Tissue Culture Method of Patchouli

Abstract

The present invention relates to the field of plant breeding and tissue culture, and in particular to a patchouli tissue culture method. The present invention prepares patchouli protoplasts by combining a cellulase R10 mutant and a pectinase Y23, and performs protoplast fusion under the induction of PEG, which can significantly improve the yield and vitality of the protoplasts, and improve the fusion rate and seedling rate of the protoplasts, providing a new direction for the cultivation of new varieties of patchouli, and having broad application prospects in the industry.

Description

Technical Field

[0001] The invention relates to the field of plant breeding and tissue culture, and in particular to a patchouli tissue culture method. Background Art

[0002] Patchouli (Pogostemon cablin) is a plant of the genus Pogostemon in the Lamiaceae family. It is a local medicinal material in Guangdong. It has the effects of aromatic turbidity, stopping vomiting, and relieving heat. Modern pharmacological studies have shown that patchouli can regulate gastrointestinal function and has antibacterial effects. It is the main raw material for many Chinese patent medicines. In addition, the patchouli alcohol in patchouli oil has a unique and pleasant smell and can make the fragrance last long. It is widely used in daily necessities and cosmetics.

[0003] Protoplasts have the ability to regenerate into complete plants. They are ideal materials for organelle separation, transgenic and gene function analysis, and are also essential materials for cultivating new varieties in a cell hybridization manner. In terms of Chinese herbal medicine variety screening and germplasm improvement, protoplast fusion is one of the effective ways to induce cell hybridization and increase chromosome group ploidy. At present, plant protoplast preparation and fusion are mostly prepared by enzymatic hydrolysis and PEG-induced protoplast fusion, and enzyme solution composition, enzymatic hydrolysis time and permeabilization agent composition are the main factors affecting the preparation of protoplasts by enzymatic hydrolysis. Therefore, the preparation of protoplasts and tissue culture are the key to the cultivation of patchouli. The invention patent with publication number CN1027422A cultivates new varieties by the method of protoplast fusion for new varieties of patchouli, but the improvement of protoplast activity and fusion efficiency is still a problem that needs to be solved. Summary of the invention

[0004] The purpose of the present invention is to provide a method for cultivating a new variety of patchouli, and a method for obtaining the new variety by using protoplast fusion technology. The method adopts a cell enzymatic hydrolysis method to obtain protoplasts, and the protoplasts are fused by inducing polyethylene glycol, and the obtained fusion products are placed in a culture medium for culture, and the obtained cell clusters are transferred to a somatic embryo induction culture medium to induce somatic embryos until complete regenerated seedlings are obtained, and the regenerated seedlings are then transferred to a culture medium that promotes plant development to obtain a new variety.

[0005] The enzymatic hydrolysate is a combination of cellulase and pectinase.

[0006] Preferred is a combination of a cellulase mutant and a pectinase.

[0007] The preferred mutation positions of the cellulase are one or more of Q20, R72, V125, and G193.

[0008] More preferably, the cellulase mutation is one or more of Q20A, R72D, V125P, and G193S.

[0009] More preferably, the mutation sites of the cellulase are Q20A / R72D or Q20A / G193S.

[0010] The preferred cellulase is R10, and its sequence is shown in SEQ ID NO. 1. The pectinase is Y23, both of which are commercially available products.

[0011] The amino acid sequence of the enzyme mutant Q20A / R72D is shown in SEQ ID NO.3.

[0012] The amino acid sequence of the enzyme mutant Q20A / V125P is shown in SEQ ID NO.4.

[0013] The amino acid sequence of the enzyme mutant Q20A / G193S is shown in SEQ ID NO.5.

[0014] The PEG mass concentration of the fusion solution used to promote fusion can be 30%-50%.

[0015] The present invention has the following advantages:

[0016] The method for culturing patchouli tissue of the present invention is simple and efficient and does not require complicated processes.

[0017] The method of the invention can effectively improve the yield and vitality of patchouli protoplasts, and significantly improve the fusion rate thereof, thereby improving the embryo formation rate and seedling formation rate of patchouli tissue culture.

[0018] The protoplast fusion method of the present invention is pollution-free and does not involve the introduction of exogenous genes, and has broad application prospects in the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the enzyme activity of different enzymes; R10-wt is the wild type, R10-mut1 is the presence of Q20A / R72D mutations relative to the wild type, R10-mut2 is the presence of Q20A / V125P mutations relative to the wild type, and R10-mut3 is the presence of Q20A / G193S relative to the wild type.

[0020] Figure 2 Protoplast yield and viability for different enzyme combinations.

[0021] Figure 3 The fusion rate and multimerization fusion rate of protoplasts prepared by different enzyme combinations. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0023] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as the first indicated unless otherwise specified; the reagents and materials involved are all obtained from commercial sources unless otherwise specified.

[0024] The Lactococcus lactis NZ9000 competent cells used in the present invention were used for plasmid construction, and the lactic acid bacteria starter used in the experiment was purchased from Beijing Chuanxiu Technology Co., Ltd.

[0025] The medium for screening recombinant bacteria was LA solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar powder, and 37°C for culturing recombinant bacteria.

[0026] The composition of LB liquid medium was: 1 g peptone, 0.5 g yeast extract, 1 g sodium chloride, and 100 mL distilled water.

[0027] Example 1 Design of cellulase R10 gene and construction of recombinant cloning vector

[0028] The mature peptide of cellulase R-10 was obtained through GenBank (accession number: GenBank: ACY46934.1), and its sequence is shown in SEQ ID NO.1. The lactic acid bacteria expression system codon was optimized, and the primers with restriction sites were designed using Primer 5.0 software. The restriction sites were SnaB I and EcoR I. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the optimized cellulase R10 gene sequence was obtained by PCR. Its specific sequence is shown in SEQ ID NO.2.

[0029] Using pPIC9K-R10 as a template, PCR amplification was performed to obtain the target gene R10. The original plasmid 368-PGK was digested with two restriction endonucleases, SnaB I and EcoR I, to expose the sticky ends at both ends of the vector. The cellulase obtained by PCR amplification was also digested with SnaB I and EcoR I and then recovered. The cellulase R10 recovered after digestion treatment and the vector pHBM-368-PGK fragment were mixed and reacted overnight at 16 ℃ using T4 DNA ligase.

[0030] Example 2 Construction of cellulase R-10 mutant and recombinant strain

[0031] According to the amino acid map, a group of amino acids that may affect the spatial structure of the R10 enzyme were screened and mutated. The variants were Q20A / R72D, Q20A / V125P, and Q20A / G193S. The nucleotide sequences of Q20A / R72D, Q20A / V125P, and Q20A / G193S were obtained by site-directed mutagenesis. The wt and its variants were successfully cloned into the plasmid pHBM-368-PGK, respectively. The successfully constructed recombinant plasmids were transformed into Lactococcus lactis NZ9000 competent cells, recorded as R10-wt and R10-mut1, R10-mut2, and R10-mut3. The recombinant single colony was picked, cultured in the culture medium, and the colony PCR was identified. After restriction digestion identification, sequencing verification was performed, and the wild-type and mutant engineering bacteria were selected for preservation.

[0032] Example 3 Expression of Cellulase R10 by Recombinant Engineering Bacteria

[0033] Three strains of each type were selected and cultured in liquid LB medium (containing 5μg / mL erythromycin), and cultured at 30℃ overnight. The next day, 5% inoculation was inoculated into 100mL LB liquid medium (containing 5μg / mL erythromycin), and cultured at 30℃ until OD600=0.5. 100μL 0.3M CuSO4 was added to the clean bench after filtration and sterilization, and cultured at 30℃ for 4h, and then cultured at 16℃ for 24h. 90μL of the supernatant of the recombinant strain cell wall liquid was taken respectively, mixed with 30μL 4×ProteinLoading Buffer, and boiled at 100℃ for 10min. Centrifuged at 4℃, 12,000rpm for 5min, and the sample treatment liquid was collected. The enzyme activity was detected by colorimetry.

[0034] like Figure 1 As shown, the average extracellular enzyme activities of R10-wt, R10-mut1, R10-mut2, and R10-mut3 were 82.3U / L, 147.1U / L, 27.9U / L, and 91.6U / L, respectively. The results of the extracellular enzyme activity determination of cellulase R10 showed that the recombinant Lactococcus lactis successfully achieved the secretory expression of cellulase R10, and the enzyme activity of R10-Q20A / R72D increased by about 1.78 times compared with the wild type, while R10-Q20A / V125P almost caused the enzyme activity to be lost, which was only 0.34 times that of the wild type, and the enzyme activity of R10-Q20A / G193S was slightly higher than that of the wild type.

[0035] Example 4 Preparation and fusion of protoplasts

[0036] (1) Liquid culture medium: MS medium was used as the basic culture medium, and 1.0 mg 6-BA and 30 g sucrose were added per liter of the above basic culture medium. The pH was 5.5;

[0037] Somatic embryo induction medium: MS medium was used as the basic medium. 1.0 mg 6-BA, 30 g sucrose and 6.5 g agar powder were added to each liter of the above basic medium. The pH was 5.5.

[0038] Protoplast culture medium: MS medium was used as the basic medium, 1.0 mg 6-BA was added per liter of the above basic medium, and 60 g mannitol and 1 g MES were added, with a pH of 5.5;

[0039] Growth medium: MS medium was used as the basic medium, 30 g sucrose and 6.5 g agar powder were added to each liter of the above basic medium, and the pH was 5.5;

[0040] High pH high calcium wash: 0.05 M CaCl2, 0.4 M mannitol, and 0.05 M glycine-NaOH buffer, pH 10.5.

[0041] (2) Callus induction and suspension culture: Take commercially available patchouli as explants and inoculate them in MS+0.05 2,4-D+0.5 KT (in mg / L) solid culture medium. Induce patchouli callus at a light intensity of 1500 lx, (25±2)℃, and light intensity of 12 h / d. Select pale yellow callus and transfer it to liquid culture medium for suspension culture. The rotation speed is 135 r / min, the inoculum size is 70 g FW / L, and the subculture cycle is 24 days.

[0042] (3) Protoplast preparation: Take the leaves of sterile patchouli seedlings that have been subcultured for 12 days and are located in the third pair of unfolded leaves below the terminal bud (leaf size is about 1.2 cm × 1.5 cm), remove the large veins, and immerse the leaf fragments in enzyme solutions with different concentrations of pectinase and cellulase (Table 1). The fresh weight of the leaves and the volume of the enzyme solution are 1:20. Each group of enzyme solutions is prepared with an aqueous solution containing 9wt% mannitol, 0.1wt% MES, and 0.02wt% CaCl2, pH 5.7, and filtered for sterilization. Incubate at (26±1)℃ on a low-speed shaker at a speed of 40 r / min in the dark for 10 h. Starting from 4 h, check the protoplast yield every 2 h to determine the best enzyme solution combination for protoplast isolation.

[0043] Table 1 Protoplast enzymatic hydrolysis combination (enzyme concentration in W / V)

[0044] Enzyme solution number Combination 1 Combination 2 Combination 3 Enzyme composition 0.25% pectinase Y-23 + 2% cellulase R10-wt 0.25% pectinase Y-23 + 2% cellulase R10-mut1 0.25% pectinase Y-23 + 2% cellulase R10-mut3

[0045] (4) Protoplast purification and collection: The enzymatic hydrolysate was passed through 40, 100, and 200 mesh sieves in turn, and the filtrate was collected and centrifuged at 1000 r / min for 3 min to remove the supernatant. The precipitate was mixed with an appropriate amount of CPW-4M solution and centrifuged again to remove the supernatant. After repeating three times, the precipitate was transferred to the protoplast culture medium for storage.

[0046] (5) Determination of yield and vitality: The protoplast solution obtained in the above steps was dripped onto a blood cell counting plate and the number of protoplasts was calculated. The density and yield were calculated using the following formula. The experiment was repeated no less than 3 times. During microscopic examination, different visual fields were measured ≥ 5 times. The average of the 5 measurements was taken for each sample (the same below).

[0047] Protoplast density (pieces / mL) = total number of protoplasts in 5 large grids × 5 × 10000 × dilution multiple; the calculation formula for protoplast yield (pieces / g) is as follows:

[0048] Protoplast yield = (protoplast density × volume after dilution) / cell biomass (g)

[0049] Protoplast activity = number of fluorescent protoplasts under dark field / number of protoplasts under bright field

[0050] (6) Protoplast fusion: The high pH and high calcium method was used to fuse protoplasts. The separated and purified protoplasts were suspended in CPW-4M solution, centrifuged and washed, and placed in a culture dish for 15 minutes. The density was about 3×10 5 / mL, slowly add 30% (w / v) PEG 6000 fusion solution, the amount added is 1.5 times the volume of the protoplast suspension, let it stand for 5 minutes, then add high pH high calcium washing solution twice the volume of the fusion solution, let it stand for 20 minutes, centrifuge the mixed solution and remove the supernatant, repeat 3 times, examine under the microscope and measure the cell diameter of normal morphology, calculate the polymerization rate and polymerization rate, the formula is as follows:

[0051] Aggregation rate = (number of protoplasts in two-cell aggregation / total number of protoplasts) × 100%.

[0052] Multiplication rate = (number of protoplasts in aggregates of 3 or more cells / total number of protoplasts) × 100%.

[0053] (7) The successfully fused protoplasts were transferred to somatic embryo induction medium and cultured using 1.0 mg / L 6-BA to induce somatic embryos.

[0054] (8) The somatic embryos were transferred to a somatic embryo induction medium and subcultured every 15 days until the embryos germinated and regenerated seedlings were obtained. The regenerated seedlings with a height of 1.5 cm were transferred to a patchouli growth medium for culture to obtain patchouli plant seedlings with somatic cell fusion.

[0055] Example 5 Results and Analysis

[0056] Table 2 Characterization of protoplasts prepared by various enzymatic hydrolysate combinations

[0057] Combination 1 Combination 2 Combination 3 <![CDATA[Yield (×10 5 individuals / g)]]> 8.9±0.3a 18.2±0.8b 10.3±0.6a vitality(%) 70.2±3.3a 92.5±4.8c 80.4±3.9b Polymerization rate (%) 55.13±1.44a 69.26±1.67b 58.47±1.55a Polymerization rate (%) 10.25±0.23a 16.22±0.55b 12.36±0.34a

[0058] Note: Different letters abc indicate significant differences (P<0.05).

[0059] See also Figure 2 As shown in Table 2, the yield and activity of protoplasts prepared by different enzymatic hydrolysis combinations were quite different. The yield and activity of protoplasts prepared by combination 2, i.e., 0.25% pectinase Y-23 + 2% cellulase R10-Q20A / R72D hydrolysis solution, were significantly different from those of other bacteria (P<0.05), and the yield could reach 18.2±0.8×10 5 The protoplast yield of combination 3, i.e., 0.25% pectinase Y-23 + 2% cellulase R10-Q20A / G193S enzymatic hydrolysate, was also higher than that of combination 1, reaching 10.3±0.6×10 5 / g, but there was no significant difference compared with combination 1, but there was a significant improvement in vitality (P<0.05), with an average vitality of 80.4±3.9%. Therefore, using the enzyme combination described in combination 2 to prepare protoplasts can significantly increase its yield and vitality, and using the enzyme combination described in combination 3 to prepare protoplasts can significantly increase its vitality.

[0060] See also Figure 3 As shown in Table 2, there are great differences in the yield and vitality of protoplasts using different enzymatic hydrolysis combinations. The fusion rate and multi-fusion rate of protoplasts prepared by combination 2, i.e., 0.25% pectinase Y-23 + 2% cellulase R10-Q20A / R72D enzymatic hydrolysis solution combination, are significantly improved (P<0.05), with the polymerization rate reaching 69.26±1.67% and the multi-fusion rate reaching 16.22±0.55%. The fusion rate and multi-fusion rate of protoplasts prepared by combination 3, i.e., 0.25% pectinase Y-23 + 2% cellulase R10-Q20A / G193S enzymatic hydrolysis solution combination, are also higher than those of combination 1, reaching 58.47±1.55% and 12.36±0.34%, but there is no significant difference compared with combination 1. That is, the preparation of protoplasts using the enzymatic hydrolysis solution of combination 2 can significantly improve its fusion rate.

[0061] Therefore, the above enzyme mutant combination obtained by the present invention has a good effect on the preparation of protoplasts by enzymatic hydrolysis, can solve the problem of low fusion rate in the cultivation of new varieties of patchouli protoplasts, and has broad application value in the cultivation of new patchouli varieties.

[0062] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A tissue culture method for Patchouli, characterized in that: The cellulase R10 mutant and pectinase Y23 were combined to prepare patchouli protoplasts, and the protoplasts were fused under the induction of PEG fusion solution, and induced to form embryos and seedlings. The cellulase R10 mutant has a Q20A / R72D or Q20A / G193S mutation relative to the wild type, the amino acid sequence of the wild type cellulase R10 is shown in SEQ ID NO.1, the amino acid sequence of the enzyme mutant having Q20A / R72D relative to the wild type is shown in SEQ ID NO.3, and the amino acid sequence of the enzyme mutant having Q20A / G193S relative to the wild type is shown in SEQ ID NO.5; The enzymatic hydrolysis used a combination of 0.25% pectinase Y23 + 2% cellulase R10 mutant.

2. The tissue culture method of Patchouli according to claim 1, characterized in that: The mass concentration of PEG in the PEG fusion solution is 30%-50%.

3. The tissue culture method of Patchouli according to claim 1, characterized in that: The embryo induction medium component is based on MS medium, 1.0 mg 6-BA, 30 g sucrose and 6.5 g agar powder are added to each liter of the above basic medium, and the pH is 5.5-6.

0.

4. The tissue culture method of Patchouli according to claim 1, characterized in that: The culture medium component for inducing seedlings is based on MS culture medium, 30g of sucrose and 6.5g of agar powder are added to each liter of the basic culture medium, and the pH value is 5.5-6.

0.

5. The tissue culture method of Patchouli according to claim 1, characterized in that: The method for protoplast fusion is a high pH high calcium method; the high pH high calcium method is: the separated and purified patchouli protoplasts are suspended in CPW-4M solution, centrifuged and washed, and allowed to stand in a culture dish for 15 minutes, with a density of 3×10 5 / mL, add 30% PEG 6000 fusion solution in an amount of 1.5 times the volume of the patchouli protoplast suspension, let stand for 5 minutes, then add high pH high calcium washing solution in an amount of 2 times the volume of the fusion solution, let stand for 10-40 minutes, centrifuge the mixed solution and remove the supernatant; wherein the high pH high calcium washing solution is composed of 0.05M CaCl2, 0.4M mannitol and 0.05M glycine-NaOH buffer, pH 10.5.

Images