Method for promoting bougainvillea speetabilis tissue culture seedling proliferation

Abstract

The invention discloses a method for promoting bougainvillea speetabilis tissue culture seedling proliferation. The method comprises the following steps: (1) seed disinfection; (2) performing sterile germination; (3) inducing cluster buds; (4) rooting culture; and (5) transplanting tissue culture seedlings. The bud culture medium MS0 selected during sterile germination comprises the following components: 600mg / L of complex mineral elements, 1.6 mg / L of aspartic acid, 1.6 mg / L of glycine, 1.2 mg / L of vitamin B1, 0.8 mg / L of vitamin B6 and 0.8 mg / L of thidiazuron. A slave bud proliferation culture medium MS1 selected during cluster bud induction comprises the following components: MS, 4.5 mg / L of 6-BA, 0.2 mg / L of IBA, 30g / L of sugar and 6g / L of agar; wherein MS is an MS culture medium; the 6-BA is 6-benzyladenine, and the 6- IBA is 3-indolebutyric acid. The bougainvillea speetabilis tissue culture seedling propagation method is high in propagation and propagation coefficient, early in rooting time and high in survival rate.

Description

Technical Field

[0001] This invention relates to the field of bougainvillea tissue culture technology, specifically a method for promoting the proliferation of bougainvillea tissue culture seedlings. Background Technology

[0002] Bougainvillea, also known as paper flower, comes in a variety of varieties, is highly adaptable, easy to cultivate, and offers both beautiful flowers and foliage. With its diverse flower colors and long blooming period, it has high ornamental value and is widely used in landscaping. Furthermore, it contains chemical substances that have positive effects on human health, possessing certain medicinal value, antiviral properties, and environmental protection benefits. Currently, bougainvillea is the city flower of 18 cities including Beihai, Wuzhou, Shenzhen, Zhuhai, Zhongshan, Jiangmen, Xiamen, Huizhou, and Haikou. With societal progress and increasingly higher demands for urban ecological environments, bougainvillea, as an important landscaping plant, has a large market demand. However, most bougainvillea is propagated using conventional methods such as cuttings and layering, which have long production cycles and cannot meet market demand.

[0003] Currently, domestic research on bougainvillea mainly focuses on cultivation management, biological characteristics, and landscaping applications. Bougainvillea plants have very small flower tubes, resulting in limited natural pollination opportunities, low pollen activity, and difficulty in fruit set, especially cultivated varieties which are extremely difficult to propagate in my country. Asexual reproduction methods such as cuttings, grafting, and layering are the primary propagation methods. However, conventional propagation of bougainvillea suffers from disadvantages such as a low proliferation coefficient, long rooting time, and seasonal limitations. Furthermore, for some rare varieties and those difficult to root from cuttings, such as variegated Angus and spotted Red Bougainvillea, traditional seed propagation methods are insufficient to obtain a large number of seedlings in a short period.

[0004] Therefore, it is essential to study a method to promote the proliferation of bougainvillea tissue culture seedlings to meet market demand. Summary of the Invention

[0005] The purpose of this invention is to provide a method for promoting the proliferation of bougainvillea tissue culture seedlings, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for promoting the proliferation of bougainvillea tissue culture seedlings, comprising the following steps:

[0007] (1) Seed disinfection:

[0008] Remove the wings from the seeds, soak them in a neutral detergent for 10-15 minutes, rinse them under running water for 20-30 minutes, then disinfect them with 75% alcohol for 30 seconds and mercuric chloride for 8 minutes.

[0009] The disinfected seeds were pretreated by soaking them in a mixed aqueous solution of 5% sodium hypochlorite, 10 mg / L GGR (Double Gill) No. 6 and 20-25 mg / L gibberellin (GA4+7) for 10 min, and then rinsed 5 times with sterile water.

[0010] (2) Aseptic germination

[0011] The pretreated seeds were sown in germination medium MSO and cultured.

[0012] (3) Induction of clustered buds

[0013] When the buds are 1-2 cm tall, replace the budding medium MS0 with the bud proliferation medium MS1.

[0014] (4) Rooting culture

[0015] When the buds are 3-4 cm tall, replace the bud proliferation medium MS1 with the rooting medium MS2.

[0016] (5) Transplanting of tissue culture seedlings

[0017] When the buds are 8-10cm tall and have 5-6 leaves, clean the MS2 culture medium from the roots of the bougainvillea and immediately transplant them into a mixture of peat moss and perlite (3:1). Place a shade net on the soil and cover it with a film to retain moisture. Once 2-3 new leaves have grown, remove the film and begin hardening off the seedlings.

[0018] Preferably, in this technical solution, the bud culture medium MSO comprises: 600 mg / L of compound minerals, 1.6 mg / L of aspartic acid, 1.6 mg / L of glycine, 1.2 mg / L of vitamin B1, 0.8 mg / L of vitamin B6, and 0.8 mg / L of thidiazuron.

[0019] Preferably, in this technical solution, the composite mineral element contains at least K. + Na + Ca + Mg + Fe + Cu + IP + and Zn + .

[0020] Preferably, in this technical solution, the components of the bud proliferation medium MS1 include: MS, 4.5 mg / L 6-BA, 0.2 mg / L IBA, 30 g / L sugar and 6 g / L agar; wherein, MS is MS medium; 6-BA is 6-benzyladenine; and IBA is 3-indolebutyric acid.

[0021] Preferably, in this technical solution, the rooting medium MS2 comprises: 1 / 2 MS and 0.5 mg / L NAA.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The bougainvillea tissue culture seedling propagation method of this invention has a high propagation coefficient, early rooting time, and high survival rate. It allows for the study of the growth and differentiation patterns of the cultured parts without seasonal influence or interference from other parts of the plant. Furthermore, it allows for the use of various culture medium conditions to influence their growth and differentiation, enabling the production of a large number of high-quality seedlings with consistent genotypes in a short period. The seedling production cycle is short, production costs are reduced, and it allows for rapid, large-scale propagation of superior seedlings. Attached Figure Description

[0024] Figure 1 This is a seed status diagram of the method of the present invention;

[0025] Figure 2 This is a diagram showing the condition of the bottle after bud induction using the method of the present invention;

[0026] Figure 3 This is a diagram showing the external condition of the bottle after bud induction using the method of the present invention;

[0027] Figure 4 This is a diagram showing the root condition after rooting according to the method of the present invention;

[0028] Figure 5 This is a diagram showing the root and seedling condition during transplanting using the method of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments; the same or similar concepts or processes may not be described again in some embodiments.

[0031] The present invention provides a method for promoting the proliferation of bougainvillea tissue culture seedlings, comprising the following steps:

[0032] (1) Seed disinfection:

[0033] Remove the seed wings, soak them in a neutral detergent for 10 - 15 min first, then rinse under running water for 20 - 30 min, and then disinfect with 75% alcohol for 30 s and with mercuric chloride for 8 min;

[0034] Pre - treat the disinfected seeds by soaking them in a mixed aqueous solution of 5% sodium hypochlorite, 10 mg / L of GGR (Giggle) No. 6 and 20 - 25 mg / L of gibberellin (GA4 + 7) for 10 min, and then rinse with sterile water 5 times;

[0035] (2), Sterile germination

[0036] Sow the pre - treated seeds on the germination medium MS0 for cultivation;

[0037] (3), Induction of cluster buds

[0038] When the bud height is 1 - 2 cm, change the germination medium MS0 to the shoot proliferation medium MS1;

[0039] (4), Rooting culture

[0040] When the bud height is 3 - 4 cm, change the shoot proliferation medium MS1 to the rooting medium MS2;

[0041] (5), Transplanting of tissue - cultured seedlings

[0042] When the bud height is 8 - 10 cm and there are 5 - 6 leaves, wash the medium MS2 from the roots of Bougainvillea spectabilis, and immediately transplant it into peat soil + perlite (3:1). Place a shade net and cover it with a film to keep moisture. When 2 - 3 new leaves grow, the film can be removed for hardening management.

[0043] In this invention, several groups of media are optimized as follows:

[0044] Composition of the germination medium MS0: Compound mineral elements 600 mg / L, aspartic acid 1.6 mg / L, glycine 1.6 mg / L, vitamin B1 1.2 mg / L, vitamin B6 0.8 mg / L and thidiazuron 0.8 mg / L.

[0045] Compound mineral elements: At least include potassium (K + ), sodium (Na + ), calcium (Ca + ), magnesium (Mg + ), iron (Fe + ), copper (Cu + ), inorganic phosphorus (IP + ) and zinc (Zn + ), which are used to provide mineral nutrition for seed germination, promote seed germination, and ensure the fertility required for subsequent growth to improve the survival rate. Tianke - Diligao

[0046] Thiadiazon synergizes with vitamins B1 and B6, promoting seed cell division and germination. It also accelerates the absorption of complex minerals and glycine by activating cell surfaces, providing ample nutrition for subsequent germination and root development. Furthermore, by promoting seed cell division and accelerating the absorption and utilization of nutrients such as vitamins B1, B6, complex minerals, glycine, and aspartic acid, it enhances seed germination, protein synthesis, provides nitrogen during periods of stress, strengthens resistance, and prevents weak or underdeveloped embryos.

[0047] The components of the shoot proliferation medium MS1 are: MS, 4.5 mg / L 6-BA, 0.2 mg / L IBA, 30 g / L sugar and 6 g / L agar; where MS is MS medium; 6-BA is 6-benzyladenine; and IBA is 3-indolebutyric acid.

[0048] Based on the germination medium MS0, the bud proliferation medium MS1 is used in combination to allow vitamin B1 and vitamin B6 to further promote the utilization of sugars by the cotyledons, which can accelerate germination and stem and leaf growth. 6-Benzyladenine, by stimulating root cell division and elongation, works together with the endogenous hormone 3-indolebutyric acid to further promote and increase stem and leaf cell division, and together with agar, promotes the growth of root surface area, thereby improving the efficiency of water and nutrient absorption.

[0049] Rooting medium MS2: 1 / 2 MS, 0.5 mg / L NAA.

[0050] This invention selected three bottles from each of the three color systems—purple, red, and pink—for seedling propagation experiments. The specific varieties selected are as follows:

[0051] Three varieties of bougainvillea in the purple series: Large-flowered Night-Shining Purple, Golden Tortoise Purple, and Golden-edged Angus.

[0052] Three varieties of red bougainvillea: Zhangzhou Red, Rouge Red, and Variegated Red.

[0053] Three types of pink bougainvillea: batik pink, royal bougainvillea, and golden light pink.

[0054] Experimental location: Flower Base, Guangxi Zhuang Autonomous Region Forestry Research Institute

[0055] Seed harvesting time: November to March of the following year

[0056] Test period: November 2023 - March 2025

[0057] The experiment included: (1) aseptic germination test; (2) cluster bud induction test; (3) rooting culture test; and (4) tissue culture seedling transplantation test.

[0058] Partial process recording photos: Figures 1-5 .

[0059] (1) Aseptic germination test

[0060] In November 2023, 30 seeds (30 bottles) of each variety were selected and the sterilized seeds were sown in germination medium MS0 for dark culture. The germination status at 15 days and 20 days was compared with and without the pretreatment of the present invention, and the data were statistically recorded and compiled into Table 21 below.

[0061] Table 1 Results of Pretreatment Germination Rate Experiment

[0062]

[0063]

[0064] As shown in the table above, the average germination rate of bougainvillea seeds sown in germination medium MS0 after pretreatment was 90.00% on day 10, significantly higher than the average germination rate of 57.41% for bougainvillea seeds sown directly in germination medium MS0 after sterilization without pretreatment. On day 20, the germination rate of the pretreated seeds reached 98.89%, also significantly higher than the germination rate of 83.33% for the untreated seeds. This indicates that bougainvillea seeds sown in germination medium MS0 after sterilization and pretreatment according to this invention germinate earlier and have a higher germination rate.

[0065] Meanwhile, in November 2023, the pre-treated seeds were sown on germination medium for dark culture. Three germination media, MS, WPM and MS0, were used for the experiment (due to research funding constraints, the medium comparison experiment was conducted simultaneously with the experiment in Table 1, and the data recorded in Table 1 of the MS0 experiment were compared with the experimental data of MS and WPM). Among them, the MS medium was from Shanghai Kanglang Biotechnology Co., Ltd., and the WPM medium was from Haibo Biotechnology Co., Ltd. 90 bottles were treated for each color system (i.e., 30 bottles for each variety), and the germination was observed at 10d, 15d and 20d. The germination rate was counted and compiled into Table 2 below.

[0066] Table 2. Germination rate test results of different germination media

[0067]

[0068]

[0069] As shown in the table above, on day 10, the average germination rate in MS was 21.48%, in WPM it was 24.07%, and in MS0 it was 34.44%; on day 15, the average germination rate in MS was 71.11%, in WPM it was 78.15%, and in MS0 it was 90.00%; on day 20, the average germination rate in MS was 83.70%, in WPM it was 93.70%, and in MS0 it was 98.89%. This indicates that bougainvillea seeds sown in germination medium MS0 germinate earlier and have a higher germination rate.

[0070] Based on the experiment in Table 2, the seedlings germinating in different germination media were observed and recorded for a period of time. When the seedlings grew 1-2 true leaves, the survival rate of the seed seedlings was calculated (survival rate = number of surviving seedlings / total number of seedings * 100%), and the results were compiled into Table 3.

[0071] Table 3. Survival rate statistics of seedlings when they have grown 1-2 true leaves.

[0072]

[0073]

[0074] As shown in Table 3 above, the average survival rate of bougainvillea seeds was 80.00% in MS germination medium, 89.63% in WPM germination medium, and 97.78% in MS0 germination medium. This indicates that the survival rate of bougainvillea seeds planted in MS0 germination medium was significantly higher. The survival rate of the red series bougainvillea varieties reached as high as 98.89%.

[0075] (2) Induction of clustered buds

[0076] Based on the aseptic germination experiment, when the shoots reached a height of 1-2 cm, 20 seedlings of each variety were selected and replaced with shoot proliferation medium MS1. MS and WPM were used as control groups for shoot proliferation. The shoot induction experiment was then conducted. After 30 days, shoot growth was observed, the average number of shoots proliferated was recorded, and the shoot proliferation coefficient (proliferation coefficient = number of proliferating shoots / number of seed shoots) was calculated, as shown in Tables 4, 5, and 6.

[0077] As shown in Table 4, the growth status of the buds

[0078]

[0079]

[0080] Table 5 shows the statistical results of the average number of shoot proliferations.

[0081]

[0082] Table 6 Results of Bud Proliferation Coefficient Calculation

[0083]

[0084] The statistical results in Tables 4 and 5 show that the average bud proliferation coefficient of the bud proliferation medium MS was 5.29, the average bud proliferation coefficient of the WPM bud proliferation medium was 5.47, and the average bud proliferation coefficient of the bud proliferation medium MS1 was as high as 5.93.

[0085] (3) Rooting culture experiment;

[0086] Based on the bud induction experiment, when the buds reached a height of 3-4 cm, two control groups were designed. The bud proliferation medium was replaced with rooting medium, and the light intensity was gradually increased. After 20 days, the rooting status was observed, and the average rooting rate was calculated (rooting rate (%) = number of rooted plants / number of seedlings x 100). The first control group E1 consisted of 5 bottles / plants of each variety where the bud proliferation medium was replaced with ordinary rooting medium (1 / 2 MS medium) for comparison. The second control group E2 consisted of 5 bottles / plants of each variety where the bud proliferation medium MS1 was replaced with rooting medium MS2 and compared with ordinary rooting medium (1 / 2 MS medium).

[0087] Table 7 Statistical results of the first control group E1

[0088]

[0089] Table 8. Rooting status of the first control group E1

[0090]

[0091]

[0092] As shown in Tables 7 and 8, when using the same ordinary rooting medium (1 / 2 MS medium), the seedlings grown from the shoot proliferation medium MS1 had the earliest rooting time, a rooting rate of 91.11%, and shorter, thicker roots with fewer lateral roots, resulting in the best rooting effect.

[0093] Table 9 Statistical results of E1 in the second control group

[0094]

[0095] Table 10 Rooting status of the second control group E1

[0096]

[0097] As shown in Tables 9 and 10, when seedlings grown on the bud proliferation medium MS1 were used with ordinary rooting medium (1 / 2 MS medium), the average rooting rate was 95.56%; when using rooting medium MS2, the average rooting rate was 98.89%, with earlier rooting time, thicker and shorter roots, fewer lateral roots, and the best rooting effect.

[0098] (4) Tissue culture seedling transplantation experiment.

[0099] After rooting for about 20 days, the bougainvillea seedlings were transferred to a greenhouse for 4-5 days of hardening off. When the buds reached 8-10 cm in height and had 5-6 leaves, the MS2 culture medium around the roots of the bougainvillea was cleaned off, and the seedlings were immediately transplanted into a mixture of peat moss and perlite (3:1). A shade net was placed on the seedlings, and a film was used to maintain moisture. For the first two weeks after transplanting, watering was necessary during the day, and the leaves were sprayed with water daily. Once 2-3 new leaves had grown, the film was removed for further hardening off. Ten seedlings of each variety were used, with three replicates (MS2-TS1, MS2-TS2, and MS2-TS3). After 30 days, the survival rate of the crape myrtle seedlings under different treatments was calculated (survival rate % = number of surviving seedlings / number of seedlings transplanted * 100%).

[0100] Table 11 Survival rate after transplanting

[0101]

[0102] As shown in Table 11, the survival rate of transplanted crops using the method of the present invention is over 98.89%.

[0103] In summary, the bougainvillea tissue culture seedling propagation method of the present invention has a high propagation coefficient, early rooting time, and high survival rate. It allows for the study of the growth and differentiation patterns of the cultured parts without seasonal influence or interference from other parts of the plant. Furthermore, it allows for the use of various culture medium conditions to influence their growth and differentiation, enabling the production of a large number of high-quality seedlings with consistent genotypes in a short period. The seedling production cycle is short, production costs are reduced, and it allows for rapid, large-scale propagation of superior seedlings.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for promoting the proliferation of bougainvillea tissue culture seedlings, characterized in that, Includes the following steps: (1) Seed disinfection: Remove the wings from the seeds, soak them in a neutral detergent for 10-15 minutes, rinse them under running water for 20-30 minutes, then disinfect them with 75% alcohol for 30 seconds and mercuric chloride for 8 minutes. The disinfected seeds were pretreated by soaking them in a mixed aqueous solution of 5% sodium hypochlorite, 10 mg / L GGR (Double Gill) No. 6 and 20-25 mg / L gibberellin (GA4+7) for 10 min, and then rinsed 5 times with sterile water. (2) Aseptic germination The pretreated seeds were sown in germination medium MSO and cultured. (3) Induction of clustered buds When the buds are 1-2 cm tall, replace the budding medium MS0 with the bud proliferation medium MS1. (4) Rooting culture When the buds are 3-4 cm tall, replace the bud proliferation medium MS1 with the rooting medium MS2. (5) Transplanting of tissue culture seedlings When the buds are 8-10cm tall and have 5-6 leaves, clean the MS2 culture medium from the roots of the bougainvillea and immediately transplant them into a mixture of peat moss and perlite (3:1). Place a shade net on the soil and cover it with a film to retain moisture. Once 2-3 new leaves have grown, remove the film and begin hardening off the seedlings.

2. The method for promoting the proliferation of bougainvillea tissue culture seedlings according to claim 1, characterized in that, The bud culture medium MSO consists of: 600 mg / L of complex minerals, 1.6 mg / L of aspartic acid, 1.6 mg / L of glycine, 1.2 mg / L of vitamin B1, 0.8 mg / L of vitamin B6, and 0.8 mg / L of thidiazuron.

3. The method for promoting the proliferation of bougainvillea tissue culture seedlings according to claim 2, characterized in that, The complex mineral element contains at least K. + Na + Ca + Mg + Fe + Cu + IP + and Zn + .

4. The method for promoting the proliferation of bougainvillea tissue culture seedlings according to claim 1, characterized in that, The components of the shoot proliferation medium MS1 include: MS, 4.5 mg / L 6-BA, 0.2 mg / L IBA, 30 g / L sugar and 6 g / L agar; wherein, MS is MS medium; 6-BA is 6-benzyladenine; and IBA is 3-indolebutyric acid.

5. The method for promoting the proliferation of bougainvillea tissue culture seedlings according to claim 1, characterized in that, The rooting medium MS2 consists of: 1 / 2 MS and 0.5 mg / L NAA.

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