A method for rapidly inducing callus and proliferation by using swallow flower seeds

Abstract

The application provides a method for rapidly inducing callus and proliferation by using swallow flower seeds. The swallow flower seeds are directly induced to callus for the first time, the induction time is short, the callus can be induced 30 days after the swallow flower seeds are used as explants and are sterilized, and the induction rate reaches 58.48%. The aseptic rate of the seeds is 95.56% 5 days after sterilization, the aseptic rate during the callus induction period is 99.17%, and the aseptic rate during the callus subculture period is 100%. The callus has a fast growth speed and good activity, the proliferation rate of 2mm callus clumps is 81.54%, the callus browning rate during the subculture period is only 5.36%, and the callus can be proliferated for a long time. The application has the advantages of easy operation, low cost, high efficiency, easy access to materials, and no season limitation, and can provide a large amount of swallow flower callus in a short period, thereby laying a foundation for subsequent establishment of a high-efficiency elite propagation system and a genetic transformation system of swallow flower.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for rapidly inducing and proliferating callus tissue using seeds of the iris. Background Technology

[0002] *Iris laevigata* Fisch., a perennial wetland plant belonging to the genus *Iris* in the family Iridaceae, is native to high-altitude wetlands in Northeast and Southwest my country and is widely distributed in Japan, Korea, and Russia. Due to its large, blue-purple flowers (flowering from May to June) and its strong cold and waterlogging tolerance, *Iris laevigata* is an important ornamental plant for wetland ecological restoration and waterscape creation. However, its traditional propagation methods have significant limitations: seed propagation requires three years to flower; as a large wetland plant, division propagation is inconvenient, with a low division coefficient and susceptibility to root rot, making it difficult to meet the needs of large-scale production and breeding. Establishing rapid propagation pathways is crucial for plant research, breeding, application, and commercial production. Tissue culture is an effective means to rapidly increase the propagation coefficient of *Iris laevigata* and is also a necessary foundation for its genetic engineering breeding. However, current research on rapidly establishing tissue culture systems for *Iris laevigata* is limited, making the development of tissue culture propagation pathways for *Iris laevigata* both necessary and urgent.

[0003] Callus tissue carries all the genetic information of the entire plant, possesses the totipotency of secondary metabolite biosynthesis and plant regeneration, and is the basic material required for genetic transformation. However, Iris is a monocotyledonous plant without a cambium, and faces two major challenges in callus induction: First, the regeneration capacity of Iridaceae is lower than that of Amaryllidaceae, Araceae and Liliaceae within the monocotyledonous class (Wei Xiaoyu et al. Research progress on tissue culture rapid propagation technology of Iris[J]. Molecular Plant Breeding, 2024, 22(19):6500-6513.), making callus induction difficult and time-consuming. Second, existing explants have spatial and temporal limitations. Flower stem nodes and young leaves have high contamination rates and serious endophytic fungal contamination, and the operation window is short. Aseptic hypocotyls, aseptic leaf sheaths, and aseptic root segments rely on the supply of aseptic seedlings. Furthermore, existing explants all suffer from long callus induction cycles and low induction rates (Liu Yujia. Establishment of the tissue culture system and preliminary study on the genetic transformation system of *Iris tectorum* [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 *Iris tectorum* 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 comparison, *Iris tectorum* seeds have three unique advantages as explants: First, year-round availability. *Iris tectorum* seeds are available year-round, and the problem of seed germination difficulties has been overcome (DB23 / T 3054-2021, Technical Specification for Seedling Raising through Stratification of *Iris tectorum* Seeds [S]), thus the availability of materials is not limited by time or space; Second, high sterilization success rate. *Iris tectorum* seeds are easy to remove the seed coat, and the seeds are hard, capable of withstanding multiple washes and long-term sterilization at high concentrations; Third, embryogenic cells have stronger dedifferentiation potential, which can rapidly induce callus tissue. This invention is the first to achieve direct callus induction from *Ilex cornuta* seeds. The induction time is short, with callus induced within 30 days after sterilization, achieving an induction rate of 58.48%. The sterility rate during induction is 99.17%, and the sterility rate during subculture is 100%. Through optimized methods, a low browning rate (5.36%) was obtained. The callus grows rapidly and has good activity. The proliferation rate of 2mm callus clusters is 81.54%, which can be used for long-term proliferation, providing a large amount of callus. This lays a technical foundation for rapid propagation of *Ilex cornuta* through tissue culture, gene editing breeding, and industrial production of secondary metabolites. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapidly establishing and proliferating seed-induced callus of *Iris tectorum* using seeds as explants, through steps such as explant acquisition, explant disinfection, callus induction, and callus proliferation. This method can rapidly obtain large quantities of bright yellow, well-preserved callus, which is beneficial for subsequent redifferentiation to form adventitious buds and further cultivation into complete plants, thereby achieving rapid propagation of *Iris tectorum* and providing a foundation for the subsequent establishment of a genetic transformation system for *Iris tectorum*.

[0006] To achieve the above-mentioned technology, the technical solution adopted by the present invention is: a method for rapidly inducing callus tissue and proliferation using seeds of *Iris tectorum*, comprising the following steps:

[0007] Step 1: Explant acquisition. Select the seeds of the iris that were harvested and stratified in the current year as explants. Remove the seed coat and rinse them clean for later use.

[0008] Step 2: Explant disinfection. Take the clean seeds treated in Step 1, and wash the peeled seeds with detergent and water for 5 minutes. Rinse thoroughly with running tap water for 30 minutes. In a sterile container, wash the seeds with 75% ethanol solution for 1 minute, rinse three times with sterile water for 1 minute each time, and then disinfect them by shaking in 4% NaClO solution for 25 minutes. Rinse five times with sterile water for 1 minute each time. Place the disinfected seeds on sterile filter paper.

[0009] Step 3: Callus induction. The seeds obtained in Step 2 were pressed into the callus induction medium. 90mm culture dishes were used, with 30 seeds distributed in each dish. The dishes were sealed and cultured in a dark environment at 25±1℃. After 5 days, culture dishes without fungal contamination were selected, and seeds without bacterial growth were inoculated into the callus induction medium and cultured in the dark for 25 days.

[0010] Step 4: Callus proliferation. The sterile seeds with robust callus obtained in Step 3 were subcultured into the callus proliferation medium. After 30 days, the callus was cut off, placed on sterile filter paper, and the browned tissue was removed. The callus was cut into small pieces with a diameter of 2 mm and placed on the callus proliferation medium. The medium was then cultured at 26°C in the dark. Subculture was performed 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 used in this invention produces the following beneficial effects during the callus induction process: the explants are easy to obtain and disinfect, the sterility rate is high during induction, the induction speed is fast, the obtained callus tissue has good activity, can proliferate continuously, and there are no endophytic bacteria during the proliferation period. Attached Figure Description

[0014] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings:

[0015] Appendix Figure 1 This is an image of the obtained explant.

[0016] Appendix Figure 2 The image shows callus tissue obtained after placing Iris tectorum seeds in callus induction medium for 25 days.

[0017] Appendix Figure 3 This is a diagram of the callus tissue on day 1 after the fourth subculture.

[0018] Appendix Figure 4 This is a diagram of the callus tissue 30 days after its fourth subculture. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings to aid in understanding the content of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and researchers in the art can achieve the results by following conventional tissue culture methods. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0021] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for rapidly inducing callus tissue and proliferation using Iris tectorum seeds.

[0022] The relevant culture medium preparation methods in this embodiment of the invention are as follows:

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

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

[0025] The methods for calculating sterility rate, induction rate, proliferation rate, and browning rate in this embodiment are as follows:

[0026] Disinfection sterility rate = number of seeds that did not grow bacteria 5 days after disinfection / total number of seeds × 100%.

[0027] Induction period sterility rate = number of seeds that did not grow bacteria after 25 days of callus induction / total number of seeds × 100%.

[0028] Subculture sterility rate = Number of seeds that did not grow bacteria 30 days after the first subculture / Total number of seeds × 100%.

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

[0030] Proliferation rate = Number of callus blocks with a diameter of 2 mm that showed a significant increase in volume after 30 days of subculture / Total number of callus tissues × 100%.

[0031] Browning rate = Number of browned callus tissues after 30 days of subculture / Total number of callus tissues × 100%.

[0032] This invention provides a method for rapidly inducing callus and proliferation using seeds of the iris. The method includes the following steps:

[0033] (1) Explant Acquisition

[0034] Take healthy, plump *Iris tectorum* seeds after stratification, remove the seed coat, and rinse under tap water for 5 minutes. (See attached image.) Figure 1 It refers to the state of the obtained explant.

[0035] (2) Explant disinfection

[0036] Using conical flasks as containers, each flask was filled with one-sixth of its volume of explants and clean water was added. The flasks were sealed with gauze and shaken vigorously for 5 minutes. Then, they were rinsed under running tap water for 30 minutes to ensure thorough cleaning. Subsequently, in a sterile work area, within 10 cm of the air vent under the alcohol lamp, the explants were transferred to new containers using sterile conical flasks. The explants were then washed with a 75% ethanol solution (enough to submerge the explants) and shaken for 1 minute, followed by rinsing three times with sterile water for 1 minute each time. The explants were then placed in a 4% NaClO solution and disinfected by intermittent vigorous shaking for 25 minutes, followed by rinsing five times with sterile water for 1 minute each time. The disinfected explants were then placed on sterile filter paper to absorb excess moisture.

[0037] (3) Callus induction

[0038] Gently pick up the seeds with tweezers and press them into the callus induction medium. Use 90mm petri dishes, placing approximately 15 seeds in each dish. Seal the petri dishes and incubate them in a dark environment at 25±1℃. After 5 days, select petri dishes without fungal contamination, and use tweezers to pick out the bacteria-free seeds. Place them on sterile filter paper to remove any remaining culture medium from the seed surface, then inoculate them into the callus induction medium and incubate in the dark for 25 days. Figure 2 The image shows callus tissue obtained after placing Iris tectorum seeds in callus induction medium for 25 days.

[0039] The sterility rate during disinfection, the sterility rate during the induction period, and the induction rate were statistically analyzed separately.

[0040] Statistics show that the sterility rate after 5 days of disinfection 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] Healthy, sterile *Iris tectorum* seeds with callus tissue were subcultured into callus proliferation medium. Nutrients from the seed endosperm continuously supplied the callus tissue for growth. After 30 days, robust callus tissue with a bright yellow color and clear secretions was selected. Using a new scalpel, only the callus tissue was removed, carefully trimming away the browned portions. The callus tissue pieces were then cut in half lengthwise, ultimately resulting in small pieces with a diameter of 2 mm. These pieces were placed on callus proliferation medium, with approximately 55 pieces distributed across each 90 mm dish. The medium was incubated at 25 ± 1 °C in the dark, and subcultured every 30 days. The sterility rate, proliferation rate, and browning rate were recorded. Figure 3 This is a diagram of the callus tissue on day 1 after the 4th subculture. (Attached) Figure 4 This is a diagram of the callus tissue on day 30 after the fourth subculture.

[0043] Statistics show that the sterility rate during the subculture period was 100%, the proliferation rate was 81.54%, and the browning rate was 5.36%.

[0044] This invention provides a method for rapidly inducing and proliferating callus tissue using *Ilex cornuta* seeds. The seeds are used as explants; sterile seeds are obtained after disinfection, and then callus tissue is induced. The induction rate is 58.48%, the sterility rate during subculture is 100%, the proliferation rate of 2mm callus sections is 81.54%, and the browning rate is 5.36%. This invention is easy to master, low-cost, and highly efficient, laying the foundation for establishing a high-efficiency seed propagation system and a genetic transformation system. The experimental materials are not limited by season, are easy to obtain and preserve, and can achieve the goal of rapid year-round tissue culture propagation of *Ilex cornuta*.

[0045] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for rapidly inducing callus and proliferation using seeds of *Iris tectorum*, characterized in that, Includes the following steps: Step 1: Explant acquisition: Seeds of *Iris tectorum* were selected as explant material; Step 2: Explant disinfection: Wash the seeds of the iris flower with dish soap and water for 5 minutes, rinse with running tap water for 30 minutes to thoroughly clean them, and then wash them with 75% ethanol solution for 1 minute in a sterile table. Rinse them with sterile water 3 times for 1 minute each time, and then put them in 4% NaClO solution to disinfect for 25 minutes. Rinse them with sterile water 5 times for 1 minute each time. Place the disinfected seeds on sterile filter paper to absorb the moisture. Step 3: Callus induction: Inoculate sterilized Iris tectorum 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 *Iris tectorum* with callus tissue into 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, only the callus tissue is taken and cut into callus tissue blocks with a diameter of 2 mm and inoculated into callus proliferation medium. The pH of all the above culture media was adjusted to 5.8-6.0, the temperature of the culture room was 25±1℃, and the culture was carried out in the dark.

2. The method for rapidly inducing callus and proliferation using *Iris tectorum* seeds according to claim 1, characterized in that... The iris seeds mentioned in step 1 refer to iris seeds that were harvested in the current year, stratified to promote germination, and then had their seed coats removed.

3. The method for rapidly inducing callus and proliferation using *Iris tectorum* seeds according to claim 1, characterized in that... The callus induction described in step 3 refers to inoculating the sterilized seeds from claim 1 into the callus induction medium, culturing them in a dark environment at a temperature of 25±1℃ for 5 days, selecting culture dishes without fungal contamination, selecting seeds without bacterial growth, inoculating them into the callus induction medium, and culturing them in the dark for 25 days to produce *Echinochloa crus-galli* seeds with callus tissue.

4. The method for rapidly inducing callus and proliferation using *Iris tectorum* seeds according to claim 1, characterized in that... The callus proliferation described in step 4 refers to inoculating the *Iris tectorum* seeds with callus tissue as described in claim 3 into a callus proliferation medium, utilizing the nutrients in the seed endosperm to continuously supply the callus growth. After 30 days, select robust callus tissue with bright yellow color and clear secretions, and use a brand-new scalpel to cut off only the callus tissue. After carefully removing the browned part, cut the callus tissue block in half in sequence, and finally cut it into small pieces with a diameter of 2 mm. Place them on the callus proliferation medium and culture them at 25±1℃ in the dark. Subculture once every 30 days.

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