Method for rapidly inducing vegetative propagation of bougainvillea speetabilis

Abstract

The method mainly comprises the following steps: disinfecting seeds, inoculating the disinfected seeds to a culture medium for seed embryo germination: MS + 30g / L sucrose + 3g / L plant gel, then inoculating the seeds to an optimal callus culture medium: MS + 2mg / L 6BA + 0.2 mg / L 2, 4-D + 30g / L sucrose + 3g / L plant gel, carrying out dark culture for 20-30 days, then transferring the seeds to an optimal differential culture medium: MS + 2mg / L 6BA + 0.2 mg / L 2, 4-D + 0.2 mg / L sucrose + 3g / L plant gel, and carrying out dark culture for 20-30 days. The method comprises the following steps: adding 4, 4-D, 30g / L sucrose and 3g / L plant gel into a culture medium, transferring the culture medium to an illumination condition of 2500lx, an illumination duration of 16 hours and a temperature of 25 DEG C, and culturing for 20-30 days to obtain differentiated adventitious buds of about 1cm. The efficient bougainvillea speetabilis tissue culture regeneration system established by the invention can provide important technical reference for subsequent bougainvillea speetabilis genetic transformation and functional research.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for tissue culture regeneration of bougainvillea. Background Art

[0002] Bougainvillea is a plant of the genus Bougainvillea in the family Nyctaginaceae. There are about 1,000 cultivars worldwide, which are widely distributed in Asian countries (China, India, South Korea, Malaysia, the Philippines, Singapore, Thailand, etc.), as well as Australia, tropical America, etc. (Chen, 2019; Rastogi et al., 2019). It is a widely popular ornamental plant with important economic value. It can be used in parks, desert gardens, home and institutional gardens in the form of shrubs, hedges, shaped plants, potted plants, bonsai, hanging baskets, wind curtains, landscapes and climbing on trellises, trees, river banks and difficult-to-maintain hillsides. It is known as the "glory of the garden". Therefore, Bougainvillea has attracted the attention of plant lovers and breeders around the world (Datta, 2022).

[0003] According to the survey, bougainvillea is the provincial flower of Hainan Province and the city flower of Sanya City. However, the development of the bougainvillea industry in Hainan Province is relatively slow. The main reasons are: 1) Because people have more or less defects in the growth of a large number of bougainvillea varieties in Hainan, these varieties cannot be promoted on a large scale; 2) At the same time, because bougainvillea has the characteristics of regional climate and is only suitable for growth in the south, the application range of bougainvillea is limited. 3) There are few independent bougainvillea varieties in China, and most of the patents are in the hands of foreign scientific research institutions and companies, which has a great impact on the profits of the bougainvillea industry (Yang Guangsui et al., 2018). Due to the difficulty of hybrid breeding of bougainvillea, the extremely low fruiting rate, and the low activity of embryos, it is difficult to cultivate new varieties and breed seedlings through hybrid breeding (Zhou Qun et al., 2016). It is imperative to find other ways to breed. In order to change this situation, we need to carry out targeted transformation of existing bougainvillea varieties, so establishing a molecular breeding improvement system for bougainvillea is a necessary means to solve this situation.

[0004] At present, the propagation methods of Bougainvillea at home and abroad are still limited to cuttings, grafting and other methods. Through tissue culture regeneration, it is only limited to using the stem segments with buds (Gong Wei et al., 2005; Chen Xuan et al., 2024), stem tips with buds (Guo Haibin and Lei Jiajun, 2006), and seeds (Pan Mei et al., 2017) of Bougainvillea as explants, and by adjusting the types and concentrations of auxins and cytokinins in the culture medium at different stages, a Bougainvillea rapid propagation system is established to obtain complete plants. At present, there is still no research on the induction of callus tissue and further differentiation induction of callus tissue. It will provide a new way for the functional study of key genes of Bougainvillea and genetic engineering breeding, provide a new experimental material for the functional study of important genes of Bougainvillea and the fixed-point molecular improvement of important traits, and also provide reference and reference for the cultivation and expansion of Bougainvillea callus. The Bougainvillea embryonic callus induction method provided by the present invention is not only suitable for the study of the genetic transformation system of Bougainvillea, but also suitable for the rapid propagation of Bougainvillea. Summary of the invention

[0005] The present invention aims to provide a method for inducing bougainvillea embryonic callus to plant regeneration and a culture medium thereof. The method is convenient and simple, and the whole process only requires two culture media and the same culture environment to complete the tissue culture regeneration of bougainvillea.

[0006] The object of the present invention is achieved by the following scheme: a method for inducing tissue culture regeneration of Bougainvillea and a culture medium, comprising the following steps: Step S1, obtaining the hypocotyl for inducing callus tissue: picking bougainvillea fruits, taking their seeds, selecting plump and uncontaminated seeds, first washing the surface dust with detergent, rinsing with running water for 20-30 min, then moving the seeds to a sterile bottle on a clean bench, first washing with sterile water 3 times, each time for 1 min; then disinfecting with 1% sodium hypochlorite solution for 25-30 min; after disinfection, rinsing with sterile water 5 times, each time for 1 min; after disinfection, inoculating into a medium for seed embryo germination: MS+30g / L sucrose+3g / L plant gel; Step S2, callus induction: cut the hypocotyl obtained in S1 into small segments of 0.5-1 cm, transfer to callus induction medium MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L phytagel, and culture in the dark for 20-30 days; Step S3, inducing adventitious buds from callus: taking out the callus obtained in S2, transferring it to the most suitable differentiation medium: MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L plant gel, and moving it to a light bar for 20-30 days to obtain differentiated adventitious buds of about 1-2 cm; Step S4, seedling cultivation: the adventitious buds obtained in S3 are cut from the callus tissue and inoculated into a seedling cultivation medium for seedling cultivation: MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L plant gel, and adventitious buds of about 2-3 cm in height are obtained after about 30 days; Step S5, rooting culture: the adventitious buds obtained in S4 were cut from the callus tissue and inoculated into a rooting medium for rooting culture: 1 / 2MS + 1.25 mg / L NAA + 0.75 mg / L IBA + 30 g / L sucrose + 3 g / L plant gel, and regenerated plants of Bougainvillea were obtained after about 30 days; Step S6, hardening and transplanting: the tissue culture seedlings obtained in S5 are opened and hardened for about 8-10 days, then the plant gel at the roots is washed off, and the seedlings are transplanted into nutrient soil and cultured for about 30 days to obtain healthy plants.

[0007] In the above steps S1 to S5, the temperature in the culture environment is 25-28° C. and the relative humidity is 50-60%. In steps S3 to S5, the illumination duration is 16 h / d and the illumination intensity is 2500 lx.

[0008] The pH values ​​of the seed germination medium, callus induction medium, differentiation medium, seedling growth medium and rooting medium are all 5.8.

[0009] The present invention uses the hypocotyl of sterile bougainvillea seedlings as explants to rapidly induce callus tissue, differentiate seedlings, and then perform rooting culture on the seedlings to obtain in vitro regenerated plants. The most important thing about the present invention is that embryonic callus with differentiation ability is obtained from the embryo regeneration pathway, which achieves rapid reproduction and shortens its breeding period, providing a technical reference for the construction of a genetic transformation system of bougainvillea in the future. The method of the present invention is simple and fast, and the variety of bougainvillea in the present invention is Bougainvillea 'Light violet'. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the process of hypocotyl induction of Bougainvillea seeds.

[0011] Figure 2 This is the callus induction of hypocotyls of Bougainvillea bougainvillea; scale bar: 1 cm.

[0012] Figure 3 This is the adventitious buds differentiated from the callus of Bougainvillea; scale bar: 1 cm.

[0013] Figure 4 To cultivate the strong seedlings and rooting of the adventitious buds of Bougainvillea.

[0014] Figure 5 To harden and transplant the tissue culture seedlings of Bougainvillea. DETAILED DESCRIPTION

[0015] The following is further described in detail through specific implementation methods: Terms involved in this invention: 6-BA is 6-benzyladenine; IBA is indolebutyric acid; 2,4-D is 2,4-dichlorophenoxyacetic acid; KT is kinetin 6-glycosaminopurine; NAA is naphthylacetic acid; IAA is 3-indoleacetic acid; MS basic medium is 4.4 g / L; 1 / 2MS medium is 2.2 g / L; the reagents used in the embodiments of the present invention can be obtained from commercial channels.

[0016] Example 1 Bougainvillea glabra Preparation method of 'Light violet' by Bougainvillea glabra 'Mrs. Eva White' was selected from the female parent whose stamens were removed and bagged before the pollen was immature. When mucus was observed to ooze out from the top of the stigma of the carpel, Bougainvillea glabra The mature pollen of 'NewRiver' was pollinated with a pollination stick and bagged. The seeds were collected after the bracts and flowers completely withered.

[0017] This embodiment provides a method and culture medium for regeneration of bougainvillea tissue culture, comprising the following steps: Step S1: Obtaining hypocotyls for inducing callus Pick the fruits of Bougainvillea, take its seeds, select plump and uncontaminated seeds, first wash the surface dust with detergent, rinse with running water for 20-30 min, then move the seeds to a sterile bottle on a clean bench, wash with sterile water 3 times, 1 min each time; then disinfect with 1% sodium hypochlorite solution for 25-30 min; rinse with sterile water 5 times after disinfection, 1 min each time; place on sterile filter paper to dry, and then inoculate into seed embryo germination medium MS + 30g / L sucrose + 3g / L plant gel, pH 5.8; culture conditions: temperature 25-28℃, relative humidity 50-60%, dark culture for 10-14 d, obtain sterile seedlings with a height of about 4-5 cm, remove the roots and cotyledons of the seedlings, and obtain the hypocotyls of induced callus tissue ( Figure 1 ).

[0018] Step S2: Callus induction The hypocotyls of step S1 were cut into small segments of about 0.5-1 cm in length, and transferred to callus induction medium MS+2mg / L 6BA+0.2 mg / L 2,4-D+30g / L sucrose+3g / L phytogel, pH 5.8, and 6-9 explants were inoculated in each culture dish. The culture dish used was a plastic culture dish with a diameter of 90 mm and a height of 20 mm. The culture conditions were: temperature 25-28°C, relative humidity 50-60%, dark culture for 20-30 days to obtain callus, the callus induction rate was counted, and the callus growth status was observed and recorded. During this period, a new callus induction medium was replaced every 15 days.

[0019] The present invention tested the induction effects of six different hormone ratio culture media on the callus of bougainvillea hypocotyls, and found that all of them could induce callus, but the effects were significantly different (Table 1). Figure 2 D) and 2 mg / L 6BA + 0.2 mg / L 2,4-D ( Figure 2 E) The induction effect was obvious, and 2 mg / L 6BA+0.2 mg / L2,4-D could differentiate into buds at 25 days of induction. 0.5 mg / L 6BA+1 mg / L KT ( Figure 2 A), 0.5 mg / L 6BA+2 mg / L 2,4-D ( Figure 2 B), 0.5 mg / L 6BA+4 mg / L 2,4-D ( Figure 2 C) After 25 days of induction, callus formation was poor, while 2.5 mg / L 2,4-D alone ( Figure 2 F) Induce rooting.

[0020] Table 1 Effects of different culture media on the induction of hypocotyl callus

[0021] Step S3: Inducing adventitious buds from callus tissue The callus obtained in step 2 was transferred to the differentiation medium MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30g / L sucrose+3g / L plant gel, pH 5.8, 6-9 per culture dish, the culture dish used was a plastic culture dish with a diameter of 90 mm and a height of 20 mm. The culture conditions were: temperature 25-28°C, relative humidity 50-60%, and 16 h photoperiod. The budding points were differentiated after about 10 days of culture, and adventitious buds were obtained after 30 days. During this period, the new callus induction medium was replaced every 15 days.

[0022] The present invention tested the induction effect of four different hormone ratio culture media on adventitious buds and found that 0.5 mg / L 6BA+1 mg / L KT ( Figure 3 A), 2 mg / L KT+0.2 mg / L NAA+0.2 mg / L IAA ( Figure 3 B) and 0.5 mg / L KT+0.05mg / L NAA+0.05 mg / L IAA ( Figure 3 C) can only make the callus continue to grow but cannot induce differentiation of adventitious buds, 2 mg / L 6BA+0.2 mg / L 2,4-D ( Figure 3 D) can induce adventitious buds with short induction time, high efficiency and an average induction differentiation rate of 90% (Table 2), making it the best medium for inducing adventitious buds.

[0023] Table 2 Effect of different culture media on induction of adventitious buds

[0024] Step S4, seedling strengthening: The healthy adventitious buds grown in step S3 are cut from the base and inoculated into a seedling strengthening medium MS + 2 mg / L 6BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L plant gel, at a temperature of 25-28°C, a relative humidity of 50-60%, and a photoperiod of 16 h. After about 30 days, adventitious buds with 8-10 true leaves are obtained ( Figure 4 AB); Step S5, rooting culture The adventitious buds obtained in S4 were inoculated into five different hormone ratio culture media for rooting of the adventitious buds. The culture conditions were: temperature 25-28°C, relative humidity 50-60%, 16 h photoperiod. After about 30 days of culture, it was found that 1 / 2MS+1.25 mg / L NAA+0.75 mg / LIBA+30 g / L sucrose+3 g / L plant gel could induce rooting of the adventitious buds. The average rooting rate was 95% (Table 3), and the root system grew robustly ( Figure 4 C, D).

[0025] Table 3 Effects of different culture media on rooting of adventitious buds

[0026] The robust adventitious buds grown in step S3 are cut off from the base, the excess callus tissue is removed, and the buds are inoculated into the rooting medium, 4-6 per tissue culture bottle, and the culture conditions are: temperature 25-28°C, relative humidity 50-60%, and 16 h photoperiod. The tissue culture seedlings obtained in step S3 were opened and hardened for about 10 days ( Figure 5A), then wash off the plant gel at the roots, transplant them into sterilized soil, and culture them for about 30 days to obtain healthy plants ( Figure 5 B).

Claims

1. A method for rapidly inducing asexual reproduction of Bougainvillea, characterized in that: The following steps are involved: S1. Select full and uncontaminated seeds, sterilize them, and inoculate them on the culture medium for embryo germination; S2, cutting the hypocotyl obtained in S1 into small segments of 0.5-1 cm, and transferring them into callus induction medium, wherein the callus induction medium is MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L phytagel; S3, taking out the callus tissue obtained in S2 and transferring it to a differentiation medium, wherein the differentiation medium is MS+2 mg / L 6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L phytagel; S4, cutting the adventitious buds obtained in S3 from the base and inoculating them into a culture medium, wherein the culture medium is MS+2 mg / L6BA+0.2 mg / L 2,4-D+30 g / L sucrose+3 g / L plant gel; S5. Inoculate the adventitious buds obtained in S4 into a rooting medium, wherein the culture medium is: 1 / 2MS+1.25mg / L NAA+0.75mg / L IBA+30g / L sucrose+3g / L plant gel, and obtain regenerated plants of Bougainvillea bougainvillea.

2. The method according to claim 1, characterized in that: The culture medium described in step S1 is MS+30 g / L sucrose+3 g / L plant gel.

3. The method according to claim 1, characterized in that: The culture conditions of the callus induction medium in step S2 are: temperature 25-28° C., relative humidity 50-60%, and dark culture for 20-30 days.

4. The method according to claim 1, characterized in that: The culture conditions of the differentiation medium in step S3 are: temperature 25-28° C., relative humidity 50-60%, and 16 h photoperiod.

5. The method according to claim 1, characterized in that: The culture conditions of the culture medium in step S4 are: temperature 25-28° C., relative humidity 50-60%, and 16 h photoperiod.

6. The method according to claim 1, characterized in that: The culture conditions of the culture medium in step S5 are: temperature 25-28° C., relative humidity 50-60%, and 16 h photoperiod.

7. The method according to any one of claims 1 to 6, characterized in that: The method for asexual reproduction of Bougainvillea serrata also comprises the following steps: S6. Open the lid of the culture bottle and train the seedlings outdoors for 8-10 days; S7. Clean the culture medium of S6 seedlings and finally transplant them into the soil.

8. The method according to any one of claims 7, characterized in that: The pH of the culture medium in steps S1-S5 is 5.

8.

9. The method according to any one of claims 8, characterized in that: The variety of the bougainvillea is Bougainvillea 'Light violet'.

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