Method for acquiring regeneration seedlings of dove trees through tissue culture by taking dove tree leaves as explants

[0036] Example 1

[0037] In this example, young Davidia involucrata leaves were collected from adult Davidia involucrata plants in Yinchanggou Scenic Area, Pengzhou City, Sichuan Province (see figure 1 ), the sterilization operation of the young Davidia involucrata leaves is as follows: the detergent (white cat brand) is made into a dilution with tap water, the volume ratio of the detergent to the tap water is 1:1000, and the collected young The leaves of Davidia involucrata were soaked in the detergent diluent for 10 minutes, then taken out and rinsed with tap water for 1 h to clean the surface debris, then sterilized with ethanol with a mass concentration of 75% under aseptic conditions for 30 seconds, and then put in a 0.1% mass concentration Sterilize in mercury chloride aqueous solution for 4 minutes, then take it out and rinse it with sterile water 5 times to complete the sterilization. Cut the sterilized Davidia involucrata leaves with scissors to a size of about 1 cm 2 The small pieces are used for inoculation.

[0038] In this embodiment, the steps of the method for obtaining regenerated seedlings of Davidia involucrata by tissue culture using Davidia involucrata leaves as explants are as follows:

[0039] (1) Obtained callus from leaves of Davidia involucrata

[0040] Use a 100ml Erlenmeyer flask, fill each bottle with about 40ml callus induction medium, inoculate 5-6 small pieces of sterilized Davidia involucrata leaves (with the back of the leaf facing up), and cultivate for 25 days in the dark to obtain recovery of Davidia involucrata leaves. Wounded tissue (see figure 2 ), the callus induction medium uses MS as the basic medium, and each liter of callus induction medium contains 1.5 mg of indole butyric acid, 0.5 mg of 6-benzylamino adenine, 20 g of sucrose and 5 g of agar powder, Its pH value is controlled at 5.8;

[0041] (2) Callus proliferation

[0042] The callus of Davidia involucrata leaves obtained in step (1) was inoculated on the callus proliferation medium and cultured in the dark for 18 days to enlarge the callus (see image 3 ), the callus proliferation medium is based on MS, and each liter of callus proliferation medium contains 1.5 mg of indole butyric acid, 1 mg of 6-benzylamino adenine, 20 g of sucrose and 5 g of agar powder. The pH value is controlled at 5.8;

[0043] (3) Inducing clumping buds

[0044] Cut the callus of Davidia involucrata leaves after proliferation in step (2) and inoculate it on the bud differentiation medium, and cultivate it for 25 days under the light conditions of 1500~2000lx light intensity and 10h/d light time to induce clumping buds. The bud differentiation medium uses MS as the basic medium, and each liter of bud differentiation medium contains 0.5 mg of indole butyric acid, 3 mg of 6-benzylamino adenine, 20 g of sucrose and 5 g of agar powder, and its pH value is controlled at 5.8;

[0045] (4) Proliferation of cluster buds

[0046] After cutting the clump buds obtained in step (3), inoculate them on a bud multiplication medium, and cultivate them for 25 days under the light conditions of 1500~2000lx light intensity and 10h/d light time to make the clump buds grow. Based on MS as the basic medium, each liter of bud proliferation medium contains 0.1mg of 6-benzylamino adenine, 0.1mg of gibberellin, 20g of sucrose and 5g of agar powder, and its pH value is controlled at 5.8;

[0047] (5) Induced rooting

[0048] Cut the shoots with a height of 2.5 cm or more from the multiplication buds in step (4), inoculate them in the first rooting medium and cultivate for 3 days, then transfer to the second rooting medium and cultivate for 25 days to get roots. For transplantable Davidia involucrata regenerated seedlings, the light conditions when cultured with the above two rooting media are: light intensity of 1500~2000lx, light time of 10h/d, and the first rooting medium is based on 1/2MS medium Medium, each liter of the first rooting medium contains 5 mg of auxin indole acetic acid, 15g of sucrose and 5g of agar powder, and its pH value is controlled at 5.8. The second rooting medium is cultured on the basis of 1/2MS medium The second rooting medium contains 2g activated carbon, 15g sucrose and 5g agar powder per liter of the second rooting medium, and its pH value is controlled at 5.8.

[0049] In the above steps (1) to (5), the ambient temperature is controlled at 22°C, and the ambient humidity is controlled at 70%.

[0050] (6) Transplant

[0051] Transfer the regenerated Davidia involucrata seedlings that can be transplanted after rooting to natural room temperature for 4 days, take out the seedlings, rinse the second rooting medium with tap water, soak them in a 1000-fold diluted carbendazim solution for 1 hour, and then transplant them under high pressure Sterilized nutrient soil: vermiculite mass ratio = 2:1 on the cultivation substrate.

[0052] In this example, the cluster bud differentiation rate was 50%, the rooting rate was 100%, and the transplanting survival rate was 91%.

[0053] Example 2

[0054] In this example, young Davidia involucrata leaves were collected from adult Davidia involucrata plants in Emeishan Scenic Area, Emeishan City, Sichuan Province, and the sterilization operation of the young Davidia involucrata leaves was the same as in Example 1. Cut the sterilized Davidia involucrata leaves with scissors to a size of about 1 cm 2 The small pieces are used for inoculation.

[0055] In this embodiment, the steps of the method for obtaining regenerated seedlings of Davidia involucrata by tissue culture using Davidia involucrata leaves as explants are as follows:

[0056] (1) Obtained callus from leaves of Davidia involucrata

[0057] Use a 100ml Erlenmeyer flask, fill each bottle with about 40ml callus induction medium, inoculate 5~6 small pieces of sterilized Davidia involucrata leaves (with the back of the leaf facing up), and cultivate for 28 days in the dark to obtain recovery of Davidia involucrata leaves. Wound tissue, the callus induction medium uses MS as the basic medium, and each liter of callus induction medium contains 2 mg of indole butyric acid, 1 mg of 6-benzylamino adenine, 30 g of sucrose and 5.2 g of agar powder. Its pH value is controlled at 6.0;

[0058] (2) Callus proliferation

[0059] The callus of Davidia involucrata leaves obtained in step (1) was inoculated on a callus proliferation medium, and cultured in the dark for 21 days to enlarge the callus. The callus proliferation medium was cultured on the basis of MS Base, each liter of callus proliferation medium contains 2.5 mg of indole butyric acid, 1.5 mg of 6-benzylamino adenine, 30 g of sucrose and 5.2 g of agar powder, and the pH value is controlled at 6.0;

[0060] (3) Inducing clumping buds

[0061] The callus of Davidia involucrata leaves after step (2) proliferation is cut and inoculated on the bud differentiation medium, and cultivated for 30 days under light conditions of 1500~2000lx light intensity and 12h/d light time to induce clumping buds (see Figure 4 , 5 ), the bud differentiation medium is based on MS, and each liter of bud differentiation medium contains 0.33 mg of indole butyric acid, 5 mg of 6-benzylamino adenine, 30 g of sucrose and 5.2 g of agar powder, and its pH value is controlled At 6.0;

[0062] (4) Proliferation of cluster buds

[0063] Cut the cluster buds obtained in step (3) and inoculate them on the bud multiplication medium (see Image 6 ), cultivate for 30 days under the light conditions of light intensity of 1500~2000lx and light time of 12h/d to make cluster buds grow (see Figure 7 ), the bud proliferation medium is based on MS, and each liter of bud proliferation medium contains 0.25 mg of 6-benzylamino adenine, 0.5 mg of gibberellin, 30 g of sucrose and 5.2 g of agar powder, and its pH value is controlled At 6.0;

[0064] (5) Induced rooting

[0065] Cut off the shoots with a height of 2.5 cm or more from the cluster buds after step (4), and inoculate them in the first rooting medium for 4 days (see Figure 8 ), then transfer to the second rooting medium and cultivate the regenerated Davidia involucrata seedlings that can be transplanted after rooting for 30 days. The light conditions when culturing with the above two rooting media are: light intensity 1500~2000lx, light time 12h /d, the first rooting medium uses 1/2MS medium as the basic medium, and each liter of the first rooting medium contains 10 mg of auxin indole butyric acid, 20 g of sucrose and 5.2 g of agar powder, and its pH value is controlled At 6.0, the second rooting medium uses 1/2MS medium as the basic medium, and each liter of the second rooting medium contains 1 g of activated carbon, 15 g of sucrose and 5.2 g of agar powder, and the pH value is controlled at 6.0.

[0066] In the above steps (1) to (5), the ambient temperature is controlled at 24°C, and the ambient humidity is controlled at 60%.

[0067] (6) Transplant

[0068] Transfer the regenerated Davidia involucrata seedlings that can be transplanted after rooting to natural room temperature for 4 days, take out the seedlings, rinse the second rooting medium with tap water, soak them in a 1000-fold diluted carbendazim solution for 1 hour, and then transplant them under high pressure Sterilized nutrient soil: vermiculite mass ratio = 2:1 on the cultivation substrate.

[0069] In this example, the cluster bud differentiation rate was 53%, the rooting rate was 100%, and the transplanting survival rate was 94%.

[0070] Example 3

[0071] In this example, young Davidia involucrata leaves were collected from adult Davidia involucrata plants in Longcanggou Nature Reserve, Yingjing County, Ya'an City, Sichuan Province, and the sterilization operation of the young Davidia involucrata leaves was the same as in Example 1. Cut the sterilized Davidia involucrata leaves with scissors to a size of about 1 cm 2 The small pieces are used for inoculation.

[0072] In this embodiment, the steps of the method for obtaining regenerated seedlings of Davidia involucrata by tissue culture using Davidia involucrata leaves as explants are as follows:

[0073] (1) Obtained callus from leaves of Davidia involucrata

[0074] Use a 100ml Erlenmeyer flask, fill each bottle with about 40ml callus induction medium, inoculate 5-6 small pieces of sterilized Davidia involucrata leaves (with the back of the leaf facing up), and cultivate for 30 days in the dark to obtain recovery of Davidia involucrata leaves. Wounded tissue (see figure 2 ), the callus induction medium is based on MS, and each liter of callus induction medium contains 2.5 mg of indole butyric acid, 1.5 mg of 6-benzylamino adenine, 30 g of sucrose and 5.8 g of agar powder , Its pH value is controlled at 6.1;

[0075] (2) Callus proliferation

[0076] The callus of Davidia involucrata leaves obtained in step (1) was inoculated on a callus proliferation medium and cultured for 28 days in the dark to enlarge the callus. The callus proliferation medium was cultured on the basis of MS Base, each liter of callus proliferation medium contains 2.5 mg of indole butyric acid, 2.5 mg of 6-benzylamino adenine, 30 g of sucrose and 5.8 g of agar powder, and its pH value is controlled at 6.1;

[0077] (3) Inducing clumping buds

[0078] The callus of Davidia involucrata leaves after the proliferation in step (2) is cut and inoculated on the bud differentiation medium, and cultured for 28 days under the light conditions of 1500~2000lx light intensity and 14h/d light time to induce clumping buds. The bud differentiation medium uses MS as the basic medium, and each liter of bud differentiation medium contains 1 mg of indole butyric acid, 10 mg of 6-benzylamino adenine, 30 g of sucrose and 5.8 g of agar powder, and its pH value is controlled at 6.1;

[0079] (4) Proliferation of cluster buds

[0080] After cutting the clump buds obtained in step (3), inoculate them on a bud proliferation medium, and cultivate them for 28 days under the light conditions of 1500~2000lx light intensity and 14h/d light time to make clump buds grow. Based on MS as the basic medium, each liter of bud proliferation medium contains 0.5mg of 6-benzylamino adenine, 0.5mg of gibberellin, 30g of sucrose and 5.8g of agar powder, and its pH value is controlled at 6.1;

[0081] (5) Induced rooting

[0082] Cut the shoots with a height of 2.5cm or more from the multiplication buds in step (4), inoculate them in the first rooting medium and cultivate for 6 days, then transfer them to the second rooting medium and cultivate for 28 days to get roots. Transplantable dovetree regenerated seedlings (see Picture 9 , 10 11), the light conditions when culturing with the above two rooting media are: light intensity is 1500~2000lx, light time is 14h/d, the first rooting medium uses 1/2MS medium as the basic medium, One liter of the first rooting medium contains 15mg of auxin naphthalene acetic acid, 20g of sucrose and 5.8g of agar powder, and its pH value is controlled at 6.1. The second rooting medium uses 1/2MS medium as the basic medium, and each liter of The two rooting medium contains 1g of activated carbon, 20g of sucrose and 5.8g of agar powder, and its pH value is controlled at 6.1.

[0083] In the above steps (1) to (5), the ambient temperature is controlled at 26°C, and the ambient humidity is controlled at 90%.

[0084] (6) Transplant

[0085] Transfer the regenerated Davidia involucrata seedlings that can be transplanted after rooting to natural room temperature for 4 days, take out the seedlings, rinse the second rooting medium with tap water, soak them in a 1000-fold diluted carbendazim solution for 1 hour, and then transplant them under high pressure Sterilized nutrient soil: Vermiculite mass ratio = 2:1 on the cultivation substrate (see Picture 12 ).

[0086] In this embodiment, the cluster bud differentiation rate is 50%, the rooting rate is 100%, and the transplanting survival rate is 90%.