A method for in vitro regeneration of quinoa inflorescences

Abstract

The application provides a quinoa inflorescence in vitro regeneration method. Specifically, the application first induces culture of quinoa inflorescences with a specific length, and can obtain sterile, large, stable and relatively easy-to-repeat high-efficiency regenerated quinoa plants.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation, and specifically to a method for in vitro regeneration of quinoa inflorescences. Background Technology

[0002] Quinoa belongs to the subfamily Chenopodiaceae of the family Amaranthaceae and is a dicotyledonous plant. Native to Bolivia, Ecuador, and Peru in South America, quinoa is distributed from sea level to altitudes of 4000 meters and is characterized by its cold resistance, drought tolerance, tolerance to poor soil conditions, and salt tolerance.

[0003] In the 1980s, when NASA was searching for food suitable for closed-loop ecological life support systems for long-term human space missions, quinoa stood out after more than a decade of research on thousands of existing foods due to its comprehensive nutritional components. Subsequently, its nutritional value was confirmed and promoted by the Food and Agriculture Organization of the United Nations (FAO), gradually gaining recognition and acceptance worldwide. Some quinoa varieties can be used as ideal silage for cattle and sheep. Therefore, quinoa cultivation has broad prospects. However, as a crop with a small planting area, short cultivation history, and limited public awareness in my country, its variety selection has significant shortcomings, and its production suffers from many deficiencies (inability to withstand high temperatures, humidity, and herbicides, low yield, etc.), severely restricting its promotion and industrialization. More rapid and efficient molecular breeding is needed to improve existing varieties. Furthermore, efficient in vitro regeneration systems are essential for the support of genetic transformation and molecular breeding.

[0004] Therefore, it is of great significance to study how to establish an efficient in vitro regeneration system for quinoa.

[0005] Therefore, there is an urgent need in this field to develop an efficient in vitro regeneration system for quinoa. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient in vitro regeneration system for quinoa.

[0007] Another objective of this invention is to provide an efficient method for in vitro regeneration of quinoa inflorescences, in order to solve the problems existing in the background art and provide technical support for quinoa variety improvement and the development of the quinoa industry.

[0008] The first aspect of this invention provides a method for the in vitro regeneration of quinoa, comprising the steps of:

[0009] (a) Select quinoa inflorescences, wherein the length of the inflorescences is 2-20 mm, preferably 3-15 mm, more preferably 5-10 mm;

[0010] (b) The inflorescence is induced to produce quinoa plants.

[0011] In another preferred embodiment, the inflorescence does not include the outermost mature portion.

[0012] In another preferred embodiment, step (a) further includes the step of disinfecting the inflorescence.

[0013] In another preferred embodiment, step (b) includes the following sub-steps:

[0014] (b1) The inflorescence is cultured to obtain callus tissue;

[0015] (b2) The callus tissue is subjected to a budding treatment to obtain adventitious buds; and

[0016] (b3) The adventitious buds are subjected to rooting treatment to obtain quinoa plants.

[0017] In another preferred embodiment, in steps (b1) and (b2), the inflorescence is placed on MS medium containing 5-200 g / L (preferably, 10-100 g / L, more preferably, 20-50 g / L) of sucrose, 0.1-40 mg / L (preferably, 0.5-20 mg / L, more preferably, 0.5-10 mg / L) of 6-benzylaminopurine, 0.01-4 mg / L (preferably, 0.05-2 mg / L, more preferably, 0.08-1 mg / L) of 2,4-D (dichlorophenoxyacetic acid), and 0.5-50 g / L (preferably, 1-20 g / L, more preferably, 2-10 g / L) of plant gel, and cultured at a pH of 5-7 (preferably, 5.5-6) ​​to obtain callus and adventitious shoots.

[0018] In another preferred embodiment, in steps (b1) and (b2), the culture conditions are selected from one or more of the following groups:

[0019] (i) The temperature is 20-30°C, preferably 22-27°C;

[0020] (ii) The light intensity is 2000-4000 Lux, preferably 2600-3500 Lux;

[0021] (iii) The illumination time is 10-25 hours, preferably 14-18 hours;

[0022] (iv) The incubation time is 8-20 hours, preferably 10-18 hours.

[0023] In another preferred embodiment, in step (b3), the adventitious buds are placed on MS medium containing 5-200 g / L (preferably, 10-100 g / L, more preferably, 20-50 g / L) of sucrose, 10-500 g / L (preferably, 40-400 mg / L, more preferably, 60-200 g / L) of banana, and 0.5-50 g / L (preferably, 1-20 g / L, more preferably, 2-10 g / L) of plant gel, and cultured at a pH of 5-7 (preferably, 5.5-6) ​​to obtain adventitious roots.

[0024] In another preferred embodiment, the amount of MS medium added is 1-10 g / L, more preferably 2-6 g / L.

[0025] In another preferred embodiment, in step (b3), the obtained adventitious roots are placed on MS medium containing 5-200 g / L (preferably 10-100 g / L, more preferably 20-50 g / L) of sucrose and 0.5-50 g / L (preferably 1-20 g / L, more preferably 2-10 g / L) of plant gel and cultured at 22-27°C for 10-20 days to obtain quinoa plants regenerated in vitro.

[0026] In another preferred embodiment, in step (b3), the adventitious buds are placed on MS medium containing 5-200 g / L (preferably 10-100 g / L, more preferably 20-50 g / L) of sucrose and 0.5-50 g / L (preferably 1-20 g / L, more preferably 2-10 g / L) of plant gel to obtain quinoa plants.

[0027] In another preferred embodiment, in step (b3), the culture conditions are selected from one or more of the following groups:

[0028] (i) The temperature is 20-30°C, preferably 22-27°C;

[0029] (ii) The light intensity is 2000-4000 Lux, preferably 2600-3500 Lux;

[0030] (iii) The duration of illumination is 10-25 hours, preferably 12-18 hours;

[0031] (iv) The culture time is 10-60 days, preferably 10-30 days.

[0032] In another preferred embodiment, the method further includes the step of hardening off the plants obtained in step (b) and then transplanting them into cultivation soil for further cultivation.

[0033] In another preferred embodiment, the temperature for cultivation in the soil is 20-30°C, more preferably 22-27°C.

[0034] In another preferred embodiment, the substrate of the cultivation soil comprises vermiculite and garden soil.

[0035] In another preferred embodiment, the ratio of vermiculite to garden soil is 1:1.

[0036] In another preferred embodiment, the growth conditions for hardening off and indoor transplanting are each independently selected from one or more of the following groups:

[0037] (i) The temperature is 20-30°C, preferably 22-27°C;

[0038] (ii) The light intensity is 2000-4000 Lux, preferably 2600-3500 Lux;

[0039] (iii) The illumination time is 10-25h, preferably 14-18h.

[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0041] Figure 1 The inflorescence of quinoa to be selected is shown, bar = 1 cm.

[0042] Figure 2 The image shows the state of the inflorescence after disinfection under the present invention, bar = 0.5 cm.

[0043] Figure 3 The callus tissue produced by the inflorescence under the present invention is shown, bar = 0.5 cm.

[0044] Figure 4 This demonstrates the induction of adventitious buds from inflorescence callus tissue under the present invention, bar = 1 cm.

[0045] Figure 5 The image shows the rooting of adventitious buds in a rooting pre-culture medium of the present invention, bar = 1 cm.

[0046] Figure 6 This invention demonstrates a comparison of adventitious shoots on rooting pre-med medium and rooting medium.

[0047] Figure 7 Quinoa in the rooting medium of this invention, bar = 1 cm

[0048] Figure 8 The image shows the regenerated seedlings of this invention transplanted into the soil 30 days after planting, with a bar value of 3 cm.

[0049] Figure 9 The image shows seeds from a regenerated seedling produced under this invention, bar = 0.5 cm. Detailed Implementation

[0050] Through extensive and in-depth research and screening, the inventors, for the first time, induced culture of quinoa inflorescences of a specific length, obtaining a large quantity of stable and easily reproducible quinoa plants with high efficiency for regeneration. Based on this, the present invention was completed.

[0051] culture medium

[0052] The MS medium used in this paper is the most widely used medium in the field. It was designed by Murashige and Skoog in 1962 for tobacco cell culture. Its characteristics include high concentrations of inorganic salts and ions, making it a relatively stable ion balance solution. It has a high nitrate content, and the quantity and ratio of nutrients are appropriate to meet the nutritional and physiological needs of plant cells. Therefore, it has a wide range of applications and is used as the basic medium for rapid propagation of most plant tissue cultures (Murashige and Skoog A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 1962.15(3):473.). Unless otherwise specified, MS medium or MS solid medium can be prepared using formulas known in the art.

[0053] In this invention, the culture medium used can be constructed based on the conventional culture medium for quinoa plants in the field, with the addition of a certain concentration of sucrose, 6-BA (6-benzylaminopurine), 2,4-D (dichlorophenoxyacetic acid), and plant gel, thereby obtaining a culture medium for inducing callus and adventitious shoots (i.e., inflorescence induction differentiation medium), and the pH of the culture medium is 5-7.

[0054] In this invention, the culture medium used can be constructed based on the conventional culture medium for quinoa plants in the field, with the addition of a certain concentration of sucrose, banana, and plant gel to obtain a rooting culture medium with a pH of 5-7.

[0055] In a preferred embodiment, the inflorescence induction differentiation medium is: MS medium (catalog number, MSP09-50LT, purchased from Caisson) with added sucrose (catalog number, 10021418, purchased from Hushi), 6-benzylaminopurine (catalog number, B130, purchased from Phytotechlab), 2,4-D (dichlorophenoxyacetic acid) (catalog number, D309, purchased from Phytotechlab), and plant gel (catalog number, P8169, purchased from Sigma).

[0056] In a preferred embodiment, the rooting pre-medium is MS medium (catalog number, MSP09-50LT, purchased from Caisson) with added sucrose (catalog number, 10021418, purchased from Hushi), banana (purchased from imported sweet bananas from the Philippines), and plant gel (catalog number, P8169, purchased from Sigma).

[0057] In a preferred embodiment, the rooting medium is MS medium (catalog number, MSP09-50LT, purchased from Caisson) with added sucrose (catalog number, 10021418, purchased from Hushi) and plant gel (catalog number, P8169, purchased from Sigma).

[0058] 6-Benzylaminopurine

[0059] As used in this article, the terms “6-benzylaminopurine”, “6-benzyladenine”, “6-benzyladenine”, “6-phenylmethyladenine”, “6-BA”, “benzyladenine”, “BA”, etc., can be used interchangeably.

[0060] 6-Benzylaminopurine is a broad-spectrum plant growth regulator that can promote plant cell growth, inhibit the decomposition of chlorophyll, nucleic acids, and proteins, increase amino acid content, delay leaf senescence, and transport amino acids, auxins, and inorganic salts to the treated parts. It has a variety of effects and is widely used in all stages of agriculture, fruit trees, and horticultural crops from germination to harvest.

[0061] 6-Benzylaminopurine can be obtained by conventional methods, such as commercial purchase or preparation using conventional methods.

[0062] sucrose

[0063] As used in this article, the terms “sucrose,” “sugar,” and “Sugar” are used interchangeably with these terms.

[0064] Sucrose is the main component of sugar and is a disaccharide formed by the condensation and dehydration of the hemiacetal hydroxyl groups of one molecule of glucose and one molecule of fructose. In tissue culture, sucrose serves as a carbon source, providing energy. It helps create a relatively stable osmotic pressure, preventing plant cell dehydration and poor growth. Using sucrose can also reduce microbial contamination to some extent. The concentration of sucrose influences the type and number of vascular bundles in callus tissue to a certain extent.

[0065] Those skilled in the art can obtain sucrose using conventional methods, such as purchasing it from the market or producing it using conventional methods.

[0066] 2,4-D (dichlorophenoxyacetic acid)

[0067] As used herein, the terms “2,4-D” and “dichlorophenoxyacetic acid” are used interchangeably.

[0068] 2,4-D is an auxin analogue, a synthetic plant hormone with similar physiological effects to auxin (IAA). It can be used to regulate plant growth and is a commonly used auxin analogue for inducing callus formation.

[0069] Those skilled in the art can obtain 2,4-D using conventional methods, such as purchasing it from the market or preparing it using conventional methods.

[0070] Plant gel

[0071] As used in this article, the terms “plant gel,” “pyhtal,” and “phytagel” are used interchangeably.

[0072] Plant gel is an agar substitute produced by fermentation of bacteria (Pseudomonas elodea). It contains substances such as glucuronic acid, rhamnose, and glucose. It is colorless, transparent, and highly resilient, and is a major component in the preparation of plant tissue culture media and microbial culture media.

[0073] Those skilled in the art can obtain plant gels using conventional methods, such as purchasing them from the market or preparing them using conventional methods.

[0074] In vitro regeneration method of quinoa

[0075] This invention provides a method for the in vitro regeneration of quinoa, comprising the following steps:

[0076] (a) Select quinoa inflorescences, wherein the length of the inflorescences is 2-20 mm, preferably 3-15 mm, more preferably 5-10 mm;

[0077] (b) The inflorescence is induced to produce quinoa plants.

[0078] In a preferred embodiment, the present invention provides an efficient method for in vitro regeneration of quinoa inflorescences, the method comprising the following steps:

[0079] (1) Selection and sterilization of inflorescences: Select young, pale green inflorescences from plants free from pests and diseases. Rinse them with tap water for 2-3 hours. Place the rinsed inflorescences in a sterilized Erlenmeyer flask and rinse them three times with sterilized distilled water (1 min, 3 min, and 5 min respectively). Discard the distilled water and then soak them in 75% anhydrous ethanol for 30-40 seconds. Immediately afterward, rinse them three times with sterilized distilled water (1 min, 3 min, and 5 min respectively). Discard the distilled water and then pour in an 8% sodium hypochlorite solution diluted at a 1:2 ratio and soak for 3-6 minutes. Finally, rinse them three times with sterilized distilled water (1 min, 3 min, and 5 min respectively). Then, use sterile forceps to remove the inflorescences and place them on filter paper lined with autoclaved filter paper to absorb the moisture from the surface of the inflorescences. Use sterile scissors to cut the inflorescences to a size of 5-10 mm, while also cutting off the outermost mature part of the inflorescence.

[0080] (2) Callus and adventitious shoot induction: The sterilized inflorescences from step (1) were transferred to the induction differentiation medium, with 9 materials placed in each culture dish. The culture dishes containing the materials were then moved to the culture room. Culture environment: temperature 22-27℃, light intensity 2600-3500 Lux, light duration 14-18 hours; culture for 12-16 days. The composition of the inflorescence induction differentiation medium was: MS medium 4.43 g / L, sucrose 20-50 g / L, 6-BA (6-benzylaminopurine) 1.0-5.0 mg / L, 2,4-D (dichlorophenoxyacetic acid) 0.1-0.5 mg / L, plant gel 3-8 g / L, and the pH of the medium was adjusted to 5.5-6.0.

[0081] (3) Rooting induction: After the inflorescence in step (2) has grown callus tissue, and then adventitious buds of 1-2 cm have grown from the callus tissue, cut off the adventitious buds with a sterile scalpel (while retaining part of the callus tissue); use sterile forceps to transfer the cut adventitious buds to a conical flask containing rooting pre-medium, and then move the conical flask containing the material to the culture room. Culture environment: temperature 22-27℃, light intensity 2600-3500 Lux, light duration 14-18 hours; culture for 10-20 days. (During this period, unless the culture medium is contaminated, it is generally not necessary to change the culture medium). The components of the rooting pre-medium are: MS medium 4.43 g / L, sucrose 25 g / L, banana 80-130 g / L, plant gel 3-8 g / L, and the pH of the culture medium is adjusted to 5.5-6.0. After 10-20 days, adventitious roots are obtained. Then, using sterile forceps, the quinoa seedlings that have developed adventitious roots are transferred to a rooting medium.

[0082] (4) Hardening off and indoor transplanting: After rooting induction in step (3), open the mouth of the conical flask containing the material in step (3), add distilled water to the culture medium until it just covers the surface of the culture medium, and change the distilled water 1-2 times a day to begin hardening off. Hardening off environment: temperature 22-27℃, light intensity 2600-3500 Lux, light duration 14-18 hours; harden off for 2-3 days. Then, remove the tissue culture seedlings with strong root growth after hardening off from the culture medium and rinse the roots with tap water (to wash away any residual culture medium); then transplant them into flower pots filled with seedling soil (the seedling soil is a mixture of vermiculite and garden soil in a 1:1 ratio), seal the opening of the flower pot with plastic film, and cut a few holes in the plastic film. Water regularly. Cultivation environment: Temperature 22-27℃, light intensity 2600-3500 Lux, light duration 14-18 hours; remove the plastic film after 3-5 days and move to the growing room or field for growth.

[0083] The quinoa obtained by the method of the present invention has the advantages of high phenotypic uniformity and good stability of regenerated plants.

[0084] The main advantages of this invention include:

[0085] (1) This invention is the first to induce culture of quinoa inflorescences of a specific length, which can obtain quinoa plants with high phenotypic uniformity and good stability of regenerated plants.

[0086] (2) This invention is the first to study the sterilization of quinoa inflorescences and obtained a high frequency of sterile inflorescences.

[0087] (3) This invention is the first to use quinoa inflorescences as starting material to obtain callus tissue, adventitious buds, and regenerated plants through induced culture. The in vitro regeneration method of quinoa inflorescences established by this invention provides technical support for the genetic transformation and molecular breeding of quinoa, and promotes the innovation of quinoa varieties and the development of the quinoa industry.

[0088] (4) The method of the present invention can rapidly and massively propagate quinoa plants in vitro with good repeatability.

[0089] (5) The method of the present invention can produce a large number of high-quality quinoa seedlings in a short time, and the occurrence of such seedlings is not limited by the season and can be produced all year round.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0092] Unless otherwise stated, all materials and reagents used in the embodiments of this invention are commercially available products.

[0093] Example 1

[0094] Quinoa inflorescence disinfectant:

[0095] Prepare 75% anhydrous ethanol by diluting it with water at a ratio of 3:1, based on conventional anhydrous ethanol (product number 10009218, Shanghai type).

[0096] Prepare a 1:2 sodium hypochlorite solution based on conventional sodium hypochlorite (product number 80010428, Shanghai type).

[0097] Preparation of Quinoa Inflorescence Culture Medium

[0098] Preparation of inflorescence differentiation induction medium:

[0099] A quinoa inflorescence differentiation induction medium was prepared based on the formula of standard MS medium (catalog number MSP09-50LT, purchased from Caisson, concentration 4.43 g / L) without adjustment of the components, with the addition of 30 g / L sucrose, 4 g / L plant gel, 1.0 mg / L 6-BA and 0.1 mg / L 2,4-D. The pH was adjusted to 5.8, and the medium was sterilized at 121°C for 15-25 minutes.

[0100] Preparation of rooting pre-culture medium:

[0101] Prepare a pre-rooting medium based on the formula of standard MS medium (catalog number MSP09-50LT, purchased from Caisson, concentration 4.43 g / L) without adjusting the components, adding 30 g / L sucrose, 4 g / L plant gel, and 100 g / L banana (imported from the Philippines). Adjust the pH to 5.8 and sterilize at 121℃ for 15-25 minutes.

[0102] Preparation of rooting medium:

[0103] Based on the formulation of standard MS medium (catalog number, MSP09-50LT, purchased from Caisson, concentration 4.43 g / L) without adjustment of the medium composition, 30 g / L sucrose and 4 g / L plant gel were added. The pH was adjusted to 5.8, and the medium was sterilized at 121°C for 15-25 minutes.

[0104] Meanwhile, commercially available MS medium was used as a control medium.

[0105] Example 2: Preparation of regenerated plants

[0106] In this embodiment, the quinoa inflorescence culture medium prepared in Example 1 and the control MS culture medium were used to regenerate the inflorescences (or control segment materials) prepared by a specific process.

[0107] Each experimental condition was repeated three times. The specific experimental method is as follows:

[0108] (1) Selection and sterilization of inflorescences: Select tender young inflorescences that are free from pests and diseases and have a light green color. Figure 1 First, rinse the inflorescence with tap water for 3 hours. Then, place the rinsed inflorescence in a sterilized Erlenmeyer flask and rinse three times with sterile distilled water, 1-5 minutes each time. Discard the distilled water, then soak in 75% anhydrous ethanol for 20-40 seconds. Immediately afterward, rinse three times with sterile distilled water, 1-5 minutes each time. Discard the distilled water, then pour in a 1:2 diluted sodium hypochlorite solution and soak for 3-5 minutes. Finally, rinse three times with sterile distilled water, 1-5 minutes each time. Next, use sterile forceps to remove the inflorescence and place it on filter paper lined with autoclaved filter paper to absorb excess moisture. Use sterile scissors to cut the inflorescence to a size of 5-10 mm, while also removing the outermost mature part of the inflorescence. Figure 2 ).

[0109] (2) Callus and adventitious shoot induction: The sterilized inflorescences from step (1) were transferred to the differentiation induction medium described in Example 1. Nine materials were placed in each culture dish, and then the culture dishes containing the materials were moved to the culture room. Culture environment: temperature 26±1℃, light intensity 3000Lux, light duration 16 hours; culture for 14 days.

[0110] (3) Rooting induction: After the inflorescence in step (2) first grows callus tissue ( Figure 3 Then, after adventitious buds grow from the callus tissue, 1-2 cm long buds are cut off with a sterile scalpel (while preserving some callus tissue). Figure 4 Using sterile forceps, transfer the cut adventitious buds into the conical flask containing the rooting pre-medium described in Example 1, and then move the conical flask containing the material to the culture room. Figure 5 Culture environment: temperature 26±1℃, light intensity 3000 Lux, light duration 12-18 hours; culture for 10-20 days. After adventitious roots have grown, transfer the quinoa seedlings to the conical flasks containing the rooting medium described in Example 1 (without changing the medium during this period). Figure 6 ).

[0111] (4) Hardening off and indoor transplanting: After rooting induction in step (3), open the rooting medium bottle in the plastic box containing the materials from step (3), add distilled water to the medium until it just covers the surface of the medium, and change the distilled water 1-2 times a day to begin hardening off. Hardening off environment: temperature 23±1℃, light intensity 3000Lux, light duration 12-18 hours; hardening off for 3-5 days. Then, take the tissue culture seedlings with strong root growth after hardening off out of the medium and rinse the roots with tap water (to wash away any residual medium from the roots); then transplant them into flower pots filled with seedling soil (the seedling soil is a mixture of vermiculite and garden soil in a 1:1 ratio), seal the opening of the flower pot with plastic film, and cut a few holes in the plastic film. Water regularly. Cultivation environment: temperature 23±1℃, light intensity 3000Lux, light duration 14h; after 5 days, remove the plastic film and move to the growing room for further growth. Figure 8 Vibrant new quinoa seeds can be obtained after 50-80 days. Figure 9 ).

[0112] Experimental results:

[0113] When using conventional MS medium (control medium) and inflorescences of 5-10 mm as plant material, it is impossible to obtain regenerated inflorescence plants. In contrast, quinoa inflorescences prepared using the specific process of this invention and placed in a specific medium can significantly improve the yield of regenerated plants. Furthermore, the phenotype is highly consistent with the parental lines, with an adventitious bud induction rate of approximately 70% and a rooting rate of approximately 70%. Compared to the control group using other media, the regenerated plants in the quinoa inflorescence medium group exhibit superior morphology.

[0114] Example 3: Preparation of regenerated plants

[0115] The method is the same as in Example 2, except for step (3). Step (3) of Example 3 is as follows:

[0116] (3) Rooting induction: After the inflorescence in step (2) first grows callus tissue ( Figure 3 Then, after adventitious buds grow from the callus tissue, 1-2 cm long buds are cut off with a sterile scalpel (while preserving some callus tissue). Figure 4 Using sterile forceps, transfer the cut adventitious buds to an Erlenmeyer flask containing the rooting medium described in Example 1. Then, move the Erlenmeyer flask containing the material to the culture room. After 20-50 days, adventitious roots will grow from the adventitious buds. Figure 7 Culture environment: temperature 26±1℃, light intensity 3000Lux, light duration 12-18 hours; culture for 10-20 days.

[0117] Experimental results:

[0118] We obtained quinoa inflorescences by growing quinoa indoors. Figure 1 After sterilization experiments, and using sterile scalpels and forceps, sterile quinoa inflorescences were obtained. Figure 2 Aseptic quinoa inflorescences were placed on differentiation medium to obtain callus tissue from the inflorescences. Figure 3 After 30-50 days of treatment, the callus tissue eventually forms adventitious buds. Figure 4 When transferring adventitious buds to rooting medium, we performed different treatments. In Examples 2 and 3, we observed that adventitious buds that underwent rooting pre-culture produced better numbers and lengths of regenerated roots compared to those that did not undergo rooting pre-culture. Figure 5 6, 7). On the 20th day of the rooting experiment, 20 materials were selected for statistical analysis (Table 1). During the hardening-off and transplanting process, the quinoa adventitious buds that had undergone rooting pre-culture showed good growth after transplanting. Figure 8 After 30-50 days, we harvested fresh, more vigorous quinoa seeds. Figure 9 Quinoa inflorescences prepared using the specific process of this invention and placed in a specific culture medium can significantly improve the yield of regenerated plants, increase the number of adventitious buds forming roots, the average root length of adventitious buds, and shorten the rooting time to 6-8 days. Furthermore, they exhibit high phenotypic consistency with the parent plants, with an adventitious bud induction rate of approximately 70% and a rooting rate of approximately 70%. Compared to the control group using other culture media, the regenerated plants in the quinoa inflorescence culture medium group show superior morphology.

[0119] Table 1

[0120] Number of adventitious roots Average length of adventitious roots (cm) Example 2 14 13.4 Example 3 9 8.4

[0121] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for the in vitro regeneration of quinoa, characterized in that, Including the following steps: (a) Select quinoa inflorescences, the length of which is 2-20 mm; (b) The inflorescence is induced to produce quinoa plants; The inflorescence does not include the outermost mature part; Step (b) includes the following sub-steps: Callus and adventitious bud induction: Sterilized inflorescences were transferred to differentiation induction medium, with 9 samples placed in each petri dish. The petri dishes containing the samples were then moved to a culture room. The culture environment was: temperature 26±1℃, light intensity 3000 Lux, light duration 16 hours; cultured for 14 days. Rooting induction: After callus tissue emerged from the inflorescence, and then adventitious buds of 1-2 cm grew from the callus, the adventitious buds were cut off with a sterile scalpel while retaining some callus tissue. The cut adventitious buds were transferred to Erlenmeyer flasks containing rooting pre-medium using sterile forceps. The Erlenmeyer flasks containing the samples were then moved to a culture room. The culture environment was: temperature 26±1℃, light intensity 3000 Lux, light duration 12-18 hours; cultured for 10-20 days. After adventitious roots developed, the quinoa seedlings were transferred to Erlenmeyer flasks containing rooting medium. The medium was not changed during this period. The inflorescence induction differentiation medium is MS medium composed of 5-30 g / L sucrose, 0.1-1 mg / L 6-benzylaminopurine, 0.1-4 mg / L 2,4-D (dichlorophenoxyacetic acid), and 0.5-4 g / L plant gel. The rooting pre-medium is MS medium composed of 5-30 g / L sucrose, 10-100 g / L banana and 0.5-4.43 g / L plant gel; The rooting medium is MS medium composed of 5-30 g / L sucrose and 0.5-4 g / L plant gel.

2. The method as described in claim 1, characterized in that, The inflorescence is 5-10 mm in length.

3. The method as described in claim 1, characterized in that, The method further includes the step of hardening off the plants obtained in step (b) and then transplanting them into cultivation soil for further cultivation; the substrate of the cultivation soil includes vermiculite and garden soil.

4. The method as described in claim 3, characterized in that, The temperature for cultivation in the soil is 20-30℃; The growth conditions for seedling hardening are as follows: (i) The temperature is 20-30℃; (ii) Illumination intensity is 2000-4000 Lux; (iii) The illumination time is 10-25h.

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