A method for rapidly inducing asexual reproduction of bougainvillea

Abstract

The present invention specifically discloses a method for tissue culture regeneration of bougainvillea through asexual reproduction and its culture medium, which mainly includes the following steps: After disinfecting the seeds, inoculate them into the culture medium for embryo germination: MS + 30 g / L sucrose + 3 g / L phytagel, and then inoculate them into the optimal callus culture medium: MS + 2 mg / L 6-BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel. After dark culture for 20 - 30 days, transfer them to the optimal differentiation culture medium: MS + 2 mg / L 6-BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel, and move them to a culture condition with a light intensity of 2500 lx, a light duration of 16 h, and a temperature of 25 °C for 20 - 30 days to obtain adventitious buds with a differentiation of about 1 cm. The efficient tissue culture regeneration system of bougainvillea established by the present invention can provide important technical references for subsequent genetic transformation and functional research of bougainvillea.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a method for tissue culture and regeneration of Bougainvillea spectabilis Willd. Background Art

[0002] Bougainvillea spectabilis Willd. is a plant of the genus Bougainvillea in the family Nyctaginaceae. There are about 1000 cultivated varieties globally. It is a widely popular ornamental plant and has important economic value. It can be applied in parks, desert gardens, home and institutional gardens in the forms of shrubs, hedges, shaped plants, potted plants, bonsai, hanging baskets, wind curtains, landscapes, and climbing on pergolas, trees, riverbanks, and difficult-to-maintain slopes, etc., and has the laudatory name of "Garden Glory". Therefore, Bougainvillea spectabilis Willd. has attracted the attention of plant lovers and breeders around the world (Datta, 2022).

[0003] Surveys show that Bougainvillea spectabilis Willd. is the provincial flower of Hainan Province and also the city flower of Sanya City. Due to the difficulties in cross-breeding of Bougainvillea spectabilis Willd., the seed setting rate is extremely low, and the activity of the embryo is not high. It is difficult to cultivate new varieties and breed seedlings through cross-breeding means (Zhou Qun et al., 2016). It is imperative to find other breeding methods. In order to change this situation, we need to perform targeted modification on the existing Bougainvillea spectabilis Willd. varieties. Therefore, establishing a molecular breeding improvement system for Bougainvillea spectabilis Willd. is a necessary means to solve this situation.

[0004] Currently, the breeding methods of Bougainvillea spectabilis Willd. at home and abroad still remain at methods such as cutting and grafting. Through the method of tissue culture and regeneration, it only stays at using the stem segments with bud points (Gong Wei et al., 2005; Chen Xuan et al., 2024), shoot tips with bud points (Guo Haibin and Lei Jiajun, 2006), seeds (Pan Mei et al., 2017) of Bougainvillea spectabilis Willd. as explants, and establishing a rapid propagation system of Bougainvillea spectabilis Willd. to obtain complete plants by adjusting the types and concentrations of auxin and cytokinin in the culture medium at different stages. Currently, there is still no research on the induction of callus and further differentiation induction of callus. It will provide a new way for the functional research of key genes of Bougainvillea spectabilis Willd. and genetic engineering breeding, provide a new experimental material for the functional research of important genes of Bougainvillea spectabilis Willd. and targeted molecular improvement of important traits, and at the same time provide reference for the culture and propagation of Bougainvillea spectabilis Willd. callus. The method for inducing embryogenic callus of Bougainvillea spectabilis Willd. provided by the present invention is not only applicable to the research of the genetic transformation system of Bougainvillea spectabilis Willd., but also applicable to the rapid propagation of Bougainvillea spectabilis Willd. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for inducing embryogenic callus of Bougainvillea spectabilis Willd. to plant regeneration and its culture medium. The method is convenient and simple, and the whole process only requires 2 culture media and the same culture environment to complete the tissue culture and regeneration of Bougainvillea spectabilis Willd.

[0006] The object of the present invention is achieved through the following solutions: A method and culture medium for inducing tissue culture regeneration of bougainvillea, comprising the following steps:

[0007] Step S1, obtaining hypocotyls for inducing callus: Pick bougainvillea fruits, take their seeds, select plump and pollution-free seeds. First, wash the surface dust with detergent and rinse with running water for 20 - 30 min. Then, transfer the seeds to a sterile bottle in a laminar flow hood, wash them 3 times with sterile water for 1 min each time; disinfect with 1% sodium hypochlorite solution for 25 - 30 min; after disinfection, rinse with sterile water 5 times for 1 min each time; after disinfection, inoculate onto the medium for seed embryo germination: MS + 30 g / L sucrose + 3 g / L phytagel;

[0008] Step S2, callus induction: Cut the hypocotyls obtained in S1 into segments of 0.5 - 1 cm, transfer them to the callus induction medium MS + 2 mg / L 6 - BA + 0.2 mg / L 2,4 - D + 30 g / L sucrose + 3 g / L phytagel, and culture them in the dark for 20 - 30 d;

[0009] Step S3, inducing adventitious buds from callus: Take out the callus obtained in S2 and transfer it to the optimal differentiation medium: MS + 2 mg / L 6 - BA + 0.2 mg / L 2,4 - D + 30 g / L sucrose + 3 g / L phytagel, and culture it under light conditions for 20 - 30 d to obtain adventitious buds about 1 - 2 cm in differentiation;

[0010] Step S4, strong seedling culture: Cut the adventitious buds obtained in S3 from the callus and inoculate them into the strong seedling medium for strong seedling culture: MS + 2 mg / L 6 - BA + 0.2 mg / L 2,4 - D + 30 g / L sucrose + 3 g / L phytagel, and obtain adventitious buds about 2 - 3 cm high after about 30 d;

[0011] Step S5, rooting culture: Cut the adventitious buds obtained in S4 from the callus and inoculate them into the rooting medium for rooting culture: 1 / 2MS + 1.25 mg / L NAA + 0.75 mg / L IBA + 30 g / L sucrose + 3 g / L phytagel, and obtain regenerated plants of bougainvillea after about 30 d;

[0012] Step S6, acclimatization and transplantation: Uncover the tissue - cultured seedlings obtained in S5 for acclimatization for about 8 - 10 d, then wash off the phytagel on the roots and transplant them into nutrient soil, and cultivate for about 30 d to obtain robust plants.

[0013] In the culture environment in the above steps S1 to S5, the temperature is 25 - 28 °C, the relative humidity is 50 - 60%, and in steps S3 to S5, the light duration is 16 h / d and the light intensity is 2500 lx.

[0014] The pH values of the seed germination medium, callus induction medium, differentiation medium, seedling strengthening medium, and rooting medium are all 5.8.

[0015] The present invention uses the hypocotyl of aseptic seedlings of Bougainvillea as explants, quickly induces callus, differentiates seedlings, and then conducts rooting culture on the small seedlings to obtain in vitro regenerated plants. The most important thing in the present invention is to obtain embryogenic callus with differentiation ability from the embryo regeneration pathway, realize rapid propagation, shorten its breeding years, provide technical reference for the construction of the genetic transformation system of Bougainvillea in the future, and the method of the present invention is simple and fast. The variety of Bougainvillea in the present invention is Bougainivillea ‘Light violet’. Description of the Drawings

[0016] Figure 1 It is the induction process of the hypocotyl of Bougainvillea seeds.

[0017] Figure 2 It is the induction of callus from the hypocotyl of Bougainvillea; Scale bar: 1 cm.

[0018] Figure 3 It is the differentiation of adventitious buds from the callus of Bougainvillea; Scale bar: 1 cm.

[0019] Figure 4 It is the seedling strengthening and rooting culture of adventitious buds of Bougainvillea.

[0020] Figure 5 It is the acclimatization and transplantation of tissue culture seedlings of Bougainvillea. Detailed Embodiment

[0021] The following is a further detailed description through specific embodiments:

[0022] The terms involved in the present invention:

[0023] 6-BA is 6-benzyladenine; IBA is indolebutyric acid; 2,4-D is 2,4-dichlorophenoxyacetic acid; KT is kinetin 6-glycoaminopurine; NAA is naphthaleneacetic acid; IAA is 3-indoleacetic acid; MS basal medium is 4.4 g / L; 1 / 2MS medium is 2.2 g / L; The reagents and the like used in the examples of the present invention can all be obtained through commercial channels.

[0024] Example 1 Preparation Method of Bougainivillea glabra ‘Light violet’

[0025] Using Bougainvillea glabra ‘Mrs.Eva White’ as the female parent, the stamens were removed and the flower was bagged before the pollen matured. When it was observed that there was mucus secreted from the top of the carpel stigma, mature pollen of Bougainvillea glabra ‘New River’ was selected, and pollination was carried out with a pollination stick and then bagged. After the bracts and flowers completely withered, the seeds were collected.

[0026] Example 2 Propagation method of Bougainivillea ‘Light violet’

[0027] This example provides a method and medium for tissue culture and regeneration of Bougainvillea, including the following operating steps:

[0028] Step S1: Obtain the hypocotyl for inducing callus

[0029] Pick the fruits of Bougainvillea, take their seeds, and select plump and pollution-free seeds. First, wash the surface dust with detergent and rinse with running water for 20 - 30 min. Then transfer the seeds to a sterile bottle in a laminar flow hood, wash them 3 times with sterile water, 1 min each time; then disinfect them with 1% sodium hypochlorite solution for 25 - 30 min; after disinfection, rinse them 5 times with sterile water, 1 min each time; place them on sterile filter paper to dry, and then inoculate them into the seed embryo germination medium MS + 30 g / L sucrose + 3 g / L phytagel, pH 5.8; the culture conditions are: temperature 25 - 28 °C, relative humidity 50 - 60%, dark culture for 10 - 14 d to obtain sterile seedlings with a height of about 4 - 5 cm, remove the roots and cotyledons of the seedlings to obtain the hypocotyl for inducing callus ( Figure 1 ).

[0030] Step S2: Induction of callus

[0031] Cut the hypocotyls obtained in step S1 into small segments about 0.5 - 1 cm in length, transfer them to the callus induction medium MS + 2 mg / L 6 - BA + 0.2 mg / L 2,4 - D + 30 g / L sucrose + 3 g / L phytagel, pH 5.8, inoculate 6 - 9 explants in each petri dish, and the petri dishes used are plastic petri dishes with a diameter of 90 mm and a height of 20 mm. The culture conditions are: temperature 25 - 28 °C, relative humidity 50 - 60%, dark culture for 20 - 30 d to obtain callus, count the induction rate of callus and observe and record the callus growth status, and replace the fresh callus induction medium every 15 d during this period.

[0032] The present invention tested the induction effects of six different hormone - ratio media on the hypocotyl callus of Bougainvillea and found that callus could be induced in all cases, but there were obvious differences in the effects (Table 1). 2 mg / L 6 - BA + 0.5 mg / L 2,4 - D ( Figure 2D) and 2 mg / L 6-BA + 0.2 mg / L 2,4-D( Figure 2 E) had an obvious induction effect, and buds could differentiate from 2 mg / L 6-BA + 0.2 mg / L 2,4-D on the 25th day of induction. 0.5 mg / L 6-BA + 1 mg / L KT( Figure 2 A), 0.5 mg / L 6-BA + 2 mg / L 2,4-D( Figure 2 B), 0.5 mg / L 6-BA + 4 mg / L 2,4-D( Figure 2 C) had poor callus induction effect after 25 days of induction, while using 2.5 mg / L 2,4-D alone( Figure 2 F) induced rooting.

[0033] Table 1 Induction effect of different media on hypocotyl callus

[0034]

[0035]

[0036] Step S3: Adventitious buds are induced from the callus

[0037] Transfer the callus obtained in the second step to the differentiation medium MS + 2 mg / L 6-BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel, pH 5.8, 6 - 9 in each petri dish. The used petri dish is a plastic petri dish with a diameter of 90 mm and a height of 20 mm. The culture conditions are: temperature 25 - 28 °C, relative humidity 50 - 60%, 16 h photoperiod. Buds differentiate after culturing for about 10 d, and adventitious buds are obtained after 30 d. During this period, the new callus induction medium is replaced every 15 d.

[0038] The present invention tested the induction effect of four different hormone ratio media on adventitious buds and found that 0.5 mg / L 6-BA + 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 + 0.05 mg / L NAA + 0.05 mg / L IAA( Figure 3 C) could only make the callus continue to grow but could not induce the differentiation of adventitious buds, while 2 mg / L 6-BA + 0.2 mg / L 2,4-D( Figure 3 D) could induce adventitious buds, with a short induction time, high efficiency, and an average induction and differentiation rate of 90% (Table 2), which was the best medium for inducing adventitious buds.

[0039] Table 2 Induction effect of different media on adventitious buds

[0040]

[0041] Step S4, strong seedling stage: Cut the robust adventitious buds obtained in step S3 from the base, and inoculate them into the strong seedling medium MS + 2 mg / L 6-BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel. The temperature is 25 - 28 °C, the relative humidity is 50 - 60%, and the photoperiod is 16 h. After about 30 days, adventitious buds with 8 - 10 true leaves are obtained ( Figure 4 A - B);

[0042] Step S5, rooting culture: Inoculate the adventitious buds obtained in S4 into five different hormone - ratio media for rooting of adventitious buds. The culture conditions are: temperature 25 - 28 °C, relative humidity 50 - 60%, photoperiod 16 h. After culturing for about 30 days, it is found that 1 / 2MS + 1.25 mg / L NAA + 0.75 mg / L IBA + 30 g / L sucrose + 3 g / L phytagel can induce adventitious buds to root, and the average rooting rate is 95% (Table 3), and the root system grows robustly ( Figure 4 C, D).

[0043] Table 3 Rooting effects of different media on adventitious buds

[0044]

[0045] Cut the robust adventitious buds obtained in step S3 from the base, remove the excess callus, and inoculate them into the rooting medium, 4 - 6 in each tissue culture bottle. The culture conditions are: temperature 25 - 28 °C, relative humidity 50 - 60%, photoperiod 16 h. The present invention tests step S6, acclimatization and transplantation

[0046] Acclimatize the tissue - cultured seedlings obtained in step S3 by opening the caps for about 10 days ( Figure 5 A), then wash off the phytagel on the roots, transplant them into the sterilized soil, and cultivate for about 30 days to obtain robust plants ( Figure 5 B).

Claims

1. A method for rapidly inducing asexual reproduction of bougainvillea, characterized in that, The bougainvillea variety is Bougainivillea ‘Light violet’. Bougainivillea ‘Light violet’ is obtained by pollinating Bougainvillea glabra 'Mrs.Eva White' as the female parent with mature pollen of Bougainvillea glabra ‘NewRiver’. The method includes the following steps: S1. Select plump and pollution-free seeds. After disinfection, inoculate them onto the medium for embryo germination. S2. Cut the hypocotyls obtained in S1 into segments of 0.5 - 1 cm and transfer them to the callus induction medium, which is MS + 2 mg / L 6BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel. S3. Take out the callus obtained in S2 and transfer it to the differentiation medium, which is MS + 2 mg / L 6BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel. S4. Cut the adventitious buds obtained in S3 from the base and inoculate them into the medium, which is MS + 2 mg / L 6BA + 0.2 mg / L 2,4-D + 30 g / L sucrose + 3 g / L phytagel. S5. Inoculate the adventitious buds obtained in S4 into the rooting medium. The medium is: 1 / 2MS + 1.25 mg / L NAA + 0.75 mg / L IBA + 30 g / L sucrose + 3 g / L phytagel, and obtain the regenerated plants of bougainvillea. And the pH of the medium in steps S1 - S5 is 5.

8.

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

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 d.

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, wherein: The culture conditions of the 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 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 the bougainvillea also includes the following steps: S6. Open the lid of the culture bottle outdoors for acclimatization for 8 - 10 days. S7. Wash the seedlings in S6 with the medium and finally transplant them into the soil.

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