0013] The technology adopted by the present invention has the following beneficial effects in the callus induction process: convenient acquisition and disinfection of explants, high sterility rate during induction, fast induction speed, good activity of the obtained callus, sustainable proliferation, and no endophytes during proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments, and the advantages and implementation modes of the present invention will become more obvious. The contents shown in the accompanying drawings are only used to explain the present invention and do not constitute any limitation to the present invention in any sense. In the accompanying drawings:

[0015] Attached Figure 1 is a picture of the obtained explant.

[0016] Attached Figure 2 This is the callus tissue picture obtained after the seeds of the Swallow's Nest were placed on the callus induction medium for 25 days.

[0017] Attached Figure 3 This is the status of callus tissue on the first day after the fourth subculture.

[0018] Attached Figure 4 This is the status of callus tissue 30 days after the fourth subculture. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments and drawings to help understand the content of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and researchers in the field can achieve the results by following conventional tissue culture methods; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0021] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for rapidly inducing callus and proliferation using seeds of spatholobi.

[0022] The preparation method of the relevant culture medium in the embodiment of the present invention is as follows:

[0023] The callus induction medium was MS + 1.0 mg / L 6-BA + 0.4 mg / L NAA + 1.0 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar, with a pH of 5.8-6.0.

[0024] The callus proliferation medium was MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar, with a pH of 5.8-6.0.

[0025] The calculation methods of sterility rate, induction rate, proliferation rate and browning rate in the embodiment are as follows:

[0026] Sterility rate of disinfection = number of seeds without bacteria 5 days after disinfection / total number of seeds × 100%.

[0027] The sterility rate during the induction period = the number of seeds without bacteria after 25 days of callus induction / the total number of seeds × 100%.

[0028] The sterility rate during the subculture period = the number of seeds that did not grow bacteria 30 days after the first subculture / the total number of seeds × 100%.

[0029] Induction rate = number of seeds induced to callus / number of seeds germinated × 100%.

[0030] Proliferation rate = the number of callus pieces with a diameter of 2 mm that increased significantly in volume 30 days after subculture / the total number of callus pieces × 100%.

[0031] Browning rate = number of calli that showed browning after subculture for 30 days / total number of calli × 100%.

[0032] The embodiment of the present invention provides a method for rapidly inducing callus and proliferation using seeds of spatholobi, which includes the following steps:

[0033] (1) Explant acquisition

[0034] Take the strong and full seeds of the stratified flower, remove the seed coat and rinse them under tap water for 5 minutes for later use. Figure 1 is the explant status obtained.

[0035] (2) Explant disinfection

[0036] Use conical flasks as containers, place one-sixth of the volume of explants in each conical flask and add clean water, seal it with gauze, shake it quickly for 5 minutes, and then place it under the tap to rinse for 30 minutes to thoroughly wash it. Then, in the sterile table, within 10 cm from the air outlet under the alcohol lamp, use a sterile conical flask as a container to transfer the explants to a new container. Use a 75% ethanol solution that has submerged the explants to shake and wash for 1 minute, rinse with sterile water 3 times, 1 minute each time, then place the explants in a 4% NaClO solution, shake and disinfect intermittently for 25 minutes, rinse with sterile water 5 times, 1 minute each time, and place the disinfected explants on sterile filter paper to absorb the moisture.

[0037] (3) Callus induction

[0038] Use tweezers to gently pick up the seeds and press them into the callus induction medium. Use a 90mm petri dish and place about 15 seeds in each dish. After sealing the dish, culture it in a dark environment at a temperature of 25±1℃. After 5 days, select a petri dish that is not contaminated with fungi, use tweezers to pick out the seeds that do not grow bacteria, place them on sterile filter paper, remove the culture medium remaining on the surface of the seeds, and then inoculate them into the callus induction medium and culture them in the dark for 25 days. Figure 2 This is the callus tissue picture obtained after the seeds of the Swallow's Nest were placed on the callus induction medium for 25 days.

[0039] The sterilization rate, induction period sterility rate and induction rate were counted separately.

[0040] According to statistics, the 5-day disinfection sterility rate was 95.56%, the sterility rate during the induction period was 99.17%, and the induction rate was 58.48%.

[0041] (4) Callus proliferation

[0042] Subculture the well-growing and sterile seeds of the Chinese nightshade with callus tissue to the callus proliferation medium, and use the nutrients in the seed endosperm to continuously supply the callus tissue growth. After 30 days, select the callus tissue with strong growth, bright yellow color and clear secretions. Use a brand new scalpel to cut off only the callus tissue, carefully remove the browned part, cut the callus tissue blocks in half one by one, and finally cut them into small pieces with a diameter of 2mm. Place them on the callus proliferation medium, and place about 55 pieces of callus tissue in each culture dish (90mm). Culture them at 25±1℃ in the dark and subculture them every 30 days. The sterility rate, proliferation rate and browning rate were calculated. Figure 3 This is the callus tissue condition on the first day after the fourth subculture. Figure 4 This is the status of callus tissue 30 days after the fourth subculture.

[0043] According to statistics, the sterility rate during the subculture period was 100%, the proliferation rate was 81.54%, and the browning rate was 5.36%.

[0044] The embodiment of the present invention provides a method for rapidly inducing callus and proliferation using a spatholobi seed. The seeds are used as explants, sterilized seeds are obtained, and callus is induced to form. The induction rate is 58.48%, the sterility rate during subculture is 100%, the proliferation rate of 2 mm callus cuttings is 81.54%, and the browning rate is 5.36%. The technology of the present invention is easy to master, low in cost and high in efficiency, and lays the foundation for the subsequent establishment of an efficient and improved variety expansion system and a genetic transformation system. The test materials are not restricted by seasons and are easy to obtain and preserve, so the goal of rapid tissue culture and propagation of spatholobi throughout the year can be achieved.

[0045] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for rapidly inducing callus and proliferation using seeds of spatholobi, characterized in that: The following steps are involved: Step 1: Explant acquisition: Select the seeds of Swallowtail as explant material; Step 2: Explant disinfection: Disinfect the swallowtail flower seeds; Step 3: Callus induction: Inoculate the sterilized spatholobi seeds into callus induction medium: MS + 1.0 mg / L 6-BA + 0.4 mg / L NAA + 1.0 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar; Step 4: Callus proliferation: Inoculate the seeds of the Chinese nightshade with callus into a callus proliferation medium: MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar. After culturing for 30 days, take only the callus and cut it into callus blocks with a diameter of 2 mm and inoculate it into a callus proliferation medium; The pH values ​​of all the above culture media were adjusted to 5.8-6.0, the temperature of the culture chamber was 25±1°C, and the culture was carried out in dark.

2. The method for rapidly inducing callus and proliferating callus using seeds of Hibiscus sibiricus according to claim 1, characterized in that The jasmine seeds described in step 1 refer to the jasmine seeds that are harvested in the current year and have their seed coats removed after stratification and germination.

3. The method for rapidly inducing callus and multiplying callus using seeds of Hibiscus sibiricus according to claim 1, characterized in that The disinfection treatment described in step 2 refers to shaking and cleaning the swallowtail flower seeds in claim 2 with detergent water for 5 minutes, rinsing with tap water for 30 minutes, and thoroughly cleaning them. In a sterile table, shaking and cleaning them with 75% ethanol solution for 1 minute, rinsing with sterile water 3 times, each time for 1 minute, and then placing them in 4% NaClO solution and shaking for 25 minutes for disinfection, rinsing with sterile water 5 times, each time for 1 minute, and placing the disinfected seeds on sterile filter paper to absorb moisture.

4. The method for rapidly inducing callus and multiplying callus using seeds of Hibiscus sibiricus according to claim 1, characterized in that The callus induction described in step 3 refers to inoculating the disinfected seeds in claim 3 into a callus induction medium, culturing them in a dark environment and a culture room at a temperature of 25±1°C. After 5 days, a culture dish without fungal contamination is selected, seeds without bacteria are selected, and inoculated into the callus induction medium. The seeds are cultured in the dark for 25 days to produce the jasmine seeds with callus tissue.

5. The method for rapidly inducing callus and multiplying callus using seeds of Hibiscus sibiricus according to claim 1, characterized in that The callus proliferation described in step 4 refers to inoculating the callus seeds with callus tissue in claim 4 into a callus proliferation medium, using the nutrients in the seed endosperm to continuously supply the callus tissue growth, and after 30 days, selecting the callus tissue with strong growth, bright yellow color, and clear secretions, and cutting only the callus tissue with a brand new scalpel. After carefully removing the browned part, the callus tissue blocks are cut in half one by one, and finally cut into small pieces with a diameter of 2 mm, placed on a callus proliferation medium, and cultured at 25±1°C in the dark, and subcultured once every 30 days.

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