Rapid propagation culture medium for guava tissue culture and method thereof

Abstract

The invention discloses a rapid propagation culture medium for guava tissue culture and a method thereof, and belongs to the field of agricultural production. The rapid propagation culture medium comprises the following culture media: a primary culture medium for primary culture, wherein the primary culture medium comprises the following components: an MS culture medium, NAA, 6-BA, corn steep liquor, soybean protein hydrolysate, agar and cane sugar; the proliferation culture medium for proliferation culture comprises the following components: an MS culture medium, NAAL, sterol, corn steep liquor, soybean protein hydrolysate, agar and cane sugar, the rooting culture medium for rooting culture comprises the following components: a 1 / 2 MS culture medium, NAA, IAA, orange juice, soybean protein hydrolysate, agar and cane sugar. According to the method disclosed by the invention, by optimizing the components and dosage of the culture medium in different stages of tissue culture, stem growth and bud proliferation in a proliferation stage can be simultaneously improved, and growth indexes in other stages are also remarkably improved, so that a foundation is laid for rapidly producing a large number of high-quality guava seedlings.

Description

Technical Field

[0001] This invention relates to the field of agricultural production, and in particular to a rapid propagation culture medium and method for guava tissue culture. Background Technology

[0002] Guava (scientific name: *Psidium guajava* L.) is a tree belonging to the genus *Psidium* in the family Myrtaceae. It is a plant with edible, medicinal, and ornamental value. Medicinally, the fruit, seeds, leaves, roots or root bark, and bark of the guava can all be used. Studies have shown that guava is rich in various important aromatic secondary metabolites. These polyphenols are what give guava its multiple effects, such as softening blood vessels, lowering blood sugar and lipids, anti-oxidation, and anti-tumor properties. It also has significant therapeutic effects on symptoms such as diarrhea, enteritis, gastrointestinal ulcers, eczema, and cough. Therefore, the propagation and widespread cultivation of guava has broad market prospects.

[0003] Currently, guava cultivation methods include seed propagation, cuttings, grafting, and tissue culture. However, seed propagation suffers from severe variability, while cuttings and grafting, although more advantageous than seed propagation, have a longer propagation speed and a longer breeding cycle to obtain a large number of high-quality seedlings. Therefore, tissue culture has become the primary choice for shortening the plant's propagation cycle. However, most guava tissue culture techniques optimize the use of axillary buds as explants in combination with disinfection and hormone combinations to improve guava survival rates. Furthermore, multiple studies have found that only using mature axillary bud disinfection methods can effectively reduce browning rates and increase budding rates. Young and over-mature axillary buds lack differentiation capabilities, which is detrimental to reducing browning rates and increasing budding rates. Moreover, the collection of mature axillary buds is time-limited, making it difficult to carry out tissue culture techniques at any time. There are also related techniques for rapid propagation using stem segments as explants. For example, Chen Guangcai et al. published "A Preliminary Study on Guava Tissue Culture," which found that NAA can promote the growth of guava stem segments but inhibit bud growth, making it difficult to quickly obtain a large number of high-quality guava seedlings. However, propagation using stem segments to induce clustered buds results in less variation and is suitable for the preservation and propagation of superior varieties. Therefore, exploring a rapid tissue propagation method that can promote stem segment growth while also increasing bud proliferation is essential for quickly obtaining a large number of superior guava seedlings. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid propagation culture medium and method for guava tissue culture, in order to solve the problems existing in the prior art. By optimizing the composition and dosage of the culture medium at different stages of tissue culture, it is possible to simultaneously improve the stem segment growth and bud proliferation at the proliferation stage, and also significantly improve the growth indicators at other stages. This lays the foundation for the rapid production of a large number of high-quality guava seedlings.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a rapid propagation culture medium for guava tissue culture, comprising the following culture media:

[0007] The primary culture medium used for initial culture consists of the following components: MS medium + NAA 0.15-0.25 mg / L + 6-BA 0.8-1.2 mg / L + corn steep liquor 1.5-2.5 mg / L + hydrolyzed soybean protein 3.5-5 mg / L + agar 15 g / L + sucrose 20 g / L;

[0008] The proliferation medium used for proliferation culture consists of the following components: MS medium + NAA 0.1-0.25 mg / L + sterol 1.0-1.2 mg / L + corn steep liquor 2.0-3 mg / L + hydrolyzed soybean protein 3-5 mg / L + agar 15 g / L + sucrose 20 g / L;

[0009] The rooting medium used for rooting culture consists of 1 / 2 MS medium + NAA 0.1-0.25 mg / L + IAA 0.2-0.4 mg / L + orange juice 3-7 g / L + hydrolyzed soybean protein 4-7 mg / L + agar 15 g / L + sucrose 20 g / L.

[0010] Preferably, the primary culture medium comprises MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L;

[0011] The proliferation medium consists of MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 3 mg / L + agar 15 g / L + sucrose 20 g / L;

[0012] The rooting medium consists of 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

[0013] This invention also provides a rapid propagation method for guava tissue culture, comprising the following steps:

[0014] After sterilizing the guava stem segments, they were inoculated into a primary culture medium for primary culture. The components of the primary culture medium included: MS medium + NAA 0.15-0.25 mg / L + 6-BA 0.8-1.2 mg / L + corn steep liquor 1.5-2.5 mg / L + hydrolyzed soybean protein 3.5-5 mg / L + agar 15 g / L + sucrose 20 g / L.

[0015] Stem segments with similar growth and thickness from the initial culture were selected and inoculated into a proliferation medium for proliferation culture. The components of the proliferation medium included: MS medium + NAA 0.1-0.25 mg / L + sterol 1.0-1.2 mg / L + corn steep liquor 2.0-3 mg / L + hydrolyzed soybean protein 3-5 mg / L + agar 15 g / L + sucrose 20 g / L;

[0016] Select robust propagation seedlings, take stem segments, and inoculate them with rooting medium for rooting culture. The components of the rooting medium include 1 / 2 MS medium + NAA 0.1-0.25 mg / L + IAA 0.2-0.4 mg / L + orange juice 3-7 g / L + hydrolyzed soybean protein 4-7 mg / L + agar 15 g / L + sucrose 20 g / L.

[0017] Preferably, the disinfection method is as follows: after the guava seeds have sprouted, take seedlings with 4-5 nodes, rinse them with water, and retain the terminal bud and the 2-3 nodes below it. First, soak them in 75% ethanol for 30 seconds, then treat them with 0.1‰ mercuric chloride solution for 8 minutes, then treat them with 75% ethanol for 10 seconds, and finally rinse them with sterile water 2-3 times.

[0018] Preferably, the primary culture medium comprises MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

[0019] Preferably, the proliferation medium comprises MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 3 mg / L + agar 15 g / L + sucrose 20 g / L.

[0020] Preferably, the rooting medium comprises 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

[0021] Preferably, the conditions for the primary culture, proliferation culture and rooting culture are: 25±2℃, 1000 lux, 8h light / 16h darkness.

[0022] Preferably, the guava includes the golden guava.

[0023] The present invention discloses the following technical effects:

[0024] This invention optimizes the disinfection method and the composition and dosage of the culture medium at different tissue culture stages, thereby significantly reducing the contamination rate and increasing the number of leaves, nodes, seedling height, and internode length during the initial culture process. It also improves the induction rate during the proliferation culture stage and simultaneously increases the number of axillary buds and seedling height in guava. Furthermore, it increases the number of roots, root length, and root diameter during the rooting culture stage and significantly reduces the number of days required for rooting. This is of great significance for efficiently and rapidly obtaining a large number of guava seedlings.

[0025] Regarding the disinfection method, this invention reduces the dosage of mercuric chloride to 1‰, which not only reduces the contamination rate but also plays an important role in reducing mercuric chloride residue and browning of guava stem segments.

[0026] For the initial culture stage, the primary culture medium can promote stem elongation and growth by adding plant hormones NAA and 6-BA to MS medium. Corn steep liquor is rich in a variety of organic nitrogen compounds, such as small peptides and free amino acids, while soybean hydrolysate is rich in a variety of amino acids, including many essential amino acids. Adding these to the primary culture medium can synergistically provide nutrition to guava with plant hormones while also promoting stem elongation and growth.

[0027] For the proliferation culture stage, the simultaneous addition of plant hormones NAA and sterols to MS medium can promote plant biomass and stress resistance, laying the foundation for increasing cell proliferation and rapidly inducing axillary bud growth. At the same time, the addition of corn steep liquor and soy protein to the medium can meet nutritional requirements and synergistically reduce the negative impact of the two plant hormones on plant growth, further promoting the formation of axillary buds, thus laying the foundation for rapidly obtaining a large number of guava seedlings.

[0028] For the rooting stage, the simultaneous addition of NAA and IAA to 1 / 2 MS medium can induce root growth, while the addition of orange juice can increase the demand for vitamins and minerals, affecting the thickness of the root system. Soy hydrolyzed protein can meet the needs of guava stem segments for a variety of amino acids, promote cell analysis and protein synthesis, and thus balance growth regulators and nutrient sources to meet the rooting conditions of guava, significantly improving the rooting effect.

[0029] The optimization of guava culture media at each stage is not done in isolation, but rather through synergistic effects. By balancing different nutrients and growth regulators in terms of nutrition and metabolism through different primary culture, proliferation culture, and rooting culture, guava can rapidly produce a large number of high-quality guava seedlings under specific culture media and conditions, thus meeting market demand. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In the prior art, there are many techniques for guava tissue culture, such as CN105961205B "A Tissue Culture Method for Improving the Survival Rate of Pearl Guava", which discloses that the tissue culture method includes the following steps: (1) Disinfection of the culture room: Sterilize the culture room with closed doors and windows 12-14 hours before culture; (2) Selection of explants: Select the terminal bud of the pearl guava tree as the explant, with an explant length of 1cm-2cm and a diameter of 0.4cm-0.8cm; (3) Sterilization of explants: After rinsing with water, soaking in 75% alcohol and draining, the explants are sterilized by soaking in a plant treatment solution with a mass concentration of 8%-10% for 20min-25min. The composition and weight percentage of the plant treatment solution are as follows: 30%-50% Ganoderma lucidum extract, 10%-15% Polygonum hydropiper extract, 5%-15% garlic extract, 10%-15% onion extract, 5%-10% Agastache rugosa extract, 5%-15% cactus extract, and 5%-10% Spirulina extract; (4) Initial induction culture: The sterilized explants are inoculated into the induction culture medium and induction culture is carried out in an incubator at a temperature of 24℃-26℃. White LED lights are used for illumination at a light intensity of 500Lx-800Lx for 12h-16h; (5) Subculture: The explants that have grown new shoots in step (4) are transferred to Subculture was carried out in an incubator in a subculture medium at a temperature of 24℃-26℃. White LED lights were used for illumination at a light intensity of 700Lx-1400Lx for 18-20 hours. The culture medium was changed continuously as the explants grew. After 5-7 generations of subculture, the rooted seedlings were transferred to nutrient cups with culture medium for further cultivation. (6) Hardening: The guava seedlings in the nutrient cups were placed in a greenhouse for hardening at a temperature of 22℃-26℃ and under sunlight. The induction culture medium in step (4) consisted of 15%-20% cassava alcohol waste liquid with a COD content of 300mg / L-800mg / L. The subculture medium in step (5) consists of: 15-20% cassava alcohol waste liquid with a COD of 500mg / L-1000mg / L, sucrose (5g / L-8g / L), Ganoderma lucidum extract (3g / L-5g / L), Spirulina extract (8g / L-10g / L), Polygonum hydropiper extract (2g / L-4g / L), agar (4.0g / L-7.0g / L), and naphthaleneacetic acid (0.3mg / L-0.6mg / L);This patent utilizes a specially designed plant extract for explant sterilization and induction culture, and employs cassava alcohol waste liquid to provide abundant organic matter to promote explant growth during subculture, thereby increasing the survival rate of guava. However, the use of mature axillary buds as explants in this patent is not conducive to timely collection and use, and is subject to time constraints; furthermore, the specific plant extract requires the preparation of multiple plant extracts, which is cumbersome and complex.

[0036] For example, the literature "Preliminary Study on Guava Tissue Culture" discloses that, using aseptic guava stem segments as material, the effects of growth regulators and MS concentration on bud germination and rooting of aseptic guava stem segments were studied. The results showed that, using MS as the basal medium, the optimal NAA concentration for the growth of aseptic guava seedlings was 0.4 mg·L⁻¹. -1 The highest values ​​were observed for the number of leaves (7.59), number of nodes (3.95), seedling height (5.10 cm), and internode length (1.41 cm); the 6-BA concentration was 5.0 mg·L⁻¹. -1 At that time, the stem segment had the highest bud induction rate (100%) and a relatively high number of buds induced (2.44); 1 / 2MS + NAA 0.05 mg·L -1 The optimal method for guava stem segment rooting was achieved, with a rooting rate of 100%, 2.30 roots per segment, and a root length of 4.64 cm. In guava tissue culture, NAA promoted stem segment growth but inhibited bud induction. It can be seen that this study, through screening and optimization of NAA and 6-BA dosages, obtained a scheme that promotes both stem segment growth and rooting in guava. However, it cannot simultaneously promote both bud and stem segment growth, which limits the rapid production of a large number of guava seedlings.

[0037] Therefore, based on the shortcomings of existing guava tissue culture technology, this invention proposes a tissue culture method that can simultaneously promote bud growth and stem segment growth, so as to quickly obtain a large number of guava seedlings to meet the demand for guava seedlings.

[0038] Unless otherwise specified, all culture medium components involved in the following examples can be routinely purchased through commercial channels.

[0039] Example 1: A method for guava tissue culture

[0040] (1) Explant disinfection

[0041] After the seeds of the Golden Guava have sprouted, take seedlings with 4-5 nodes, rinse them clean with water, and keep the terminal bud and the 2-3 nodes below it. First, soak them in 75% ethanol for 30 seconds, then treat them with 0.1‰ mercuric chloride solution for 8 minutes, then treat them with 75% ethanol for 10 seconds, and finally rinse them with sterile water 2-3 times.

[0042] (2) Initial cultivation

[0043] Under aseptic conditions, the base of the stem segment with the apical bud was removed, leaving one node. The segment was then transversely inoculated into the primary culture medium (MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L) and cultured at 25 ± 2℃, 1000 lux, under 8 h light / 16 h dark conditions. The stem segment growth was recorded after 30 days of culture.

[0044] (3) Proliferation culture

[0045] Stem segments with similar growth and thickness from the initial culture were selected and cut into 1.5 cm long segments containing only one node. These segments were inoculated onto proliferation medium (MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 3 mg / L + agar 15 g / L + sucrose 20 g / L) for axillary bud induction and proliferation. Four stem segment explants were inoculated per bottle, with five replicates. Culture conditions were the same as for the initial culture. After 40 days, the number of seedlings induced from each stem segment, seedling height, and induction rate were recorded.

[0046] Induction rate (%) = Number of germinating stem segments / Total number of inoculated stem segments × 100.

[0047] (4) Rooting culture

[0048] Take robust propagation seedlings, and select stem segments with 2-3 nodes at the top of the seedling (1.5 cm high). Inoculate these segments onto rooting medium (1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L). Inoculate 4 stem segments per bottle, repeating 5 times. Culture conditions are the same as the initial culture conditions. After 30 days of culture, record the rooting rate, number of roots, total root length, and root thickness.

[0049] Example 2: A method for guava tissue culture

[0050] (1) Explant disinfection

[0051] After the seeds of the Golden Guava have sprouted, take 4-5 nodes of seedlings, rinse them with water, and keep the terminal bud and the 2-3 nodes below it. Soak them in 75% ethanol for 30 seconds, then treat them with 0.1‰ mercuric chloride solution for 8 minutes, then treat them with 75% ethanol for 10 seconds, and finally rinse them with sterile water 2-3 times.

[0052] (2) Initial cultivation

[0053] Under aseptic conditions, the base of the stem segment with the apical bud was removed, leaving one segment. The segment was then transversely inoculated into primary culture medium (MS medium + NAA 0.25 mg / L + 6-BA 1.2 mg / L + corn steep liquor 2.0 mg / L + hydrolyzed soybean protein 4.0 mg / L + agar 15 g / L + sucrose 20 g / L). The segment was cultured at 25 ± 2 ℃, 1000 lux, under 8 h light / 16 h dark conditions. The growth of the stem segment was recorded after 30 days of culture.

[0054] (3) Proliferation culture

[0055] Stem segments with consistent growth and thickness genes from the initial culture were selected and cut into 1.5 cm long segments containing only one node. These segments were inoculated onto proliferation medium (MS medium + NAA 0.25 mg / L + sterol 1.0 mg / L + corn steep liquor 3.0 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L) for axillary bud induction and proliferation. Four stem segment explants were inoculated per bottle, with five replicates. Culture conditions were the same as for the initial culture. After 40 days, the number of seedlings induced from each stem segment, seedling height, and induction rate were recorded.

[0056] Induction rate (%) = Number of germinating stem segments / Total number of inoculated stem segments × 100.

[0057] (4) Rooting culture

[0058] Take robust propagation seedlings, and select stem segments with 2-3 nodes at the top of the seedling (1.5 cm high). Inoculate these segments onto rooting medium (1 / 2 MS medium + NAA 0.25 mg / L + IAA 0.40 mg / L + orange juice 7 g / L + hydrolyzed soybean protein 7 mg / L + agar 15 g / L + sucrose 20 g / L). Inoculate 4 stem segments per bottle, repeating 5 times. The culture conditions are the same as the initial culture conditions. After 30 days of culture, record the rooting rate, number of roots, total root length, and root thickness.

[0059] Example 3: A method for guava tissue culture

[0060] (1) Explant disinfection

[0061] After the seeds of the Golden Guava have sprouted, take 4-5 nodes of seedlings, rinse them with water, and keep the terminal bud and the 2-3 nodes below it. Soak them in 75% ethanol for 30 seconds, then treat them with 0.1‰ mercuric chloride solution for 8 minutes, then treat them with 75% ethanol for 10 seconds, and finally rinse them with sterile water 2-3 times.

[0062] (2) Initial cultivation

[0063] Under aseptic conditions, the base of stem segments with apical buds was removed, leaving one segment. These segments were then inoculated transversely onto primary culture medium (MS medium + NAA 0.15 mg / L + 6-BA 0.8 mg / L + corn steep liquor 1.5 mg / L + hydrolyzed soybean protein 3.5 mg / L + agar 15 g / L + sucrose 20 g / L). Five stem segments were inoculated per culture flask, for a total of 50 segments. The culture was carried out at 25 ± 2℃, 1000 lux, with an 8-hour light / 16-hour dark cycle. Stem segment growth was assessed after 30 days of culture.

[0064] (3) Proliferation culture

[0065] Stem segments with consistent growth and thickness genes from the initial culture were selected and cut into 1.5 cm long segments containing only one node. These segments were inoculated onto proliferation medium (MS medium + NAA 0.1 mg / L + sterol 1.0 mg / L + corn steep liquor 2.0 mg / L + hydrolyzed soybean protein 4.0 mg / L + agar 15 g / L + sucrose 20 g / L) for axillary bud induction and proliferation. Four stem segment explants were inoculated per bottle, with five replicates. Culture conditions were the same as for the initial culture. After 40 days, the number of seedlings induced from each stem segment, seedling height, and induction rate were recorded.

[0066] Induction rate (%) = Number of germinating stem segments / Total number of inoculated stem segments × 100.

[0067] (4) Rooting culture

[0068] Take robust propagation seedlings, and select stem segments with 2-3 nodes at the top of the seedling (1.5 cm high). Inoculate these segments onto rooting medium (1 / 2 MS medium + NAA 0.1 mg / L + IAA 0.20 mg / L + orange juice 3 g / L + hydrolyzed soybean protein 4 mg / L + agar 15 g / L + sucrose 20 g / L). Inoculate 4 stem segments per bottle, repeating 5 times. Culture conditions are the same as the initial culture conditions. After 30 days of culture, record the rooting rate, number of roots, total root length, and root thickness.

[0069] Comparative Example 1

[0070] Compared with Example 1, the difference is as follows: (1) Explant disinfection: After the seeds of Prunus yunnanensis are sown and the seedlings emerge, take 4-5 nodes of seedlings, rinse them with water, and retain the terminal bud and the 2-3 nodes below it. First, soak them in 75% ethanol for 30 seconds, then treat them with 0.1% mercuric chloride solution for 8 minutes, and finally rinse them with sterile water 2-3 times. Other methods and steps are the same as in Example 1.

[0071] Comparative Example 2

[0072] Compared with Example 1, the difference is that the initial culture medium was MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps were the same as in Example 1.

[0073] Comparative Example 3

[0074] Compared with Example 1, the difference is that the initial culture medium was MS medium + NAA 0.2 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0075] Comparative Example 4

[0076] Compared with Example 1, the difference is that the initial culture medium was MS medium + NAA 0.4 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0077] Comparative Example 5

[0078] Compared with Example 1, the difference is that the initial culture medium was MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps were the same as in Example 1.

[0079] Comparative Example 6

[0080] Compared with Example 1, the difference is that the initial culture medium was MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps were the same as in Example 1.

[0081] Comparative Example 7

[0082] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.2 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0083] Comparative Example 8

[0084] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0085] Comparative Example 9

[0086] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.4 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0087] Comparative Example 10

[0088] Compared with Example 1, the difference is that the proliferation medium is MS medium + 1.2 mg / L sterol + 15 g / L agar + 20 g / L sucrose. All other methods and steps are the same as in Example 1.

[0089] Comparative Example 11

[0090] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.4 mg / L + sterol 1.2 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0091] Comparative Example 12

[0092] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0093] Comparative Example 13

[0094] Compared with Example 1, the difference is that the proliferation medium is MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0095] Comparative Example 14

[0096] Compared with Example 1, the difference is that the rooting medium is 1 / 2 MS medium + NAA 0.2 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0097] Comparative Example 15

[0098] Compared with Example 1, the difference is that the rooting medium is 1 / 2 MS medium + IAA 0.35 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0099] Comparative Example 16

[0100] Compared with Example 1, the difference is that the rooting medium is 1 / 2 MS medium + NAA 0.4 mg / L + IAA 0.35 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0101] Comparative Example 17

[0102] Compared with Example 1, the difference is that the rooting medium is 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0103] Comparative Example 18

[0104] Compared with Example 1, the difference is as follows: 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L. All other methods and steps are the same as in Example 1.

[0105] Results and Analysis:

[0106] 1. After disinfecting the explants according to the disinfection method of Example 1, stem segments with similar growth and thickness and retaining one node were obtained and inoculated into MS medium for culture. Fifty stem segments were repeated, with four segments inoculated into each medium, and the browning rate was observed. After treatment with the disinfection method of Comparative Example 1, the contamination rate was observed using the same culture method. The results are shown in Table 1.

[0107] Table 1. The impact of different disinfection methods on contamination rate

[0108] Disinfection methods Number of vaccinations (individuals) Pollution rate (%) Example 1 50 0 Comparative Example 1 50 10

[0109] As can be seen from Table 1, the disinfection method of the present invention is more conducive to the growth and reproduction of guava. The inventors speculate that this may be based on the differences in guava varieties, which have different sensitivities to ethanol and mercuric chloride. The disinfection method of the present invention can not only reduce the amount of mercuric chloride used, but also prevent contamination from affecting the later reproduction process.

[0110] 2. The growth of stem segments after 30 days of initial culture following the methods of Example 1 and Comparative Examples 2-6 is shown in Table 2.

[0111] Table 2. Effects of different primary culture media components on stem segment growth.

[0112]

[0113]

[0114] Note: Different letters in the same column indicate significant differences compared to Example 1.

[0115] As shown in Table 2, compared to Example 1, changing the type or dosage of plant hormones, and omitting corn steep liquor or hydrolyzed soybean protein, significantly increased the number of leaves, nodes, seedling height, and internode length. This indicates that the primary culture medium in the primary culture chamber can only exert a synergistic effect by combining specific components and dosages to significantly improve the above indicators and achieve the best results.

[0116] 3. After propagation culture according to the methods of Example 1 and Comparative Examples 7-13, the number of seedlings induced to germinate, seedling height and induction rate of each stem segment were counted after 40 days of culture. The results are shown in Table 3.

[0117] Table 3 Effects of different proliferation media on guava proliferation culture

[0118] Grouping Induction rate (%) number of proliferations Seedling height (cm) Example 1 100a 4.78±0.10a 0.45±0.02a Comparative Example 7 75.12±0.23c 2.41±0.02c 0.26±0.03b Comparative Example 8 88.33±0.31b 3.47±0.01b 0.32±0.01b Comparative Example 9 65.78±0.17c 2.12±0.01c 0.23±0.02b Comparative Example 10 68.75±0.58c 2.19±0.03c 0.25±0.01b Comparative Example 11 77.59±0.37c 2.31±0.02c 0.27±0.02b Comparative Example 12 87.55±0.25b 3.41±0.02b 0.30±0.02b Comparative Example 13 90.17±0.11b 3.56±0.02b 0.33±0.01b

[0119] As can be seen from Table 3, the selection of components and dosage of the proliferation medium of the present invention directly affects the induction rate, number of proliferations and seedling height in the proliferation stage, with the most significant impact on seedling height. The above data indicate that the proliferation medium can only exert a synergistic effect when specific components and dosages are combined to significantly improve the induction rate, number of proliferations and seedling height in the above proliferation stage, thus achieving the best results.

[0120] 4. After rooting culture according to the methods of Example 1 and Comparative Examples 14-18, the rooting rate, number of roots, root length and root thickness were counted after 30 days (with Example 1 as the control, to observe whether there are roots with the same thickness as the thickest root in Example 1). The results are shown in Table 4.

[0121] Table 4. Effects of different rooting media on guava rooting culture

[0122]

[0123]

[0124] Table 4 shows that the plant hormones NAA and IAA in the rooting medium have little effect on the rooting rate, but their dosage does affect the rooting rate. The components and dosage of the rooting medium have a significant impact on the number of roots, root length, and root diameter. The experimental data indicate that the rooting medium can only exert a synergistic effect by combining specific components and dosages, significantly improving the rooting rate, number of roots, root length, and root diameter.

[0125] 5. After rooting culture according to the experimental methods of the above embodiments and comparative examples, the seedlings were transferred to a greenhouse growth substrate (soil, peat, humus and perlite in a mass ratio of 3:1:1:1) for conventional hardening-off. The survival rate was counted after 40 days. The results are shown in Table 5.

[0126] Table 5 Survival Rate Statistics

[0127]

[0128]

[0129] As can be seen from the above experimental data, the experimental methods of Examples 1-3 of this invention can achieve a 100% survival rate of seedlings 30 days after transplanting. The disinfection method and the adjustment of the culture medium components and dosage during the primary culture, proliferation culture and rooting culture not only directly affect the changes in growth indicators at each stage, but also affect the survival rate in the later stage. Based on the inventor's many years of experience in tissue culture, it is possible that the changes in the culture medium components at different stages affect the guava's absorption and utilization of nutrients, which may lead to certain changes in nutrient storage and metabolism. Therefore, the survival rate of guava is affected after transplanting in the later stage.

[0130] 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 rapid propagation culture medium for guava tissue culture, characterized in that, The following culture media are included: The primary culture medium used for initial culture consists of the following components: MS medium + NAA 0.15-0.25 mg / L + 6-BA 0.8-1.2 mg / L + corn steep liquor 1.5-2.5 mg / L + hydrolyzed soybean protein 3.5-5 mg / L + agar 15 g / L + sucrose 20 g / L; The proliferation medium used for proliferation culture consists of the following components: MS medium + NAA 0.1-0.25 mg / L + sterol 1.0-1.2 mg / L + corn steep liquor 2.0-3 mg / L + hydrolyzed soybean protein 3-5 mg / L + agar 15 g / L + sucrose 20 g / L; The rooting medium used for rooting culture consists of 1 / 2 MS medium + NAA 0.1-0.25 mg / L + IAA 0.2-0.4 mg / L + orange juice 3-7 g / L + hydrolyzed soybean protein 4-7 mg / L + agar 15 g / L + sucrose 20 g / L.

2. The rapid propagation culture medium for guava tissue culture as described in claim 1, characterized in that, The components of the primary culture medium include MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L; The proliferation medium consists of MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 3 mg / L + agar 15 g / L + sucrose 20 g / L; The rooting medium consists of 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

3. A rapid propagation method for guava tissue culture, characterized in that, Includes the following steps: After sterilizing the guava stem segments, they were inoculated into a primary culture medium for primary culture. The components of the primary culture medium included: MS medium + NAA 0.15-0.25 mg / L + 6-BA 0.8-1.2 mg / L + corn steep liquor 1.5-2.5 mg / L + hydrolyzed soybean protein 3.5-5 mg / L + agar 15 g / L + sucrose 20 g / L. Stem segments with similar growth and thickness from the initial culture were selected and inoculated into a proliferation medium for proliferation culture. The components of the proliferation medium included: MS medium + NAA 0.1-0.25 mg / L + sterol 1.0-1.2 mg / L + corn steep liquor 2.0-3 mg / L + hydrolyzed soybean protein 3-5 mg / L + agar 15 g / L + sucrose 20 g / L; Select robust propagation seedlings, take stem segments, and inoculate them with rooting medium for rooting culture. The components of the rooting medium include 1 / 2 MS medium + NAA 0.1-0.25 mg / L + IAA 0.2-0.4 mg / L + orange juice 3-7 g / L + hydrolyzed soybean protein 4-7 mg / L + agar 15 g / L + sucrose 20 g / L.

4. The method as described in claim 3, characterized in that, The disinfection method is as follows: After the guava seeds are sown and the seedlings emerge, take the seedlings that have grown 4-5 nodes, rinse them clean with water, and retain the terminal bud and the 2-3 nodes below it. First, soak them in 75% ethanol for 30 seconds, then treat them with 0.1‰ mercuric chloride solution for 8 minutes, treat them with 75% ethanol for 10 seconds, and finally rinse them with sterile water 2-3 times.

5. The method as described in claim 3, characterized in that, The components of the primary culture medium include MS medium + NAA 0.2 mg / L + 6-BA 1.0 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

6. The method as described in claim 3, characterized in that, The proliferation medium consists of MS medium + NAA 0.2 mg / L + sterol 1.2 mg / L + corn steep liquor 2.5 mg / L + hydrolyzed soybean protein 3 mg / L + agar 15 g / L + sucrose 20 g / L.

7. The method as described in claim 3, characterized in that, The rooting medium consists of 1 / 2 MS medium + NAA 0.2 mg / L + IAA 0.35 mg / L + orange juice 5 g / L + hydrolyzed soybean protein 5 mg / L + agar 15 g / L + sucrose 20 g / L.

8. The method as described in claim 3, characterized in that, The conditions for the initial culture, proliferation culture and rooting culture were: 25±2℃, 1000 lux, 8h light / 16h darkness.

9. The method as described in claim 3, characterized in that, The guava mentioned includes the Golden Guava.

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