An induction medium for inducing embryogenic callus in Ili black iris and its application
By using adventitious roots of Ili black iris as explants, combined with specific culture media and growth regulators, the problems of browning and low induction rate in black iris callus culture were solved, achieving efficient and uniform callus production, which is suitable for industrial production.
Patent Information
- Application Number
- CN202610552550.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies for black iris callus culture suffer from problems such as severe explant browning, low callus induction rate, slow growth, high browning rate, and uneven growth, making it difficult to meet the needs of industrial production.
Using adventitious roots of Ili black iris as explants, combined with an induction medium consisting of MS medium, sucrose, agar, 6-benzylaminopurine, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, proline, and silver nitrate, and through dark culture and specific plant growth regulator ratios, efficient callus induction and anti-browning were achieved.
The induction rate of embryogenic callus from Ili black iris was increased to 64.5%-90.6%, and the browning rate was controlled below 10%-14%. The obtained callus was vigorous and uniform, and suitable for research such as genetic transformation and germplasm preservation.
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Figure CN122296244A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of industrial biotechnology, and in particular to an induction medium for inducing embryogenic callus in Ili black iris and its application. Background Technology
[0002] Black Iris ( Iris tectorum Iris Maxim. is a herbaceous plant belonging to the genus Iris in the family Iridaceae. It is rich in flavonoids, polysaccharides, and phenolic acids, and has anti-inflammatory, antioxidant, and blood-activating effects. It is also an important raw material for making perfumes and has high medicinal, ornamental, and economic value.
[0003] Due to its multiple values and unique aroma, Ili black iris (black iris from Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region) is widely used in food, pharmaceutical, and cosmetic production. Currently, the acquisition of Ili black iris and its active ingredients mainly relies on direct extraction from natural plants. However, wild resources have long growth cycles, and over-harvesting can easily cause resource damage and ecological pressure. Direct extraction also carries microorganisms, pesticide residues, and heavy metals, making safety difficult to control. Plant callus culture technology can achieve unrestricted, aseptic, and continuous production, making it an ideal way to obtain highly active and homogeneous plant raw materials. However, in the process of black iris callus culture, there are generally technical bottlenecks such as severe explant browning, low callus induction rate, and slow growth, and existing technologies cannot meet the needs of industrial production. Therefore, developing a black iris callus preparation technology with high induction rate, low browning rate, and excellent callus quality has significant application value and market prospects.
[0004] Furthermore, different types of explants exhibit significant differences in cell division capacity and regeneration potential, directly affecting callus formation efficiency. Currently, the preparation of black iris callus cells typically uses plant roots, stems, leaves, and flowers as explants. While the flowers of *Iris tectorum* (Yili black iris) are brightly colored, they are easily damaged during sterilization and turn brown, making them unsuitable as explants for callus induction. Iris adventitious roots are fibrous roots that grow from non-radicle parts such as rhizome nodes, stems, and leaves, forming the core root system for iris absorption, attachment, and reproduction. When the taproot is damaged, adventitious roots can quickly replace it, maintaining plant survival; under adverse conditions such as flooding and drought, adventitious root proliferation enhances adaptability. Currently, there is limited research on techniques for establishing callus tissue and preparing extracts using black iris adventitious roots as explants.
[0005] In view of this, the present invention aims to provide a method for improving the induction efficiency of embryogenic callus and resistance to browning of Ili black iris using adventitious roots as explants, so as to make up for the above-mentioned deficiencies. Summary of the Invention
[0006] The purpose of this invention is to provide an induction culture medium for inducing embryogenic callus in Ili black iris and its application, so as to solve the problems existing in the prior art.
[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides an induction medium for inducing embryogenic callus in *Iris iliens*, wherein the induction medium is based on MS medium and further comprises 25-35 g / L sucrose, 7-8 g / L agar, 1.0-2.0 mg / L 6-benzylaminopurine, 0.3-1.0 mg / L naphthaleneacetic acid, 0.3-1.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline, and 2.0-4.0 mg / L silver nitrate.
[0008] Optionally, the induction medium is based on MS medium and further includes 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline and 3.0 mg / L silver nitrate.
[0009] The present invention provides the use of the above-described induction medium in any of the following: (1) Improve the efficiency of embryogenic callus induction in Ili black iris; (2) Prepare products that improve the induction efficiency of embryogenic callus from Ili black iris; (3) Improve the browning resistance of embryogenic callus from Ili black iris; (4) Prepare products that improve the browning resistance of embryogenic callus of Ili black iris.
[0010] This invention provides a product for improving the induction efficiency of embryogenic callus from Ili black iris, the product comprising the aforementioned induction culture medium.
[0011] Optionally, the product may also include auxiliary materials.
[0012] This invention provides a product for improving the browning resistance of embryogenic callus from Ili black iris, the product comprising the aforementioned induction culture medium.
[0013] Optionally, the product may also include auxiliary materials.
[0014] This invention provides a method for improving the induction efficiency of embryogenic callus from Ili black iris, comprising the steps of using adventitious roots of Ili black iris as explants and inoculating the explants into an induction medium for culture. The induction medium is based on MS medium and also includes 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline and 3.0 mg / L silver nitrate.
[0015] Optionally, the culture conditions are set to dark culture at 25°C.
[0016] This invention provides a method for improving the browning resistance of embryogenic callus of Ili black iris, comprising the step of using adventitious roots of Ili black iris as explants and inoculating the explants into an induction medium for culture. The induction medium is based on MS medium and also includes 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline and 3.0 mg / L silver nitrate.
[0017] Optionally, the culture conditions are set to dark culture at 25°C.
[0018] The present invention discloses the following technical effects: This invention develops a culture medium that improves the induction efficiency and browning resistance of embryogenic callus from *Iris tectorum*. Using this induction medium to culture adventitious roots of *Iris tectorum*, the callus induction rate can reach 64.5%-90.6%, the embryogenic callus cell induction rate can reach 88%, and the browning rate is 10%-14%. Simultaneously, the dry weight of *Iris tectorum* callus cells reaches its peak on day 18. The callus tissue is yellow and granular, exhibits vigorous growth, and shows no signs of waterlogging or vitrification. It is an excellent callus tissue capable of stable subculturing and is suitable for research in genetic transformation, embryoid induction, cell suspension culture, in vitro mutagenesis, and germplasm preservation.
[0019] This invention also provides a method for improving the induction efficiency and anti-browning ability of embryogenic callus from Ili black iris, which has the following advantages: (1) Using adventitious roots as explants has significant advantages: adventitious roots themselves develop from parenchyma cells and meristematic cells. It exhibits high cell division activity and strong dedifferentiation ability. Its source is uniform and stable, with high homogeneity, easy sterilization, high sterility, absence of endogenous bacteria, mild browning, and more uniform callus induction. Using adventitious roots as explants results in uniform structure, clean surface, few endophytic bacteria, low contamination, high sterilization success rate, and good experimental reproducibility. Due to its strong meristematic ability, suitable endogenous hormones, uniform structure, and mild browning, adventitious roots are among the most efficient and stable explants for callus induction. Combined with stepwise sterilization, a sterility rate of over 90% can be achieved.
[0020] (2) The browning rate was controlled below 15% by synergistic regulation of plant growth regulators (6-benzylaminopurine + naphthaleneacetic acid + 2,4-dichlorophenoxyacetic acid), compound anti-browning system (proline + silver nitrate) and dark culture conditions.
[0021] (3) The method of the present invention is simple, the culture medium components are clear, the conditions are controllable, the operation is simple, and it is easy to standardize and scale up the culture in the laboratory.
[0022] Therefore, this invention solves the technical problems of difficult disinfection of explants of the Iris genus, severe browning, low callus induction efficiency, and poor system stability. It establishes a black iris callus culture system with unified source, simple operation, good reproducibility, and scalability, which can provide stable recipient materials and technical basis for the genetic transformation, somatic embryogenesis, in vitro preservation of germplasm, secondary metabolism regulation, and biotechnology breeding of black iris. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 The embryonic callus cells of Ili black iris in Example 1; Figure 2 The growth curve of callus cells from *Iris tectorum* in Example 2 is shown. Figure 3 For comparison, non-embryonic callus cells were used in Experiment 1. Figure 4 For comparison, non-embryonic callus cells in Experiment 2; Figure 5 For comparison, browned explants and callus cells without anti-browning agent and under dark culture treatment in Experiment 4; Figure 6 To compare the yellowing and hardening callus cells treated with anti-browning agent and light culture in Experiment 4. Detailed Implementation
[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0030] Unless otherwise specified, the methods used in this invention are all well known to those skilled in the art; the components used in this invention are all commonly purchased by those skilled in the art.
[0031] Example 1: Induction and Culture of Adventitious Root Callus from Ili Black Iris The steps for inducing and culturing adventitious root callus of *Iris tectorum* in this embodiment are as follows: Mature adventitious roots of *Iris tectorum* were disinfected with 75% (v / v) ethanol for 30 seconds, rinsed four times with sterile water, then disinfected with 2wt% sodium hypochlorite for 15 minutes, and rinsed six times with sterile water. The epidermal cells of the adventitious roots were removed, and the roots were cut into pieces to obtain 300 explants (each explant measuring 0.3 × 0.3 cm). 2The adventitious roots were inoculated into an induction medium (MS medium (manufacturer: Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number: HB8469-5) with a pH of 5.5 (sucrose 30 g / L + agar 7.5 g / L + 6-benzylaminopurine (6-BA) 1.5 mg / L + naphthaleneacetic acid (NAA) 0.3 mg / L + 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5 mg / L + proline 0.25 g / L + silver nitrate 3.0 mg / L) at 25℃ in the dark for 18 days. The adventitious roots gradually absorbed and swelled, and loose yellow callus tissue was produced around the cut and on the roots, resulting in 270 Ili black iris callus cell clusters. Figure 1 The number of embryogenic callus was 237, the number of callus formations (number of callus emergences) was 270, and the number of browned explants was 30.
[0032] Subsequently, the induction rates of callus cells and embryogenic callus cells were calculated separately. The callus cell induction rate refers to the rate at which explants form callus cells on the induction medium; the embryogenic callus induction rate is the ratio of embryogenic callus cells to the total number of callus cells generated.
[0033] The calculation formula is as follows: Callus induction rate (%) = (Number of callus tissues produced / Number of explants inoculated) × 100%; Embryogenic callus induction rate (%) = Number of embryogenic callus / Number of callus generated × 100%; Browning rate (%) = number of browned explants / number of inoculated explants × 100%.
[0034] Calculations show that the callus induction rate in this embodiment is 90%, the embryogenic callus induction rate is 88%, and the browning rate is 10%.
[0035] Example 2 Subculture of callus from Ili black iris The steps for subculturing Ili black iris callus in this embodiment are as follows: The callus tissue obtained in Example 1 was cut into 3-5 mm pieces and transferred to the same culture medium (induction medium). It was cultured in the dark at 25°C. The optimal subculture time was day 15. After two consecutive subcultures, a superior callus tissue system with stable growth rate, uniform texture, and no browning or vitrification was formed, which can be preserved and subcultured for a long time. The growth curve of Ili black iris callus cells is shown below. Figure 2 As shown, the growth pattern exhibits an "S" shape, with the callus dry weight reaching its peak on day 18.
[0036] Comparative test To obtain better embryogenic callus cells from *Iris tectorum* for suspension culture, this invention investigated the effects of different explant stem discs, leaves, and adventitious roots, as well as different hormones and concentrations in the culture medium, on callus formation. Specific experiments are as follows: Comparative Experiment 1: Induction of Embryogenic Callus Cells from Stem Discs of Ili Black Iris Using stem discs of *Iris tectorum* as explants, the stem discs were disinfected with 75% (v / v) ethanol for 30 seconds, rinsed four times with sterile water, then disinfected with 2wt% sodium hypochlorite for 20 minutes, rinsed six times with sterile water, and then cut into pieces to obtain 252 explants (each explant measuring 0.5 × 0.5 cm). 2 The cells were inoculated into an induction medium with a pH of 5.5 (MS medium was used as the basal medium, and the concentrations of other components are shown in Table 1), and induced at 25°C under no light conditions. A small amount of callus tissue appeared after 18-21 days. The experimental results (Table 1) show that only a few stem discs were induced to produce embryogenic callus tissue, which was generally pale yellow with a granular surface. Most of the callus tissue was yellow, compact, and blocky, and therefore non-embryonic (e.g., ...). Figure 3 As shown in the figure, this type of non-embryonic callus is hard in texture, grows slowly, and after subculture, the callus particles become larger and larger, and the color gradually darkens.
[0037] Table 1. Effects of different induction conditions on stem disc-induced embryogenic callus Comparative Experiment 2: Induction of Embryogenic Callus Cells from Ili Black Iris Leaves Young, terminal leaves of *Iris tectorum* were used as explants. The leaves were disinfected with 75% (v / v) ethanol for 30 seconds, rinsed four times with sterile water, then disinfected with 2wt% sodium hypochlorite for 15 minutes, rinsed six times with sterile water, and finally cut into pieces to obtain 252 explants (each explant measuring 0.5 × 0.5 cm). 2 The leaves of *Iris tectorum* were inoculated into an induction medium with a pH of 5.5 (MS medium was used as the basal medium, and the concentrations of other components are shown in Table 2). Induction was performed at 25°C under no light. The induction time for callus formation using leaves of *Iris tectorum* was longer than that using stem discs, with a small amount of callus appearing at the leaf margins after 20-25 days. Table 2 shows that when the medium contained only auxin (naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid), no callus formed on the leaves, and the leaves gradually turned brown. However, when the medium contained both cytokinin (6-BA) and auxin, callus formation occurred on the leaves. Initially, the callus induced from the leaves was a non-embryonic, yellow, water-soaked form. The induction rate of embryogenic callus was very low. As the culture time increased, the callus began to swell, becoming a swollen white (as shown in Table 2). Figure 4 As shown in the figure, the poor growth status after subculture indicates that leaves are not a good explant for inducing embryogenic callus.
[0038] Table 2 Effects of different induction conditions on leaf-induced embryogenic callus Comparative Experiment 3: Induction of Embryogenic Callus Using Adventitious Roots of Ili Black Iris as Explants Mature adventitious roots of *Iris tectorum* were disinfected with 75% (v / v) ethanol for 30 seconds, rinsed four times with sterile water, then disinfected with 2wt% sodium hypochlorite for 15 minutes, and rinsed six times with sterile water. The epidermal cells of the adventitious roots were removed, and the roots were cut into pieces to obtain 252 explants (each explant measuring 0.3 × 0.3 cm). 2 The explants were inoculated into an induction medium with a pH of 5.5 (MS medium was used as the basal medium, and the concentrations of other components are shown in Table 3), and induced at 25°C in the absence of light. Table 3 shows that when the medium did not contain 6-BA, the explants showed rooting, but no callus formation occurred. When the medium contained both cytokinin and auxin, the induction rate of adventitious roots was relatively high, presumably because the adventitious roots were stimulated by plant growth regulators, resulting in more active cells at the cut site and easier dedifferentiation. Callus formation occurred in 15-18 days, initially forming slowly around the cut and then expanding outwards. The formed callus was mainly pale yellow, granular embryogenic callus (similar to...). Figure 1 (The shape shown).
[0039] Table 3. Effects of different induction conditions on adventitious root-induced embryogenic callus Comparative Experiment 4: Effects of different treatments on callus induction of adventitious roots of Ili black iris (1) The induction medium and culture conditions were the same as in Example 1, denoted as anti-browning agent + dark culture treatment. The results are shown in Table 4. The results showed that the browning rate was 14% and the callus induction rate was only 80.5%.
[0040] (2) No silver nitrate and proline anti-browning components were added to the induction medium. The other culture conditions were the same as in Example 1. This was recorded as no anti-browning agent added + dark culture treatment. The results are shown in Table 4 and Figure 5 As shown. The results showed that the explants and callus were severely browned ( Figure 5 The browning rate reached 53.5%, and the callus induction rate was only 24.5%, indicating a significant decrease in culture effectiveness.
[0041] (3) The induction medium was the same as in Example 1, but the culture conditions were changed to 16h light / 8h dark, with the other conditions remaining the same. This was recorded as anti-browning agent + light culture treatment, and the results are shown in Table 4. The results showed that although browning was somewhat alleviated, the callus tissue exhibited yellowing, aging, and hardening. Figure 6 The growth rate and uniformity decreased, and the callus induction rate was only 41%.
[0042] Table 4. Effects of different treatments on callus induction from adventitious roots of Iris tectorum in Yili Therefore, using adventitious roots of *Iris tectorum* as explants, the induction rate of embryogenic callus reached over 85%. By employing a specific ratio of plant growth regulators (6-benzylaminopurine + naphthaleneacetic acid + 2,4-dichlorophenoxyacetic acid) and a composite anti-browning regulation system (proline + silver nitrate), high-efficiency culture of *Iris tectorum* callus with high induction rate, low browning rate, and uniform growth was achieved. The optimal induction medium consisted of MS basal medium, 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-BA, 0.3 mg / L NAA, 0.5 mg / L 2,4-D, 0.25 g / L proline, and 3.0 mg / L silver nitrate, providing well-preserved callus tissue for large-scale culture of *Iris tectorum* cells.
[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An induction culture medium for inducing embryogenic callus in *Iris tectorum*, characterized in that, The induction medium is based on MS medium and also includes 25-35 g / L sucrose, 7-8 g / L agar, 1.0-2.0 mg / L 6-benzylaminopurine, 0.3-1.0 mg / L naphthaleneacetic acid, 0.3-1.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline, and 2.0-4.0 mg / L silver nitrate.
2. The use of the induction medium according to claim 1 in any of the following: (1) Improve the efficiency of embryogenic callus induction in Ili black iris; (2) Prepare products that improve the induction efficiency of embryogenic callus from Ili black iris; (3) Improve the browning resistance of embryogenic callus from Ili black iris; (4) Prepare products that improve the browning resistance of embryogenic callus of Ili black iris.
3. A product for improving the efficiency of embryogenic callus induction in *Iris tectorum*, characterized in that, The product includes the induction culture medium as described in claim 1.
4. The product according to claim 3, characterized in that, The product also includes auxiliary materials.
5. A product for improving the browning resistance of embryogenic callus from *Iris tectorum*, characterized in that, The product includes the induction culture medium as described in claim 1.
6. The product according to claim 5, characterized in that, The product also includes auxiliary materials.
7. A method for improving the induction efficiency of embryogenic callus from *Iris tectorum*, characterized in that, The method includes the step of inoculating the adventitious roots of Ili black iris into an induction medium for culture, using the adventitious roots of Ili black iris as explants. The induction medium is based on MS medium and also includes 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline and 3.0 mg / L silver nitrate.
8. The method according to claim 7, characterized in that, The culture conditions were set at 25°C for dark culture.
9. A method for improving the browning resistance of embryogenic callus from *Iris tectorum*, characterized in that, The method includes the step of inoculating the adventitious roots of Ili black iris into an induction medium for culture, using the adventitious roots of Ili black iris as explants. The induction medium is based on MS medium and also includes 30 g / L sucrose, 7.5 g / L agar, 1.5 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.25 g / L proline and 3.0 mg / L silver nitrate.
10. The method according to claim 9, characterized in that, The culture conditions were set at 25°C for dark culture.