CN116982559B - Test tube flowering induction culture method for primula sikkimensis - Google Patents

Abstract

The invention relates to a test tube flowering induction culture method of primula, belonging to the technical field of plant tissue culture. The culture medium of the invention is Huabao No. 1 3g/L+NAA0.5mg/L+sucrose 20g/L+AC 1g/L+agar. 7g/L, pH 5.8, culture temperature 15℃, light intensity 1600lx, light time 12h/d. This is because the present invention can use the culture medium and culture method to induce primrose flowering in test tubes, has the advantages of simple experimental operation, short cycle, strong practicability, etc., and provides theoretical basis and technical support for the factory production of ornamental test tube flowers.

Description

A kind of in vitro flowering induction culture method of primrose

Technical field

The invention belongs to the technical field of plant tissue culture, and specifically relates to a test tube flowering induction culture method of primrose.

Background technique

Primula sieboldii belongs to the genus Primula in the family Primulaceae. It is a perennial herb and is mainly distributed in the three northeastern provinces and eastern Inner Mongolia. The leaves are basal, oblong or truncate, rosette-shaped, the scapes are upright, the umbels are terminal, the flowers are dense, the corolla is purplish red to light red, the flowers are brightly colored, the overall appearance is very elegant and graceful, and they are often used in indoor potted plants and garden structures. Used for landscapes and fresh cut flowers, it has high horticultural ornamental value.

In vitro flowers are a technology that uses plant tissue culture technology to cultivate plants in artificially controlled sterile environmental conditions in portable, miniature, transparent and airtight containers with good ornamental properties. Compared with traditional horticultural planting methods, it is a very novel and unique way of flower viewing and cultivation. It has the advantages of not being restricted by seasons, avoiding diseases and natural disasters, and being easy to manage. It satisfies the current young people's desire for novelty, novelty and novelty, and has broad development prospects. In vitro flowering refers to using tissue culture technology to allow plants to complete the flowering process in a closed culture container. It is an important part of in vitro flowers. In recent years, the mainstream of the market is mainly in vitro flowers with long growth cycles and long-lasting foliage and plant types. The varieties are monotonous. However, the in vitro flower cultivation technology of ornamental flowers is still limited in terms of species selection, flowering induction, flowering phenology, etc. There are difficulties. The establishment of the Primrose in vitro flowering experimental system provides a simple and controllable experimental platform for further research on physiological metabolism, gene differential expression, and in vitro hybrid germplasm innovation during the transition from vegetative growth to reproductive growth of Primula plants. It also provides a convenient and controllable experimental platform for in vitro flowers. Market diversification provides more possibilities. At present, there are no reports on the in vitro flowering induction of primrose.

Contents of the invention

In order to overcome the problems existing in the background technology, the present invention provides a method for inducing flowering of primrose in vitro. The flowering induction medium can be used to induce flowering of primrose in vitro. It has the advantages of simple experimental operation, short cycle, strong practicability, etc. , providing theoretical basis and technical support for the factory production of ornamental test-tube flowers.

In order to achieve the above objects, the present invention is achieved through the following technical solutions:

The described test tube flowering induction culture method of Primrose is cultured by the following method, which specifically includes the following steps:

1) Cut the petioles of primrose sterile seedlings into 1cm length and inoculate them on induction medium for callus induction. Adventitious buds will differentiate after 30 days of subculture. The induction medium is MS+BA0.5mg/L+NAA0. 2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8;

2) Inoculate the differentiated adventitious buds onto the proliferation medium for adventitious bud proliferation. The proliferation medium is MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC 1g/L+agar 7g/L. , pH is 5.8;

3) Inoculate the proliferated individual adventitious buds onto the rooting medium for rooting culture. The rooting medium is 1/2MS+NAA0.5mg/L+sucrose 30g/L+AC 1g/L+agar 7g/L. pH is 5.8;

4) Inoculate the rooted seedlings with 9-10 leaves onto the flowering induction medium for in vitro flowering induction. The flowering induction medium is Huabao No. 1 3g/L+NAA0.5mg/L+sucrose 20g/L+AC 1g /L + agar 7g/L, pH 5.8, culture temperature 15°C, light intensity 1600lx, light time 12h/d. Flower buds will form after 60 days of culture, and flowers will bloom for about 20 days after culture.

Preferably, the callus induction culture conditions described in step 1) are temperature 25±2°C, light intensity 1600lx, and light time 12h/d.

Preferably, the adventitious bud proliferation culture conditions described in step 2) are temperature 25±2°C, light intensity 1600lx, and light time 12h/d.

Preferably, the culture conditions for rooting culture in step 3) are temperature 25±2°C, light intensity 1600lx, and light time 12h/d.

Beneficial effects of the present invention:

Through the tissue culture technology and the reasonable preparation of culture medium components and plant growth regulators, the present invention can induce primrose to bloom in vitro, solving the problem that the mainstream of in vitro flowers in the current market is characterized by long growth cycle and long playing time for foliage and plant growth. Based on the type-based problem, through callus induction, adventitious bud proliferation, rooting culture and in-vitro flowering induction, the flower-viewing test-tube flower primrose flower is provided. It has the advantages of simple experimental operation, short cycle, strong practicability, etc., and is a flower-viewing type. Provide theoretical basis and technical support for factory production of test-tube flowers.

Description of the drawings

Figure 1 is the test tube flowering induction process of Primrose of the present invention;

In the figure, a represents primrose test tube seedlings, b represents petiole callus and induced differentiation, c represents adventitious bud proliferation, d represents rooting culture, e represents the test tube flowering plant, f represents the test tube flowering plant in a culture bottle with a height of 10cm, g means the test-tube flowering plant in a culture bottle with a height of 7cm, and h means a flowering plant in a test-tube in a culture bottle with a height of 6cm.

Implementation

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings to facilitate the understanding of the skilled person.

The test tube seedlings of Primula sieboldii (Figure 1a) applied for in the in vitro plant bank are used as materials. The materials are sterile clones established using primula seed materials collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in China. Southwest Wildlife Germplasm Resource Bank Plant In Vitro Bank. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the petiole of the primrose sterile seedling obtained as requested to 1cm in length and inoculate it on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 99.17%. After the calli were subcultured for 30 days, adventitious buds differentiated from the calli (Figure 1b), and the induction rate was 79.66%.

The differentiated adventitious buds were inoculated onto the proliferation medium (MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for adventitious bud proliferation (Figure 1c). The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the proliferation coefficient is 3.17 after 30 days.

The proliferated individual adventitious buds were inoculated into rooting medium (1/2MS+NAA0.5mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for rooting culture (Figure 1d). The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the rooting rate reaches 100% after 30 days.

Inoculate the rooted seedlings with 9-10 leaves into the flowering induction medium (Huabao No. 1 3g/L+ NAA0.5mg/L+sucrose 20g/L+AC 1g/L+agar 7g/L, pH 5.8) for test tubes Flowering induction, the diameter of the induced flowering vase is 7.0cm, the height is 11cm, the culture temperature is 15℃, the light intensity is 1600lx, and the light time is 12h/d. After 60 days of culture, flower buds will form, and the flowers will bloom for about 20 days after the culture (Figure 1e), umbels, The test tube flowering induction rate reached 73.33%.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the petiole of the primrose sterile seedling obtained as requested to 1cm in length and inoculate it on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 99.17%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 79.66%.

The differentiated adventitious buds were inoculated into the proliferation medium (MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for adventitious bud proliferation. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the proliferation coefficient is 3.17 after 30 days.

The proliferated individual adventitious buds were inoculated into rooting medium (1/2MS+NAA0.5mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for rooting culture. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the rooting rate reaches 100% after 30 days.

Inoculate the rooted seedlings with 9-10 leaves into the flowering induction medium (1/2MS+NAA0.5mg/L+activated carbon 1g/L+sucrose 20g/L+agar 6g/L, pH 5.8) for in vitro flowering induction. The flowering vase has a diameter of 7.0cm and a height of 11cm. The culture temperature is 15°C, the light intensity is 1600lx, and the light time is 12h/d. The plants have not bloomed after 90 days of culture.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the petiole of the primrose sterile seedling obtained as requested to 1cm in length and inoculate it on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 99.17%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 79.66%.

The differentiated adventitious buds were inoculated into the proliferation medium (MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for adventitious bud proliferation. The culture temperature was 25±2℃, the light intensity was 1600lx, the light time was 12h/d, and the proliferation coefficient was 3.17 after 30 days.

The proliferated individual adventitious buds were inoculated into rooting medium (1/2MS+NAA0.5mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for rooting culture. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the rooting rate reaches 100% after 30 days.

Inoculate the rooted seedlings with 9-10 leaves into the flowering induction medium (MS+NAA0.5mg/L+activated carbon 1g/L+sucrose 20g/L+agar 6g/L, pH 5.8) for in vitro flowering induction, and induce flowering vase The diameter is 7.0cm, the height is 11cm, the culture temperature is 15℃, the light intensity is 1600lx, and the light time is 12h/d. The plants have not bloomed after 90 days of culture.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the petiole of the primrose sterile seedling obtained as requested to 1cm in length and inoculate it on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 99.17%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 79.66%.

The differentiated adventitious buds were inoculated into the proliferation medium (MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for adventitious bud proliferation. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the proliferation coefficient is 3.17 after 30 days.

The proliferated individual adventitious buds were inoculated into rooting medium (1/2MS+NAA0.5mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for rooting culture. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the rooting rate reaches 100% after 30 days.

Inoculate the rooted seedlings with 9-10 leaves into the flowering induction medium (Huabao No. 1 3g/L+ NAA0.5mg/L+sucrose 20g/L+AC 1g/L+agar 7g/L, pH 5.8) for test tubes For flowering induction, the induced flowering vase has a diameter of 7.0cm and a height of 11cm. The test tube flowering culture temperature is 25°C, the light intensity is 1600lx, and the light time is 12h/d. The plants did not bloom after 90 days of culture.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings to establish sterile clones.

Cut the leaves of the primrose sterile seedlings obtained by application into 1cm length and inoculate them on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 98.48%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 28.73%. There is no significant difference between the callus induction rate and Example 1, but the adventitious bud differentiation rate is extremely significantly lower than that of Example 1.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the root tips of the primrose sterile seedlings obtained by application into 1cm length and inoculate them on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for curing. For wound induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 98.55%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 34.33%. There is no significant difference between the callus induction rate and Example 1, but the adventitious bud differentiation rate is extremely significantly lower than that of Example 1.

Apply for primula (Primula sieboldii) test tube seedlings in the in vitro plant bank as materials. The material is a sterile clone established using primula seed material collected from Heilongjiang in 2017 and stored in the form of test tube seedlings in Southwest China Wild Species Quality resource library plant in vitro library. The specific method is: use primula seeds as raw materials, sterilize the surface of the seeds and germinate the sterile seedlings aseptically to establish sterile clones.

Cut the petiole of the primrose sterile seedling obtained as requested to 1cm in length and inoculate it on the induction medium (MS+BA0.5mg/L+NAA0.2mg/L+sucrose 30g/L+agar 6g/L, pH 5.8) for callus For induction, the culture temperature was 25±2℃, the light intensity was 1600lx, and the light time was 12h/d. The statistical callus induction rate after 30 days was 99.17%. After the calli were subcultured for 30 days, adventitious buds differentiated from the callus, and the induction rate was 79.66%.

The differentiated adventitious buds were inoculated into the proliferation medium (MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for adventitious bud proliferation. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the proliferation coefficient is 3.17 after 30 days.

The proliferated individual adventitious buds were inoculated into rooting medium (1/2MS+NAA0.5mg/L+sucrose 30g/L+AC1g/L+agar 7g/L, pH 5.8) for rooting culture. The culture temperature is 25±2℃, the light intensity is 1600lx, the light time is 12h/d, and the rooting rate reaches 100% after 30 days.

Inoculate the rooted seedlings with 9-10 leaves into the flowering induction medium (Huabao No. 1 3g/L+ NAA0.5mg/L+sucrose 20g/L+AC 1g/L+agar 7g/L, pH 5.8) for test tubes Flowering induction, the diameter of the induced flowering culture bottle is 4.5cm, the height is 10cm, 7cm, and 6cm respectively, and the outer leaves are cut off. The culture temperature is 15℃, the light intensity is 1600lx, and the light time is 12h/d. Flower buds will form after 60 days of culture. Continue the culture for 20 days. The flowers on the left and right are open. The inflorescence in the culture bottle with a height of 10cm has two flowers (Figure 1f), and the inflorescence in the culture bottle with a height of 6cm and 7cm has a single flower (Figure 1g-h).

Note: Different lowercase letters indicate significant differences in the same column between treatments at the 0.05 level, capital letters indicate extremely significant differences at the 0.01 level, the same below.

Table 1 Effects of different flowering induction media on flowering induction of Primrose in vitro

Table 2 Comparison of callus induction and differentiation of different explants

Table 3 Effects of different types of culture bottles on flowering induction of primrose in vitro

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be implemented in the form and Various changes can be made to the details without departing from the scope of the invention as defined by the claims.

Claims (4)

1. A primula forbesii test tube flowering induction culture method is characterized in that: the cultivation is carried out by the following method, which specifically comprises the following steps:

1) Cutting leaf stalks of primula sikkimensis aseptic seedlings into 1cm lengths, inoculating the leaf stalks on an induction culture medium for callus induction, and differentiating adventitious buds after subculturing for 30d, wherein the induction culture medium comprises MS+BA0.5mg/L+NAA0.2mg/L+30 g/L of sucrose+6 g/L of agar, and the pH value is 5.8;

2) Inoculating the differentiated adventitious buds to a proliferation culture medium for adventitious bud proliferation, wherein the proliferation culture medium is MS+BA1.0mg/L+NAA0.2mg/L+sucrose 30 g/L+AC1g/L+agar 7g/L, and the pH is 5.8;

3) Inoculating the proliferated adventitious bud single plant to a rooting culture medium for rooting culture, wherein the rooting culture medium is 1/2MS+NAA0.5mg/L+30 g/L of sucrose+AC1g/L+7 g/L of agar, and the pH value is 5.8;

4) Inoculating rooting seedling with 9-10 leaves to flowering induction culture medium for test tube flowering induction, wherein the flowering induction culture medium is flower bud No. 1, 3g/L, NAA0.5mg/L, sucrose 20g/L, AC 1g/L and agar 7g/L, the pH is 5.8, the culture temperature is 15 ℃, the illumination intensity is 1600lx, the illumination time is 12h/d, flower buds are formed after 60d of culture, and the flowers are continuously cultured for about 20d to bloom.

2. The primula forbesii test tube flowering induction culture method according to claim 1, wherein the method comprises the following steps of: the callus induction culture condition in the step 1) is that the temperature is 25+/-2 ℃, the illumination intensity is 1600lx, and the illumination time is 12h/d.

3. The primula forbesii test tube flowering induction culture method according to claim 1, wherein the method comprises the following steps of: the proliferation culture condition of the adventitious buds in the step 2) is that the temperature is 25+/-2 ℃, the illumination intensity is 1600lx, and the illumination time is 12h/d.

4. The primula forbesii test tube flowering induction culture method according to claim 1, wherein the method comprises the following steps of: and 3) culturing conditions of rooting culture are that the temperature is 25+/-2 ℃, the illumination intensity is 1600lx, and the illumination time is 12h/d.