A method for promoting in vitro regeneration of adventitious shoots from sunflower

Abstract

The application discloses a method for promoting adventitious shoot regeneration of sunflower in vitro, and belongs to the technical field of plant tissue culture. The method comprises the following steps: taking sunflower 'YK86' sterile seedling cotyledon as an explant, inoculating the explant into a callus induction medium containing 0.01 nM exogenous HaREF1 small peptide and a bud differentiation medium in sequence after pretreatment, effectively promoting sunflower callus induction and adventitious shoot differentiation and regeneration, reducing the browning rate, and successfully inducing adventitious shoots. Through screening of the genotype and explant of sunflower in vitro regeneration, in combination with the synergistic effect of screening and adding different concentrations of HaREF1 to the callus induction medium and the bud differentiation medium, the application effectively promotes the differentiation and regeneration of sunflower adventitious shoots in vitro, solves the problems of difficult sunflower in vitro regeneration, low regeneration efficiency and instability, and provides technical support for sunflower genetic transformation, gene function verification and variety improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology. Specifically, it relates to a method for promoting the regeneration of sunflower adventitious buds in vitro, which is applicable to research and application such as propagation and preservation of sunflower varietal resources, genetic transformation and variety improvement. Background Technology

[0002] Sunflower (Helianthus annuus L.) is an annual herbaceous plant belonging to the genus Helianthus in the family Asteraceae. It has a wide range of uses and high economic value, and is an important oilseed crop worldwide. However, traditional breeding methods are inefficient, require long periods to stabilize target traits, and suffer from severe varietal homogenization, narrow genetic bases of core parents, and incompatibility in distant hybridization. These methods can no longer fully meet the demands of production for sunflower varieties and the speed of renewal. Therefore, it is urgent to establish efficient in vitro regeneration methods for sunflowers to improve the repeatability, stability, and genetic transformation efficiency of in vitro regeneration, providing technical support for sunflower biobreeding.

[0003] Sunflowers are plants that are difficult to regenerate in vitro. Their regeneration is subject to problems such as strong genotype dependence, low embryogenic callus induction rate, easy browning, and difficulty in shoot differentiation or low shoot differentiation regeneration rate. In 1974, Rogers et al. first successfully induced callus tissue from cytoplasmic male sterile lines of sunflowers, but these callus tissues only differentiated into roots (Rogers MA, Gál HL, Horner Jr HT. Callus formation and differentiation in tissue cultures of normal and cytoplasmic male sterile sorghum, pepper, sunflower and tobacco [J]. In Vitro Cell. Dev. Biol. Plant, 1974). The first reported case of obtaining regenerated sunflower plants was achieved by BAKER using cotyledons of the HA300 variety as explants, but the regeneration efficiency was low (BAKER, MCMUNOA-FERNANDEZN, CARTER CD. Improved shoot development and rooting from mature cotyledons of sunflower [J]. Plant Cell, Tissue and Organ Culture, 1999). Subsequently, Liu Haichen et al. conducted research on callus induction, shoot differentiation, and seedling formation of sunflower hypocotyls on media containing different hormone concentrations, and found that sunflower hypocotyls improved with MS supplementation of 0.03 mg·L⁻¹. -1 IAA and 1.2 mg·L-1 High rates of adventitious shoot regeneration were achieved on 6-BA medium, and adventitious shoots could grow into healthy seedlings on 1 / 2 MS medium (Liu Haichen, Guo Hongyu, Yu Peng, et al. Study on hypocotyl tissue culture and plant regeneration in sunflower [J]. Journal of Changchun University of Technology, 2005). Wang Yuanyuan et al. used anthers of 10 different genotypes of sunflower as explants to study the induction of callus tissue and the differentiation process of callus tissue by anther culture. The results showed that anthers of hybrid varieties were easier to culture than anthers of inbred lines, and the optimal hormone combination for anther callus induction was 2 mg·L⁻¹. -1 NAA+2 mg·L -1 6-BA + 2 mg·L -1 KT, the optimal combination of differentiation hormones is 2 mg / L. -1 6-BA + 0.5 mg·L -1 NAA+1 mg·L -1 KT laid the foundation for further exploration of sunflower anther culture technology (Wang Yuanyuan. Research on the establishment of sunflower in vitro regeneration system [D]. Northeast Agricultural University, 2011). There are also reports on in vitro regeneration culture of sunflower using cotyledonary nodes, embryos and protoplasts as explants to induce regeneration and obtain adventitious buds or regenerated plants, which has made a significant breakthrough in the in vitro regeneration technology of sunflower. However, it has shown problems such as low regeneration frequency and unstable regeneration, and difficulty in repeating it in the later stage.

[0004] Current research largely focuses on optimizing explants and hormone ratios for specific genotypes, lacking systematic screening of a large number of genotype materials. This results in regeneration systems with poor reproducibility and limited general applicability, leading to the absence of a mature sunflower in vitro regeneration system. Therefore, this invention aims to establish a method for promoting the in vitro regeneration of adventitious shoots from sunflowers, improving the efficiency and reproducibility of sunflower in vitro regeneration, and providing technical support for sunflower genetic transformation and functional genomics research. Summary of the Invention

[0005] To address the problems of strong genotype dependence, low differentiation and regeneration rate, and instability in current sunflower in vitro regeneration systems, the purpose of this invention is to establish a stable method that can effectively promote the in vitro regeneration of sunflower adventitious buds by screening genotypes, explants, and culture medium formulations to synergistically promote the differentiation and regeneration of sunflower adventitious buds.

[0006] To achieve the above objectives, the first objective of this invention is to provide a HaREF1 peptide that promotes the in vitro regeneration of adventitious shoots from sunflower. This peptide was designed by the inventors based on years of experience in sunflower in vitro culture, and its amino acid sequence is RVLTRRPPPPRGPISSGGGGQTN, as shown in SEQ ID NO:1. Furthermore, this invention also provides the nucleotide sequence encoding the aforementioned HaREF1 regeneration peptide.

[0007] A second objective of this invention is to provide a tissue culture medium for promoting the in vitro regeneration of adventitious shoots from sunflowers, wherein the tissue culture medium contains the HaREF1 peptide as described in claim 1.

[0008] More preferably, the tissue culture medium for promoting the regeneration of adventitious shoots in sunflower as described above is a callus induction medium, the formulation of which is: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 1 mg / L, 6-BA 2 mg / L and HaREF1 0.01 nM, pH 5~6.

[0009] More preferably, the tissue culture medium for promoting the regeneration of adventitious shoots in sunflower in vitro, as described above, is a shoot differentiation medium, the formula of which is selected from any of the following:

[0010] (1) MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 0.3 mg / L, 6-BA 0.2 mg / L, AgNO3 0.3 mg / L and HaREF1 0.01 nM, pH 5~6;

[0011] (2) MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.05 mg / L and 6-BA 1.5 mg / L, pH 5~6.

[0012] A third objective of this invention is to provide the application of the aforementioned HaREF1 peptide in promoting the in vitro regeneration of adventitious shoots in sunflowers. More preferably, the sunflower variety is YK86.

[0013] This invention compares the callus formation effect by inoculating hypocotyls and cotyledons of 53 sunflowers with different genotypes into callus induction medium, and screens out suitable genotypes and explants. Then, suitable explants of suitable genotypes are inoculated into mediums with different hormone combinations and different concentrations of HaREF1, and the callus induction efficiency on different callus induction media and the bud formation in bud differentiation media are compared. Finally, the hormone formula and HaREF1 concentration that effectively promote the in vitro regeneration of sunflowers are screened out, and stable regenerated buds are obtained, which significantly improves the regeneration efficiency of sunflowers.

[0014] Therefore, a fourth objective of this invention is to provide a method for promoting the in vitro regeneration of adventitious buds from sunflowers, the method comprising the following steps:

[0015] (1) Selection of explants: Cotyledons of aseptic sunflower seedlings with a seedling age of 4-6 days were selected as explants;

[0016] (2) Callus induction: Explants were inoculated into callus induction medium and cultured to obtain green and dense callus with differentiation potential; the formula of the callus induction medium was: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 1 mg / L, 6-BA 2 mg / L and HaREF 10.01 nM, pH 5~6;

[0017] (3) Bud formation: After the callus tissue has grown for 28-32 days, it is transferred to the bud differentiation medium for culture to obtain differentiated buds; the formula of the bud differentiation medium is: MS basal medium 4.0-4.5 g / L, sucrose 20-40 g / L, agar 5-10 g / L, NAA 0.5 mg / L, KT 0.3 mg / L, 6-BA 0.2 mg / L, AgNO3 0.3 mg / L and HaREF1 0.01 nM, pH 5-6; or MS basal medium 4.0-4.5 g / L, sucrose 20-40 g / L, agar 5-10 g / L, NAA 0.05 mg / L and 6-BA 1.5 mg / L, pH 5-6;

[0018] (4) Bud elongation and proliferation: The differentiated buds are transferred to the bud elongation and proliferation medium for culture to obtain regenerated buds; the formula of the bud elongation and proliferation medium is: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.05 mg / L, 6-BA 1.5 mg / L, activated carbon 0.5 g / L and GA3 0.5 mg / L, pH 5~6.

[0019] As a preferred embodiment of the present invention, the method for promoting the in vitro regeneration of adventitious buds of sunflower as described above, wherein the explant is the cotyledon of a 4-day sterile seedling of sunflower variety YK86, which is cut into small pieces of 0.2~0.3 cm under sterile conditions.

[0020] As a preferred embodiment of the present invention, the method for promoting the in vitro regeneration of adventitious buds of sunflower as described above includes the following method for preparing sterile sunflower seedlings: plump and intact sunflower seed kernels of variety YK86 are disinfected by soaking in 70%~75% alcohol for 1~2 min and 3% sodium hypochlorite for 8~10 min, and then inoculated with the embryo end facing down in MS solid basal medium. After culturing for 4 days, sterile seedlings are obtained. The culture conditions are a temperature of 25±2℃, a day / night photoperiod of 16 h / 8 h, and a light intensity of 2000 lx.

[0021] As a preferred embodiment of the present invention, the callus induction culture medium is formulated as follows: MS basal medium 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.5 mg / L, KT 1 mg / L, 6-BA 2 mg / L and HaREF1 0.01 nM, pH 5.8.

[0022] According to a method for promoting the in vitro regeneration of adventitious buds from sunflowers, in the bud formation step, the callus transferred to the bud differentiation medium is a dense green or yellowish-green callus that has grown for 30 days in a callus induction medium and is either non-vitrified or lightly vitrified.

[0023] As a preferred embodiment of the present invention, the bud differentiation medium is formulated as follows: MS basal medium 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.5 mg / L, KT 0.3 mg / L, 6-BA 0.2 mg / L, AgNO3 0.3 mg / L and HaREF1 0.01 nM, pH 5.8; or MS basal medium 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.05 mg / L and 6-BA 1.5 mg / L, pH 5.8.

[0024] As a preferred embodiment of the present invention, the formula of the bud elongation and proliferation medium is as follows: MS basal medium 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.05 mg / L, 6-BA 1.5 mg / L, activated carbon 0.5 g / L and GA3 0.5 mg / L, pH 5.8.

[0025] Compared with the prior art, the method for promoting the regeneration of sunflower adventitious buds in vitro provided by the present invention has the following beneficial effects.

[0026] 1. This invention screened the cotyledons and hypocotyls of 53 sunflower genotype materials, compared their callus induction efficiency and bud differentiation, and clarified that cotyledons are suitable explants for sunflower regeneration. A genotype 'YK86' with regeneration ability was obtained, and the cotyledons of this genotype can be induced to differentiate into regenerated buds on two different hormone ratios in culture media. It has wide applicability and reproducibility.

[0027] 2. This invention, through systematic screening and optimization of the hormone ratio in callus induction and bud differentiation media, and the introduction of HaREF1 in synergistic effect with growth regulators, established the optimal callus induction and bud differentiation media, and determined the optimal concentration of HaREF1 (0.01 nM). This significantly reduced the browning rate of explants, effectively promoted the dedifferentiation of sunflower cotyledons and the regeneration of adventitious buds, achieving a callus induction rate of 99.70%, a browning rate of zero, a bud differentiation rate of 12.50%, and a proliferation coefficient of 3.13. This lays the foundation and provides technical support for the establishment of a highly efficient in vitro regeneration system for sunflowers.

[0028] 3. This invention establishes a method for promoting the in vitro regeneration of adventitious buds from sunflowers, optimizes the in vitro regeneration system of sunflowers, and solves the problems of difficult, inefficient and poor reproducibility of in vitro regeneration of sunflowers, providing technical support for carrying out genetic transformation and functional genomics research on sunflowers. Attached Figure Description

[0029] Figure 1 The diagram shows the in vitro regeneration process of sunflower; A is a sterile sunflower seedling cultured for 4 days, B is a cotyledon explant, C is callus tissue formed by cotyledon dedifferentiation, D is callus tissue that has just been subcultured, E is adventitious bud formed by differentiation of green callus tissue, and F is an elongated adventitious bud.

[0030] Figure 2 This refers to callus tissue induced by dedifferentiation of the hypocotyl (A) and cotyledon (B) of sunflower.

[0031] Figure 3 A comparison of the morphological characteristics of four types of callus tissue.

[0032] Figure 4 The callus state and browning of 'YK86' cotyledon explants on different induction media.

[0033] Figure 5 The callus induction rate (A) and browning rate (B) of cotyledons under different HaREF1 concentrations.

[0034] Figure 6 Shoot differentiation and growth of 'YK86' callus on B2 (A and B) and B4 (C and D) media. Detailed Implementation

[0035] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0036] Example 1: Screening and optimization of suitable genotypes and explants

[0037] The materials used in this embodiment were 53 sunflower varietal resources (Table 1); the culture medium used was callus induction medium: MS basal medium 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.5 mg / L, KT 1 mg / L and 6-BA 2 mg / L, pH 5.8.

[0038] (1) Explant treatment: 53 shelled and plump sunflower kernels were disinfected with 70% alcohol (soaked for 1 min) and 3% sodium hypochlorite (soaked for 8-10 min), and then inoculated into MS solid medium with the embryo end facing down. After culturing for 4 days at 25±2 ℃ temperature, 75% humidity, 16 h / 8 h light-dark cycle and 2000 lx light intensity, the cotyledons and hypocotyls were harvested and cut into 0.2 cm pieces and 0.2 cm segments, respectively.

[0039] (2) Callus induction: Cotyledons and hypocotyls of 53 genotype sterile seedlings were inoculated into callus induction medium (MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.5 mg / L, KT 1 mg / L and 6-BA 2 mg / L, pH 5.8). After 2 weeks, the callus induction rate of different materials was found to be 40.00%~100.00%, of which the callus induction rate of cotyledons and hypocotyls of 3 materials (SZ8003, 4672, Bepezanckuu) reached 100%. The callus induction rate of cotyledon explants of different materials was 33.33%~100.00%, while the callus induction rate of hypocotyls was 20.00%~100.00% (see Table 1). The callus induced by hypocotyl explants was mostly loose, white and water-soaked. Figure 2 A); while the cotyledon explants induce mostly dense callus tissue that is green or yellowish-green ( Figure 2 B). Based on callus induction rate and growth status, four types were identified ( Figure 3The classification criteria are as follows: Type I callus induction rate ≥95%, and dense green; Type II callus induction rate ≥80%, and dense yellow; Type III callus induction rate ≥50%, and white water-stained; Type IV callus induction rate <50%, and prone to browning. Based on these criteria, statistical analysis of 53 materials revealed that most hypocotyls (40 samples) formed white water-stained Type III callus, with 2 samples forming Type I and 9 samples forming Type II. Cotyledon explants primarily dedifferentiated to form dense yellow Type II and Type III callus tissues, with 18 samples forming Type II and 25 forming Type III. Three genotypes (YK86, Lin Kui Hua-12, 7861-S7) could obtain Type I callus from their cotyledons, among which YK86 could form dense green callus tissue with some differentiation potential (the callus surface had small green protrusions). By comparing the callus induction rate, explant browning rate, and callus status of cotyledons and hypocotyls of 53 genotypes, it was concluded that cotyledons of YK86 are the most suitable material for in vitro regeneration and callus induction in sunflower.

[0040] Table 1: Callus induction status of 53 sunflower cotyledons and hypocotyls

[0041]

[0042] Example 2: Screening of callus induction medium and synergistic optimization of HaREF1

[0043] The material used in this embodiment was YK86 cotyledons; the callus induction medium used is shown in Table 2; the introduced HaREF1 small peptide sequence was RVLTRRPPPPRGPISSGGGGQTN (synthesized by Nanjing Genscript Biotech Co., Ltd.); and five different concentration gradients of 0, 0.01, 0.1, 1, and 10 nM were set and added to A2 medium respectively.

[0044] (1) Explant treatment: After disinfecting the plump and intact kernels of YK86 with 70% alcohol (soaking for 1 min) and 3% sodium hypochlorite (soaking for 8~10 min), the kernels were inoculated with the embryo end facing down in MS solid medium and cultured for 4 days (culture conditions were 25±2 ℃ temperature, 75% humidity, 16 h / 8 h light and dark cycle, 2000 lx light intensity). The cotyledons were then taken and cut into 0.2 cm pieces.

[0045] Table 2: Hormone composition of callus induction culture medium

[0046]

[0047] (2) Callus induction: YK86 cotyledon pieces were inoculated into callus induction medium A1-A4 (Table 2). After 2 weeks, the callus induction rate was above 90% and the browning rate was 27.60%~36.70% (see Table 3). The callus formation rate on A1 medium (4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, 0.5 mg / L NAA, 0.5 mg / L IAA, and 0.5 mg / L 6-BA) was 96.70%, and the browning rate was 27.60%, forming white, water-soaked, undifferentiated, non-embryonic calluses. The callus induction rate and browning rate on A2 medium (4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, 0.5 mg / L NAA, 1 mg / L KT, and 2 mg / L 6-BA) were comparable to those on A1 medium, at 96.70% and 24.60%, respectively, but it could form dense, green callus tissue with differentiation and regeneration potential. On A3 medium (4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, 2 mg / L IAA, and 1 mg / L 6-BA), the callus formation rate was 96.70%, and the browning rate was 27.60%, forming white, water-soaked, undifferentiated, non-embryonic calluses. The lowest callus induction rate (93.70%) and the highest explant browning rate (36.70%) were observed on 6-BA, resulting in dense, brown, and difficult-to-differentiate callus tissue. The highest callus formation rate (98.30%) was observed on A4 medium (4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, 0.5 mg / L NAA, and 2 mg / L 6-BA), but the browning rate was also high (31.10%), producing dense, yellow, and difficult-to-differentiate callus tissue. These results indicate that cotyledons exhibit the best callus induction effect on A2 medium (see Table 3 and...). Figure 4 ).

[0048] Table 3: Callus induction of 'YK86' cotyledonary explants in four culture media

[0049]

[0050] Note: ++ indicates good growth; + indicates good growth; - indicates average growth; -- indicates poor growth.

[0051] Adding different concentrations of HaREF1 peptide to A2 medium inoculated with YK86 cotyledons revealed that 0.01 nM HaREF1 not only increased callus induction rate but also significantly reduced explant browning rate. The callus induction rate increased by 5.20% compared to the control, and the browning rate decreased to zero. Figure 5Furthermore, the introduction of 0.01 nM HaREF1 into A2 medium can significantly promote the dedifferentiation of cotyledons of YK86 to form green, dense callus with differentiation potential, indicating that it can also form dense callus. Through screening sunflower callus induction media and synergistic optimization of different concentrations of HaREF1, the results show that the combination of adding 0.01 nM HaREF1 to A2 medium has the best callus formation effect.

[0052] (3) Bud formation: After 30 days of callus induction culture, green or yellowish-green callus tissue with no or mild vitrification was selected and transferred to bud differentiation medium B1-B5 (Table 4). After 4 weeks, the bud formation rates were 0.00%, 5.01%, 0.00%, 12.50%, and 0.00%, respectively (Table 5). The bud formation rate on B1 medium (4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, 0.03 mg / L NAA, and 1.2 mg / L 6-BA) was 0.00%. However, when the concentration of 6-BA was reduced to 0.2 mg / L, the concentration of NAA was increased to 0.5 mg / L, and 0.3 mg / L KT and 0.3 mg / L AgNO3 were added (B2 medium), robust green adventitious buds formed. Figure 6 A), the bud formation rate was 5.01%. Some adventitious buds could directly form complete plant morphology with terminal buds, stems, and leaves, but there were few buds or seedlings, and some leaves turned yellow and curled. Figure 6 B); Adding 1.5 mg / L 6-BA and 0.04 mg / L IAA to MS medium (B3 medium) resulted in no adventitious bud differentiation; adding 1.5 mg / L 6-BA and 0.05 mg / L NAA to MS medium (B4 medium) increased the bud formation rate to 12.50%, inducing more shoots or bud points, but the shoot points were shorter and the bud stems were slender. Figure 6 C); with the NAA concentration kept constant (0.05 mg / L) and the 6-BA dosage reduced (0.25 mg / L), no adventitious shoots differentiated (shoot formation rate 0.00%). These results indicate that adventitious shoots differentiated in both B2 and B4 media, and the addition of an appropriate amount of AgNO3 is beneficial in alleviating yellowing and curling, and forming normal seedlings.

[0053] Table 4: Hormone Ratios in Adventitious Bud Differentiation Culture Medium

[0054]

[0055] (4) Bud elongation and proliferation: Differentiated buds were transferred to bud elongation and proliferation medium (MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, NAA 0.05 mg / L, 6-BA 1.5 mg / L, activated carbon 0.5 g / L and GA3 0.5 mg / L). After 30 days of culture, the number of buds increased and the bud stems elongated, with an average proliferation coefficient of 3.13. Figure 6 D).

[0056] Table 5: Effects of different differentiation media on adventitious shoot induction

[0057]

Claims

1. A HaREF1 peptide that promotes the in vitro regeneration of adventitious shoots from sunflower, the amino acid sequence of which is RVLTRRPPPPRGPISSGGGGQTN, as shown in SEQ ID NO:

1.

2. The nucleotide sequence encoding the sunflower REF1 regeneration-promoting peptide of claim 1.

3. A tissue culture medium for promoting the in vitro regeneration of adventitious shoots from sunflowers, characterized in that, The tissue culture medium contains the HaREF1 small peptide as described in claim 1.

4. The tissue culture medium for promoting the in vitro regeneration of adventitious shoots from sunflower according to claim 1, characterized in that, The culture medium is a callus induction medium with the following formula: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 1 mg / L, 6-BA 2 mg / L and HaREF1 0.01 nM, pH 5~6.

5. The tissue culture medium for promoting the in vitro regeneration of adventitious shoots from sunflower according to claim 1, characterized in that, The tissue culture medium is a bud differentiation medium, and its formula is selected from any of the following: (1) MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 0.3 mg / L, 6-BA 0.2 mg / L, AgNO3 0.3 mg / L and HaREF1 0.01 nM, pH 5~6; (2) MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.05 mg / L and 6-BA 1.5 mg / L, pH 5~6.

6. The application of the HaREF1 peptide according to claim 1 in promoting the in vitro regeneration of adventitious shoots in sunflower.

7. The application according to claim 6, characterized in that, The sunflower in question is the YK86 sunflower variety.

8. A method for promoting the in vitro regeneration of adventitious buds from sunflowers, characterized in that, The method includes the following steps: (1) Select cotyledons of aseptic sunflower seedlings aged 4-6 days as explants; (2) The explants were inoculated into callus induction medium and cultured to obtain green dense callus with differentiation potential; the formula of the callus induction medium was: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.5 mg / L, KT 1 mg / L, 6-BA 2 mg / L and HaREF1 0.01 nM, pH 5~6; (3) After the callus tissue has grown for 28-32 days, it is transferred to the bud differentiation medium for culture to obtain differentiated buds; the formula of the bud differentiation medium is: MS basal medium 4.0-4.5 g / L, sucrose 20-40 g / L, agar 5-10 g / L, NAA 0.5 mg / L, KT 0.3 mg / L, 6-BA 0.2 mg / L, AgNO3 0.3 mg / L and HaREF1 0.01 nM, pH 5-6; or MS basal medium 4.0-4.5 g / L, sucrose 20-40 g / L, agar 5-10 g / L, NAA 0.05 mg / L and 6-BA 1.5 mg / L, pH 5-6; (4) The differentiated buds are transferred to the bud elongation and proliferation medium for culture to obtain regenerated buds; the formula of the bud elongation and proliferation medium is: MS basal medium 4.0~4.5 g / L, sucrose 20~40 g / L, agar 5~10 g / L, NAA 0.05mg / L, 6-BA 1.5 mg / L, activated carbon 0.5 g / L and GA3 0.5 mg / L, pH 5~6.

9. The method for promoting the in vitro regeneration of adventitious buds from sunflowers according to claim 8, characterized in that, The explants were cotyledons of 4-day-old seedlings of the sunflower variety YK86, cut into small pieces of 0.2-0.3 cm under aseptic conditions.

10. The method for promoting the in vitro regeneration of adventitious buds from sunflowers according to claim 8, characterized in that, The method for preparing sterile sunflower seedlings is as follows: plump and intact sunflower seed kernels of variety YK86 are disinfected by soaking in 70%~75% alcohol for 1~2 min and 3% sodium hypochlorite for 8~10 min, and then inoculated into MS solid basal medium with the embryo end facing down. After culturing for 4 days, sterile seedlings are obtained. The culture conditions are: temperature 25±2℃, day / night photoperiod 16 h / 8 h, and light intensity 2000 lx.

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