A method for inducing dedifferentiation of cinnamomum kanehirae flowers to form callus

Abstract

The application discloses a method for inducing dedifferentiation of anther of Lagerstroemia indica to form a callus. The application selects the anther of Lagerstroemia indica as an explant for dedifferentiation induction, and through a large number of experiments, the most suitable dedifferentiation culture medium formula and culture conditions are screened out, thereby providing reference value for future development of related tests. The explant inoculation contamination rate is less than 10%, the average dedifferentiation culture rate can reach 35%, and the callus cell mass develops healthily. The dedifferentiation induction efficiency of the woody Lagerstroemia indica anther is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing dedifferentiation of anther of Lagerstroemia indica to form callus. BACKGROUND

[0002] Lagerstroemia indica is a deciduous shrub or tree of the family Lythraceae, and has beautiful tree shape, colorful and changeable flowers, and a long flowering period of up to 100 days, and is a very important summer flowering plant. Lagerstroemia indica has strong adaptability and is easy to maintain, and is widely used as a summer landscaping tree with an ultra-long flowering period.

[0003] After years of breeding, new germplasm materials with specific horticultural traits can be obtained through artificial hybridization breeding technology, and new breeding methods and techniques such as cell hybridization and transgenic breeding are needed. A most basic prerequisite for carrying out Lagerstroemia indica molecular breeding is to obtain callus cell clusters.

[0004] The application with the publication number CN112493126A discloses a method for inducing and regenerating Lagerstroemia indica somatic embryos and plants, which utilizes cotyledons of Lagerstroemia indica to induce somatic embryos and establish a regeneration plant clone, and the main steps include: 1, culture of Lagerstroemia indica sterile seedlings and selection of explants; 2, induction of callus; 3, induction of embryogenic callus; 4, somatic embryo generation and development; 5, induction of somatic embryo rooting; and 6, transplanting and seedling raising.

[0005] For example, the application with the publication number CN109757370A discloses a method for establishing a Lagerstroemia indica foetida tissue culture regeneration system, which utilizes seed germination of Lagerstroemia indica foetida to obtain sterile seedlings, then cuts tender leaves as explants, and obtains callus with strong redifferentiation ability through dedifferentiation culture of the explants, and then realizes regeneration plants through redifferentiation culture and subsequent rooting culture of the callus.

[0006] Callus has two sources, one is callus induced by dedifferentiation of somatic cells, and the other is callus induced by dedifferentiation of anther tissue. Because of natural hybridization of Lagerstroemia indica, the mother plant is mostly a highly heterozygous body, and the callus cells formed by dedifferentiation of the tissue are also heterozygous cell clusters, which greatly hinders the identification of whether the cell hybridization is successful. The callus induced by dedifferentiation of anther tissue is mostly haploid or homozygous, which is extremely beneficial to the identification of the later stage of the cell hybridization experiment. SUMMARY

[0007] The present application provides a method for inducing dedifferentiation of anther of Lagerstroemia indica to form callus

[0008] The present application first provides a solid medium for inducing dedifferentiation of anther of Lagerstroemia indica to form callus, which comprises a basic medium, plant growth hormones, agar for forming the solid medium, and sucrose for providing energy, wherein the plant growth hormones comprise:

[0009]

[0010] Preferably, the basic medium comprises N6 medium, WPN medium, and improved MS organic medium, and the components are as follows in terms of final concentration:

[0011] N6 medium: KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, KH2PO4 400 mg / L, CaCL2·2H2O 166 mg / L, MgSO4·7H2O 185 mg / L, FeSO4·7H2O 27.8 mg / L, and Na2EDTA 37.3 mg / L;

[0012] WPN medium: MnSO4·4H2O 22.5 mg / L, ZnSO4·7H2O 8.6 mg / L, CuSO4·5H2O 0.25 mg / L, Na2MoO4·2H2O 0.25 mg / L;

[0013] Improved MS organic medium: myo-inositol 100 mg / L, glycine 2 mg / L, VB5 0.5 mg / L, VB6 0.5 mg / L, VB13 mg / L.

[0014] Preferably, the concentration of agar is 6 g / L. The agar is used to form the solid medium, and the concentration of agar added can be a commonly used concentration, such as 3-8 g / L.

[0015] The concentration of sucrose is 50 g / L. Sucrose is used to provide energy for the tissue induction callus process, and therefore the concentration of sucrose can be a commonly used concentration, such as 30-60 g / L.

[0016] The present application further provides a method for inducing dedifferentiation of anther of Lagerstroemia indica to form callus, which comprises the following steps:

[0017] (1) sampling the flower buds of Lagerstroemia indica as explants;

[0018] (2) disinfecting the sampled flower buds of Lagerstroemia indica;

[0019] (3) taking the anthers from the disinfected flower buds of Lagerstroemia indica and inoculating them on the solid medium;

[0020] (4) After inoculation, dark culture is carried out first, and then light culture is carried out to obtain callus.

[0021] When the flower buds of Lagerstroemia indica are collected, the parent plants are first selected, and the Lagerstroemia indica cell hybridization or the Lagerstroemia indica parent plants that need to be subjected to transgenic experiments are selected in the field investigation, and the horticultural trait data of the parent plants are registered.

[0022] In step (1), sampling is selected in a sunny morning, preferably a sunny morning after 2-3 consecutive sunny days, to reduce the pollution sources carried by the explant materials.

[0023] Preferably, in step (1), before sampling, the target flower clusters are first sprayed with alcohol disinfection, and then the Lagerstroemia indica flower buds with a diameter of more than 80% of the mature flower bud diameter and with obvious cracks at the top but not yet cracked are collected as explants, at which time the maturity of the anthers of Lagerstroemia indica is most suitable.

[0024] Preferably, in step (2), first, alcohol disinfection is performed, then sodium hypochlorite solution disinfection is performed, then mercury sulfate solution disinfection is performed, and finally sterile water is used for washing. The alcohol disinfection uses alcohol with a volume ratio concentration of 75%, after adding, shaking for 30S, and then pouring out the alcohol. The sodium hypochlorite solution uses a sodium hypochlorite solution with a concentration of 25-30mg / L, and disinfection is performed for 3-5min. The mercury sulfate solution uses a mercury sulfate solution with a concentration of 0.1%, and disinfection is performed for 2-3min.

[0025] Preferably, in step (4), the dark culture temperature is 24-26℃, and the dark culture time is 20-25 days.

[0026] Preferably, in step (4), the light culture conditions are light intensity of 4000-6000Lx, light period of 12-14h, and light culture time of 40-60 days. The light culture time is based on the induction of callus growth, and the light culture can be ended when the callus growth is in a shorter culture time and a smaller proportion.

[0027] In order to carry out Lagerstroemia indica distant hybridization experiments and cultivate new Lagerstroemia indica germplasm materials with high resistance and specific horticultural traits, the present application selects the anthers of Lagerstroemia indica as the explants for dedifferentiation induction, and through a large number of experiments, the most suitable dedifferentiation medium formula and culture conditions are screened out, providing reference value for the development of related experiments in the future. The explant inoculation contamination rate is less than 10%, the average dedifferentiation culture rate can reach 35%, and the callus cell mass develops healthily. The dedifferentiation induction efficiency of woody Lagerstroemia indica anthers is improved. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The result graph of Example 1.

[0029] Figure 2 The result graph of Example 2.

[0030] Figure 3 Resulting graph for Example 3. DETAILED DESCRIPTION

[0031] The application provides a method for inducing dedifferentiation of flower anthers of Lagerstroemia indica to form callus, which comprises the following steps:

[0032] (1) Preparation: prepare culture medium in advance, sterilize tools, and prepare sterile water.

[0033] (2) Selection of mother plants: select Lagerstroemia indica cell hybridization or Lagerstroemia indica mother plants for transgenic experiments in field investigation, and record the horticultural trait data of the mother plants.

[0034] (3) Sampling: in the morning of a sunny day, first spray 75% (V / V) disinfectant on target flower clusters, collect Lagerstroemia indica flower buds with a diameter of more than 80% of the mature flower bud diameter and an obviously cracked but not split top as the explants after 2 min, and then put the explants into a moisture-retaining low-temperature sealing bag and take them back to the laboratory.

[0035] (4) Double disinfection treatment: first batch into containers in a clean working environment, pour in 75% alcohol and shake for 30 s, pour out the alcohol, then add 25-30 mg / L sodium hypochlorite solution for disinfection for 3-5 min, then replace it with 0.1% mercury chloride solution for disinfection treatment for 2-3 min, and finally rinse with sterile water for 3-5 times for standby use.

[0036] (5) Inoculation: take out the anthers in advance with sterilized sharp forceps in a sterile environment and inoculate them on the dedifferentiation induction medium.

[0037] (6) Dark culture + light culture: the setting of culture conditions: first inoculation is dark culture, and the culture condition is 24-26°C. After 20 to 25 days, transfer to light intensity of 4000-6000 Lx, light cycle of 12-14 h for light culture for 40-60 d, and then a certain proportion of Lagerstroemia indica haploid or homozygous Lagerstroemia indica cell clusters can be obtained.

[0038] The embodiments of the application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the application, and should not be regarded as limiting the scope of the application. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not indicated, they are all conventional products that can be obtained by market purchase.

[0039] Explanation of components in the solid dedifferentiation induction medium:

[0040] N6: N6 medium commonly used for plant tissue culture, KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, KH2PO4 400 mg / L, CaCL2·2H2O 166 mg / L, MgSO4·7H2O 185 mg / L, FeSO4·7H2O 27.8 mg / L and Na2EDTA 37.3 mg / L.

[0041] WPM: WPM medium for plant tissue culture, MnSO4·4H2O 22.5 mg / L, ZnSO4·7H2O 8.6 mg / L, CuSO4·5H2O 0.25 mg / L, Na2MoO4·2H2O 0.25 mg / L;

[0042] MS organic (modified): myo-inositol 100 mg / L, glycine 2 mg / L, VB5 0.5 mg / L, VB6 0.5 mg / L, VB1 3 mg / L;

[0043] 2,4-D: 2,4-dichlorophenoxyacetic acid, 1-1.8 mg / L;

[0044] 6-BA: 6-benzylaminopurine, 0.5-1.0 mg / L;

[0045] KT: furan amino purine, 0.8-1.0 mg / L;

[0046] IBA: indolebutyric acid, 0.6-0.8 mg / L;

[0047] Agar, for making the medium into a solid medium, 6 g / L;

[0048] Sucrose, for providing energy, 50 g / L.

[0049] Example 1

[0050] On the morning of September 10, 2021, the flower buds of "Bai Xue" and "Zi Hua Gui Fei" were collected from Zhejiang Provincial Forestry Science Research Institute, and the de-differentiation tissue culture test was carried out, and the steps were as follows:

[0051] (1) Choose a sunny morning after 3 sunny days, choose flower clusters with appropriate maturity from healthy "Bai Xue" and "Zi Hua Gui Fei" mother plants, first spray 75% (V / V) disinfectant alcohol, 2 min later, collect a batch of purple glory flower buds with a diameter of more than 80% of normal flower buds, and the flower bud top is obviously cracked but not cracked, and the flower buds are used as explants, and the flower buds are brought back to the laboratory on the same day under the condition of moisture and ice bag cold treatment.

[0052] (2) On the afternoon of September 10, 2021, the containers were filled in batches in a super-clean working room, 75% alcohol was poured in and shaken for 30 seconds, the alcohol was poured out, 25 mg / L sodium hypochlorite solution was added for disinfection for 3 minutes, then 0.1% (mass / volume ratio, hereinafter the same) mercury solution was used for disinfection for 3 minutes, and finally washed with sterile water for 3 times.

[0053] (3) The surface moisture was absorbed with sterile tissue paper, the top skin was cut with a sharp scalpel blade, and the anther was quickly inoculated on the solid dedifferentiation induction medium prepared in advance, with the formula: N6 macro + WPM micro + MS organic (modified) + 2,4-D 1.5 mg / L + 6-BA 0.8 mg / L + KT 0.8 mg / L + IBA 0.6 mg / L + agar 6 g / L + sucrose 50 g / L.

[0054] Among them, the final concentration of each component in N6: KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, KH2PO4 400 mg / L, CaCL2·2H2O 166 mg / L, MgSO4·7H2O 185 mg / L, FeSO4·7H2O 27.8 mg / L and Na2EDTA 37.3 mg / L.

[0055] The final concentration of each component in WPM: MnSO4·4H2O 22.5 mg / L, ZnSO4·7H2O 8.6 mg / L, CuSO4·5H2O 0.25 mg / L, Na2MoO4·2H2O 0.25 mg / L;

[0056] The final concentration of each component in MS organic (modified): myo-inositol 100 mg / L, glycine 2 mg / L, VB5 0.5 mg / L, VB6 0.5 mg / L, VB1 3 mg / L.

[0057] (4) The anthers were cultured in a dark room at room temperature set at 24°C for 20 days, and 8% of the anthers had started to sprout. At the same time, the contamination rate of the explants was counted, and 9% of the bottles were contaminated. Then all the sprouted and unsprouted culture bottles were transferred to a culture environment with light intensity set at 4000LX and light duration of 12h, and the culture was carried out under alternating light and darkness. When the culture was carried out for 60d, the number of calli appeared was counted.

[0058] The results are as follows Figure 1As shown, the results show that the method for inducing dedifferentiation of anther callus of Lagerstroemia indica provided by the present application can effectively obtain sterile bottle seedlings of growing and healthy callus cell groups by taking the anthers of Lagerstroemia indica “Bai Xue” as explants and performing a series of treatments, the explant inoculation contamination rate is 8%, the dark culture period is 20 days, the illumination culture period is 60 days, the culture temperature is 25 DEG C, the illumination intensity is 4000 LX, and the dedifferentiation induction rate is 35%. By taking the anthers of Lagerstroemia indica “Zi Hua Gui Fei” as explants and performing a series of treatments, sterile bottle seedlings of growing and healthy callus cell groups can be effectively obtained, the explant inoculation contamination rate is 7%, and under the same culture conditions, the dedifferentiation induction rate of “Zi Hua Gui Fei” is 38%.

[0059] Example 2

[0060] On August 9, 2022, the flower buds of Lagerstroemia indica “Fen Wei Wang” were collected from the Qinling National Botanical Garden, and the dedifferentiation tissue culture test was carried out, and the steps were as follows:

[0061] (1) On a sunny morning, select the flower clusters with appropriate maturity from the healthy “Fen Wei Wang” mother plant, first spray 75% disinfectant alcohol, 3 min later, collect a batch of flower buds with a diameter of more than 80% of normal flower buds, and the flower bud top is obviously cracked but not cracked, take the anthers of Lagerstroemia indica as explants, and mail them back to the laboratory on the same day under the condition of moisture and ice bag cold package.

[0062] (2) On the afternoon of August 13, 2022, in the clean work, first batch into the container, pour in 75% (V / V) alcohol and shake for 30 s, pour out the alcohol, then add 30 mg / L sodium hypochlorite solution for disinfection for 4 min, then replace it with 0.1% mercury chloride solution for disinfection for 3 min, and finally rinse with sterile water for 4 times.

[0063] (3) Dry the surface moisture with sterile face tissue paper, cut the top skin with a sharp scalpel, and quickly inoculate it on the solid dedifferentiation induction medium prepared in advance, the formula is: N6 macro + WPM micro + MS organic (modified) + 2,4-D 1.8 mg / L + 6-BA 1.0 mg / L + KT 1.0 mg / L + IBA 0.8 mg / L + agar 6 g / L + sucrose 50 g / L. The concentrations of the base medium, agar and sucrose in the formula are the same as in Example 1, and the concentration of the plant growth hormone is changed.

[0064] (4) In the dark room at room temperature, set the culture temperature to 25 DEG C for 23 days, 9% of the anthers have begun to sprout. At the same time, the explant contamination rate was calculated, and 10% of the bottle number appeared to be contaminated. Then all the sprouted and non-sprouted culture bottles were transferred to a culture environment with an illumination intensity of 5000 LX and a light duration of 12 h, and the day and night were alternated. When the culture was 60 d, the number of calluses appeared was counted.

[0065] Results as shown in Table 1 Figure 2 The results show that the method for inducing anther de-differentiation of Lagerstroemia indica flower to obtain callus provided by the present application can effectively obtain healthy callus cell clusters of Lagerstroemia indica 'Fenweiwang' through a series of treatments. The contamination rate of the explants is 10%, the dark culture period is 23 days, the light culture period is 60 days, the culture temperature is 25°C, the light intensity is 5000LX, and the de-differentiation rate is 31%.

[0066] Example 3

[0067] On September 12, 2023, flower buds of Lagerstroemia indica 'Zi Hua Gui Fei' and Lagerstroemia indica 'Bai Xue' were collected from the nursery of Sencheng Company in Haining, Zhejiang Province, and de-differentiation tissue culture experiments were carried out. The steps are as follows:

[0068] (1) Choose a sunny morning after 3 consecutive sunny days, and select flower clusters with appropriate maturity from healthy Lagerstroemia indica 'Zi Hua Gui Fei' mother plants. Spray 75% (V / V) disinfectant alcohol for 2 minutes, then collect a batch of flower buds with a diameter of more than 80% of normal flower buds, and the top of the flower bud is obviously cracked but not cracked. Lagerstroemia indica flower buds as explants, and mail them back to the laboratory on the same day under the condition of moist and ice bag cold wrapping.

[0069] (2) On the afternoon of September 13, 2023, in the clean working room, first batch into the container, pour in 75% alcohol and shake for 30s, pour out the alcohol, then add 25mg / L sodium hypochlorite solution for disinfection for 3min, then change to 0.1% mercury chloride solution for disinfection for 2min, and finally rinse with sterile water for 5 times.

[0070] (3) Dry the surface moisture with sterile face tissue, cut the top skin with a sharp scalpel, and quickly inoculate it on the solid de-differentiation induction medium prepared in advance, with the formula: N6 macro + WPM micro + MS organic (modified) + 2,4-D 1.0mg / L + 6-BA 0.5mg / L + KT 0.8mg / L + IBA 0.6mg / L + agar 6g / L + sucrose 50g / L. The concentrations of the base medium, agar and sucrose in the formula are the same as in Example 1, and the concentrations of the plant growth hormones are changed.

[0071] (4) In the dark room at room temperature, set the temperature to 26°C and culture for 25 days. There are 13% of the anthers that have started to sprout. At the same time, the contamination rate of the explants is calculated, and 7% of the bottles are contaminated. Then, all the sprouted and non-sprouted culture bottles are transferred to a culture environment with a light intensity of 6000LX and a light duration of 14h, and the day and night are alternated. When the culture is 60d, the number of calluses that appear is counted.

[0072] The results are shown in Table 1 Figure 3As shown, the results show that the method for inducing dedifferentiation of anther of Lagerstroemia indica to obtain callus provided by the application can effectively obtain healthy callus cell groups of Lagerstroemia indica 'Zi Hua Gui Fei' under aseptic conditions through a series of treatments, the contamination rate of the explant inoculation is 7%, the dark culture period is 25 days, the light culture period is 60 days, the culture temperature is 26 DEG C, the light intensity is 6000 LX, and the induction rate is 38%. The anther of Bai Xue is used as the dedifferentiation explant, and under the same culture conditions, the dedifferentiation rate is 40%.

Claims

1. A type of crape myrtle ( Lagerstroemia indica A solid culture medium for inducing anther dedifferentiation to form callus, comprising a basal culture medium, plant growth hormones, agar for forming the solid culture medium, and sucrose for providing energy, characterized in that... The plant growth hormone is: 2,4-D 1~1.8 mg / L 6-BA 0.5~1.0 mg / L KT 0.8~1.0 mg / L IBA 0.6~0.8 mg / L; The basal culture medium consists of N6 medium, WPN medium, and modified MS organic medium, and the components are as follows, based on their final concentrations at the time of use: N6 medium: KNO3 2830 mg / L, (NH4)2SO4 463 mg / L, KH2PO4 400 mg / L, CaCl2•2H2O 166 mg / L, MgSO4•7H2O 185 mg / L, FeSO4•7H2O 27.8 mg / L and Na2EDTA 37.3 mg / L; WPN medium: MnSO4•4H2O 22.5mg / L, ZnSO4•7H2O 8.6mg / L, CuSO4•5H2O 0.25mg / L, Na2MoO4•2H2O 0.25mg / L; Improved MS organic medium: inositol 100 mg / L, glycine 2 mg / L, VB5 0.5 mg / L, VB6 0.5 mg / L, VB1 3 mg / L; The concentration of agar was 6 g / L; the concentration of sucrose was 50 g / L.

2. A method for inducing dedifferentiation of crape myrtle anthers to form callus, characterized in that, Includes the following steps: (1) Sample crape myrtle flower buds as explants; (2) Disinfect the sampled crape myrtle buds; (3) Take out the anthers from the sterilized crape myrtle buds and inoculate them on the solid culture medium described in claim 1; (4) After inoculation, the tissue is first cultured in the dark and then cultured in the light to obtain callus tissue.

3. The method for inducing dedifferentiation of crape myrtle anthers to form callus according to claim 2, characterized in that, In step (1), sampling is performed on a sunny morning.

4. The method for inducing dedifferentiation of crape myrtle anthers to form callus according to claim 2, characterized in that, In step (1), during sampling, the target flower clusters are first sprayed with alcohol for disinfection, and then crape myrtle flower buds with a diameter of more than 80% of the diameter of mature flower buds and obvious but not cracked at the top of the flower bud are collected as explants.

5. The method for inducing dedifferentiation of crape myrtle anthers to form callus according to claim 2, characterized in that, In step (2), first disinfect with alcohol, then disinfect with sodium hypochlorite solution, then disinfect with mercuric chloride solution, and finally rinse with sterile water.

6. The method for inducing dedifferentiation of crape myrtle anthers to form callus according to claim 2, characterized in that, In step (4), the temperature for dark culture is 24~26℃ and the time for dark culture is 20~25 days.

7. The method for inducing dedifferentiation of crape myrtle anthers to form callus according to claim 2, characterized in that, In step (4), the conditions for light culture are light intensity of 4000~6000 Lx, photoperiod of 12~14h, and light culture time of 40~60 days.

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