A method for isolating proplast of honeysuckle flower by embryogenic callus

Abstract

The application discloses a method for separating protoplasts of honeysuckle by using embryogenic callus, which comprises the following steps: D. Enzymolysis, embryogenic callus of honeysuckle is taken, filtered into a sterile centrifuge tube by using a cell screen, horizontal centrifugation is carried out for 5 min, and the supernatant is discarded; 10 mL of enzymolysis solution is added, the callus cells are resuspended by gently sucking and beating with a pipette gun, and the resuspension is subjected to shock enzymolysis at 25 DEG C in dark conditions for 2-4 h; E. Protoplast extraction, after the enzymolysis is completed, an equal volume of pre-cooled W5 solution is added to the enzymolysis solution, and the resuspension is subjected to shock for 3 min; the enzymolysis solution is filtered by using a cell screen; horizontal centrifugation is carried out for 5 min, and the supernatant is discarded; 5 ml of pre-cooled W5 solution is added to resuspend the protoplasts, and the resuspension is subjected to ice bath for 30 min; horizontal centrifugation is carried out for 5 min, and the supernatant is discarded; 5 ml of pre-cooled MMG solution is added to resuspend the protoplasts, horizontal centrifugation is carried out for 5 min, the supernatant is discarded, and the sediment is the protoplasts. The method for preparing the protoplasts of honeysuckle by using the embryogenic callus of honeysuckle has a short enzymolysis time, and the yield of the protoplasts of honeysuckle is as high as 20.40 x 10 6 6 / g, and the activity coefficient is as high as 92%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a method for isolating Lonicera japonica protoplasts from embryogenic callus. BACKGROUND

[0002] Lonicera japonica is a native plant in China, widely distributed in the North China Plain, the southeast coast and the southwest inland areas of China's subtropical and temperate monsoon regions. As a medicinal material, Lonicera japonica has the effects of clearing heat and detoxifying, and is mainly used for treating warm disease fever, dysentery, and carbuncle, etc. Modern research has proved that Lonicera japonica contains chlorogenic acid, luteolin glycoside and other pharmacologically active ingredients, and has strong inhibitory effect on many pathogenic bacteria such as pneumococcus, meningococcus, greenish pyocyanin, tubercle bacillus, hemolytic streptococcus, and staphylococcus aureus, and upper respiratory tract infection pathogenic viruses, etc. In addition, it can also enhance immunity, anti-early pregnancy, protect liver, anti-tumor, anti-inflammatory, antipyretic and inhibit intestinal absorption of cholesterol, etc. Its clinical use is very wide. Lonicera japonica is one of the main components of Shuanghuanglian Oral Liquid, Jinhua Qinggan Granules and Lianhua Qingwen Capsules.

[0003] Plant protoplast refers to the protoplast part without cell wall, which is called "naked cell". The protoplast still has cell activity, and can synthesize cell wall again and regenerate into a complete plant under certain conditions. Due to the lack of cell wall obstruction, protoplast is more deformable, and is often used for cell fusion and somatic cell hybridization, which is an effective means to overcome distant hybridization incompatibility. In addition, protoplast is more susceptible to absorption of external genetic material, and is widely used in subcellular localization, transformation, protein interaction, and other physiological and biochemical and molecular genetic fields.

[0004] At present, the preparation of Lonicera japonica protoplast is mainly completed by leaf, such as CN111471640A discloses a method for isolating and culturing Lonicera japonica protoplast and a special culture medium. The Lonicera japonica protoplast is obtained through a series of steps such as explant disinfection, explant pretreatment, protoplast separation, protoplast purification and protoplast culture. The enzymolysis time of the leaf is 8h, the protoplast yield is 5.73x10 6 6 / g, and the protoplast activity is 85.4%. However, the leaf of Lonicera japonica is thin and soft, and the mesophyll cells are less. Therefore, a large number of leaves need to be collected to obtain the required amount of protoplast. In addition, the mesophyll cells in the leaf are not easy to release, and a long enzymolysis time is needed, which causes great damage to the cells, and the yield and activity of the obtained protoplast are low.

[0005] Embryogenic callus is a kind of callus with embryogenic cells, which can form a complete plant through somatic embryogenesis. The somatic embryogenesis pathway is mostly single cell origin, and each embryogenic cell has the potential to develop into a complete plant. Compared with ordinary callus, embryogenic callus has the following advantages when preparing protoplasts:

[0006] 1. Embryogenic callus proliferates rapidly in liquid medium, which can obtain a large amount of preparation materials in a short time, improve the yield of protoplasts, and shorten the preparation period;

[0007] 2. After liquid culture, the structure of embryogenic callus is loose, and the cells in the tissue can fully contact with the enzyme solution, which can shorten the enzyme digestion time, reduce the damage of enzyme to cells, improve the enzyme digestion efficiency, and obtain protoplasts with strong activity;

[0008] 3. Embryogenic callus has strong regeneration ability, and the probability of regenerating a complete plant from the obtained protoplasts is high;

[0009] 4. Embryogenic callus has strong ability to accept exogenous DNA, and the transformation efficiency of exogenous DNA is high after preparation into protoplasts, which is an ideal material for genetic transformation.

[0010] However, the conditions for inducing embryogenic callus are harsh, and the embryogenic callus grows slowly on solid medium and often grows mixed with ordinary callus. If not separated and purified in time, it is easy to be lost. These difficulties in induction, separation and proliferation make it difficult to apply embryogenic callus to prepare protoplasts. SUMMARY

[0011] In order to solve the above technical problems, the purpose of the present application is to provide a method for separating protoplasts of honeysuckle by using embryogenic callus. In this method, the embryogenic callus induction rate and proliferation rate of honeysuckle are high, the yield of protoplasts prepared from honeysuckle embryogenic callus is high, and the activity coefficient is high.

[0012] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0013] A method for separating protoplasts of honeysuckle by using embryogenic callus, comprising the following steps:

[0014] D. Enzymolysis

[0015] Take the embryogenic callus of honeysuckle, filter to 50 mL sterile centrifuge tube with high-temperature sterilized 80-mesh cell sieve, RCF 100 x g horizontal centrifugation for 5 min, discard the supernatant; add 10 mL enzyme solution, and gently suck and beat with a pipette to resuspend the callus cells, and then perform enzymatic hydrolysis at 25 DEG C in the dark for 2-4 h; wherein the mass concentration of D-mannitol in the enzyme solution is 11%, the mass concentration of cellulase is 1%, the mass concentration of dissociation enzyme is 0.3%, the concentration of MES is 5 mM, the mass concentration of BSA is 0.1%, and the concentration of CaCl2 is 10 mM;

[0016] E. Protoplast extraction

[0017] After the enzymatic hydrolysis is completed, an equal volume of pre-cooled W5 solution is added to the enzyme solution, and the solution is shaken at 30 rpm for 3 min; the enzyme solution is filtered after being washed with a pre-cooled W5 solution through a 200-mesh cell sieve; the solution is centrifuged at 100 x g for 5 min, and the supernatant is discarded; 5 ml of pre-cooled W5 solution is added to resuspend the protoplasts, and the solution is ice-bathed for 30 min; the solution is centrifuged at 100 x g for 5 min, and the supernatant is discarded; 5 ml of pre-cooled MMG solution is added to resuspend the protoplasts, and the solution is centrifuged at 100 x g for 5 min, and the supernatant is discarded; and the protoplasts are obtained by centrifugation.

[0018] Preferably, in step D, the ratio of the embryogenic callus of honeysuckle to the enzyme solution is 0.2 g / 10 mL.

[0019] Preferably, in step D, the enzymatic hydrolysis time is 2 h.

[0020] Preferably, in step E, the components of the W5 solution are: NaCl 0.154 M, CaCl2 0.125 M, KCl 2.5 mM, and MES 5 mM.

[0021] Preferably, in step E, the components of the MMG solution are: mannitol 0.5 M, MES 0.01 M, and MgCl2 0.01 M.

[0022] Preferably, the embryogenic callus of honeysuckle is obtained by the following steps:

[0023] A. Sterilization

[0024] The flower buds of honeysuckle with a length of 2.5-3.0 cm are collected, soaked in a detergent solution for 10 min, gently rubbed during the soaking, then washed with running water, and then soaked in a 0.1% NaClO solution for 5 min; washed with running water for 1 h; after the water is absorbed, the flower buds are irradiated with ultraviolet light for 20 min on an ultraclean workbench; then sterilized in 75% alcohol for 1 min; and finally washed with sterile water for 3 times;

[0025] B. Embryogenic callus induction

[0026] On the super-clean bench, the sterilized honeysuckle flower bud is absorbed with sterile filter paper to remove the surface moisture, the ovary is cut off, and is inoculated into the embryogenic callus induction medium, 6-8 ovaries are inoculated into each bottle of medium; the closed petals are opened, the anthers are taken out, and are inoculated into the embryogenic callus induction medium, 15-20 anthers are inoculated into each bottle of medium; the inoculated culture bottle is placed in an artificial climate chamber for light-free culture, the temperature is 25 DEG C, and the humidity is 45-55 %;

[0027] C. Embryogenic callus liquid proliferation

[0028] After 20d of ovary induction culture, the embryogenic callus tissue of honeysuckle with tender yellow color and vigorous proliferation is selected, is inoculated into a 100ml conical flask, each bottle contains 30ml of liquid proliferation medium, and 4g is inoculated into each bottle of medium;

[0029] After 40d of anther induction culture, the embryogenic callus tissue of honeysuckle with fresh and tender color and loose granular shape is selected, is inoculated into a 100ml conical flask, each bottle contains 30ml of liquid proliferation medium, and 3g is inoculated into each bottle of medium;

[0030] The conical flask is placed on a shaker with a rotation speed of 120 rpm for light-free culture, the temperature is 25 DEG C, and the humidity is 45-55 %.

[0031] Preferably, in step C, the embryogenic callus tissue of honeysuckle obtained by inoculation of ovary induction is gently crushed, so that the tissue block in the liquid proliferation medium has a diameter of less than 0.5cm.

[0032] Preferably, in step B, the components of the embryogenic callus induction medium are as follows: MS 4.4g / L, sucrose 30g / L, hydrolyzed casein 2g / L, agar 7.5g / L, 2,4-D 2mg / L, 6-BA 1mg / L, AgNO3 2mg / L, and pH is 5.8-6.0.

[0033] Preferably, in step C, the components of the liquid proliferation medium are as follows: MS 4.4g / L, sucrose 30g / L, hydrolyzed casein 2g / L, 2,4-D 0.2mg / L, 6-BA 0.1mg / L, and pH is 5.8-6.0.

[0034] The present application has the following beneficial effects due to the adoption of the above technical solutions:

[0035] The application establishes a method for selecting ovary and anther of honeysuckle to induce embryogenic callus of honeysuckle, the induction rate of embryogenic callus of honeysuckle is 67.31% and 55.36% respectively, and the callus obtained by induction is embryogenic callus, the induction rate is high, and the proliferation efficiency is high, reaching 93.02%; the application also initiatively uses embryogenic callus of honeysuckle to prepare honeysuckle protoplast, the enzymolysis time is short, so that the yield of honeysuckle protoplast is as high as 20.40×10 6 / g, and the activity coefficient is as high as 92%. DETAILED DESCRIPTION

[0036] The following detailed description of several specific embodiments of the application, but it should be understood that the scope of protection of the application is not limited by the specific embodiments.

[0037] Example 1:

[0038] A method for separating honeysuckle protoplast by using embryogenic callus, comprising the following steps:

[0039] A. Disinfection

[0040] Collect honeysuckle flower buds with a length of 2.5-3.0 cm, soak in a detergent solution for 10 min, gently rub during the period, then rinse with running water, then soak in 0.1% NaClO solution for 5 min; rinse with running water for 1 h; after absorbing water, irradiate with ultraviolet light for 20 min on a clean bench; then disinfect in 75% alcohol for 1 min; finally rinse with sterile water for 3 times.

[0041] B. Embryogenic callus induction

[0042] On the clean bench, absorb the surface water of the disinfected honeysuckle flower buds with sterile filter paper, cut off the ovary and inoculate into embryogenic callus induction medium, 6-8 ovules per bottle of medium; open the closed petals, take out the anthers, inoculate into the embryogenic callus induction medium, 15-20 anthers per bottle of medium; put the inoculated culture bottle into an artificial climate chamber for dark culture, temperature 25℃, humidity 45-55%; the composition of the embryogenic callus induction medium is: MS 4.4g / L, sucrose 30g / L, hydrolyzed casein 2g / L, agar 7.5g / L, 2,4-D 2mg / L, 6-BA 1mg / L, AgNO3 2mg / L, pH 5.8-6.0.

[0043] C. Liquid proliferation of embryogenic callus

[0044] After 20 days of ovary induction culture, select the embryogenic callus of honeysuckle with tender yellow color and vigorous proliferation, inoculate into 100 ml conical flask containing 30 ml liquid proliferation medium; gently crush the tissue, so that the tissue fragments in the liquid proliferation medium are less than 0.5 cm in diameter, and inoculate 4 g of the tissue per flask;

[0045] After 40 days of anther induction culture, select the embryogenic callus of honeysuckle with tender color and loose granular shape, inoculate into 100 ml conical flask containing 30 ml liquid proliferation medium, and inoculate 3 g of the callus per flask;

[0046] Place the conical flask on a shaker rotating at 120 rpm for dark culture at a temperature of 25℃ and a humidity of 45-55%, and the composition of the liquid proliferation medium is: MS 4.4 g / L, sucrose 30 g / L, hydrolyzed casein 2 g / L, 2,4-D 0.2 mg / L, 6-BA 0.1 mg / L, and pH 5.8-6.0.

[0047] D. Enzymatic hydrolysis

[0048] Take 0.2 g of embryogenic callus of honeysuckle, filter it into a 50 mL sterile centrifuge tube through an 80-mesh cell sieve with high-temperature sterilization, centrifuge at RCF 100×g for 5 min, and discard the supernatant; add 10 mL of enzymatic hydrolysis solution, and gently suck and beat it with a pipette to resuspend the callus cells, and perform enzymatic hydrolysis at 25℃ in the dark for 2-4 h with 30 rpm shaking; in the enzymatic hydrolysis solution, the mass concentration of D-mannitol is 11%, the mass concentration of cellulase is 1%, the mass concentration of lytic enzyme is 0.3%, the concentration of MES is 5 mM, the mass concentration of BSA is 0.1%, and the concentration of CaCl2 is 10 mM.

[0049] E. Protoplast extraction

[0050] After enzymatic hydrolysis is completed, add an equal volume of pre-cooled W5 solution to the enzymatic hydrolysis solution, and shake at 30 rpm for 3 min; filter the enzymatic hydrolysis solution after rinsing a 200-mesh cell sieve with pre-cooled W5 solution; centrifuge at RCF 100×g for 5 min, and discard the supernatant; resuspend the protoplasts by adding 5 ml of pre-cooled W5 solution, and ice-bath for 30 min; after centrifugation at RCF 100×g for 5 min, discard the supernatant; resuspend the protoplasts by adding 5 ml of pre-cooled MMG solution, centrifuge at RCF 100×g for 5 min, discard the supernatant, and the sediment is the protoplasts; the composition of the W5 solution is: NaCl 0.154 M, CaCl2 0.125 M, KCl 2.5 mM, and MES 5 mM; the composition of the MMG solution is: mannitol 0.5 M, MES 0.01 M, and MgCl2 0.01 M.

[0051] F. Protoplast yield detection

[0052] Add 2 ml of pre-cooled MMG solution, resuspend the protoplasts to obtain protoplast suspension, use a pipette to suck 20 μL of protoplast suspension, carefully inject into the counting chamber of 16x25 type blood cell counting plate. Slowly put the cover glass from one side of the counting chamber to avoid air bubbles, so that the suspension fills the counting chamber evenly. Move the blood cell counting plate under the optical microscope to observe and calculate the protoplast yield. Repeat counting 3 times for each sample.

[0053] Protoplast yield (number / g) = number of protoplasts in 100 small squares / 100x400x10 4 (number / ml) x 2 ml / 0.2 g.

[0054] G. Protoplast activity detection

[0055] Use a pipette to suck 9 μL of protoplast suspension onto a glass slide, add 1 μL of 0.1% FDA solution. After standing for 3 min, carefully cover the cover glass to avoid air bubbles, and place it under a fluorescence microscope for observation. Active protoplasts appear green fluorescent, count the number of fluorescent cells and total cells in the field of view, repeat 3 times. Activity coefficient = number of fluorescent cells / total number of cells.

[0056] Example 2:

[0057] Effect of different disinfection methods on the contamination rate of honeysuckle flower buds

[0058] Experimental materials: 2.5-3.0 cm honeysuckle flower buds

[0059] Experimental method: The honeysuckle flower buds were disinfected by the following 5 methods: the method in Example 1, without NaClO solution soaking, without UV irradiation, without NaClO solution soaking and UV irradiation, and without dishwashing liquid soaking, NaClO solution soaking and UV irradiation. The disinfected honeysuckle flower buds were inoculated on MS solid medium, and the contamination rate was counted after 5 days, where the contamination rate = number of contaminations / inoculation number x 100%, and the results are shown in Table 1:

[0060] Table 1: Effect of different disinfection methods on the contamination rate of honeysuckle flower buds

[0061]

[0062]

[0063] Experimental results: As shown in Table 1, due to the presence of more fluff on the flower bud surface of honeysuckle, more microorganisms are attached, and after collection, it should be cleaned with dishwashing liquid first to remove most of the microorganisms. Compared with the two disinfection methods of dishwashing liquid immersion and NaClO solution immersion, the ultraviolet disinfection time is the longest, and the effect of killing fungi is good. A3, A4 and A5 groups without ultraviolet disinfection appear more white fungal hyphae pollution. The combined use of dishwashing liquid immersion, NaClO solution immersion and ultraviolet irradiation can kill all microorganisms, and the pollution rate is reduced to 0.

[0064] Example 3:

[0065] Effect of different explant materials on embryogenic callus induction rate;

[0066] In order to explore the effect of different honeysuckle explant materials on embryogenic callus induction rate, five groups of explant materials of ovary, anther, filament, leaf and stem segment were set in turn while other experimental adjustments remained unchanged.

[0067] The specific operation is as follows: L1: cancel the flower bud after detoxification, cut off the ovary at the base of the flower bud in the clean bench, and inoculate it into the embryogenic callus induction medium. 6-8 are inoculated into each embryogenic callus induction medium, a total of 52 ovules are inoculated. L2: cancel the flower bud after detoxification, open the closed petals in the clean bench, take out the anther and inoculate it into the embryogenic callus induction medium. 15-20 are inoculated into each embryogenic callus induction medium, a total of 112 anthers are inoculated. L3: cancel the flower bud after detoxification, open the closed petals in the clean bench, remove the anther, cut the filament, and inoculate it into the embryogenic callus induction medium. 15-20 are inoculated into each embryogenic callus induction medium, a total of 96 filaments are inoculated. L4: cancel the leaf after detoxification, cut it into small pieces of about 0.5 cm in length perpendicular to the main vein in the clean bench, and inoculate it into the embryogenic callus induction medium. 3-4 pieces are inoculated into each embryogenic callus induction medium, a total of 49 leaves are inoculated. L5: cancel the stem segment after detoxification, cut it into small pieces of about 1-1.5 cm in length in the clean bench, and inoculate it into the embryogenic callus induction medium. 6-8 segments are inoculated into each medium, a total of 51 stem segments are inoculated. Put the inoculated L1 to L5 culture bottles into the artificial climate chamber for dark culture, the temperature is 25℃, the humidity is 45-55%, and after 2 months, the induction rate is counted. The composition of the embryogenic callus induction medium used is: MS 4.4g / L, sucrose 30g / L, hydrolyzed casein 2g / L, agar 7.5g / L, 2,4-D 2mg / L, 6-BA 1mg / L, AgNO32mg / L, pH 5.8-6.0. The results are shown in Table 2:

[0068] Table 2: Effect of different honeysuckle explant materials on embryogenic callus induction rate

[0069] Group L1 L2 L3 L4 L5 Inoculation material Ovary Anther Filament Leaf Stem segment Inoculation number 52 112 96 49 51 Embryogenic callus induction number 35 62 0 12 11 Embryogenic callus induction rate 67.31％ 55.36％ 0.00％ 24.49％ 21.57％

[0070] Experimental results: As shown in Table 2, L1 and L2 respectively used ovary and anther as the explant inoculation material of honeysuckle to induce embryogenic callus, and the induction rates were 67.31% and 55.36% respectively, and the induced callus was embryogenic callus. Although the induction rates of callus of leaves and stems were not low, the induced callus was a mixture of ordinary callus and embryogenic callus, most of which was ordinary callus, and the yield of embryogenic callus was low, only 24.49% and 21.57%. The filaments of honeysuckle had no differentiation ability, and all died after inoculation, without inducing any callus.

[0071] Example 4:

[0072] Taking ovary as an example, the effect of different embryogenic callus induction media on the induction rate of embryogenic callus was explored;

[0073] The specific operation is: the embryogenic callus induction medium of M1-M4 is respectively: M1: take 4.4g MS powder, dissolve in 1L deionized water, add 30g sucrose, 2g hydrolyzed casein, 7.5g agar, boil to dissolve the agar to form a uniform liquid, add 2,4-D, 6-BA, AgNO3, respectively, so that the final concentration reaches 2mg / L, 1mg / L, 2mg / L, cool to 40℃, then adjust the pH to 5.8-6.0 using 1M NaOH. M2: take 4.4g MS powder, dissolve in 1L deionized water, add 30g sucrose, 7.5g agar, boil to dissolve the agar to form a uniform liquid, add 2,4-D, 6-BA, AgNO3, respectively, so that the final concentration reaches 2mg / L, 1mg / L, 2mg / L, cool to 40℃, then adjust the pH to 5.8-6.0 using 1M NaOH. M3: take 4.4g MS powder, dissolve in 1L deionized water, add 30g sucrose, 2g hydrolyzed casein, 7.5g agar, boil to dissolve the agar to form a uniform liquid. Add 2,4-D, 6-BA, AgNO3, respectively, so that the final concentration reaches 0.2mg / L, 0.1mg / L, 2mg / L. Cool to 40℃, then adjust the pH to 5.8-6.0 using 1M NaOH. M4: take 4.4g MS powder, dissolve in 1L deionized water, add 30g sucrose, 2g hydrolyzed casein, 7.5g agar, boil to dissolve the agar to form a uniform liquid. Add 2,4-D, 6-BA, respectively, so that the final concentration reaches 2mg / L, 1mg / L. Cool to 40℃, then adjust the pH to 5.8-6.0 using 1M NaOH. The embryogenic callus induction medium of M1-M4 is respectively divided into culture bottles, sterilized at 121℃ for 20min, and cooled to room temperature for standby. The flower buds are removed from the ovaries at the base of the flower buds in the clean bench, and then inoculated into the embryogenic callus induction medium of M1-M4, 6-8 per bottle of medium, 51-55 ovaries per medium, respectively. After 2 months, the induction rate is counted respectively, and the results are shown in Table 3:

[0074] Table 3: Comparison of embryogenic callus induction rates of different embryogenic callus induction media (ovaries as explants)

[0075]

[0076]

[0077] Analysis of experimental results: As shown in Table 3, compared with M1, in M2, the induction rate of embryogenic callus was very low, only 5.88%, because the hydrolyzed casein was absent in the embryogenic callus induction medium, while the induction rate of embryogenic callus in M1 was 67.27%, therefore, the hydrolyzed casein was a very important organic nitrogen source in the process of embryogenic callus induction. The appropriate hormone concentration was also very crucial for the induction rate of embryogenic callus of honeysuckle, in M3, when the hormone concentration was too low, the induction rate of embryogenic callus also decreased, only 25.93%. In M4, when silver nitrate was absent in the embryogenic callus induction medium, a certain proportion of embryogenic callus browned and died, thereby affecting the final induction rate, the induction rate decreased to only 43.64%, therefore, silver nitrate was an important component to prevent callus browning.

[0078] Example 4:

[0079] Exploring the influence of different initial inoculation amounts on the proliferation efficiency of embryogenic callus from ovary and anther;

[0080] Specific operation: B1, for embryogenic callus from ovary, after 20d of ovary induction culture, select embryogenic callus with tender yellow color and vigorous proliferation, inoculate into 100ml conical flask, each containing 30ml liquid proliferation medium, gently crush the tissue, so that the tissue fragments in the medium are less than 0.5cm in diameter, inoculate about 3, 4, 5g in each medium; B2, for embryogenic callus from anther, after 40d of anther induction culture, select embryogenic callus with fresh color and loose granular shape, inoculate into 100ml conical flask, each containing 30ml liquid medium, gently crush the tissue, so that the tissue fragments in the medium are less than 0.5cm in diameter, inoculate about 3, 4, 5g in each medium. Place the above 6 conical flasks in B1 and B2 groups on a shaker with a rotation speed of 120rpm, cultivate in the dark, temperature 25℃, humidity 45-55%, after 15d, count the weight of embryogenic callus, the results are shown in Table 4 below. The composition of the liquid proliferation medium is: MS 4.4g / L, sucrose 30g / L, hydrolyzed casein 2g / L, 2,4-D 0.2mg / L, 6-BA 0.1mg / L, pH 5.8-6.0.

[0081] Table 4: Comparison of the proliferation efficiency of embryogenic callus from ovary and anther with different initial inoculation amounts

[0082]

[0083] Analysis of experimental results: According to the results in Table 4, the proliferation rate of embryogenic callus in liquid medium is closely related to the initial inoculation amount, and the optimal initial inoculation amount is different for embryogenic callus from different explants. For embryogenic callus from ovary, the optimal proliferation efficiency is achieved by inoculating 4 g in 30 ml of liquid medium, and the proliferation efficiency can reach 83.87% in 15 days. For embryogenic callus induced from anther, the optimal proliferation efficiency is achieved by inoculating 3 g in 30 ml of liquid medium, and the proliferation efficiency is 93.02% in 15 days. Too much or too little initial inoculation amount of embryogenic callus will significantly reduce the proliferation efficiency.

[0084] Example 5

[0085] Influence of different plant materials and enzymolysis time on the yield and activity coefficient of protoplasts.

[0086] Specific operation: 0.2 g of embryogenic callus cultured for 20 days was gently crushed with sterile tweezers, filtered into a 50 ml sterile centrifuge tube with a high-temperature sterilized 80-mesh cell sieve, centrifuged at a relative centrifugal force (RCF) of 100 x g for 5 min, and the supernatant was discarded; enzyme solution was added, and the callus cells were resuspended by gently sucking with a pipette. The protoplasts were enzymatically digested at 25°C in the dark for 2 h, 3 h, and 4 h at 30 rpm, respectively, and were recorded as D1-D3, and the yield and activity coefficient of protoplasts were calculated. The 3rd-5th fully expanded leaves below the terminal bud of honeysuckle were cut off, and 0.2 g of the leaves was weighed. The leaves were cut into small pieces as much as possible, and enzyme solution was added. The leaf fragments were uniformly dispersed and fully contacted with the enzyme solution by gently sucking with a pipette. The protoplasts were enzymatically digested at 25°C in the dark for 2 h, 3 h, and 4 h at 30 rpm, respectively, and were recorded as D4-D6, and the yield and activity coefficient of protoplasts were calculated. In the enzyme solution of this example, the mass concentration of D-mannitol was 11%, the mass concentration of cellulase was 1%, the mass concentration of dissociation enzyme was 0.3%, the concentration of MES was 5 mM, the mass concentration of BSA was 0.1%, and the concentration of CaCl2 was 10 mM. The detection operation and calculation method of protoplast yield refer to steps F in Example 1. The detection operation and calculation method of activity coefficient refer to steps G in Example 1.

[0087] Table 5: Influence of different plant materials and enzymolysis time on the yield and activity coefficient of protoplasts

[0088]

[0089] Result analysis: when the enzymolysis time is 1h, the embryogenic callus and the tender leaf blade all do not start enzymolysis. As can be seen from table 5, using embryogenic callus as the material to prepare protoplast is better than using tender leaf blade in terms of yield and activity coefficient. The main reason is that the cell division in embryogenic callus is vigorous, the structure is loose, and the cell is easy to release and enzymolysis. Through the comparison of enzymolysis time, it is found that the leaf blade needs to be enzymolyzed for 3h to reach the highest protoplast yield 9.6x10 6 / g, and the embryogenic callus can obtain the highest protoplast yield 20.40x10 6 / g in 2h. In addition, the activity of the protoplast prepared by using embryogenic callus is obviously higher than that of the protoplast prepared by using tender leaf blade, and the activity of the protoplast prepared by using embryogenic callus can be as high as 0.91. Therefore, compared with using tender leaf blade to prepare protoplast, using embryogenic callus to prepare protoplast of honeysuckle can greatly shorten the preparation time and improve the activity of protoplast, which is a high-efficiency method for preparing protoplast of honeysuckle.

[0090] Example 6:

[0091] Explore the effect of different enzyme preparations on the efficiency of protoplast preparation.

[0092] Specific operation:

[0093] The embryogenic callus cultured for 20d is 0.2g, which is gently crushed into larger particles with sterile forceps, filtered into a 50ml sterile centrifuge tube with a high-temperature sterilized 80-mesh cell sieve, centrifuged at a relative centrifugal force (RCF) of 100xg for 5min, and the supernatant was discarded; add enzyme solution, wherein the enzyme solution is a combination of one or more of 1% cellulase, 0.30% macerate enzyme and 0.50% pectinase, see table 6. Gently suck and beat with a pipette gun to resuspend the embryogenic callus cells, and shake at 25℃ in the dark for 2h at 30rpm. Calculate the yield and activity coefficient of protoplast, and the detection operation and calculation method of protoplast yield refer to steps F in example 1. The detection operation and calculation method of activity coefficient refer to steps G in example 1.

[0094] Table 6: Effect of different enzyme preparations on the efficiency of protoplast preparation

[0095] Group X1 X2 X3 Cellulase 1％ 1％ 1％ Macerozyme 0.30％ 0 0 Pectolyase 0 0.50％ 0 Protoplast yield (x 10 6 Individuals / g) 19.87 17.87 10.53 Activity coefficient 0.92 0.60 0.73

[0096] Result analysis: as can be seen from table 6, the combination of cellulase and macerate enzyme has the best effect, not only the protoplast yield is the highest, which is 19.87x10 6 / g, but also the activity is the best, which is 0.92. Although the combination of cellulase and pectinase has good enzymolysis effect and can obtain a large amount of protoplast (17.87x10 6The yield of protoplasts was 10.53×10 6 cells / g.

[0097] All features described in the specification, the claims appended hereto, and / or the figures attached hereto, either individually or in any combination, are important features of the present invention.

Claims

1. A method for isolating honeysuckle protoplasts using embryogenic callus, characterized in that, Includes the following steps: A. Disinfection Collect honeysuckle flower buds with a length of 2.5-3.0 cm, soak them in dish soap solution for 10 minutes, gently rubbing them during the process, then rinse them thoroughly with running water, and then soak them in 0.1% NaClO solution for 5 minutes; rinse them again with running water for 1 hour; after drying, irradiate them with ultraviolet light on a clean bench for 20 minutes; then disinfect them in 75% alcohol for 1 minute; finally, rinse them 3 times with sterile water. B. Embryonic callus induction After sterilizing, the surface moisture of the honeysuckle flower buds is absorbed, the ovary is cut off, and the ovary is inoculated into embryogenic callus induction medium. The closed petals are opened, the anthers are removed, and the anthers are inoculated into embryogenic callus induction medium. The components of the embryogenic callus induction medium are: MS 4.4 g / L, sucrose 30 g / L, hydrolyzed casein 2 g / L, agar 7.5 g / L, 2,4-D 2 mg / L, 6-BA 1 mg / L, AgNO3 2 mg / L, pH 5.8-6.

0. C. Fluid proliferation of embryonic callus Select pale yellow, vigorously proliferating honeysuckle embryogenic callus tissue, inoculate it into liquid proliferation medium, and place it on a shaker at 120 rpm for incubation in the dark at 25℃ and 45-55% humidity. The liquid proliferation medium consists of: MS 4.4 g / L, sucrose 30 g / L, hydrolyzed casein 2 g / L, 2,4-D 0.2 mg / L, 6-BA 0.1 mg / L, and pH 5.8-6.

0. D. Enzymatic hydrolysis Take 0.2g of honeysuckle embryogenic callus tissue, filter it through an 80-mesh cell sieve sterilized at high temperature into a 50mL sterile centrifuge tube, centrifuge horizontally at 100×g for 5min, and discard the supernatant; add 10mL of enzymatic hydrolysis solution, gently aspirate with a pipette to resuspend the callus cells, and hydrolyze at 30rpm for 2-4h under dark conditions at 25℃; wherein, the enzymatic hydrolysis solution contains 11% D-mannitol, 1% cellulase, 0.3% sorbitase, 5 mM MES, 0.1% BSA, and 10mM CaCl2; E. Protoplast extraction After enzymatic hydrolysis, add an equal volume of pre-chilled W5 solution to the hydrolysate and shake at 30 rpm for 3 min. Rinse the 200-mesh cell sieve with pre-chilled W5 solution and filter the hydrolysate. Centrifuge horizontally at 100×g for 5 min and discard the supernatant. Resuspend the protoplasts in 5 ml of pre-chilled W5 solution and incubate on ice for 30 min. Centrifuge horizontally at 100×g for 5 min and discard the supernatant. Resuspend the protoplasts in 5 ml of pre-chilled MMG solution, centrifuge horizontally at 100×g for 5 min and discard the supernatant. The precipitate is the protoplast.

2. The method for isolating honeysuckle protoplasts using embryogenic callus according to claim 1, characterized in that, In step D, the ratio of honeysuckle embryogenic callus to enzymatic hydrolysate is 0.2 g / 10 mL.

3. The method for isolating honeysuckle protoplasts using embryogenic callus according to claim 1, characterized in that, In step D, the enzymatic hydrolysis time is 2 hours.

4. The method for isolating honeysuckle protoplasts using embryogenic callus according to claim 1, characterized in that, In step E, the composition of the W5 solution is: NaCl 0.154M, CaCl2 0.125M, KCl 2.5mM, MES 5mM.

5. The method for isolating honeysuckle protoplasts using embryogenic callus according to claim 1, characterized in that, In step E, the MMG solution contains: 0.5M mannitol, 0.01M MES, and 0.01M MgCl2.

6. A method for isolating honeysuckle protoplasts using embryogenic callus according to any one of claims 1-5, characterized in that, The honeysuckle embryogenic callus was obtained by the following steps: A. Disinfection Collect honeysuckle flower buds with a length of 2.5-3.0 cm, soak them in dish soap solution for 10 minutes, gently rubbing them during the process, then rinse them thoroughly with running water, and then soak them in 0.1% NaClO solution for 5 minutes; rinse them again with running water for 1 hour; after drying, irradiate them with ultraviolet light on a clean bench for 20 minutes; then disinfect them in 75% alcohol for 1 minute; finally, rinse them 3 times with sterile water. B. Embryonic callus induction On a clean bench, the surface moisture of the sterilized honeysuckle flower buds is absorbed with sterile filter paper. The ovaries are cut off and inoculated into embryogenic callus induction medium, with 6-8 ovaries inoculated per bottle of medium. The closed petals are opened, the anthers are removed, and inoculated into embryogenic callus induction medium, with 15-20 anthers inoculated per bottle of medium. The inoculated culture bottles are placed in an artificial climate chamber for cultivation in the dark at a temperature of 25℃ and a humidity of 45-55%. C. Fluid proliferation of embryonic callus After ovary induction culture for 20 days, pale yellow and vigorously proliferating honeysuckle embryogenic callus tissue was selected and inoculated into 100ml Erlenmeyer flasks, each containing 30ml of liquid proliferation medium; 4g of the culture medium was inoculated into each flask. After 40 days of anther induction culture, fresh-colored, loose, granular honeysuckle embryogenic callus tissue was selected and inoculated into 100ml Erlenmeyer flasks containing 30ml of liquid proliferation medium, with 3g of the tissue inoculated into each flask.

7. The method for isolating honeysuckle protoplasts using embryogenic callus according to claim 6, characterized in that, In step C, when inoculating the honeysuckle embryogenic callus obtained by ovary induction, the tissue is gently crushed so that the diameter of the tissue fragments in the liquid proliferation medium is less than 0.5 cm.

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