Sugarcane regeneration-promoting small peptide ScREF1 and application thereof

Abstract

The application discloses a sugarcane regeneration-promoting small peptide ScREF1 and application thereof, and belongs to the technical field of plant biotechnology and sugarcane tissue culture. The amino acid sequence of the ScREF1 is shown as SEQ ID NO. 1. The application provides a high-activity sugarcane-specific regeneration-promoting small peptide ScREF1, and provides a matching small peptide mother liquor preparation and callus induction / differentiation medium adding process, and the optimal application concentration is determined in stages. The ScREF1 provided by the application can overcome the defects of existing sugarcane tissue culture, such as serious callus browning, weak embryogenic callus proliferation, low regeneration efficiency and strong genotype dependence. The application can be widely applied to factory-like rapid propagation of sugarcane elite varieties, cultivation of virus-free seedlings, genetic transformation of transgenic materials, creation of CRISPR gene editing materials, and significantly improves the efficiency of sugarcane molecular breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and sugarcane tissue culture technology, and in particular to a sugarcane regeneration-promoting peptide ScREF1 and its applications. Background Technology

[0002] sugar cane( Saccharum officinarum Sugarcane (L.) is a globally important sugar and energy crop, and its industrial development is crucial for ensuring sugar security and bioenergy supply. Tissue culture technology is the core foundation of modern biotechnology systems such as rapid propagation of improved sugarcane varieties, virus-free rejuvenation, transgenic breeding, and gene editing. The efficiency of callus induction and regeneration is key to the effectiveness of this technology. Currently, sugarcane tissue culture regeneration faces challenges such as easy browning and waterlogging during explant-induced callus induction, weak callus proliferation and differentiation capabilities, and difficulties in regenerating some superior genotypes (i.e., genotype dependence). These issues have become one of the key bottlenecks restricting the progress of sugarcane biobreeding.

[0003] In recent years, Regeneration Factor 1 (REF1) peptides have been identified as primary signaling molecules for plant damage-induced regeneration. They activate downstream signaling pathways by binding to the receptor kinase PORK1, initiating tissue repair and organ regeneration processes, and this function is conserved across the plant kingdom. However, due to differences in REF1 homologous peptide sequences, receptor affinity, and endogenous expression levels among different species, exogenous application may yield varying effects, leading to a preference for endogenous peptides. Exogenous application of REF1 peptides has been shown to significantly enhance callus proliferation, regeneration capacity, and genetic transformation efficiency in crops such as tomato, soybean, and wheat. Patents related to endogenous REF1 peptides have been published for species such as American black poplar and tobacco, but their use suffers from species-specific limitations.

[0004] Currently, there are no reports, either domestically or internationally, on the discovery, activity verification, and supporting tissue culture application systems of endogenous REF1 homologous peptides from sugarcane. There is a lack of endogenous peptides and related technical solutions that are highly efficient at promoting callus proliferation and differentiation in sugarcane. Therefore, discovering sugarcane's own REF1 homologous peptides and establishing a highly efficient sugarcane callus induction and regeneration system based on endogenous REF1 peptides is of significant industrial and scientific research value for overcoming the genotypic barriers in sugarcane tissue culture and improving breeding transformation efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a sugarcane regeneration-promoting peptide ScREF1 and its applications to solve the problems existing in the prior art. The ScREF1 provided by this invention can overcome the defects of existing sugarcane tissue culture callus browning, weak embryogenic callus proliferation, low regeneration efficiency, and strong genotype dependence. It can be widely used in the industrialized rapid propagation of improved sugarcane varieties, virus-free seedling cultivation, transgenic genetic transformation, and the creation of CRISPR gene editing materials, significantly improving the efficiency of sugarcane molecular breeding.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a sugarcane regeneration-promoting peptide ScREF1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a plant tissue culture medium for promoting plant regeneration, wherein the plant tissue culture medium contains the sugarcane regeneration-promoting peptide ScREF1.

[0008] The present invention also provides the application of the sugarcane regeneration-promoting peptide ScREF1 in promoting plant regeneration.

[0009] Optionally, the plant includes sugarcane.

[0010] Optionally, the sugarcane includes New Taiwan Sugar No. 22, Yunnan Sugar No. 05-51, Yunnan Sugar No. 08-1609, Guangdong Sugar No. 93-159, and Gui Sugar No. 42.

[0011] This invention also provides a method for promoting the proliferation, differentiation, and regeneration of plant callus tissue, comprising the following steps: Plant explants were inoculated into a basic callus induction medium and cultured to obtain embryogenic callus tissue; pale yellow and loose embryogenic callus tissue was selected and transferred to a differentiation medium for further culture. The basic callus induction medium was based on MS medium, with the addition of 2,4-D 2.0 mg / L, sucrose 30 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, and sterilized at 121℃ for 20 min. After sterilization, the medium was cooled, and sugarcane regeneration peptide ScREF1 was added. The final concentration of sugarcane regeneration peptide ScREF1 in the basic callus induction medium was 0.001-0.01 nM. The differentiation medium was based on MS medium, with the addition of 6-BA 2.0 mg / L, KT 0.5 mg / L, sucrose 20 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, and sterilized at 121℃ for 20 min. After sterilization, the medium was cooled, and sugarcane regeneration peptide ScREF1 was added. The final concentration of sugarcane regeneration peptide ScREF1 in the differentiation medium was 0.01 nM. The amino acid sequence of the sugarcane regeneration-promoting peptide ScREF1 is shown in SEQ ID NO.1.

[0012] Optionally, the plant includes sugarcane, with 6-month-old healthy sugarcane tender heart leaves as explants.

[0013] Optionally, the sugarcane includes New Taiwan Sugar No. 22, Yunnan Sugar No. 05-51, Yunnan Sugar No. 08-1609, Guangdong Sugar No. 93-159, and Gui Sugar No. 42.

[0014] Optionally, the culture conditions are dark culture at 28°C, subculture every 14 days, and continuous culture for 3-4 generations.

[0015] Optionally, the conditions for continued cultivation are 12 h light / 12 h dark, light intensity of 2000 lx, temperature of 28°C during light and 25°C during darkness.

[0016] The present invention discloses the following technical effects: (1) The ScREF1 peptide can be effective at a very low concentration of 0.001 nM; compared with the control group without the addition of the peptide, the exogenous addition of 0.001 nM and 0.01 nM of ScREF1 significantly increased the callus induction weighted index by 21.8% and 19.3%, respectively. (3) Set different application concentrations in stages. During the differentiation stage, 0.01 nM of ScREF1 was added exogenously, and the regeneration index increased by 19.4% compared with the control. Make full use of the biphasic dose effect of small peptides and avoid the imbalance of proliferation / differentiation caused by single concentration. The nanomolar ultra-low application concentration has extremely low industrialization cost.

[0017] (4) Practical verification shows that the ScREF1 and its supporting tissue culture application system provided by the present invention can significantly improve the callus induction and regeneration ability of sugarcane with multiple genotypes such as XTT22, YZ05-51, and YZ08-1609, and overcome the genotype dependence defect of traditional tissue culture. (5) It can be widely used in the industrialized rapid propagation of improved sugarcane varieties, the cultivation of virus-free seedlings, the genetic transformation of transgenic varieties, and the creation of CRISPR gene editing materials, which can significantly improve the efficiency of sugarcane molecular breeding. Attached Figure Description

[0018] 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.

[0019] Figure 1Reference chart for scoring the callus induction index (A) and differentiation index (B); Figure 2 Figure showing the callus index induced by exogenous addition of ScREF1 peptide to CIM; Figure 3 Figure 1 shows the results of adding different concentrations of ScREF1 to CIM to improve the sugarcane callus index. Figure 4 Figure showing the results of adding different concentrations of ScREF1 to CDM to improve the sugarcane regeneration index; Figure 5 The effect of ScREF1 on promoting callus proliferation and regeneration in different sugarcane varieties. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] This invention provides ScREF1, a sugarcane-specific, highly active regeneration-promoting small peptide, and provides a matching process for preparing the small peptide stock solution and adding it to the callus induction / differentiation medium, clarifying the optimal application concentration at each stage. ScREF1 is used to overcome the shortcomings of existing sugarcane tissue culture callus, such as severe browning, weak proliferation of embryogenic callus, low regeneration efficiency, and strong genotype dependence. A highly efficient callus induction and regeneration system with broad-spectrum adaptability to multiple sugarcane varieties is constructed.

[0026] Example 1 In the early screening stage of this invention, a small peptide ScREF1 was obtained, with the amino acid sequence LAPPPPPPGPPKEGCGGRGGSHN (SEQ ID NO.1).

[0027] 1. Small peptide synthesis and sterile mother liquor preparation The ScREF1 sequence was synthesized in solid phase by a peptide synthesis company with a purity ≥95%, and dispensed in 1mg / tube. The solution was dissolved in sterile ultrapure water in a laminar flow hood to prepare a 100 nM stock solution. The solution was then sterilized using a 0.22 μm aqueous filter membrane. After being dispensed in small quantities into sterile centrifuge tubes, the solution was stored at -20℃ in the dark to avoid repeated freeze-thaw cycles and inactivation.

[0028] 2. Verification of the function of ScREF1 in promoting sugarcane callus proliferation using the graded weighted index method. Basic callus induction medium (CIM): MS was used as the basal medium, with the addition of 2,4-D 2.0 mg / L, sucrose 30 g / L, plant gel 3.5 g / L and MES (2-morpholinoethanesulfonic acid) 0.5 g / L, pH 5.8, and sterilized at 121℃ for 20 min; after sterilization, the medium was cooled to 50℃ and ScREF1 small peptide stock solution was added aseptically, with the final concentration of small peptide in the medium being 0.01 nM; the blank control group CK was supplemented with an equal volume of sterile water.

[0029] Explants: Tender heart leaves of healthy 6-month-old sugarcane varieties were sterilized with 75% ethanol for 30 seconds, then sterilized with 2% sodium hypochlorite for 10 minutes, rinsed 5 times with sterile water, and cut into 0.5 cm × 0.5 cm thin slices. They were placed on the above CIM medium and cultured in the dark at 28°C. Subculture was performed every 14 days for 3-4 generations.

[0030] Evaluation method: The callus induction index was calculated using a graded weighted index method. First, the callus induction status of the explants (including browning, water staining, callus emergence, etc.) was graded into 5 levels, from poor to good, with scores ranging from 1 to 5. Figure 1The evaluation (A) involves comprehensively assessing indicators such as callus browning, callus formation rate, and callus hydration, scoring them from left to right according to callus quality, from poorest to best. The scores are as follows: 1 point for severe browning, few calluses, and severe hydration; 2 points for more than half of the area being browned and hydrated, with only a small amount of callus growing; 3 points for a mixture of browning, hydration, and healthy callus; 4 points for a small amount of browning and hydration, with a relatively large and loose callus growth; and 5 points for full, non-browning and hydration-free callus with a loose texture and the highest quality. The callus index is then calculated using a formula based on the scores.

[0031] Healing Index = ∑(number of heals per level × number of hits) / (total number of heals × highest level).

[0032] Result: From Figure 2 It is evident that the small peptide ScREF1 can significantly promote the proliferation of sugarcane callus.

[0033] 3. Screening the optimal ScREF1 peptide concentration for promoting sugarcane callus proliferation using a graded weighted index method. Plant REF1 peptides generally exhibit a typical biphasic dose-response effect, with low concentrations promoting growth and high concentrations inhibiting growth. A series of CIM mediums with gradient final concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM were prepared. Sugarcane heart leaf explants were uniformly inoculated, and after 14 days of dark culture, the optimal ScREF1 peptide concentration for promoting sugarcane callus proliferation was statistically screened using a graded weighted index method.

[0034] Evaluation method: The evaluation method is the same as that for "2. Verification of the function of ScREF1 in promoting sugarcane callus proliferation by graded weighted index method". The optimal small peptide application concentration that can significantly improve the callus index is obtained by statistical screening using graded weighted index method.

[0035] Result: From Figure 3 It can be seen that the exogenous addition of 0.001 nM and 0.01 nM of ScREF1 significantly improved the callus induction index compared with the control, by 21.8% and 19.3% respectively. Therefore, 0.001 nM was determined to be the optimal concentration of ScREF1 for promoting sugarcane callus proliferation.

[0036] 4. Screening the optimal concentration of ScREF1 for promoting callus differentiation using the graded weighted index method. Differentiation medium (CDM): MS was used as the basal medium, supplemented with 2.0 mg / L 6-BA, 0.5 mg / L KT, 20 g / L sucrose, 3.5 g / L plant gel, and 0.5 g / L MES, pH 5.8, and sterilized at 121℃ for 20 min. After sterilization, the medium was cooled to 50℃ and different volumes of ScREF1 stock solution were added, with gradient final concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM, where 0 was the blank control (CK), i.e., no small peptide was added. Culture conditions: 12 h light / 12 h dark, light intensity 2000 lx, light temperature 28℃, dark temperature 25℃, subcultured every 14 days.

[0037] Evaluation method: After 14 days of differentiation culture, the callus differentiation index was calculated using the graded weighted index method. Figure 1 (Medium B) Specifically, callus differentiation is divided into 5 levels. The overall evaluation of callus differentiation and emergence is based on these levels, from left to right, representing differentiation quality from poorest to best. Callus with a surface cell differentiation rate of less than 20% receives 1 point; callus with a surface cell redifferentiation rate of 20%-40% receives 2 points; callus with a surface cell redifferentiation rate of 41%-60% receives 3 points; callus with a surface cell redifferentiation rate of 61%-80% receives 4 points; and callus with a surface cell redifferentiation rate of over 80% receives 5 points. The optimal small peptide application concentration that significantly improves the callus differentiation index was determined through screening.

[0038] Result: From Figure 4 It is evident that 0.01 nM ScREF1 can significantly promote sugarcane callus differentiation and regeneration, increasing the callus differentiation index by 19.4% compared to the control, making it the optimal concentration for callus differentiation and regeneration.

[0039] 5. ScREF1 promotes callus proliferation and differentiation in different sugarcane varieties. To verify the effect of ScREF1 on callus induction, differentiation and regeneration of different sugarcane varieties, three sugarcane varieties with different difficulties in callus induction and regeneration and which are widely planted in production (all provided by the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences) were selected for callus induction and differentiation culture.

[0040] Standardized processing procedure for sugarcane explants: Select healthy 6-month-old seed stem tips with tender heart leaves of varieties XTT22, YZ05-51, and YZ08-1609; disinfect with 75% ethanol for 30 seconds → disinfect with 2% sodium hypochlorite for 10 minutes, rinse 5 times with sterile water, and cut into 0.5 cm × 0.5 cm thin slices.

[0041] Culture and subculture procedures: 1) Callus induction and proliferation: CIM medium (MS as the basal medium, supplemented with 2,4-D 2.0 mg / L, sucrose 30 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, sterilized at 121℃ for 20 min; after sterilization, cooled to 50℃ and aseptically added with ScREF1 peptide stock solution, the final peptide concentration in the medium was 0.001 nM) was cultured in the dark at 28℃, with fresh medium replaced every 14 days and the callus index counted. Subcultured for 3-4 generations to obtain embryogenic callus tissue; 2) Bud differentiation and regeneration: Pale yellow, loose embryogenic callus tissue was transferred to CDM medium (MS as the basal medium, supplemented with 6-BA 2.0 mg / L, KT 0.5 mg / L, sucrose 20 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, sterilized at 121℃ for 20 min). min; Sterilize and cool to 50℃, add ScREF1 small peptide stock solution, the final concentration of small peptide in the culture medium is 0.01 nM), culture under light (12 h light / 12 h dark, light intensity 2000 lx, temperature 28℃ during light and 25℃ during dark), subculture once every 14 days, and count the regeneration efficiency after one generation.

[0042] Each group was set up with no less than 6 biological replicates. The differences were determined by one-way ANOVA and Duncan's method for multiple comparisons. P < 0.05 was considered statistically significant.

[0043] Result: From Figure 5 It is evident that the optimal concentration of exogenous ScREF1 promoted callus proliferation and differentiation regeneration in all three tested varieties compared to the control.

[0044] Further validation was conducted on two sugarcane varieties, Yuetang 93-159 and Guitang 42 (both provided by the Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences). The standardized treatment process for sugarcane explants and the culture and subculture procedures were the same as above. The results showed that the optimal concentration of exogenous ScREF1 promoted callus proliferation and differentiation regeneration in both tested varieties compared with the control.

[0045] 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 sugarcane regeneration-promoting small peptide, ScREF1, characterized in that, The amino acid sequence of the sugarcane regeneration-promoting peptide ScREF1 is shown in SEQ ID NO.

1.

2. A plant tissue culture medium for promoting plant regeneration, characterized in that, The plant tissue culture medium contains the sugarcane regeneration-promoting peptide ScREF1 as described in claim 1.

3. The application of the sugarcane regeneration-promoting peptide ScREF1 as described in claim 1 in promoting plant regeneration.

4. The application as described in claim 3, characterized in that, The plant mentioned includes sugarcane.

5. The application as described in claim 4, characterized in that, The sugarcane mentioned includes New Taiwan Sugar No. 22, Yunnan Sugar No. 05-51, Yunnan Sugar No. 08-1609, Guangdong Sugar No. 93-159, and Gui Sugar No.

42.

6. A method for promoting the proliferation, differentiation, and regeneration of plant callus tissue, characterized in that, Includes the following steps: Plant explants were inoculated into a basic callus induction medium and cultured to obtain embryogenic callus tissue. Select pale yellow, loose embryogenic callus tissue and transfer it to differentiation medium for further culture; The basic callus induction medium was based on MS medium, with the addition of 2,4-D 2.0 mg / L, sucrose 30 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, and sterilized at 121℃ for 20 min. After sterilization, the medium was cooled, and sugarcane regeneration peptide ScREF1 was added. The final concentration of sugarcane regeneration peptide ScREF1 in the basic callus induction medium was 0.001-0.01 nM. The differentiation medium was based on MS medium, with the addition of 6-BA 2.0 mg / L, KT 0.5 mg / L, sucrose 20 g / L, plant gel 3.5 g / L and MES 0.5 g / L, pH 5.8, and sterilized at 121℃ for 20 min. After sterilization, the medium was cooled, and sugarcane regeneration peptide ScREF1 was added. The final concentration of sugarcane regeneration peptide ScREF1 in the differentiation medium was 0.01 nM. The amino acid sequence of the sugarcane regeneration-promoting peptide ScREF1 is shown in SEQ ID NO.

1.

7. The method as described in claim 6, characterized in that, The plant includes sugarcane, with 6-month-old healthy sugarcane tender heart leaves as explants.

8. The method as described in claim 7, characterized in that, The sugarcane mentioned includes New Taiwan Sugar No. 22, Yunnan Sugar No. 05-51, Yunnan Sugar No. 08-1609, Guangdong Sugar No. 93-159, and Gui Sugar No.

42.

9. The method as described in claim 6, characterized in that, The culture conditions were: dark culture at 28°C, subculture every 14 days, and continuous culture for 3-4 generations.

10. The method as described in claim 6, characterized in that, The conditions for continued cultivation were 12 h of light / 12 h of darkness, light intensity of 2000 lx, temperature of 28°C during light and 25°C during darkness.

Images