De-vitrification culture medium and method for vitrified callus of date palm
By using a devitrified culture medium with a specific composition and a light-protected culture method, the problem of difficult differentiation of vitrified callus tissue in date palms was successfully solved, achieving efficient restoration and utilization of the excellent characteristics of date palm seedlings and reducing production costs.
Patent Information
- Application Number
- CN202511409558.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-11-18
AI Technical Summary
Vitrified callus tissue of dates is difficult to differentiate, leading to material waste. Existing culture medium protocols are inconsistent, affecting the preservation of the superior characteristics of date seedlings and fruit features.
The vitrified fraction was isolated and transferred to devitrified medium at pH 5.6 using 1/2 MS medium, 1 mg/L 6-BA, 0.5 mg/L 2,4-D, 40 g/L sucrose, 10 g/L sorbitol, 8 g/L agar, 4 g/L activated carbon, and 1 mM reduced glutathione. The mixture was cultured in the dark, and the vitrified fraction was isolated and transferred to devitrified medium. The treatment was repeated until the sample returned to normal.
It significantly improved the recovery rate of vitrified callus of date palms, reaching 95.21%-97.12%, ensuring the maximum utilization and differentiation capacity of date palm varieties and reducing tissue culture costs.
Smart Images
Figure CN120959144A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a culture medium and method for devitrifying date palm vitrified callus. Background Technology
[0002] Date palm (Phoenix dactylifera L.) is a perennial woody plant belonging to the palm family (Arecaceae) and the Phoenix genus. It is an important food crop in arid and hot regions of the world, including the Middle East, South Asia, and North Africa, playing a vital role in the economic, cultural, and ecological well-being of people in these regions (Abeed et al., 2020; Yahyai, 2007). The development of the date palm industry, especially its cultivation, is constrained by traditional propagation methods. Traditional methods often result in the loss of the target traits of the parent plants, failing to adequately guarantee the superior characteristics of the mother plant and the fruit's features. Existing research indicates that tissue culture technology is the best and most effective means to solve the problems in date palm seedling production and ensure the renewal and extension of date palm plantations.
[0003] Date palms can be propagated from seeds, but because they are dioecious plants and highly heterozygous, this method of reproduction can lead to the loss of the target traits of the parent plants in production, failing to guarantee the superior characteristics of the mother plant and the fruit's features. Tissue culture technology is an effective way to solve the problem of date palm seedling propagation. During plant tissue culture, callus vitrification is a common phenomenon. Vitrified tissue appears transparent or translucent and water-soaked, fragile and easily broken, also known as hyperhydration. Vitrified tissue often exhibits low differentiation ability and difficulty in differentiating into buds. In production, vitrified material is often discarded, resulting in significant waste of research materials. As an introduced species, date palm resources in China are relatively scarce in both variety and quantity, making date palm material particularly valuable. Controlling vitrified callus tissue to restore its proliferative and differentiation growth ability can maximize the preservation and utilization of tissue culture materials, and its application in production practice can greatly reduce tissue culture costs.
[0004] The use of plant growth regulators is crucial throughout the rapid callus propagation process, with control of vitrification in date palms primarily occurring during the proliferation stage. However, different explant sources and parent date palm varieties lead to variations in endogenous hormone levels within the callus. Different concentrations of growth regulators in the culture medium result in differences in differentiation capacity among explants from different sources. In experimental studies on date palm callus culture and rapid propagation, the plant growth regulator 6-BA has shown a significant impact on callus proliferation and induction. Currently, research findings on date palm somatic embryogenesis vary among researchers. The required culture medium composition and induction time differ between varieties. Different research groups, including Veramendi et al., Al-Khayri et al., and Bajaj et al., have proposed different culture medium regimens. Al-Khayri and Al-Bahrany conducted a study examining the effects of water stress on date palm callus, concluding that varietal seedlings exhibit higher tolerance to water stress compared to conventionally cultivated seedlings. Therefore, it is evident that variety type also influences factors affecting vitrification regulation during the research process.
[0005] Therefore, providing a culture medium and method for devitrifying date palm vitrified callus is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides a culture medium and method for devitrifying date palm vitrified callus.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A devitrification culture medium for date palm vitrified callus, wherein the devitrification culture medium comprises the following components:
[0009] 1 / 2 MS medium, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, 40 g / L sucrose, 10 g / L sorbitol, 8 g / L agar, 4 g / L activated carbon, pH 5.6.
[0010] Furthermore, the devitrified culture medium also includes: 1 mM reduced glutathione.
[0011] A method for devitrifying date palm vitrified callus includes the following steps:
[0012] (1) When vitrified date palm callus appears on the differentiation medium of normal culture, separate the vitrified part from the normal tissue in a laminar flow hood. If the vitrified callus is large, use sterile forceps to divide it into appropriate sizes and remove it from the culture medium. Transfer it to the devitrified culture medium until the vitrified callus returns to normal. If there is too much water in the bottle during this period, the culture bottle can be replaced and the operation is the same as above.
[0013] (2) Culture of treated callus: The date callus that has been restored to normal after treatment in step (1) is transferred to differentiation medium in a clean bench for subculture. After 5-7 days, the original vitrified date callus can differentiate normally.
[0014] (3) If the devitrified date palm callus treated in step (3) re-vitrifies during subculture, repeat steps (1) and (2).
[0015] Furthermore, the culture conditions in step (1) are a temperature of 25±2℃ and a relative humidity of 50%-70%.
[0016] Furthermore, in step (1), the culture conditions are light-protected culture, and the culture time is 20-30 days.
[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:
[0018] The devitrification culture medium of this invention has a strong positive regulatory effect on vitrified date palm callus, achieving an average vitrification recovery rate of over 95.21% in the experimental group. This ensures maximum utilization of the valuable date palm variety in callus induction, effectively promoting the devitrification of vitrified date palm callus with significant results. The originally vitrified date palm callus, after treatment with this method, can continue to differentiate normally, eventually forming complete plants. Vitrified date palm callus can be regulated into normal tissue without incurring additional costs. After 20-30 days of controlled culture, it can resume growth and possess the ability to proliferate and differentiate into embryogenic callus. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0020] Figure 1 This refers to the morphological changes of vitrified callus tissue before and after regulation in Example 1 of the present invention. Figure 1A represents vitrified callus cells; Figure 1 B represents callus cells in the regulatory process; Figure 1 C represents the callus cells that have recovered and are now functioning normally. Detailed Implementation
[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] Example 1
[0023] A method for devitrifying date palm vitrified callus includes the following steps:
[0024] (1) When vitrified date palm callus appears on the differentiation medium of normal culture, the vitrified part is separated from the normal tissue in a laminar flow hood. If the vitrified callus is large, it is divided into appropriate sizes with sterile forceps and removed from the culture medium and transferred to devitrified culture medium until the vitrified callus returns to normal.
[0025] Devitrified medium components: 1 / 2 MS medium, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, 40 g / L sucrose, 10 g / L sorbitol, 8 g / L agar, 4 g / L activated carbon, pH 5.6;
[0026] The culture conditions were 25±2℃, 60% relative humidity, and cultured in the dark for 22 days.
[0027] If there is too much water in the bottle during the process, you can replace the culture bottle and follow the same procedure as above.
[0028] (2) Culture of treated callus: The date callus that has been restored to normal after treatment in step (1) is transferred to differentiation medium in a clean bench for subculture. After 5 days, the original vitrified date callus can differentiate normally.
[0029] Differentiation medium components: MS medium, 30 g / L sucrose, 2 mg / L 6-BA, 0.2 mg NAA, 5 g / L agar, pH 5.6;
[0030] (3) If the devitrified date palm callus treated in step (3) re-vitrifies during subculture, repeat steps (1) and (2).
[0031] Example 1: The devitrification culture medium has a strong positive regulatory effect on vitrified date palm callus, enabling the average vitrification recovery rate of the experimental group to reach 95.21%. This ensures that the precious date palm variety achieves maximum utilization in callus induction and can effectively promote the devitrification of vitrified date palm callus, with significant results.
[0032] The morphological changes of vitrified callus tissue before and after regulation in Example 1 were observed. Results are shown below. Figure 1 ,in Figure 1 A represents vitrified callus cells; Figure 1 B represents callus cells in the regulatory process; Figure 1 C represents the callus cells that have recovered and are now functioning normally.
[0033] Morphological index observation: Callus and embryogenic callus were selected as test materials. The tissue samples were sectioned using paraffin sectioning technique, stained with safranin and fast green, and the morphology and structure of the cells and tissues were observed. Experimental procedure: dewaxing to water - fast green staining - safranin staining - dehydration and mounting - microscopic examination.
[0034] The changes in reactive oxygen species (ROS) levels before and after regulation of vitrified callus in Example 1 were detected separately, and the results are shown in Table 1:
[0035] Table 1. Changes in reactive oxygen species in callus tissue under different treatments.
[0036]
[0037] Table 1 shows that significant differences were observed in vitrified callus tissue before and after regulation. Catalase (CAT) changed with the substrate hydrogen peroxide, showing a trend of first decreasing and then increasing, as did glutathione reductase (GR), which is related to the scavenging of reactive oxygen species. Superoxide dismutase (SOD) was extremely active during vitrification of callus tissue, with activity nearly twice that of normal callus in its steady state, showing a trend of first increasing and then decreasing before and after vitrification regulation. This indicates that when callus tissue is subjected to water stress, SOD activity increases significantly, catalyzing the conversion of superoxide anions to H2O2. However, vitrified callus tissue cannot rely solely on enzyme regulation to complete the relevant dismutation reaction to produce H2O2 and respond normally to oxidative stress. The contents of CAT and GR were both lower than the steady state, indicating an imbalance in free radical content and an imbalance within the tissue's peroxide protective enzyme system, further pushing the tissue towards death. In multiple comparisons of CAT and SOD activities, healthy date palm callus and regulated date palm callus showed no significant difference, while the differences in GR and H2O2 were small, both of which were significantly different from vitrified callus.
[0038] In summary, after regulation, the reactive oxygen species content in vitrified callus tends to stabilize and balance, and physiological indicators basically return to normal.
[0039] In experiments investigating the protective enzymatic system-related indicators of vitrified callus, this invention found that the CAT and GR activities of date palm vitrified callus contradicted the conclusions of existing techniques regarding vitrification-related physiological indicators in carnation and garlic tissue culture seedlings. However, the H2O2 content was consistent with the rapid decrease in hydrogen peroxide during the later observation period, and the SOD content was consistent with the changes observed in blueberry and garlic test-tube seedlings after vitrification in existing techniques. Further research and analysis suggest that the main reason might be that the date palm callus, cultured in the dark, had not yet differentiated into chloroplasts. Chloroplasts are the primary reaction site for the non-enzymatic GR pathway and are also important participants in enzymatic reaction systems such as CAT, leading to differences in related physiological indicators between the date palm callus vitrification regulation test and existing vitrified callus.
[0040] Example 2
[0041] Devitrification medium components: 1 / 2 MS medium, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, 40 g / L sucrose, 10 g / L sorbitol, 8 g / L agar, 4 g / L activated carbon, 1 mM reduced glutathione (GSH), pH 5.6. The remaining steps are the same as in Example 1.
[0042] After 25 days of light-protected culture, the vitrified date palm callus returned to normal, with an average vitrification recovery rate of 97.12%. The callus was firmer and its differentiation potential was improved.
[0043] The results of Example 2 show that GSH directly activates non-enzymatic antioxidant pathways. In dark culture lacking chloroplasts, the chloroplast-dependent GR enzymatic pathway fails, and exogenous GSH provides a large amount of reducing power, directly participating in the ASA-GSH cycle. GSH both enhances the efficiency of endogenous GR as a substrate, compensating for insufficient enzyme activity, and directly removes H2O2 through non-enzymatic reactions. In vitrified tissues, SOD excessively generates H2O2, but CAT / GR cannot remove it in time. The addition of GSH improves the efficiency of H2O2 removal, preventing H2O2 accumulation from triggering hydroxyl radical bursts and protecting the integrity of the membrane system.
[0044] Comparative Example 1
[0045] Devitrification medium components: MS medium, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, 40 g / L sucrose, 10 g / L sorbitol, 8 g / L agar, 4 g / L activated carbon, pH 5.6.
[0046] After 26 days of culture in the dark, the original vitrified date palm callus returned to normal, with an average vitrification recovery rate of 43.75%, which was significantly lower than that in Example 1.
[0047] Analysis of the results of Comparative Example 1 showed that the total MS concentration of 40 mM NH4 was... + Too high a concentration of NH4 + Passive diffusion into the cell lowers the vacuolar osmotic potential, causing a large influx of water. NH4 + Assimilation consumes a large amount of ATP and carbon skeleton, shifts sugar metabolism to energy supply, and weakens cell wall synthesis.
[0048] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A culture medium for devitrifying date palm vitrified callus, characterized in that, The devitrification medium comprises the following components: 1 / 2 MS medium, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, 40 g / L sucrose, 10 g / L sorbitol, 8 g / L agar, 4 g / L activated carbon, pH 5.
6.
2. The culture medium according to claim 1, characterized in that, The devitrified culture medium also includes: 1 mM reduced glutathione.
3. A method for devitrifying date palm vitrified callus, characterized in that, Includes the following steps: (1) When vitrified date palm callus appears on the differentiation medium of normal culture, the vitrified part is separated from the normal tissue in a laminar flow hood. If the vitrified callus is large, it is divided into appropriate sizes with sterile forceps and removed from the culture medium and transferred to the devitrification culture medium of claim 1 until the vitrified callus returns to normal. If there is too much water in the bottle during this period, the culture bottle can be replaced and the operation is the same as above. (2) Culture of treated callus: The date callus that has been restored to normal after treatment in step (1) is transferred to differentiation medium in a clean bench for subculture. After 5-7 days, the original vitrified date callus can differentiate normally. (3) If the devitrified date palm callus treated in step (3) re-vitrifies during subculture, repeat steps (1) and (2).
4. The method for devitrifying date palm vitrified callus according to claim 3, characterized in that, The culture conditions in step (1) are a temperature of 25±2℃ and a relative humidity of 50%-70%.
5. The method for devitrifying date palm vitrified callus according to claim 3, characterized in that, The culture conditions in step (1) are light-protected culture, and the culture time is 20-30 days.