Method for culturing lavender anther

Abstract

The invention discloses a lavender anther culture method, and relates to the technical field of plant tissue culture. The method comprises the following steps: step 1, collecting anther; step 2, disinfecting and inoculating anther; step 3, carrying out callus culture on the anther; step 4, rooting culture of anther regeneration seedlings; and step 5, hardening and transplanting anther regeneration seedlings. According to the method, the lavender anther is collected, and the callus and regenerated seedlings are subjected to induction culture, so that the callus induction rate and the seedling rate are increased, and the problems that the callus induction rate is low and the seedlings cannot be induced in the current lavender anther culture process are solved; according to the technical scheme, technical support is provided for lavender haploid breeding through the anther culture technology.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for culturing lavender anthers. Background Art

[0002] Lavender is an important specialty economic crop in Xinjiang, and its planting area accounts for 95% of the country's lavender planting area. my country has a 70-year history of lavender cultivation, but the number of cultivated varieties is small. The reason is that conventional breeding technology has the characteristics of long cycle and cumbersome operation. Therefore, the development of simple, easy and efficient breeding technology is the key to improve the efficiency of lavender breeding.

[0003] Anther tissue culture technology can produce haploid plants. After doubling, pure lines can be quickly obtained, and new germplasm with excellent traits can be bred. However, this technology has many problems, such as difficulty in establishment, easy browning of callus tissue, and low regeneration frequency. Therefore, it is urgent to establish an efficient and stable lavender anther culture system in order to provide technical support for lavender germplasm innovation and genetic improvement.

[0004] Anther culture is a method of using plant tissue culture technology to inoculate anthers containing pollen in the mononuclear marginal stage on an artificial culture medium through aseptic operation techniques to change the developmental program of pollen microspores in the anthers, induce their differentiation, and continuously undergo mitosis to form cell clusters, and then form a mass of undifferentiated thin-walled tissue - callus (or differentiate into embryoids, and then differentiate the callus into a complete plant.

[0005] At present, in the prior art, there are problems in the culture process of lavender anthers, such as low anther callus induction rate and failure to induce seedlings. Summary of the invention

[0006] In view of this, in order to solve the problems existing in the prior art, an embodiment of the present invention provides a method for culturing lavender anthers, which improves the callus induction rate and seedling rate through the method of lavender anther collection, callus induction and proliferation culture, thereby effectively providing technical support for lavender haploid breeding using anther culture technology.

[0007] To achieve the above object, the present invention mainly provides the following technical solutions:

[0008] The embodiment of the present invention provides a method for culturing lavender anthers, the method comprising the following steps:

[0009] Step 1: Collection of anthers

[0010] At 10:00-11:00 a.m. on a sunny day, we took the flower buds from healthy and pest-free lavender plants, put them in an ice box and quickly brought them back to the laboratory. Through the observation of anther morphological characteristics and cytological characteristics, we found that the length of the flower buds was 4.72-5.82 mm, the calyx was dark purple as a whole, and the anther color was dark yellow. Most of the pollen microspores were in the uninucleate marginal stage of development.

[0011] Step 2: Disinfection and inoculation of anthers

[0012] At 10:00-11:00 a.m. on a sunny day, take the budding spike and place it in an ice bag, quickly bring it back to the laboratory, remove the complete bud from the spike with tweezers, put the bud into a filter and rinse it with tap water for 8-10 minutes, then disinfect it with 75% ethanol for 30 seconds in a clean bench, then rinse it with sterile water for 3-4 times, then treat the bud with different disinfectants for 1.5-2 minutes, finally rinse it with sterile water for 3-4 times, take out the anther, and inoculate it into the anther callus induction medium;

[0013] Step 3: Callus culture of anthers

[0014] The inoculated anthers of "French Blue" and "Miscellaneous Flowers" lavender were cultured in the dark for 7 days and then cultured in the light. After 25 days of culture, the obtained anther callus was transferred to the callus proliferation medium. After 3 to 4 subcultures, the callus was induced to grow regenerated seedlings.

[0015] Step 4: Rooting culture of anther regeneration seedlings

[0016] When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to the rooting medium and cultured in the culture room under light for 30-40 days.

[0017] Step 5: Hardening and transplanting of anther regenerated seedlings

[0018] When the roots of the regenerated seedlings grow to 1-2 cm, take the regenerated seedlings out of the rooting medium, wash off the medium on the roots with warm water at 28°C, transplant the regenerated seedlings into the sterilized seedling medium, and culture them under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, transplant them to the field.

[0019] Furthermore, the varieties of lavender include French blue lavender and mixed flower lavender.

[0020] Further, in step 2, the anthers of French blue lavender and mixed lavender were treated with 10% NaClO and 0.1% HgCl 2 Disinfectant treatment.

[0021] Furthermore, in step 3 and step 4, the illumination conditions for light culture in the culture room are 2400 lx, the light cycle is 16 h / d, and the culture temperature is 25°C.

[0022] Further, in step 2, the callus induction medium of the anthers of the mixed flower lavender is MS + 0.1-0.2 mg / L 2,4-D + 0.5-1.5 mg / L 6-BA, 30 g / L sucrose, 6 g / L agar, and the pH is 5.8-6.0; the callus induction medium of the anthers of the French blue lavender is MS + 0.05-0.15 mg / L NAA + 2.5-3.5 mg / L 6-BA, 30 g / L sucrose, 6 g / L agar, and the pH is 5.8-6.0.

[0023] Further, in step 3, the callus proliferation medium of the anthers of the mixed flower lavender is MS + 0.35-0.45mg / LNAA + 1.5-2.5mg / L 6-BA, 30g / L sucrose, 7g / L agar, and the pH is 5.8-6.0; the callus proliferation medium of the anthers of the French blue lavender is MS + 0.05-0.15mg / L NAA + 0.35-0.45mg / L TDZ, 30g / L sucrose, 7g / L agar, and the pH is 5.8-6.0.

[0024] Furthermore, in step 4, the anther regeneration seedling rooting culture medium is 1 / 2MS+0.15-0.25 mg / L NAA, and the pH is 5.8-6.0.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The method of collecting lavender anthers and adopting callus induction culture and proliferation culture methods of the present invention improves the callus induction rate and seedling rate, thereby effectively providing technical support for lavender haploid breeding using anther culture technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the cytological characteristics of lavender pollen microspores at various developmental stages. In the figure, A is a microspore mother cell (10×40), B is a microspore in the tetrad stage (10×40), C is a microspore in the middle uninucleate stage (10×40), D is a microspore in the uninucleate marginal stage (10×40), and E is a mature pollen grain (10×40);

[0028] Figure 2These are different types of callus tissues induced by anthers of French blue lavender. In the figure, AB shows the first type of callus tissue, which is white and relatively compact; C shows the second type of callus tissue, which is yellow-green, with a yellow surface and is relatively loose; DE shows the third type of callus tissue, which is purple or lavender, and has a relatively compact tissue;

[0029] Figure 3 These are the regenerated seedlings induced by anthers of French blue lavender. In the figure, A shows the callus tissue growing regenerated buds; B shows the regenerated buds growing leaves; C shows the number of leaves of the regenerated seedlings gradually increasing; D shows the height of the regenerated seedlings growing to 1-2 cm; E shows the regenerated seedlings transferred to the rooting medium;

[0030] Figure 4 These are different types of callus tissues induced by the anthers of lavender. In the figure, Figure A shows the first type of callus tissue, which is purple or light purple and has a relatively compact tissue; Figure B shows the second type of callus tissue, which is yellow-green, with a yellow surface and is relatively loose; Figure C shows the third type of callus tissue, which is white and has a relatively compact tissue; Figures D and E show the fourth type of callus tissue, which is green (or light green) and has a relatively compact tissue;

[0031] Figure 5 These are regenerated seedlings induced by anthers of lavender. In the figure, A shows callus tissue growing regenerated buds; B shows regenerated buds growing leaves; C shows the number of leaves of regenerated seedlings gradually increasing; D shows the height of regenerated seedlings growing to 1-2 cm; E shows the regenerated seedlings transferred to rooting medium;

[0032] Figure 6 This is the process of hardening and transplanting the anther-regenerated seedlings of the miscellaneous lavender. In the figure, Figure A shows the initial situation of the anther-regenerated seedlings of the miscellaneous lavender after transplanting; Figure B shows the plant situation of the anther-regenerated seedlings of the miscellaneous lavender 3 months after transplanting;

[0033] Figure 7 It is the transplantation of French blue lavender anther regeneration seedlings. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, technical solutions, features and effects of the present invention application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0035] The lavender varieties used in the technical solution of the present invention are French blue lavender and mixed flower lavender, both of which are lavender varieties in the prior art and are collected in Yining County, Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China.

[0036] Example 1

[0037] This embodiment is to design a method for establishing an efficient culture system using "French Blue" lavender anthers as explants, including the following technical solutions:

[0038] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0039] The flower buds of the French Blue lavender at the developmental stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest were used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of pollen in the mononuclear marginal stage, the anthers at different developmental stages were pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the base of the bud from bottom to top and take out the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0040] 2. Lavender anther culture:

[0041] (1) At 10:00-11:00 a.m. on a sunny day, take the budding spike and place it in an ice bag and quickly bring it back to the laboratory. Use tweezers to remove the complete buds from the spike, put the buds into a filter and rinse with tap water for 8-10 minutes, then disinfect them with 75% ethanol for 30 seconds in a clean bench, then rinse with sterile water for 3-4 times, then treat with 10% NaClO disinfectant for 1.5-2 minutes, and finally rinse with sterile water for 3-4 times, then remove the anthers and inoculate them into anther callus induction medium.

[0042] (2) In this experiment, the inoculated French Blue lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 2 As shown in the figure, after the anthers of "French Blue" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 3 As shown in AD.

[0043] (3) In this experiment, when the anthers of "French Blue" lavender were inoculated in the callus medium of MS+0.1 mg / L NAA+2.5 mg / L 6-BA, the anther callus induction rate reached 10.83%.

[0044] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 3 As shown in E.

[0045] (5) Figure 7 As shown, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0046] Example 2

[0047] This embodiment is to design a method for establishing an efficient culture system using "French Blue" lavender anthers as explants, including the following technical solutions:

[0048] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0049] The flower buds of the "French Blue" lavender at the developmental stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest were used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of pollen in the mononuclear marginal stage, the anthers at different developmental stages were pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the bottom to the top from the base of the bud to remove the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0050] 2. Lavender anther culture:

[0051] (1) At 10:00-11:00 a.m. on a sunny day, take the budding spike and place it in an ice bag and quickly bring it back to the laboratory. Use tweezers to remove the complete buds from the spike, put the buds into a filter and rinse with tap water for 8-10 minutes, then disinfect them with 75% ethanol for 30 seconds in a clean bench, then rinse with sterile water for 3-4 times, then treat with 10% NaClO disinfectant for 1.5-2 minutes, and finally rinse with sterile water for 3-4 times, then remove the anthers and inoculate them into anther callus induction medium.

[0052] (2) In this experiment, the inoculated French Blue lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 2 As shown in the figure, after the anthers of "French Blue" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 3 As shown in AD.

[0053] (3) In this experiment, when the anthers of French Blue lavender were inoculated in MS+0.1 mg / L NAA+3.0 mg / L 6-BA callus medium, the anther callus induction rate reached 14.17%.

[0054] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 3 As shown in E.

[0055] (5) Figure 7 As shown, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0056] Example 3

[0057] This embodiment is to design a method for establishing an efficient culture system using "French Blue" lavender anthers as explants, including the following technical solutions:

[0058] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0059] The flower buds of the "French Blue" lavender at the developmental stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest were used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of pollen in the mononuclear marginal stage, the anthers at different developmental stages were pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the bottom to the top from the base of the bud to remove the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0060] 2. Lavender anther culture:

[0061] (1) At 10:00-11:00 a.m. on a sunny day, take the budding spike and place it in an ice bag and quickly bring it back to the laboratory. Use tweezers to remove the complete buds from the spike, put the buds into a filter and rinse them with tap water for 8-10 minutes. Then, in a clean bench, first disinfect them with 75% ethanol for 30 seconds, then rinse them with sterile water for 3-4 times, then treat them with 10% NaClO disinfectant for 1.5-2 minutes, and finally rinse them with sterile water for 3-4 times, then remove the anthers and inoculate them into anther callus induction medium.

[0062] (2) In this experiment, the inoculated French Blue lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 2 As shown in the figure, after the anthers of "French Blue" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 3 As shown in AD.

[0063] (3) In this experiment, when the anthers of "French Blue" lavender were inoculated in the callus medium of MS+0.1 mg / L NAA+3.5 mg / L 6-BA, the anther callus induction rate reached 11.67%.

[0064] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 3 As shown in E.

[0065] (5) Figure 7 As shown, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0066] Example 4

[0067] This embodiment is to design a method for establishing an efficient culture system using anthers of "miscellaneous flowers" lavender as explants, including the following technical solutions:

[0068] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0069] The flower buds of the "mixed flower" lavender at the development stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest are used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of mononuclear marginal stage pollen, the anthers at different developmental stages are pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the bottom to the top from the base of the bud to remove the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0070] 2. Lavender anther culture:

[0071] (1) On a sunny day between 10:00 and 11:00 a.m., take the budded flower spikes and place them in an ice bag and quickly bring them back to the laboratory. Before anther inoculation, remove the complete flower buds from the flower spikes with tweezers, put the flower buds into a filter and rinse them with tap water for 8 to 10 minutes, then disinfect them with 75% ethanol for 30 seconds in a clean bench, then rinse them with sterile water for 3 to 4 times, and then rinse the "mixed flower" lavender with 0.1% HgCl 2 Treat with disinfectant for 1.5 to 2 minutes, rinse with sterile water 3 to 4 times, remove the anthers, and inoculate them into anther callus induction medium.

[0072] (2) In this experiment, the inoculated “mixed flower” lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 4As shown in the figure, after the anthers of "Miscellaneous Flower" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 5 As shown in AD.

[0073] (3) In this experiment, when the anthers of “mixed flower” lavender were inoculated in MS+0.15 mg / L 2,4-D+0.5 mg / L 6-BA callus medium, the anther callus induction rate reached 14.17%.

[0074] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 5 As shown in E.

[0075] (5) Figure 6 As shown in A, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0076] Example 5

[0077] This embodiment is to design a method for establishing an efficient culture system using anthers of "miscellaneous flowers" lavender as explants, including the following technical solutions:

[0078] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0079] The flower buds of the "mixed flower" lavender at the development stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest are used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of mononuclear marginal stage pollen, the anthers at different developmental stages are pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the bottom to the top from the base of the bud to remove the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0080] 2. Lavender anther culture:

[0081] (1) On a sunny day between 10:00 and 11:00 a.m., take the budded flower spikes and place them in an ice bag and quickly bring them back to the laboratory. Before anther inoculation, remove the complete flower buds from the flower spikes with tweezers, put the flower buds into a filter and rinse them with tap water for 8 to 10 minutes, then disinfect them with 75% ethanol for 30 seconds in a clean bench, then rinse them with sterile water for 3 to 4 times, and then rinse the "mixed flower" lavender with 0.1% HgCl 2 Treat with disinfectant for 1.5 to 2 minutes, rinse with sterile water 3 to 4 times, remove the anthers, and inoculate them into anther callus induction medium.

[0082] (2) In this experiment, the inoculated “mixed flower” lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 4 As shown in the figure, after the anthers of "Miscellaneous Flower" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 5 As shown in AD.

[0083] (3) In this experiment, when the anthers of “mixed flower” lavender were inoculated in MS+0.15 mg / L 2,4-D+1.0 mg / L 6-BA callus medium, the anther callus induction rate reached 15.00%.

[0084] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 5 As shown in E.

[0085] (5) Figure 6 As shown in A, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0086] Example 6

[0087] This embodiment is to design a method for establishing an efficient culture system using anthers of "miscellaneous flowers" lavender as explants, including the following technical solutions:

[0088] 1. Determination of the uninucleate phase of lavender pollen microspores:

[0089] The flower buds of the "mixed flower" lavender at the development stage when the proportion of pollen microspores in the mononuclear marginal stage is the highest are used as experimental materials. In order to determine the development status of pollen microspores at different developmental stages of lavender anthers and the proportion of mononuclear marginal stage pollen, the anthers at different developmental stages are pressed and observed. Use tweezers and dissecting needles to cut the calyx and petals from the bottom to the top from the base of the bud to remove the anthers. Drop a drop of acetic acid magenta stain on the slide, put 3 to 4 anthers into the stain, and gently tap the anthers with tweezers. After the anthers break, the pollen microspores are released; after standing for 2 to 3 minutes, tilt the cover glass at 45° and slowly cover it, absorb the excess stain with filter paper, bake it on an alcohol lamp for 3 to 4 seconds, and observe it under a microscope. Through the observation of anther morphological characteristics and pollen cytological characteristics, the bud length is 4.72-5.82 mm, the calyx is dark purple as a whole, and the anther color is dark yellow. Most of the pollen microspores are in the uninucleate stage of development. Figure 1 shown.

[0090] 2. Lavender anther culture:

[0091] (1) On a sunny day between 10:00 and 11:00 a.m., take the budded flower spikes and place them in an ice bag and quickly bring them back to the laboratory. Before anther inoculation, remove the complete flower buds from the flower spikes with tweezers, put the flower buds into a filter and rinse them with tap water for 8 to 10 minutes, then disinfect them with 75% ethanol for 30 seconds in a clean bench, then rinse them with sterile water for 3 to 4 times, and then rinse the "mixed flower" lavender with 0.1% HgCl 2 Treat with disinfectant for 1.5 to 2 minutes, rinse with sterile water 3 to 4 times, remove the anthers, and inoculate them into anther callus induction medium.

[0092] (2) In this experiment, the inoculated “mixed flower” lavender anthers were cultured in the dark for 7 days and then cultured in the light, which was beneficial to improve the callus induction rate. Figure 4 As shown in the figure, after the anthers of "Miscellaneous Flower" lavender were cultured in callus induction medium for 25 days, the obtained callus was transferred to callus proliferation medium. After 3 to 4 subcultures, the callus grew regenerated buds. At this time, the light conditions were 2400lx, the light cycle was 16h / d, and the culture temperature was 25℃. Figure 5 As shown in AD.

[0093] (3) In this experiment, when the anthers of “mixed flower” lavender were inoculated in MS+0.15 mg / L 2,4-D+1.5 mg / L 6-BA callus medium, the anther callus induction rate reached 21.67%.

[0094] (4) When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to 1 / 2MS + 0.2 mg / L NAA rooting medium and cultured in the light room for 30-40 days. Figure 5 As shown in E.

[0095] (5) Figure 6 As shown in A, when the roots of the regenerated seedlings grow to 1-2 cm, the regenerated seedlings are taken out from the rooting medium, the medium on the roots is washed off with warm water at 28°C, the regenerated seedlings are transplanted into the sterilized seedling medium, and cultured under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, they are transplanted to the field.

[0096] After culturing each example in the dark for 7 days, the induction rate of anther callus was counted, wherein induction rate (%) = (number of explants producing callus / total number of inoculated explants) × 100%, as shown in Table 1.

[0097] Table 1 Effects of different dark culture times on anther callus induction rate

[0098]

[0099] The light culture conditions have different effects on the callus induction effect of lavender anthers. As shown in Table 1, the callus induction rate of the lavender anthers of "French Blue" in Example 1-3 was the lowest when dark cultured for 0 days, and the callus induction rate was the highest when dark cultured for 7 days; the callus induction rate of the lavender anthers of "Miscellaneous Flowers" in Example 4-6 showed a trend of first decreasing and then increasing with the extension of dark culture time. When dark cultured for 0 days and 7 days, the callus induction rate could reach 30.83%. Comprehensive analysis showed that the callus induction effect was better when the lavender anthers of "French Blue" in Example 1-3 and "Miscellaneous Flowers" in Example 4-6 were cultured in the dark for 7 days and then cultured in light.

[0100] The effects of different hormone ratios on the anther callus induction rate in each embodiment were statistically analyzed, wherein the induction rate (%) = (the number of explants producing callus / the total number of explants inoculated) × 100%; the browning rate (%) = (the number of browned anthers / the number of inoculated anthers) × 100%, as shown in Table 2.

[0101] Table 2 Effects of different hormone ratios on anther callus induction rate

[0102] Example NAA(mg / L) 2,4-D (mg / L) 6-BA(mg / L) Induction rate (%) Browning rate (%) Example 1 0.1 0 2.5 10.83 99.17 Example 2 0.1 0 3.0 14.17 96.67 Example 3 0.1 0 3.5 11.67 100.00 Example 4 0 0.15 0.5 14.17 97.5 Example 5 0 0.15 1.0 15.00 98.33 Example 6 0 0.15 1.5 21.67 94.17

[0103] It can be seen from the above examples that in Example 2, when the hormone ratio of 0.1 mg / L NAA and 3.0 mg / L 6-BA was used, the anther callus induction rate reached a maximum of 14.17%, and the browning rate dropped to a minimum of 96.67%; in Example 6, when the hormone ratio of 0.15 mg / L 2,4-D and 1.5 mg / L 6-BA was used, the anther callus induction rate reached a maximum of 21.67%, and the browning rate dropped to a minimum of 94.17%.

[0104] For matters not covered in the embodiments of the present invention, those skilled in the art can select from the prior art.

[0105] The above disclosure is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of and change or replace within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the above claims.

Claims

1. A method for cultivating lavender anthers, characterized in that: The method comprises the following steps: Step 1: Collection of anthers At 10:00-11:00 a.m. on a sunny day, we took the flower buds from healthy and pest-free lavender plants, put them in an ice box and quickly brought them back to the laboratory. Through the observation of anther morphology and cytological characteristics, we found that the flower bud length was 4.72-5.82 mm, the calyx was dark purple as a whole, and the anther was dark yellow. Most of the pollen microspores were in the uninucleate marginal stage of development. Step 2: Disinfection and inoculation of anthers Use tweezers to remove the complete flower buds from the flower spikes, put the flower buds into the filter and rinse with tap water for 8-10 minutes, then disinfect with 75% ethanol for 30 seconds in the clean bench, then rinse with sterile water for 3-4 times, then treat the lavender flower buds with different disinfectants for 1.5-2 minutes, finally rinse with sterile water for 3-4 times, remove the anthers, and inoculate them into callus induction medium; Step 3: Callus culture of anthers The inoculated lavender anthers were cultured in the dark for 7 days, and then cultured in the light. The callus obtained by the anther induction culture was inoculated into a callus proliferation medium. After 3 to 4 subcultures, the callus was induced to grow into regenerated seedlings. Step 4: Rooting culture of anther regeneration seedlings When the height of the lavender regenerated seedlings grows to 1-2 cm, they are transferred to the rooting medium and cultured in the culture room under light for 30-40 days before rooting. Step 5: Hardening and transplanting of anther regenerated seedlings When the roots of the regenerated seedlings grow to 1-2 cm, take the regenerated seedlings out of the rooting medium, wash off the medium on the roots with warm water at 28°C, transplant the regenerated seedlings into the sterilized seedling medium, and culture them under light in a greenhouse at 28-30°C for 30-40 days. When the regenerated seedlings grow well, transplant them to the field.

2. A method for cultivating lavender anthers as claimed in claim 1, characterized in that: The varieties of lavender include French blue lavender and mixed flower lavender.

3. A method for cultivating lavender anthers as claimed in claim 2, characterized in that: In step 2, anthers of French blue lavender and mixed lavender are treated with 10% NaClO and 0.1% HgCl2 disinfectants, respectively.

4. A method for cultivating lavender anthers as claimed in claim 1, characterized in that: In step 3 and step 4, the illumination conditions for light culture in the culture room are 2400 lx, the light cycle is 16 h / d, and the culture temperature is 25°C.

5. A method for cultivating lavender anthers as claimed in claim 2, characterized in that: In step 2, the callus induction medium of the anthers of the mixed flower lavender is MS + 0.1-0.2 mg / L 2,4-D + 0.5-1.5 mg / L 6-BA, 30 g / L sucrose, 6 g / L agar, and the pH is 5.8-6.0; the callus induction medium of the anthers of the French blue lavender is MS + 0.05-0.15 mg / LNAA + 2.5-3.5 mg / L 6-BA, 30 g / L sucrose, 6 g / L agar, and the pH is 5.8-6.

0.

6. A method for cultivating lavender anthers as claimed in claim 2, characterized in that: In step 3, the callus proliferation medium of the anthers of the mixed flower lavender is MS+0.35-0.45mg / L NAA+1.5-2.5mg / L 6-BA, 30g / L sucrose, 7g / L agar, and the pH is 5.8-6.0; the callus proliferation medium of the anthers of the French blue lavender is MS+0.05-0.15mg / L NAA+0.35-0.45mg / L TDZ, 30g / L sucrose, 7g / L agar, and the pH is 5.8-6.

0.

7. A method for cultivating lavender anthers as claimed in claim 1, characterized in that: In step 4, the anther regeneration seedling rooting culture medium is 1 / 2MS+0.15-0.25mg / LNAA, and the pH is 5.8-6.0.

Images