Culture method for pepper tissue regeneration and application

Abstract

The invention provides a culture method for pepper tissue regeneration and application. The culture method comprises the following steps: S1) inoculating an explant of pepper into an adventitious bud induction culture medium for adventitious bud induction culture to obtain adventitious buds; the adventitious bud induction culture medium comprises auxin and cytokinin; the auxin comprises one or more of indoleacetic acid, indolebutyric acid or naphthylacetic acid; the cytokinin comprises trans-zeatin and / or zeatin; wherein the adventitious bud culture medium comprises any one of the following hormone combinations: a) zeatin and indoleacetic acid; b) trans-zeatin and indolebutyric acid; c) zeatin and naphthylacetic acid; s2) inoculating the adventitious buds into a rooting culture medium to obtain tissue culture seedlings; s3) performing seedling hardening and transplanting on the tissue culture seedlings to obtain regenerated plants of the peppers. The problem that in-vitro regeneration success rate of pepper tissues is low in the prior art can be solved. The method is suitable for pepper tissue regeneration.

Description

Technical Field

[0001] The invention relates to the field of pepper tissue regeneration, and in particular to a culture method and application of pepper tissue regeneration. Background Art

[0002] Annual pepper (Capsicum annuum L.) is a plant of the genus Capsicum in the Solanaceae family. As a widely grown vegetable and industrial raw material crop worldwide, it is highly favored for its rich nutritional value and unique flavor. However, traditional pepper breeding and propagation methods have many limitations, such as long breeding cycles, low propagation efficiency, and poor genetic stability, which make it difficult to meet the urgent needs of the modern pepper industry for high-yield, high-quality, and disease-resistant varieties. As an efficient and controllable means of plant propagation and improvement, tissue culture technology can provide strong technical support for the rapid propagation, variety improvement, and genetic engineering research of peppers.

[0003] Pepper is a stubborn regeneration plant. During the adventitious bud induction stage, the bud differentiation rate of explants is often not high. Many explants may only remain at the callus or adventitious bud primordium stage and cannot further develop into complete plants. For example, in some studies, the adventitious bud induction rate of pepper was only 19.5%. The low regeneration frequency limits the application efficiency of pepper tissue culture technology in rapid propagation and genetic engineering improvement.

[0004] At the same time, pepper tissue culture has strong genotype specificity. Peppers of different varieties and genotypes have significant differences in their explant regeneration ability. This genotype dependence requires optimizing culture conditions for different varieties when conducting large-scale pepper variety improvement and regeneration, which increases the complexity and cost of research and application.

[0005] In addition, the pepper tissue culture regeneration process still has many technical bottlenecks such as long culture cycle, poor genetic stability, difficulty in optimizing hormone ratio and low genetic transformation efficiency, which seriously restricts the widespread application and development of pepper tissue culture technology. In summary, the pepper tissue culture regeneration system in the prior art makes the success rate of pepper tissue in vitro regeneration low. Summary of the invention

[0006] The main purpose of the present invention is to provide a culture method and application of pepper tissue regeneration to solve the problem of low success rate of in vitro regeneration of pepper tissue in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a culture method for pepper tissue regeneration is provided, which culture method comprises: S1) inoculating pepper explants into an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious buds; the adventitious bud induction medium comprises auxin and cytokinin; the auxin comprises one or more of indoleacetic acid, indolebutyric acid or naphthylacetic acid; the cytokinin comprises trans-zeatin and\or zeatin; wherein the adventitious bud medium comprises any one of the following hormone combinations: a) trans-zeatin and indoleacetic acid; b) trans-zeatin and indolebutyric acid; c) zeatin and naphthylacetic acid; S2) inoculating the adventitious buds into a rooting medium to obtain tissue culture seedlings; S3) hardening and transplanting the tissue culture seedlings to obtain regenerated plants of pepper.

[0008] Further, the hormone combination: a) includes: 0.1-10 mg / L trans-zeatin and 0.05-2 mg / L indoleacetic acid; b) includes: 0.1-10 mg / L trans-zeatin and 0.05-2 mg / L indolebutyric acid; c) includes: 0.1-10 mg / L zeatin and 0.05-1 mg / L naphthylacetic acid; Preferably, the hormone combination: a) includes: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indoleacetic acid; b) includes: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indolebutyric acid; c) includes 0.5-5 mg / L zeatin and 0.05-0.4 mg / L naphthylacetic acid.

[0009] Furthermore, the adventitious bud induction medium also includes 4.25-4.75 g / L MS medium, 28-32 g / L sucrose and 4.0-5.0 g / L plant gel, and the pH of the adventitious bud induction medium is 5.80-5.86.

[0010] Furthermore, the conditions for adventitious bud induction culture are a temperature of 25-30°C, a light intensity of 3200-3700Lx, a relative humidity of 45-55%, and a photoperiod of 14h light and 8h darkness; preferably, the time for adventitious bud induction culture is 25-35d.

[0011] Furthermore, the explant includes a cotyledon explant and / or a hypocotyl explant; the method for obtaining the cotyledon explant includes: i-1) sterilizing pepper seeds and inoculating them in a germination medium for sterile seedling germination culture to obtain sterile pepper seedlings; i-2) cutting off both ends of the cotyledons of the sterile seedlings to obtain cotyledon explants; the area of ​​the cotyledon explant is 4-6 mm×3-5 mm;

[0012] The method for obtaining hypocotyl explants comprises: ii-1) sterilizing pepper seeds and inoculating them into a germination medium for sterile seedling germination culture to obtain sterile pepper seedlings; ii-2) cutting off both ends of the hypocotyl of the sterile seedling to obtain hypocotyl explants; the length of the hypocotyl explants is 5 to 7 mm.

[0013] Furthermore, the germination medium includes 2.0-3.0 g / L MS medium, 8-15 g / L sucrose and 3.5-4.5 g / L plant gel, and the pH of the germination medium is 5.80-5.86; preferably, the germination culture conditions of the sterile seedlings are: temperature of 25-30°C, light intensity of 3200-3700 Lx, relative humidity of 45-55%, light cycle of 14h light and 8h dark alternation, and culture time of 7-8d.

[0014] Furthermore, the rooting culture includes: inoculating the adventitious buds into the rooting culture medium, and performing a first rooting culture under dark conditions; after the first rooting culture is completed, performing a second rooting culture under the conditions of a light cycle of alternating 14 hours of light and 8 hours of darkness; preferably, the time of the first rooting culture is 3 to 4 days; preferably, the temperature of the first rooting culture is 25 to 30°C; preferably, the time of the second rooting culture is 12 to 15 days; preferably, the temperature of the second rooting culture is 25 to 30°C; preferably, the light intensity of the second rooting culture is 3200 to 3700Lx.

[0015] Furthermore, the rooting medium includes 4.25-4.75 g / L MS medium, 1.0-1.5 mg / L indolebutyric acid, 38-32 g / L sucrose and 8.8-9.2 g / L agar, and the pH of the rooting medium is 5.80-5.86.

[0016] Further, hardening and transplanting include: transplanting the tissue culture seedlings from the rooting culture medium to the culture matrix, and removing the film after covering the tissue culture seedlings for 15 to 20 days; preferably, the culture matrix includes culture soil; more preferably, the culture medium also includes one or more of vermiculite, pearl salt or plant fertilizer; preferably, the culture matrix includes culture soil and vermiculite; more preferably, the mass ratio of culture soil to vermiculite is 1:0.5 to 1.2; preferably, the conditions for hardening and transplanting culture are: temperature of 25 to 30°C, light intensity of 3200 to 3700Lx, relative humidity of 45 to 55%, light cycle of 14h light and 8h dark alternation, and culture time of 60 to 75d.

[0017] In order to achieve the above object, according to a second aspect of the present invention, there is provided an application of the above-mentioned pepper tissue regeneration culture method in pepper breeding; preferably, pepper breeding includes pepper genetic transformation or pepper variety regeneration.

[0018] By applying the technical scheme of the present invention, the cotyledon explant or hypocotyl explant of pepper is inoculated into an adventitious bud induction medium containing a hormone combination of specific auxin and cytokinin, which can promote the rapid formation of callus, improve the induction efficiency of adventitious buds, and further improve the success rate of pepper tissue regeneration. Then, the adventitious buds are inoculated into a rooting medium for induction, and after obtaining the rooted tissue culture seedlings, the seedlings can be hardened, and the regenerated plants of pepper are finally harvested. The culture method of pepper tissue regeneration of the present application provides powerful technical support for pepper genetic improvement, germplasm resource preservation and rapid propagation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 A schematic diagram of the sampling sites of pepper explants in Experimental Example 1 of the present application is shown.

[0021] Figure 2 The morphological diagram of adventitious buds induced by different pepper explants in Experimental Example 1 of the present application specification is shown, wherein: Figure 2 A in the middle is the morphological diagram of the induced adventitious buds of cotyledon explants. Figure 2 Middle B is a picture of the adventitious bud morphology induced from hypocotyl explants.

[0022] Figure 3 The morphological diagram of pepper adventitious buds induced by different hormone combinations in Experimental Example 2 of the present application specification is shown.

[0023] Figure 4 The morphological diagram of pepper adventitious buds induced by different hormone combinations in Experimental Example 2 of the present application specification is shown, wherein: Figure 4 A to C in the middle are the morphological pictures of adventitious buds induced by five hormone combinations in hormone combinations 1 to 3 at different concentrations;

[0024] Figure 5 The morphology of pepper adventitious buds induced by the hormone combination of 5 mg / L TZT and 0.5 mg / L IAA in Test Example 2 of the present application specification is shown.

[0025] Figure 6 The diagram shows the callus morphology of different types of peppers in Experimental Example 3 of the present application specification.

[0026] Figure 7 The diagram shows the regeneration morphology of different types of peppers in Experimental Example 3 of the present application specification.

[0027] Figure 8The morphological diagram of the tissue culture seedlings of Zhangshugang pepper in Example 1 of the present application specification is shown.

[0028] Fig. 9 The morphological diagram of the regenerated seedlings of Zhangshugang pepper in Example 1 of the present application specification is shown. DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0030] Terminology explanation:

[0031] TZT: Trans-zeatin, trans-zeatin, a cytokinin plant growth regulator.

[0032] ZT: Zeatin, zeatin, cytokinin plant growth regulator.

[0033] 6-BA: 6-Benzylaminopurine, 6-benzyladenine, a cytokinin plant growth regulator.

[0034] IAA: Indole acetic acid, indoleacetic acid, auxin growth regulator.

[0035] IBA: Indole butyric acid, indole butyric acid, auxin growth regulator.

[0036] NAA: Naphthalene acetic acid, naphthaleneacetic acid, an auxin growth regulator.

[0037] As mentioned in the background technology, the in vitro regeneration of pepper is highly dependent on the genotype, and there are problems such as long regeneration cycle and difficult bud growth during the regeneration process, which leads to a generally low success rate of in vitro regeneration of pepper tissue in the prior art. Therefore, in this application, the inventor attempts to increase the adventitious bud induction rate of in vitro regeneration of pepper tissue and further promote the formation of regenerated plants. Based on this, a series of protection schemes of this application are proposed.

[0038] In a first typical embodiment of the present application, a culture method for pepper tissue regeneration is provided, which culture method comprises: S1) inoculating pepper explants into an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious buds; the adventitious bud induction medium comprises auxin and cytokinin; the auxin comprises one or more of indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthylacetic acid (NAA); the cytokinin comprises trans-zeatin (TZT) and\or zeatin (ZT); wherein the adventitious bud medium comprises any one of the following hormone combinations: a) trans-zeatin and indoleacetic acid; b) trans-zeatin and indolebutyric acid; c) zeatin and naphthylacetic acid; S2) inoculating the adventitious buds into a rooting medium to obtain tissue culture seedlings; S3) hardening and transplanting the tissue culture seedlings to obtain regenerated plants of pepper.

[0039] There are many limitations in the in vitro regeneration system of pepper. On the one hand, the in vitro regeneration of pepper has obvious genotype dependence, and the performance of pepper tissues of different genotypes in the in vitro regeneration process is quite different. On the other hand, there are also significant differences in the in vitro regeneration of different explants, which further increases the limitations of the prior art. In addition, the "adventitious bud induction-adventitious bud elongation-adventitious bud rooting" route is commonly used in the prior art to obtain pepper regenerated plants, wherein the adventitious bud induction cycle is 8-11 weeks, the bud elongation cycle is 6-8 weeks, and the rooting induction is 7-8 weeks. It can be seen that the regeneration cycle of this route is long, resulting in low efficiency in obtaining regenerated plants, and in the regeneration process, problems of adventitious bud induction and growth difficulties often occur.

[0040] At the same time, the in vitro culture system of pepper tissue using callus induction is not mature. Although the regeneration cycle is shortened relative to the above-mentioned route, there are also problems of low callus induction rate, adventitious bud point formation rate and adventitious bud induction rate. Although there are often methods in the prior art to promote the induction effect of adventitious buds by regulating the combination and concentration of hormones such as auxin and cytokinin and plant growth regulators in the culture medium, due to the genotype dependence of the above-mentioned pepper, the methods in the prior art are difficult to apply to the in vitro regeneration of pepper tissues of various genotypes. When the in vitro regeneration system in the prior art is used to culture pepper explants, it is often difficult for callus to induce adventitious buds, resulting in a low induction rate of adventitious buds. Therefore, the success rate of in vitro regeneration of pepper tissue in the prior art is relatively low as a whole.

[0041] At present, pepper tissue culture research is mainly focused on high-spiciness peppers, sweet peppers, chili pepper series, local specialty pepper varieties, disease-resistant pepper varieties, and wild pepper varieties. Among them, the excellent specialty farmer variety "Zhangshugang" pepper has a horn-shaped fruit, green for green and red for mature fruit, beautiful fruit shape, delicious taste, unique flavor, high market price, and excellent agronomic traits such as "short plants, dense branches, compact plant type, early maturity, resistance to low temperature and weak light", which is a very valuable germplasm resource. The high-quality genome assembly of this variety was completed in 2023 and has become one of the key research targets of new genetic engineering.

[0042] The fruit of Saijiao is in the shape of a horn, the color of the green-ripe fruit is green, and the color of the old-ripe fruit is red. The red-ripe fruit has good consistency, good commercial quality, and is resistant to diseases and viruses. The fruit of the maggot pepper is in the shape of a horn, with a smooth surface, the color of the green-ripe fruit is green, and the color of the old-ripe fruit is red. The plant grows robustly, the plant type is upright and compact, the stem is sturdy, and it is resistant to lodging and has strong stress resistance. The fruit of the eight-inch red pepper is linear, the fruit surface is slightly wrinkled, the color of the green-ripe fruit is green, and the color of the old-ripe fruit is red. The plant has strong stress resistance. The fruit of the horn pepper is in the shape of a horn, the fruit is smooth, straight and shiny, the color of the green-ripe fruit is green, and the color of the old-ripe fruit is red. The plant type is compact and has good comprehensive resistance. The fruit of the Chaotian pepper is a solitary short conical fruit with the top of the fruit facing upward, the color of the green-ripe fruit is purple, and the color of the old-ripe fruit is red. The plant is sturdy and has strong stress resistance. These peppers all have excellent agronomic shapes and strong stress resistance, and are suitable for promotion and cultivation. However, there are no reports of successful tissue culture regeneration of Zhangshugang, Saijiao, Maggot Pepper, Bacunhong Lazi, Yangjiao Pepper, and Chaotian Pepper in the current existing technology, which limits the scientific research and improvement of these pepper varieties.

[0043] The fruit of Zunla No. 1 is solitary conical, growing upward, the color of green ripe fruit is green, the color of old ripe fruit is red, the plant is compact, strong in stress resistance, and wide in adaptability. The genome sequencing of this variety was completed in 2014. Although there are reports of successful in vitro regeneration of Zunla No. 1 in the prior art, the in vitro regeneration system of the prior art still has the problems of complex regeneration path and long regeneration cycle, which also limits the scientific research and improvement of Zunla No. 1 variety. The present application develops a new culture method for pepper tissue regeneration, which selects pepper cotyledon explants or hypocotyl explants for adventitious bud induction culture in the above-mentioned adventitious bud induction medium. The adventitious bud induction medium contains any one of the above-mentioned hormone combinations of auxin and cytokinin. Through the synergistic effect of the specific hormone combination, it can promote the rapid formation of healthy callus tissue, and there is no need to replace the culture medium. The callus tissue can continue to be cultured in the above-mentioned adventitious bud culture medium, and healthy adventitious buds can be successfully induced from the callus tissue, thereby increasing the induction rate of adventitious buds, avoiding damage to the callus tissue that may be caused during the replacement of the culture medium, and reducing the overall in vitro culture cost, shortening the cultivation cycle. In addition, when the culture method for pepper tissue regeneration of the present application is applied to the in vitro regeneration of pepper tissue of the above-mentioned variety, the adventitious bud induction rate can reach 36.07% to 80.53%, and finally, regenerated plants with excellent traits that do not vary in flowering and fruiting traits can be obtained.

[0044] Moreover, the method for in vitro regeneration of pepper tissue of the present application also has the characteristic of a short regeneration cycle. After the pepper explant is inoculated into the adventitious bud induction medium, callus tissue can be obtained in about 5 to 9 days. After continuing to induce for 20 to 30 days, adventitious bud induction can be obtained. The subsequent rooting induction cycle is 12 to 14 days, and the cycle of seedling hardening and transplanting is 55 to 60 days. Compared with the prior art, the overall pepper tissue regeneration cycle is greatly shortened, and a large number of excellent pepper tissue culture seedlings can be obtained in a short time. Pepper regeneration plants can be obtained by hardening. It is proved that the in vitro regeneration success rate of the pepper tissue regeneration culture method of the present application can maintain a high level, and the growth cycle is short, and the regenerated plant traits are stable.

[0045] At present, there are no reports of successful tissue culture regeneration of Zhangshugang, Saijiao, Maggot Pepper, Bacunhong Chili, Yangjiao Pepper, and Chaotian Pepper in the prior art. The present invention can achieve an adventitious bud induction rate of 80.53% for Zhangshugang, 50% for Saijiao, 36.07% for Maggot Pepper, 38.9% for Bacunhong Chili, 72.23% for Yangjiao Pepper, and 52.77% for Chaotian Pepper, which are much higher than the adventitious bud induction efficiency of pepper varieties reported in the prior art. In the tissue in vitro culture system of Zunla No. 1 established in patent application 202410063361.4, its callus induction rate is 88.3%, which is also lower than the effect of 100% callus induction rate achieved in this application.

[0046] Compared with the in vitro regeneration method of pepper in the prior art, the method of in vitro regeneration of pepper tissue in the present application is more suitable for the success rate of in vitro regeneration of pepper tissue, and a large number of regenerated pepper plants can be obtained in a short time, providing strong technical support for genetic improvement, germplasm resource preservation and rapid reproduction of pepper.

[0047] In a preferred embodiment, the hormone combination: a) includes: 0.1-10 mg / L (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / L) trans-zeatin and 0.05-2 mg / L (including but not limited to 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0 b) comprising: 0.1-10 mg / L (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / L) trans-zeatin and 0.05-2 mg / L indolebutyric acid (including but not limited to 0.05, 0.06, 0.07, 0.08, 0.09 , 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 or 2 mg / L); c) including: 0.1-10 mg / L (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / L) zeatin and 0.05-1 mg / L (including but not limited to 0.05, 0.06, 0.07, 0 .08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 or 2 mg / L) naphthaleneacetic acid; preferably, the hormone combination: a) includes: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indoleacetic acid; b) includes: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indolebutyric acid; c) includes 0.5-5 mg / L zeatin and 0.05-0.4 mg / L naphthaleneacetic acid. More preferably, the adventitious bud culture medium includes 4-6 mg / L trans-zeatin and 0.4-0.6 mg / L indoleacetic acid.

[0048] Adventitious bud induction of pepper explants using an adventitious bud culture medium containing a specific hormone combination within the above-mentioned concentration range can promote the rapid formation of callus tissue and induce a large number of adventitious buds, thereby increasing the induction rate of adventitious buds, laying the foundation for further obtaining a large number of tissue culture seedlings, successfully obtaining regenerated plants with excellent traits, and promoting the success rate of in vitro regeneration of pepper tissues.

[0049] In a preferred embodiment, the adventitious bud induction medium also includes 4.25-4.75 g / L MS medium, 28-32 g / L sucrose and 4.0-5.0 g / L plant gel, and the pH of the adventitious bud induction medium is 5.80-5.86. Preferably, the adventitious bud induction medium also includes 4.43 g / L MS medium, 30 g / L sucrose and 4.5 g / L plant gel, and the pH of the adventitious bud induction medium is 5.80-5.86. Preferably, the MS medium is a liquid culture medium. Preferably, the components of the adventitious bud induction medium also include 4.43 g / L MS medium, 5 mg / L TZT, 0.5 mg / L IAA, 30 g / L sucrose, 4.5 g / L plant gel, pH 5.8.

[0050] The MS medium of the present application refers to the MS liquid medium, which is a commercially available MS medium (Phyto Tech M519), obtained by dissolving the dry powder. Those skilled in the art can select any conventional commercially available MS medium dry powder and dissolve it according to the actual situation to obtain the MS medium consistent with the culture effect of the present application.

[0051] In a preferred embodiment, the conditions for adventitious bud induction culture are a temperature of 25-30°C, a light intensity of 3200-3700Lx, a relative humidity of 45-55%, and a photoperiod of 14h light and 8h dark; preferably, the time for adventitious bud induction culture is 25-35d. Preferably, the conditions for adventitious bud induction culture are culture at 28°C, a light intensity of 3500Lx, and a relative humidity of 50%, with a photoperiod of 14h light and 8h dark alternating, and pepper adventitious buds are cultured in 30d.

[0052] The present application controls the components of the adventitious bud culture medium and the parameters of the culture conditions within the above-mentioned range, and works synergistically with the above-mentioned hormone combination to provide callus tissue and adventitious buds with suitable growth nutrients and environment, promote the rapid formation of pepper explants into callus tissue, and increase the induction rate of adventitious buds, further shorten the regeneration cycle, and improve the success rate of in vitro regeneration of pepper tissue.

[0053] In a preferred embodiment, the explant includes cotyledon explants and / or hypocotyl explants; the method for obtaining cotyledon explants includes: i-1) sterilizing pepper seeds, inoculating them in a germination medium for germination culture of sterile seedlings, and obtaining the sterile pepper seedlings; i-2) cutting off the two ends of the cotyledons of the sterile seedlings to obtain the cotyledon explants; the area of ​​the cotyledon explants is 4-6 mm × 3-5 mm; the method for obtaining hypocotyl explants includes: ii-1) sterilizing pepper seeds, inoculating them in a germination medium for germination culture of sterile seedlings, and obtaining the sterile pepper seedlings; ii-2) cutting off the two ends of the hypocotyl of the sterile seedlings to obtain the hypocotyl explants.

[0054] Preferably, the germination medium includes 2.0-3.0 g / L MS medium, 8-15 g / L sucrose and 3.5-4.5 g / L plant gel, more preferably, the components of the germination medium include 2.215 g / L MS medium, 10 g / L sucrose and 3.75 g / L plant gel, and the pH is 5.80-5.86; preferably, the conditions for the germination culture of the sterile seedlings are: temperature of 25-30°C, light intensity of 3200-3700 Lx, relative humidity of 45-55%, light cycle of 14h light and 8h dark alternation, and culture time of 7-8d. Preferably, the components of the germination medium include 2.215 g / L MS medium, 10 g / L sucrose, 3.75 g / L plant gel, pH 5.8, and the germination culture conditions are 28°C, 3500 Lx light intensity, 50% relative humidity, and a light cycle of alternating 14 h light and 8 h dark, and sterile seedlings with unfolded cotyledons are obtained after 7 days.

[0055] The explant acquisition method of the present application is simple to operate and easy to obtain. By controlling the composition of the germination medium and the conditions of the germination culture, it can promote the germination and growth of sterile seedlings, which is beneficial to the subsequent formation and acquisition of explants for pepper tissue culture.

[0056] In a preferred embodiment, rooting culture includes: inoculating adventitious buds into a rooting culture medium, and performing a first rooting culture under dark conditions; after the first rooting culture is completed, performing a second rooting culture under a light cycle of alternating 14 hours of light and 8 hours of darkness; preferably, the time of the first rooting culture is 3 to 4 days; preferably, the temperature of the first rooting culture is 25 to 30°C; preferably, the time of the second rooting culture is 12 to 15 days; preferably, the temperature of the second rooting culture is 25 to 30°C; preferably, the light intensity of the second rooting culture is 3200 to 3700Lx.

[0057] In a preferred embodiment, the rooting medium includes 4.25-4.75 g / L MS medium, 1.0-1.5 mg / L indolebutyric acid, 38-32 g / L sucrose and 8.8-9.2 g / L agar, preferably 4.43 g / L MS medium, 1 mg / L IBA, 30 g / L sucrose and 9 g / L agar, and the pH is 5.80-5.86. Preferably, the components of the rooting medium include 4.43 g / L MS medium, 1 mg / L IBA, 30 g / L sucrose, 9 g / L agar, pH 5.8, and the rooting culture conditions are 28 ° C in the dark for 4 days, followed by 28 ° C, 3500 Lx light intensity, relative humidity 50%, with a light cycle of 14 h light and 8 h dark alternating, and 14 days to induce rooting.

[0058] The present application controls the components of the rooting medium and the culture conditions within the above range, which can promote the formation and growth of the roots of the adventitious buds, and is conducive to further promoting the formation of adventitious buds with excellent regeneration plants. The culture conditions of each step in the present application are tissue culture conditions optimized for peppers, and can also be flexibly adjusted according to different actual conditions in actual applications.

[0059] In a preferred embodiment, hardening and transplanting include: transplanting the tissue culture seedlings from the rooting medium to the culture matrix, and removing the film after covering the tissue culture seedlings for 15 to 20 days; preferably, the culture matrix includes culture soil; more preferably, the culture medium also includes one or more of vermiculite, pearl salt or plant fertilizer; preferably, the culture matrix includes culture soil and vermiculite; more preferably, the mass ratio of culture soil and vermiculite is 1:0.5 to 1.2, more preferably 1:1. Preferably, the conditions for hardening and transplanting are: temperature of 25 to 30 ° C, light intensity of 3200 to 3700 Lx, relative humidity of 45 to 55%, light cycle of 14h light 8h dark alternation, and culture time of 60 to 75d. Preferably, the culture soil includes but is not limited to any commercially available nutrient soil for plant cultivation. The components of the above-mentioned matrix include but are not limited to any fertilizer, plant growth regulator and fertilizer known to those skilled in the art that can be used for plant cultivation.

[0060] The purpose of hardening seedlings is to make tissue culture seedlings gradually adapt to the external environment and enhance their survival ability. The present application plants tissue culture seedlings in the above-mentioned culture matrix, providing a suitable growth environment for the root seedlings of tissue culture seedlings, enabling them to develop stronger root systems, laying the foundation for subsequent growth into excellent regenerated plants. The above-mentioned culture matrix can also be selected from other commercially available conventional pepper seedling cultivation substrates; the above-mentioned covering film can be selected from plastic wrap, and can also be replaced with other light-transmitting materials that can provide a moisturizing and breathable environment, such as plastic film, seedling cover cup, etc., and technicians in this field can flexibly select according to actual conditions.

[0061] The present application cultivates 7 pepper varieties, namely Zhangshugang, Saijiao, Gujiao, Bacunhong Lazi, Yangjiaojiao, Chaotianjiao or Zunla No. 1, through the method of in vitro regeneration culture of pepper tissue of adventitious bud induction-rooting induction-seedling hardening and transplanting of the present application, and can successfully induce a large number of adventitious buds, and the obtained regenerated plants have excellent traits, and the flowering and fruiting traits have not changed. This shows that the induction of pepper tissue by the method of in vitro regeneration of pepper tissue of the present application can obtain in vitro regeneration plants of pepper tissue with excellent traits in a short time, which not only shortens the in vitro regeneration cycle of pepper tissue, but also improves the success rate of in vitro regeneration of pepper tissue. In the second typical embodiment of the present application, a method for cultivating the above-mentioned pepper tissue regeneration is provided for use in pepper breeding; preferably, pepper breeding includes genetic transformation of pepper or regeneration of pepper varieties. In addition, it can also be used for other possible applications, such as screening mutants by using the regenerated plants obtained by the cultivation method.

[0062] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0063] Unless otherwise specified, the reagents in the examples of this application are conventional commercially available products.

[0064] The reagent information of the examples of the present application is shown in Table 1.

[0065] Table 1

[0066] Reagent name Production Company Part Number Trans-zeatin TZT Sigma-Aldrich Z0876-50MG Zeatin ZT Sigma-Aldrich Z0164-50MG 6-Benzyl adenine 6-BA Sigma-Aldrich V900316-25G Indolebutyric acid IBA Sigma-Aldrich I5386-25G NAA Sigma-Aldrich N0640-25G IAA Sigma-Aldrich I2886-25G MS medium PhytoTech M519 sucrose Sinopharm Shanghai Trial 10021463 Plant Gel Solarbio P8170-500g Agar LABLEAD QZ02 Nutrient soil Lu Qing Luqing YYT-50L Vermiculite Green Garden Green Garden ZS-70L-White Bag

[0067] Experimental Example 1 Comparison of adventitious bud regeneration ability of different explants

[0068] Experimental materials: Cotyledon explants and hypocotyl explants taken from sterile seedlings of pepper in Zhangshugang.

[0069] 1. Preparation of Sterile Seedlings

[0070] Select seeds of the pepper variety Zhangshugang with bright color and full grains, soak the seeds in sterile water in a triangular bottle for 15 minutes, discard the sterile water, add 75% ethanol to disinfect the seeds on the surface for 30 seconds, discard the 75% ethanol, add 2% sodium hypochlorite solution to soak for 10 minutes, discard the 2% sodium hypochlorite solution, rinse the seeds 3-5 times with sterile water, absorb the sterile water on the seed surface with filter paper, and dry the seeds.

[0071] The seeds were inoculated on germination medium (1 / 2MS medium, supplemented with 10 g / L sucrose, 3.75 g / L phytagel, pH 5.8), and cultured at 28°C, 3500 Lx light intensity, 50% relative humidity, with a photoperiod of 14 h light and 8 h dark alternating. After 7 days, sterile seedlings with unfolded cotyledons were obtained.

[0072] 2. Explant Preparation

[0073] (1) Cotyledon explants: Select the cotyledons of sterile seedlings, remove the two ends with a blade, and cut into 4-6 mm × 3-5 mm leaves with wounds on both ends. Make sure the wounds are neat. This is the cotyledon explant. The specific parts of the sample are as follows: Figure 1 As shown;

[0074] (2) Hypocotyl explants: Select the hypocotyls of sterile seedlings and cut them into 5-7 mm hypocotyls with wounds on both ends. Make sure the wounds are neat. These are the hypocotyl explants. The specific parts of the sample are as follows: Figure 1 shown.

[0075] 3. Effects of different explants on adventitious bud regeneration

[0076] The above cotyledon explants and hypocotyl explants were selected and inoculated with the front side upward in an adventitious bud induction medium (MS medium, supplemented with 2mg / L TZT, 0.4mg / L IAA, 30g / L sucrose, 4.5g / L phytagel, pH 5.8), and cultured at 28°C, 3500Lx light intensity, 50% relative humidity, with a photoperiod of 14h light and 8h dark alternating. Pepper adventitious buds were cultured after 30 days, and the growth status of the adventitious buds was observed.

[0077] The morphology of adventitious buds induced by different explants Figure 2 As shown, A is the morphology of the induced adventitious buds of the cotyledon explants, and B is the morphology of the induced adventitious buds of the hypocotyl explants. A large number of callus tissues and adventitious buds were induced at the incisions at both ends of the cotyledon explants, and the regenerated buds grew well; for the hypocotyl explants, the incision at the upper end of the morphology directly induced regenerated buds (less callus tissue), the bud morphology was incomplete, and the shoot apical meristem failed to form.

[0078] In view of the better bud differentiation status of cotyledon explants, cotyledons were selected as explants in subsequent experiments to explore an efficient in vitro regeneration system for pepper.

[0079] Experimental Example 2 Effects of different hormone concentration ratios on adventitious bud induction and regeneration

[0080] Experimental materials: Cotyledon explants taken from sterile seedlings of Zhangshugang pepper.

[0081] 1. Preliminary test:

[0082] The cotyledon explants were inoculated with the front side facing upward in an adventitious bud induction medium (MS medium, supplemented with different cytokinins (TZT, ZT, 6-BA), different concentrations of auxins (IAA, IBA, NAA), 30 g / L sucrose, 4.5 g / L plant gel, pH 5.8), wherein the types of hormones and concentration levels were set as shown in Table 2, and a total of 81 hormone combinations were set for the hormone concentration ratio. The culture was carried out at 28°C, 3500 Lx light intensity, and 50% relative humidity with a light cycle of 14 h light and 8 h dark alternating, and the adventitious bud induction rate was counted. The concentration settings of different hormones are shown in Table 2.

[0083] Adventitious bud induction rate = (number of explants forming regenerated buds / number of inoculated explants) × 100%.

[0084] Table 2

[0085]

[0086]

[0087] The test results showed that callus appeared after 9 days of induction culture of cotyledon explants, and adventitious buds of pepper were induced after 20-30 days of continuous culture. The adventitious bud induction rates of each hormone combination group are shown in Table 3.

[0088] Table 3

[0089]

[0090]

[0091]

[0092] Note: In the table, if two groups do not have any common letters in the results in the significance level column, it means that the difference between the two groups is significant; if two or more groups share the same letters in the results, it means that the difference between these groups is not significant at the significance level of α=0.05.

[0093] As can be seen from Table 3, the adventitious bud induction efficiency of the treatments with 6-BA and various auxin ratios (hormone combination group numbers 19-27, 46-54, 73-81) was low.

[0094] Morphology of pepper adventitious buds induced by different hormone combinations Figure 3 , Figure 4 shown.

[0095] Figure 3 It is shown in the results that when the cytokinin concentration is low (0.1-0.5 mg / L) and the auxin concentration is high (1 mg / L), cotyledon explants are prone to differentiate into roots, which is not conducive to the formation and normal development of adventitious buds.

[0096] Figure 4 In the figure, the red arrows indicate the regenerated buds, and A to C are the morphological pictures of adventitious buds induced by 5 of the 3 hormone combinations (numbered 4-8, 31-33, 35-36, 67-71) at different concentrations. Figure 4 It can be seen that among the hormone combinations of TZT and IAA, TZT and IBA, and ZT and NAA, the efficiency of inducing adventitious buds from pepper cotyledons is high, and the induction rate of adventitious buds is 25%-66.6%.

[0097] 2. Refinement test

[0098] In order to further refine the hormone concentration combinations of TZT and IAA, TZT and IBA, and ZT and NAA, a total of 60 hormone combinations were set in a cross-combination manner, and the combination method is shown in Table 4. The remaining parameters were the same as in the preliminary test.

[0099] Table 4

[0100]

[0101] The statistical results of the adventitious bud induction rate of the refinement test are shown in Table 5. The results show that the hormone combination of 5mg / L TZT and 0.5mg / L IAA has a high efficiency in inducing adventitious buds from cotyledons, with an induction rate of 80.53%. The morphology of the cotyledon explants of this combination is as follows Figure 5 As shown in the figure (the part pointed by the red arrow is the regenerated bud), the bud point developed normally. Subsequent experiments used a hormone combination of 5mg / L TZT and 0.5mg / L IAA to explore the efficiency of adventitious bud induction and regeneration of different pepper genotypes.

[0102] Table 5

[0103]

[0104]

[0105]

[0106] Note: In the table, if two groups do not have any common letters in the results in the significance level column, it means that the difference between the two groups is significant; if two or more groups share the same letters in the results, it means that the difference between these groups is not significant at the significance level of α=0.05.

[0107] Test Example 3

[0108] The sterile seedling cotyledon explants of Zhangshugang, Saijiao, Chongjiao, Bacunhong Lazi, Yangjiaojiao, Chaotianjiao and Zunla No. 1 peppers were inoculated with the front side facing up into an induction medium (4.43 g / L MS medium, supplemented with 5 mg / L TZT, 0.5 mg / L IAA, 30 g / L sucrose, 4.5 g / L phytogel, pH 5.8), and cultured at 28°C, 3500 Lx light intensity, 50% relative humidity, with a photoperiod of 14 h light and 8 h dark alternating. Callus was obtained after 9 days, and culture was continued. Adventitious buds of pepper were cultured after 20-30 days. The callus induction rate, bud point formation rate, and adventitious bud induction rate were counted (the total number of cotyledon explants induced and counted for each variety of pepper was 36).

[0109] Callus induction rate = (number of explants forming callus / number of inoculated explants) × 100%;

[0110] Bud formation rate = (number of explants forming buds / number of inoculated explants) × 100%;

[0111] Adventitious bud induction rate = (number of explants forming regenerated buds / number of inoculated explants) × 100%.

[0112] In the previous study of this application, the in vitro regeneration of pepper was classified and observed, which can be divided into four types, namely, callus difficult to form, callus difficult to sprout, abnormal bud development, normal bud development, and regeneration performance such as Figure 6 (The part pointed by the red arrow is the regenerated bud) as shown in the figure. The specific classification criteria are as follows:

[0113] Callus formation is difficult: callus induction rate <30%;

[0114] Callus is difficult to sprout: callus induction rate is 30-100%, bud formation rate is <30%;

[0115] Abnormal bud development: callus induction rate 100%, bud formation rate 30-100%, adventitious bud induction rate <30%;

[0116] Bud development is normal: callus induction rate is 100%, bud formation rate is 100%, adventitious bud induction rate is 30-100%;

[0117] The statistical results are shown in Table 6. The adventitious buds of Zhangshugang, Saijiao, Qujiao, Bacunhong Lazi, Yangjiaojiao, Chaotianjiao, and Zunla No. 1 all belong to the type with normal bud development. The regeneration morphology of each variety of pepper is as follows: Figure 7 shown.

[0118] Table 6

[0119]

[0120] Note: In Table 6, if two groups do not have any common letters in the results, it means that the difference between the two groups is significant; if two or more groups share the same letters in the results, it means that the difference between these groups is not significant at the significance level of α=0.05.

[0121] Example 1

[0122] The pepper tissue in vitro method of the present application was used to verify the in vitro regeneration culture of the above 7 varieties of pepper explants. The steps are as follows:

[0123] (1) Preparation of cotyledon explants: Take the cotyledons of sterile pepper seedlings, remove the two ends with a blade, and cut them into 5 mm × 3 mm cotyledon blocks with wounds on both ends, ensuring that the wounds are neat. This is the cotyledon explant.

[0124] (2) Adventitious bud induction: Cotyledon explants were taken and inoculated with the upper side facing upward into an adventitious bud induction medium, and cultured at 28°C, 3500Lx light intensity, 50% relative humidity, with a photoperiod of alternating 14 h light and 8 h dark; wherein the formula of the adventitious bud induction medium is: MS + 5 mg / L TZT + 0.5 mg / L IAA + 30 g / L sucrose + 4.5 g / L phytogel, pH 5.8.

[0125] (3) Rooting culture: When the induced adventitious buds grow to 1-2 cm in height, they are cut into individual plants and transferred to a rooting medium. They are cultured in the dark at 28°C for 4 days, and then cultured at 28°C, 3500Lx light intensity, and 50% relative humidity with a photoperiod of 14 h light and 8 h dark alternating; wherein the rooting medium is: MS medium + 1 mg / L IBA + 30 g / L sucrose + 9 g / L agar, pH 5.8. The rooting morphology of the adventitious buds of the Zhangshugang pepper in this example is shown in the figure below. Figure 8 shown.

[0126] (4) Hardening and transplanting: Transplant the rooted tissue culture seedlings into a moist substrate, cover the seedlings with plastic wrap to maintain humidity, and make appropriate holes in the film for gas exchange; when the leaves of the new seedlings unfold, the hardening is successful. At this time, remove the plastic wrap and continue culturing to obtain regenerated plants. The substrate of this embodiment uses a 1:1 mixture of conventional horticultural nutrient soil and vermiculite. The culture conditions are: the temperature is 25°C, the light intensity is 3500Lx, the relative humidity is maintained at 45-55%, and the photoperiod is 14h of light and 8h of darkness. The morphology of the regenerated seedlings of Shugang pepper in this embodiment is shown in the figure below. Fig. 9 shown.

[0127] The results show that:

[0128] Using the pepper tissue in vitro culture method of the present application, the cotyledon explants of the above varieties can be cultured into adventitious buds in 20-30 days in the adventitious bud induction medium, with a callus formation rate of 100%, a bud point formation rate of 100%, an adventitious bud induction rate of 32% to 80.65%, and normal bud point development (as shown in Table 5 and Table 5). Figure 6 , Figure 7 As shown), among them, the variety with the best adventitious bud induction rate is Zhangshugang pepper. Moreover, the pepper tissue in vitro regeneration system of the present application has a short regeneration cycle, with adventitious bud induction of 30 days, rooting induction of 14 days, and hardening and transplanting of the seedlings for a total of 60 days before the fruits of the regenerated seedlings can be harvested.

[0129] Example 2

[0130] Zhangshugang, Saijiao, Chongjiao, Bacunhonglazi, Yangjiaojiao, Chaotianjiao and Zunla No. 1 were cultured in vitro using an adventitious bud induction medium containing 2 mg / L TZT and 0.2 mg / L IAA hormone combination. There were 36 explants in each variety group, and the rest of the culture method and culture medium were consistent with Example 1. The adventitious bud induction rate of each variety is shown in Table 7.

[0131] Table 7

[0132] Material Adventitious bud induction rate (%) Zhangshu Port 27.67 Pepper 8.33 Maggot Pepper 16.67 Eight-inch red pepper 16.67 Sheep horn pepper 22.22 Pepper 13.89 Zun La No. 1 19.44

[0133] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: by utilizing the culture method for in vitro regeneration of pepper tissue of the present application and culturing in an adventitious bud induction medium containing a specific hormone combination, the induction rate of adventitious buds can be improved, and pepper regenerated seedlings with excellent traits can be obtained in a short time. The operation is simple, the regeneration cycle is short, and the success rate of tissue regeneration is high, which provides technical support for in-depth genetic transformation, trait improvement and molecular breeding of pepper.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for cultivating pepper tissue regeneration, characterized in that: The culture method comprises: S1) inoculating pepper explants into an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious buds; The adventitious bud induction medium comprises auxin and cytokinin; The auxin includes one or more of indoleacetic acid, indolebutyric acid or naphthylacetic acid; The cytokinin includes trans-zeatin and / or zeatin; Wherein, the adventitious bud culture medium includes any one of the following hormone combinations: a) the trans-zeatin and the indoleacetic acid; b) the trans-zeatin and the indolebutyric acid; c) the zeatin and the naphthylacetic acid; S2) inoculating the adventitious buds into a rooting medium to obtain tissue culture seedlings; S3) hardening and transplanting the tissue culture seedlings to obtain the regenerated plants of the pepper.

2. The culture method according to claim 1, characterized in that In the hormone combination: The a) comprises: 0.1-10 mg / L of trans-zeatin and 0.05-2 mg / L of indoleacetic acid; The b) comprises: 0.1-10 mg / L of trans-zeatin and 0.05-2 mg / L of indolebutyric acid; The c) comprises: 0.1-10 mg / L of zeatin and 0.05-1 mg / L of naphthaleneacetic acid; Preferably, in the hormone combination: The a) comprises: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indoleacetic acid; The b) comprises: 1-10 mg / L trans-zeatin and 0.1-2 mg / L indolebutyric acid; The c) comprises 0.5-5 mg / L zeatin and 0.05-0.4 mg / L naphthyl acetic acid.

3. The culture method according to claim 1, characterized in that The adventitious bud induction culture medium also includes 4.25-4.75 g / L MS culture medium, 28-32 g / L sucrose and 4.0-5.0 g / L plant gel, and the pH of the adventitious bud induction culture medium is 5.80-5.

86.

4. The culture method according to claim 3, characterized in that The conditions for the adventitious bud induction culture are a temperature of 25-30°C, a light intensity of 3200-3700Lx, a relative humidity of 45-55%, and a photoperiod of 14h light and 8h dark alternately; Preferably, the adventitious bud induction culture time is 25-35 days.

5. The culture method according to claim 1, characterized in that The explants include cotyledon explants and / or hypocotyl explants; The method for obtaining the cotyledon explant comprises: i-1) sterilizing the pepper seeds and inoculating them into a germination medium for sterile seedling germination culture to obtain sterile pepper seedlings; i-2) cutting off both ends of the cotyledon of the sterile seedling to obtain the cotyledon explant; the area of ​​the cotyledon explant is 4-6 mm×3-5 mm; The method for obtaining the hypocotyl explant comprises: ii-1) sterilizing the pepper seeds and inoculating them into a germination medium for sterile seedling germination culture to obtain sterile pepper seedlings; ii-2) cutting off both ends of the hypocotyl of the sterile seedling to obtain the hypocotyl explant; the length of the hypocotyl explant is 5 to 7 mm.

6. The culture method according to claim 5, characterized in that: The germination medium comprises 2.0-3.0 g / L MS medium, 8-15 g / L sucrose and 3.5-4.5 g / L plant gel, and the pH of the germination medium is 5.80-5.86; Preferably, the conditions for germination culture of the sterile seedlings are: temperature of 25-30°C, light intensity of 3200-3700Lx, relative humidity of 45-55%, photoperiod of 14h light and 8h dark alternation, and culture time of 7-8d.

7. The culture method according to claim 1, characterized in that: The rooting culture comprises: inoculating the adventitious buds into the rooting medium, and performing a first rooting culture under dark conditions; After the first rooting culture is completed, a second rooting culture is carried out under the condition of a photoperiod of alternating 14 h light and 8 h dark; Preferably, the first rooting culture time is 3 to 4 days; Preferably, the temperature of the first rooting culture is 25-30°C; Preferably, the second rooting culture time is 12 to 15 days; Preferably, the temperature of the second rooting culture is 25-30°C; Preferably, the light intensity of the second rooting culture is 3200-3700 Lx.

8. The culture method according to claim 1, characterized in that: The rooting culture medium comprises 4.25-4.75 g / L MS culture medium, 1.0-1.5 mg / L indolebutyric acid, 38-32 g / L sucrose and 8.8-9.2 g / L agar, and the pH value of the rooting culture medium is 5.80-5.

86.

9. The culture method according to claim 1, characterized in that: The seedling hardening and transplanting comprises: transplanting the tissue culture seedlings from the rooting medium to a culture matrix, and covering the tissue culture seedlings with a film for 15 to 20 days, and then removing the film; Preferably, the culture matrix includes culture soil; More preferably, the culture medium further comprises one or more of vermiculite, pearl salt or plant fertilizer; Preferably, the culture matrix includes culture soil and vermiculite; More preferably, the mass ratio of the culture soil to the vermiculite is 1:0.5-1.2; Preferably, the conditions for hardening and transplanting culture are: temperature of 25-30° C., light intensity of 3200-3700 Lx, relative humidity of 45-55%, photoperiod of 14 h light and 8 h dark, and culture time of 60-75 d.

10. Use of the pepper tissue regeneration culture method according to any one of claims 1 to 9 in pepper breeding; Preferably, the pepper breeding comprises genetic transformation of pepper or regeneration of pepper varieties.

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