Efficient and universal method for preventing browning of sugarcane tissue culture

Abstract

This invention discloses a highly efficient and universal method for preventing browning in sugarcane tissue culture, belonging to the field of tissue culture technology. This invention is the first to use potassium cinnamate as an anti-browning active substance in the pretreatment and induction culture of plant tissue culture. By introducing a synergistic strategy of low-temperature pretreatment combined with potassium cinnamate soaking of explants and addition of culture medium, significant beneficial effects have been achieved in preventing browning in sugarcane tissue culture. This invention, through the synergistic application of low-temperature pretreatment and potassium cinnamate, targets and inhibits polyphenol oxidase activity, effectively blocking browning. This method significantly reduces the browning rate of different sugarcane germplasms and simultaneously improves regeneration efficiency, successfully overcoming the genotype dependence of traditional anti-browning technologies, and providing a highly efficient and universal solution for the development and utilization of sugarcane germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology, and in particular to a highly efficient and universal method for preventing browning in sugarcane tissue culture. Background Technology

[0002] Sugarcane (Saccharum spp.) is an important sugar crop. Breeding superior varieties is the foundation for the sustainable and healthy development of the sugarcane industry, while sugarcane germplasm resources are the material basis for breeding. Among the germplasm resources of the genus *Saccharum*, in addition to modern cultivated varieties, there are also many wild species and cultivated original species, such as tropical species and *Saccharum spp.*, which contain excellent genes for high yield, high sugar content, and stress resistance, and are key to genetic improvement. However, the development and utilization of these valuable germplasm resources heavily rely on efficient tissue culture technology. Currently, the browning problem of explants during the tissue culture process of *Saccharum* plants has become a major technical bottleneck restricting the development and utilization of its wild germplasm resources and cultivated original species. Existing anti-browning technologies mainly involve adding antioxidants (such as vitamin C and activated charcoal) to the culture medium, utilizing their competitive oxygen free radical properties to block the oxidation process of polyphenolic compounds. These methods have achieved certain results in some cultivated varieties, but their mechanism of action depends on the competition of reaction substrates, which has significant limitations in practical applications. The genetic differences among different sugarcane germplasm resources lead to significant variations in the types, contents, and metabolic pathways of their polyphenolic compounds. This makes it difficult for antioxidant solutions optimized for specific genotypes to produce stable effects in other materials, severely limiting the applicability of the technology across different species of sugarcane.

[0003] The root cause of these problems lies in the fact that existing technologies have failed to address the core mechanism of the browning reaction. Browning in sugarcane is essentially an enzymatic reaction catalyzed by polyphenol oxidase, an enzyme highly conserved across different germplasm resources. While adjusting the type and amount of antioxidants has attempted to alleviate browning by altering the reaction environment, it has failed to effectively inhibit the key catalytic activity of polyphenol oxidase. Furthermore, due to the complex composition and significant differences in polyphenol substrate content among different germplasm materials, simply adding antioxidants cannot achieve comprehensive protection for all substrates, resulting in insufficient technical stability. This substrate competition-based approach inevitably faces inherent limitations in addressing the diverse germplasm resources of sugarcane, including narrow applicability and unstable effectiveness.

[0004] To address the aforementioned problems, there is an urgent need in this field to develop a universal anti-browning strategy that can overcome genotype limitations. Theoretically, developing inhibitory methods targeting highly conserved polyphenol oxidases should achieve broader application, but practical implementation faces numerous challenges: firstly, it is necessary to find inhibitors that can effectively suppress enzyme activity without toxic side effects on plants; secondly, it is necessary to establish standardized treatment procedures applicable to different germplasm resources; and more importantly, it is crucial to ensure that the strategy maintains its inhibitory effect without affecting the normal growth and differentiation of explants. These technical difficulties make the development of a universal anti-browning method a key breakthrough in the development of tissue culture technology for sugarcane. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient and universal method for preventing browning in sugarcane through tissue culture, thereby addressing the problems existing in the prior art. This invention utilizes the synergistic application of low-temperature pretreatment and potassium cinnamate to target and inhibit polyphenol oxidase activity, effectively blocking browning. This method significantly reduces the browning rate of different sugarcane germplasms and simultaneously improves regeneration efficiency, successfully overcoming the genotype dependence of traditional anti-browning technologies and providing a highly efficient and universal solution for the development and utilization of sugarcane germplasm resources.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of potassium cinnamate in any of the following (1)-(2),

[0008] (1) Preparation of plant tissue culture pretreatment agent;

[0009] (2) Prepare a callus induction medium that resists browning.

[0010] The present invention also provides a plant tissue culture pretreatment agent, wherein the active ingredient comprises a potassium cinnamate solution of 100-250 mg / L.

[0011] Optionally, the active ingredient may contain a 200 mg / L potassium cinnamate solution.

[0012] The present invention also provides a callus induction culture medium for preventing browning, wherein the concentration of potassium cinnamate in the callus induction culture medium is 100-250 mg / L.

[0013] Further optionally, the concentration of potassium cinnamate in the callus induction culture medium is 200 mg / L.

[0014] Optionally, the callus induction medium is based on MS medium and supplemented with 1.0-3.0 mg / L of 2,4-D, 30 g / L of sucrose and 8.0 g / L of agar powder.

[0015] This invention also provides a method for preventing browning in sugarcane tissue culture, comprising the following steps:

[0016] (1) The explants of the sugarcane species were subjected to low-temperature pretreatment;

[0017] (2) The explants treated in step (1) were pretreated by soaking in the plant tissue culture pretreatment agent described above;

[0018] (3) The explants treated in step (2) are inoculated into the callus induction medium for culture.

[0019] Optionally, in step (2), the soaking pretreatment time is 10-20 min.

[0020] Optionally, in step (1), the conditions for the low-temperature pretreatment are: temperature 18-22℃, humidity 70%-80%, and standing in a dark environment for 14-24 hours.

[0021] Optionally, in step (1), the explant is the shoot tip tissue taken from the shoot tip of a sugarcane plant, where the shoot tip is from 3-5 nodes below the apical meristem to the +1 leaf pulvinus.

[0022] The present invention discloses the following technical effects:

[0023] The tissue culture anti-browning method provided by this invention achieves significant beneficial effects by introducing a synergistic strategy of low-temperature pretreatment, potassium cinnamate soaking of explants, and culture medium addition. This method directly targets polyphenol oxidase, a key enzyme in the browning reaction, effectively blocking the enzymatic browning process. Thus, while simplifying the operation process and culture medium composition, it achieves a superior anti-browning effect compared to conventional antioxidant strategies, completely avoiding any adverse effects on subsequent callus induction and differentiation capabilities of explants, and significantly improving the tissue culture seedling survival rate.

[0024] This invention can stably reduce browning rate and simultaneously improve regeneration efficiency for representative materials of the Sugarcane genus, including cultivated species, *Saccharum cuspidatum*, tropical species, Indian species, Chinese species, and large-stemmed wild species, with no significant interaction between species and treatments. It successfully overcomes the applicability limitations of traditional techniques due to genotype dependence, providing efficient, stable, and universally applicable technical support for the development and utilization of various germplasm resources of the Sugarcane genus. 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] The materials used in the embodiments and comparative examples of this invention are all six species of sugarcane, including the cultivated sugarcane variety Yunzhe 081609, the cut-hand-dense variety VN2, the tropical variety Black Cheliben, the Indian variety Pansahi, the Chinese variety Guilin Bamboo Sugarcane, and the large-stemmed wild variety 57NG208, hereinafter referred to as sugarcane species. All six sugarcane species were obtained from the germplasm resource bank of the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences.

[0031] Example 1

[0032] 1. Low-temperature pretreatment

[0033] Select sugarcane plants aged 6-10 months, take their tips (3-5 nodes below the apical meristem to the +1 leaf pulvinus), peel off the outer old leaves and leaf sheaths, wrap them in sterilized newspaper, and let them stand in a dark environment at 18℃ and 70% humidity for 14 hours.

[0034] 2. Potassium cinnamate pretreatment

[0035] Take a section 2-7 cm above the apical growth point, disinfect the surface with 75% alcohol, and then continue to peel off mature leaves and leaf sheaths in a clean bench, leaving the innermost 2-5 heart leaves. Cut them into single-layer leaves of 2×5 mm size and place them in a 100 mg / L potassium cinnamate aqueous solution (sterilized by vacuum filtration) and soak at room temperature for 10 min.

[0036] 3. Callus induction culture

[0037] After removing excess liquid from the leaves, they were inoculated onto callus initiation medium and cultured in the dark at 28°C for 3 weeks. The callus initiation medium consisted of MS + 2,4-D 1.0 mg / L + potassium cinnamate 100 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0038] 4. Callus subculture, differentiation, rooting and transplanting

[0039] Subsequent tissue culture steps, including callus subculture, differentiation, rooting, and transplanting, all used conventional methods, as detailed below:

[0040] Callus subculture: Callus tissue obtained after callus induction culture was inoculated into callus subculture medium and cultured in the dark at 28℃. Subculture was performed every 3 weeks for a total of 2 subcultures. The composition of the callus subculture medium was MS + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0041] Differentiation culture: Callus tissue that has undergone two subcultures was inoculated into callus differentiation medium and cultured at a constant temperature of 28℃ under light irradiation of 80 μmol / s / m². 2 The photoperiod was 14 h / 10 h (light / dark), and the cells were subcultured every 3 weeks for a total of 2 subcultures. The callus differentiation medium consisted of MS + 6-BA 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0042] Rooting induction: The seedlings obtained from differentiation culture were divided into individual plants and inoculated into rooting induction medium. The culture conditions were the same as those for differentiation culture, and the culture time was 4 weeks. The components of the rooting induction medium were MS + NAA 2.0 mg / L + sucrose 30 g / L + activated carbon 1.5 g / L + agar powder 8.0 g / L, pH 5.8.

[0043] Transplanting to seedling stage: Rooted seedlings obtained from rooting culture are transferred to a greenhouse and hardened off for 3 days before being transplanted into a seedling substrate for further cultivation. The substrate consists of peat moss and vermiculite mixed in a 7:3 volume ratio. The greenhouse has an average humidity of 75%, an average temperature of 25℃, and an average light intensity of 70 μmol / s / m². 2The photoperiod is 12 h / 12 h (light / dark).

[0044] Example 2

[0045] 1. Low-temperature pretreatment

[0046] Select sugarcane plants aged 6-10 months, take the tip (3-5 nodes below the apical meristem to the +1 leaf pulvinus), peel off the outer old leaves and leaf sheaths, wrap in sterilized newspaper, and let stand in a dark environment at 22℃ and 80% humidity for 24 hours.

[0047] 2. Potassium cinnamate pretreatment

[0048] Take a section 2-7 cm above the apical growth point, disinfect the surface with 75% alcohol, and then continue to peel off mature leaves and leaf sheaths in a clean bench, leaving the innermost 2-5 heart leaves. Cut them into single-layer leaves of 2×5 mm size and place them in a 250 mg / L potassium cinnamate aqueous solution (sterilized by vacuum filtration) and soak at room temperature for 20 min.

[0049] 3. Callus induction culture

[0050] After removing excess liquid from the leaves, they were inoculated onto callus initiation medium and cultured in the dark at 28°C for 3 weeks. The components of the callus initiation medium were MS + 2,4-D 3.0 mg / L + potassium cinnamate 250 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0051] 4. Callus subculture, differentiation, rooting and transplanting

[0052] Subsequent tissue culture steps, including callus subculture, differentiation, rooting, and transplanting, all used conventional methods, as detailed below:

[0053] Callus subculture: Callus tissue obtained after callus induction culture was inoculated into callus subculture medium and cultured in the dark at 28℃. Subculture was performed every 3 weeks for a total of 2 subcultures. The composition of the callus subculture medium was MS + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0054] Differentiation culture: Callus tissue that has undergone two subcultures was inoculated into callus differentiation medium and cultured at a constant temperature of 28℃ under light irradiation of 80 μmol / s / m². 2 The photoperiod was 14 h / 10 h (light / dark), and the cells were subcultured every 3 weeks for a total of 2 subcultures. The callus differentiation medium consisted of MS + 6-BA 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0055] Rooting induction: The seedlings obtained from differentiation culture were divided into individual plants and inoculated into rooting induction medium. The culture conditions were the same as those for differentiation culture, and the culture time was 4 weeks. The components of the rooting induction medium were MS + NAA 2.0 mg / L + sucrose 30 g / L + activated carbon 1.5 g / L + agar powder 8.0 g / L, pH 5.8.

[0056] Transplanting to seedling stage: Rooted seedlings obtained from rooting culture are transferred to a greenhouse and hardened off for 3 days before being transplanted into a seedling substrate for further cultivation. The substrate consists of peat moss and vermiculite mixed in a 7:3 volume ratio. The greenhouse has an average humidity of 75%, an average temperature of 25℃, and an average light intensity of 70 μmol / s / m². 2 The photoperiod is 12 h / 12 h (light / dark).

[0057] Example 3

[0058] 1. Low-temperature pretreatment

[0059] Select sugarcane plants aged 6-10 months, take the tip (3-5 nodes below the apical meristem to the +1 leaf pulvinus), peel off the outer old leaves and leaf sheaths, wrap in sterilized newspaper, and let stand in a dark environment at 20℃ and 75% humidity for 18 hours.

[0060] 2. Potassium cinnamate pretreatment

[0061] Take a section 2-7 cm above the apical growth point, disinfect the surface with 75% alcohol, and then continue to peel off mature leaves and leaf sheaths in a clean bench, leaving the innermost 2-5 heart leaves. Cut them into single-layer leaves of 2×5 mm size and place them in a 200 mg / L potassium cinnamate aqueous solution (sterilized by vacuum filtration) and soak at room temperature for 15 min.

[0062] 3. Callus induction culture

[0063] After removing excess liquid from the leaves, they were inoculated onto callus initiation medium and cultured in the dark at 28°C for 3 weeks. The components of the callus initiation medium were MS + 2,4-D 2.0 mg / L + potassium cinnamate 200 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0064] 4. Callus subculture, differentiation, rooting and transplanting

[0065] Subsequent tissue culture steps, including callus subculture, differentiation, rooting, and transplanting, all used conventional methods, as detailed below:

[0066] Callus subculture: Callus tissue obtained after callus induction culture was inoculated into callus subculture medium and cultured in the dark at 28℃. Subculture was performed every 3 weeks for a total of 2 subcultures. The composition of the callus subculture medium was MS + 2,4-D 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0067] Differentiation culture: Callus tissue that has undergone two subcultures was inoculated into callus differentiation medium and cultured at a constant temperature of 28℃ under light irradiation of 80 μmol / s / m². 2 The photoperiod was 14 h / 10 h (light / dark), and the cells were subcultured every 3 weeks for a total of 2 subcultures. The callus differentiation medium consisted of MS + 6-BA 2.0 mg / L + sucrose 30 g / L + agar powder 8.0 g / L, pH 5.8.

[0068] Rooting induction: The seedlings obtained from differentiation culture were divided into individual plants and inoculated into rooting induction medium. The culture conditions were the same as those for differentiation culture, and the culture time was 4 weeks. The components of the rooting induction medium were MS + NAA 2.0 mg / L + sucrose 30 g / L + activated carbon 1.5 g / L + agar powder 8.0 g / L, pH 5.8.

[0069] Transplanting to seedling stage: Rooted seedlings obtained from rooting culture are transferred to a greenhouse and hardened off for 3 days before being transplanted into a seedling substrate for further cultivation. The substrate consists of peat moss and vermiculite mixed in a 7:3 volume ratio. The greenhouse has an average humidity of 75%, an average temperature of 25℃, and an average light intensity of 70 μmol / s / m². 2 The photoperiod is 12 h / 12 h (light / dark).

[0070] Results Statistics

[0071] To verify the universality and effectiveness of this invention, six representative species of the Sugarcane genus (cultivated species Yunzhe 081609, tropical species Heicheliben, Chinese species Guilin Zhuzhe, Indian species Pansahi, Geshoumi VN2, and wild species Dagen 57NG208) were selected and cultured according to the methods described in the three embodiments above. Each treatment was replicated three times, and the culture results at key stages are recorded in Table 1, followed by analysis of variance.

[0072] Table 1. Tissue culture performance of sugarcane plants in different embodiments

[0073]

[0074] Note: Tissue culture performance data are the average of three replicates, with each replicate containing an average of 91 (83-101) uncontaminated explants. Different lowercase letters indicate significant differences in sugarcane data among different examples (P<0.05), as tested using Duncan's test. Analysis of variance was performed using a general linear model to examine the interactions between different species, different examples, and between species and examples. **, P<0.01; ***, P<0.001; ns, no significant difference.

[0075] As shown in Table 1, the browning phenomenon during tissue culture was effectively suppressed for all tested sugarcane germplasm resources using the method of this invention (Examples 1-3). Specifically, in terms of browning rate, Examples 2 and 3 showed significantly better control effects than Example 1, with Example 3 exhibiting the best conditions. This advantage was particularly evident in the easily browned varieties VN2 and 57NG208. However, in Example 2, some explants were observed to become translucent and eventually die, a significantly higher damage rate than in Examples 1 and 3. Comprehensive analysis suggests this was due to the excessively high concentration of potassium cinnamate (250 mg / L) used in this example. Furthermore, there was no statistically significant difference in callus differentiation rate among the examples, indicating that callus induction in the potassium cinnamate-containing medium provided by this invention does not negatively impact the differentiation potential during subsequent regeneration. Further analysis of the core efficiency indicators of tissue culture showed that the callus induction rate and final regeneration efficiency of all tested germplasms under the conditions of Example 3 were significantly higher than or equal to those of Examples 1 and 2, indicating that Example 3 is the optimal implementation scheme of the present invention.

[0076] To scientifically evaluate the universality of the method of this invention, an analysis of variance was performed on the data in Table 1. The results showed that different *Saccharum* germplasms themselves had extremely significant (P<0.01) or significant (P<0.05) effects on browning rate, callus induction rate, differentiation rate, and regeneration efficiency, which is consistent with the differences in biological characteristics among different species. Different examples (i.e., different treatment conditions) also had extremely significant or significant effects on browning rate, damage rate, callus induction rate, and regeneration efficiency, demonstrating the necessity of optimizing process parameters. Most importantly, the interaction between germplasm and examples did not reach a significant level on any of the detected indicators. Statistical results indicate that the technical solution provided by this invention (especially the optimal parameter combination represented by Example 3) has a stable and consistent promoting effect on the tissue culture effect of different germplasms within the *Saccharum* genus.

[0077] Based on the determination that Example 3 is the preferred solution, in order to further explore the general effects of different treatment conditions on the tissue culture effect of sugarcane plants, the key steps and core components in Example 3 were adjusted, and the following comparative examples were set up:

[0078] Comparative Example 1 (Blank Control)

[0079] This comparative example uses conventional tissue culture methods without any specific anti-browning treatment. Specifically, no low-temperature pretreatment or potassium cinnamate pretreatment is performed, and no potassium cinnamate or any other anti-browning agent is added to the subsequent callus induction medium. Apart from this, all other operations, such as explant selection, sterilization, and culture conditions, are consistent with those in Example 3.

[0080] Comparative Example 2 (Conventional Anti-browning Agent Control)

[0081] This comparative example uses common antioxidants, and differs from Example 3 in that potassium cinnamate is replaced with a mixture of 100 mg / L citric acid and 150 mg / L cysteine ​​in the explant pretreatment and culture medium.

[0082] Comparative Example 3 (Potassium Cinnamate Concentration Adjustment)

[0083] This comparative example aims to investigate the concentration effect of potassium cinnamate. The same procedures as in Example 3 were used, except that the concentration of potassium cinnamate was increased to 300 mg / L.

[0084] Comparative Analysis of Effects

[0085] Six representative species of the sugarcane genus (cultivated species *Saccharum yunnanense* 081609, tropical species *Saccharum nigra*, Chinese species *Saccharum guilinense*, Indian species *Pansahi*, *Saccharum kusnezoffii* VN2, and wild species *Saccharum dasycarpus* 57NG208) were cultured using the methods described in the three comparative examples above. Each treatment was replicated in triplicate, and the culture results at key stages are recorded in Table 2. Comparative example 0 in Table 2 represents the data from Example 3, which was used as the baseline for comparison.

[0086] Table 2. Tissue culture performance of *Saccharum* species in different comparative proportions.

[0087]

[0088] Note: Tissue culture performance data are the average of three replicates, with each replicate containing an average of 93 (86-109) uncontaminated explants. NA indicates that potassium cinnamate was not used in this comparative example. Comparative example 0, i.e., Example 3, shows the difference as a comparison with Example 3, using a two-sided Dunnette test. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

[0089] As shown in Table 2, all sugarcane germplasms in Comparative Example 1 (without anti-browning measures) exhibited a sharp increase in browning rate, which was significantly higher than that in Example 3 (P<0.001), resulting in a significant decrease in callus induction rate (P<0.05 to P<0.001), and consequently a severe loss of regeneration efficiency.

[0090] Comparative Example 2, which uses a combination of citric acid and cysteine ​​(a conventional antioxidant strategy), showed a lower browning rate than Comparative Example 1, but still significantly higher than Example 3 (P<0.01). Furthermore, the treatment effect exhibited a clear germplasm dependence: the regeneration efficiency for cultivated and tropical species was comparable to Example 3 (P>0.05), but the regeneration efficiency for Chinese, Indian, *Gynostemma pentaphyllum*, and large-stemmed wild species was significantly lower (P<0.05 to P<0.01), indicating a lack of versatility of conventional antioxidants.

[0091] When the potassium cinnamate concentration was increased to 300 mg / L (Comparative Example 3), although it strongly inhibited browning, it caused severe explant damage (damage rate 45.56%-72.85%), resulting in a decrease in callus induction rate and regeneration efficiency. For most sugarcane species (including cultivated, tropical, Chinese, and Indian species), the final regeneration efficiency of Comparative Example 3 was even lower than that of Comparative Example 1, which did not take any anti-browning measures. Based on the data from Examples 1-3, it can be seen that potassium cinnamate has a relatively strict concentration limit for its anti-browning effect in tissue culture of sugarcane species; only within a concentration range of 100-250 mg / L can toxicity be avoided and tissue culture performance improved.

[0092] 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. A browning-resistant Saccharum sp. callus induction medium, characterized by, The sugarcane callus induction medium consisted of MS as the basal medium, 100-250 mg / L potassium cinnamate, 1.0-3.0 mg / L 2,4-D, 30 g / L sucrose, and 8.0 g / L agar powder. The explants for the sugarcane callus before culture are stem tip tissues taken from the shoot tip of the sugarcane plant, wherein the shoot tip is from 3-5 nodes below the apical meristem to the +1 leaf pulvinus.

2. The callus induction medium of claim 1, wherein, The concentration of potassium cinnamate in the callus induction culture medium is 200 mg / L.

3. A method of tissue culture browning resistance in Saccharum plants, characterized by, Includes the following steps: (1) The explants of the sugarcane plant were subjected to low-temperature pretreatment; the explants were the shoot tip tissues taken from the shoot tip of the sugarcane plant, the shoot tip being from 3-5 nodes below the apical meristem to the +1 leaf pulvinus; (2) The explants treated in step (1) were pretreated by soaking in a 100-250 mg / L potassium cinnamate aqueous solution; (3) The explants treated in step (2) are inoculated into the callus induction medium described in claim 1 or 2 for culture.

4. The method of claim 3, wherein, In step (2), the soaking pretreatment time is 10-20 minutes.

5. The method of claim 3, wherein, In step (1), the conditions for the low-temperature pretreatment are: temperature 18-22℃, humidity 70%-80%, and standing in a dark environment for 14-24 hours.

6. The use of potassium cinnamate in the preparation of the browning-resistant sugarcane callus induction medium as described in claim 1 or 2.

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