Method for rapidly inducing callus and proliferating by utilizing blackberry lily seeds

Abstract

The invention provides a method for rapidly inducing calluses and proliferating by utilizing blackberry lily seeds. According to the method, the callus is directly induced by the blackberry lily seeds for the first time, the induction time is short, the callus can be induced after the blackberry lily seeds are used as explants and disinfected for 25 days, and the induction rate reaches 91.75%. After the seeds are sterilized for 5 days, the sterility rate is 56.96%, the sterility rate in the callus induction period is 94.86%, 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 3-4 mm callus mass is 93.23%, the browning rate of the callus during subculture is only 7.92%, 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 blackberry lily calluses can be provided in a short time, and a foundation is laid for follow-up establishment of a blackberry lily 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 particularly relates to a method for rapidly inducing callus and proliferation by utilizing Belamcanda chinensis seeds. Background Art

[0002] Iris domestica is a perennial herbaceous plant of the genus Iris in the family Iridaceae, widely distributed across most provinces of China. It prefers warmth and sunshine, tolerates both drought and cold, and has a relaxed soil profile, allowing it to be cultivated on hillsides and dry land. Its orange-red flowers and its tolerance to cold and drought have made it a valuable ornamental plant for ecological restoration and urban greening. Furthermore, Iris domestica has numerous medicinal benefits, including clearing heat and detoxifying, eliminating phlegm, and relieving sore throats, and is used to treat symptoms such as tonsillitis and low back pain. Establishing a rapid propagation method is crucial for the research, breeding, application, and commercial production of Iris domestica. Tissue culture technology is an effective means of rapidly improving Iris domestica's propagation efficiency and serves as a foundation for genetic engineering breeding. However, limited research exists on tissue culture systems for Iris domestica, making the development of efficient tissue culture propagation techniques particularly necessary and urgent.

[0003] Callus tissue carries all the genetic information of the entire plant and has the omnipotence of secondary metabolite biosynthesis and plant regeneration. It is also the basic material required for genetic transformation. However, as a monocot, Belamcanda 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 as explants have high contamination rates and serious endophyte contamination, and the operation window is short. Sterile hypocotyls, sterile leaf sheaths, and sterile root segments rely on the supply of sterile seedlings. In addition, existing explants all have the problems of long callus induction cycles and low induction rates.

[0004] In comparison, Belamcanda chinensis seeds have three unique advantages as explants: First, they are available year-round. Belamcanda chinensis seeds are available year-round, so the materials they use are not restricted by time or space. Second, they have a high disinfection success rate. Belamcanda chinensis seeds are hard and can withstand multiple washings and long-term disinfection at high concentrations. Third, embryonic cells have a stronger dedifferentiation potential and can quickly induce callus tissue. The present invention is the first to directly induce callus tissue from Belamcanda chinensis seeds. The induction time is short, and callus tissue can be induced 25 days after disinfection, with an induction rate of 91.75%, a sterility rate of 94.86% during induction, and a sterility rate of 100% during subculture. Through the optimized method, a low browning rate (7.92%) is achieved, and the callus tissue grows quickly and has good activity. The proliferation rate of 3-4mm callus tissue clumps is 93.23%, which can be used for long-term proliferation and provide a large amount of callus tissue, laying a technical foundation for rapid propagation of Belamcanda chinensis tissue culture, gene editing breeding, and industrial production of secondary metabolites. Summary of the Invention

[0005] The object of the present invention is to provide a method for rapidly establishing and proliferating callus tissue from Belamcanda oleracea seeds by using seeds as explants and going through the steps of explant acquisition, explant disinfection, callus induction, and callus proliferation. This method can rapidly obtain a large amount of callus tissue that is bright yellow in color and in good condition, which is conducive to subsequent redifferentiation to form adventitious buds and further cultivation into complete plants, thereby realizing rapid propagation of Belamcanda oleracea and providing a basis for the subsequent establishment of a genetic transformation system for Belamcanda oleracea.

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

[0007] Step 1: Obtain explants. Select Belamcanda seeds harvested within two years as explants, wash them and set aside.

[0008] Step 2: Explant disinfection: Take the clean seeds treated in Step 1 and shake them for 10 minutes with detergent and water. Rinse thoroughly with tap water for 30 minutes. In a sterile hood, shake and rinse with 75% ethanol solution for 1 minute. Rinse three times with sterile water for 1 minute each. Sterilize in a 5% available chlorine solution with NaClO and shake for 30 minutes. Rinse six times with sterile water for 30 seconds each. Place the disinfected seeds on sterile filter paper.

[0009] Step 3: Callus Induction: Press the seeds obtained in Step 2 into callus induction medium using 90 mm culture dishes, with approximately 15 seeds dispersed per dish. Seal the dishes and incubate in a dark room at 25 ± 1°C. After 5 days, select a dish free of fungal contamination and select seeds from it that do not show bacterial growth. Inoculate the callus induction medium and incubate in the dark for 25 days.

[0010] Step 4: Callus proliferation. The sterile seeds with robust callus obtained in step 3 were subcultured to callus proliferation medium. After 25 days, the callus was cut and placed on sterile filter paper. The browned tissue was removed and cut into small pieces with a diameter of 3-4 mm. The pieces were subcultured to callus proliferation medium and cultured at 25±1°C in the dark. The seeds were subcultured once every 25 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 + 0.5 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 used in the present invention has the following beneficial effects during callus induction: 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 conjunction with embodiments. The advantages and implementations of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings:

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

[0016] Attachment Figure 2 This is a picture of callus tissue obtained after the Belamcanda chinensis seeds were placed on callus induction medium for 25 days.

[0017] Attachment Figure 3 This is a picture of the callus tissue 25 days after the first proliferation.

[0018] Attachment Figure 4 This is the condition of the callus tissue one day after it was cut into small pieces with a diameter of 3-4 mm.

[0019] Attachment Figure 5 This is the condition of the callus tissue 25 days after it was cut into small pieces with a diameter of 3-4 mm. DETAILED DESCRIPTION

[0020] The present invention is described in further detail below with reference to the embodiments and accompanying drawings to facilitate understanding of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and researchers in this 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.

[0022] 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 Belamcanda chinensis seeds.

[0023] The preparation method of the relevant culture medium in the embodiment of the present invention is as follows:

[0024] 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.

[0025] The callus proliferation medium was MS + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar, with a pH of 5.8-6.0.

[0026] The calculation methods of sterility rate, induction rate, proliferation rate and browning rate in the examples are as follows:

[0027] Sterility rate of disinfection = number of seeds without bacteria 5 days after disinfection / total number of seeds × 100%.

[0028] The sterility rate during the induction period = the number of seeds without bacteria 25 days after callus induction / the total number of seeds × 100%.

[0029] The sterility rate during the subculture period = the number of seeds that did not grow bacteria 25 days after the first subculture / the total number of seeds × 100%.

[0030] Induction rate = number of seeds induced to callus / number of seeds germinated × 100%.

[0031] Proliferation rate = number of callus pieces with a diameter of 3-4 mm that increased significantly in volume 25 days after subculture / total number of calli × 100%.

[0032] Browning rate = number of calli that showed browning 25 days after subculture / total number of calli × 100%.

[0033] The present invention provides a method for rapidly inducing callus and proliferation using Belamcanda chinensis seeds. The method comprises the following steps:

[0034] (1) Explant acquisition

[0035] Take the strong and plump seeds of Belamcanda chinensis that have been harvested within two years, rinse them under tap water for 5 minutes and set aside. Figure 1 is the state of the explant obtained.

[0036] (2) Explant disinfection

[0037] Use Erlenmeyer flasks as containers. Place one-sixth of the explant volume in each flask and add clean water. Seal the flask with gauze and shake rapidly for 10 minutes. Then rinse thoroughly under running water for 30 minutes. Then, transfer the explants to new containers in a sterile flask. Shake and wash with a 75% ethanol solution that submerged the explants for 1 minute. Rinse with sterile water three times for 1 minute each. Place the explants in a 5% NaClO solution with intermittent, vigorous shaking for 30 minutes. Rinse with sterile water six times for 30 seconds each. Place the sterilized explants on sterile filter paper and blot dry.

[0038] (3) Callus induction

[0039] Use tweezers to gently pick up the seeds and press them into the callus induction medium. Use 90mm culture dishes and place about 15 seeds in each dish. After sealing the dishes, culture them in a dark environment at a temperature of 25±1℃. After 5 days, select a culture dish without fungal contamination and use tweezers to pick out the seeds without 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 a picture of callus tissue obtained after the Belamcanda chinensis seeds were placed on callus induction medium for 25 days.

[0040] The sterility rate of disinfection, sterility rate during induction period and induction rate were calculated respectively.

[0041] According to statistics, the 5-day disinfection sterility rate was 56.96%, the sterility rate during the induction period was 94.86%, and the induction rate was 91.75%.

[0042] (4) Callus proliferation

[0043] The well-growing and sterile seeds of Belamcanda chinensis with callus tissue were subcultured to the callus proliferation medium for the first proliferation culture. The nutrients in the seed endosperm were used to continuously supply the callus tissue growth. After 25 days, the callus tissue with strong growth, bright yellow color and clear secretion was selected. Only the callus tissue was cut off with a new scalpel. After carefully removing the browned part, the callus tissue blocks were cut in half one by one. Finally, they were cut into small pieces with a diameter of 3-4 mm and placed on the callus proliferation medium. About 100 pieces of callus tissue were scattered in each culture dish (90 mm). They were cultured at 25±1℃ and in the dark, and subcultured once every 25 days. The sterility rate, proliferation rate and browning rate were calculated. Figure 3 This is the status picture of callus tissue 25 days after the first proliferation. Figure 4This is the picture of the callus tissue after being cut into small pieces with a diameter of 3-4 mm on the first day. Figure 5 This is the condition of the callus tissue 25 days after being cut into small pieces with a diameter of 3-4 mm.

[0044] According to statistics, the sterility rate during the subculture period was 100%, the proliferation rate was 93.23%, and the browning rate was 7.92%.

[0045] The embodiment of the present invention provides a method for rapidly inducing callus and proliferation using a kind of Belamcanda chinensis seed. The seeds are used as explants, sterilized to obtain sterile seeds, and then callus is induced to form. The induction rate is 91.75%, the sterility rate during subculture is 100%, the proliferation rate of 3-4mm callus cuttings is 93.23%, and the browning rate is 7.92%. The technology of the present invention is easy to master, low in cost and high in efficiency, laying 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 season and are easy to obtain and preserve, which can achieve the goal of rapid tissue culture propagation of Belamcanda chinensis throughout the year.

[0046] The above 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 replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for rapidly inducing callus and proliferation using Belamcanda chinensis seeds, characterized in that: The following steps are involved: Step 1: Explant acquisition: Select Belamcanda chinensis seeds as explant materials; Step 2: Explant disinfection: Disinfect the seeds of Belamcanda chinensis; Step 3: Callus induction: The sterilized seeds of Belamcanda chinensis were inoculated 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 Belamcanda chinensis with callus tissue into callus proliferation medium: MS + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar. After culturing for 25 days, only the callus tissue was taken and cut into callus pieces with a diameter of 3-4 mm and inoculated into 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 the dark.

2. The method for rapidly inducing callus and proliferation using Belamcanda chinensis seeds according to claim 1, characterized in that The Belamcanda chinensis seeds described in step 1 refer to Belamcanda chinensis seeds harvested within two years.

3. The method for rapidly inducing callus and multiplying callus using Belamcanda chinensis seeds according to claim 1, characterized in that The disinfection treatment described in step 2 refers to shaking and cleaning the Belamcanda seeds in claim 2 with detergent water for 10 minutes, rinsing with tap water for 30 minutes, and thoroughly washing 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 a 5% NaClO solution of available chlorine and shaking for 30 minutes. Rinse with sterile water 6 times, each time for 30 seconds, and place the disinfected seeds on sterile filter paper to absorb moisture.

4. The method for rapidly inducing callus and multiplying callus using Belamcanda chinensis seeds 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 a callus induction medium. The seeds are cultured in the dark for 25 days to produce Belamcanda seeds with callus tissue.

5. The method for rapidly inducing callus and multiplying callus using Belamcanda chinensis seeds according to claim 1, characterized in that The callus proliferation described in step 4 refers to inoculating the Belamcanda chinensis seeds with callus tissue in claim 4 into a callus proliferation medium, using nutrients in the seed endosperm to continuously supply callus tissue growth, and after 25 days, selecting callus tissue with strong growth, bright yellow color, and clear secretions, cutting only the callus tissue with a brand new scalpel, carefully removing the browned part, and then cutting the callus tissue blocks in half in sequence, and finally cutting them into small pieces with a diameter of 3-4 mm, subculture them onto callus proliferation medium, and culture them at 25±1°C in the dark, and subculture them once every 25 days.

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