Preparation method of kale leaf protoplast and enzymatic hydrolysate

Abstract

The invention discloses a preparation method of kale leaf protoplast and enzymatic hydrolysate. The preparation method comprises the following steps: performing enzymolysis on leaves of sterile kale seedlings by using the enzymatic hydrolysate; the enzymatic hydrolysate is composed of an enzyme solution and an SCW solution, the enzyme solution comprises cellulase, pectinase, mannitol and CaCl2, and the pH value of the enzyme solution is 5.8; the SCW liquid is prepared from sorbitol, CaCl2 and MES, and the pH (Potential of Hydrogen) of the SCW liquid is 5.8; and adding 0.4 M mannitol-CPW into supernate in the enzymolysis product, centrifuging to take precipitate, repeating for three times, cleaning once by using a protoplast culture medium, and centrifuging to take precipitate which is a solution containing the kale protoplast. According to the method, a kale leaf protoplast separation system is successfully established, a theoretical basis is provided for subsequent kale protoplast fusion culture and instantaneous transformation system construction, and a new way is provided for germplasm creation and genetic improvement by using kale.

Description

Technical Field

[0001] This invention relates to the field of cruciferous crop breeding technology, and in particular to a method for preparing protoplasts from kale leaves and an enzymatic hydrolysate. Background Technology

[0002] Kale (Brassica oleracea var. acephala) is a biennial foliage herbaceous plant belonging to the genus Brassica in the family Brassicaceae, possessing extremely high nutritional and horticultural value. However, breeding efforts still face multiple challenges. For example, domestic kale varieties are mainly imported (such as F1 hybrids from Japan and the United States), lacking varieties with independent intellectual property rights, resulting in a limited germplasm resource bank and limited adaptability. Furthermore, the genetic diversity of kale is insufficiently explored: although SSR marker analysis indicates genetic diversity among different varieties (heritage coefficient 0.419–0.855), domestic research on the genetic mechanisms of key traits such as leaf shape and leaf color in kale is still in-depth, making it difficult to efficiently screen for stress-resistant or superior genes. Kale is a self-incompatible plant, requiring artificial pollination or isolated cultivation, which is time-consuming, labor-intensive, and costly. There is an urgent need to improve the stress resistance and quality of kale, as existing varieties lack sufficient resistance to common pests and diseases such as aphids. Insufficient research on the response mechanisms to stresses such as high temperatures and waterlogging leads to low yield and leaf color stability in varieties under suboptimal conditions. Therefore, establishing an efficient protoplast isolation technology for kale can provide a new approach to overcome these bottlenecks and systematically solve problems such as germplasm innovation, technological lag, and insufficient resistance in kale breeding.

[0003] Chinese patent CN115873782A discloses an enzymatic hydrolysate, kit, and method for preparing protoplasts of Brassica oleracea plants. The enzymatic hydrolysate for preparing broccoli protoplasts according to this invention includes a mixed enzyme and mannitol. The mixed enzyme is a mixture of cellulase and pectinase, with a mass ratio of cellulase to pectinase of 5–15:1, and the mass concentration of cellulase in the hydrolysate is 0.5–1.5%. The concentration of mannitol in the hydrolysate is 0.3–0.6 M. Enzymatic hydrolysis using the above-mentioned hydrolysate can yield high yields and viability of Brassica oleracea protoplasts. However, the enzymatic hydrolysis time of this patent is 13–15 hours, and pretreatment is required, making the experimental steps cumbersome and the experimental process longer. Furthermore, the hydrolysate is relatively complex, especially the CM kit, polyvinylpyrrolidone, and bovine serum albumin used in the hydrolysate, which are expensive reagents for protoplast extraction. The sucrose flotation method used requires the preparation of a sucrose solution in advance. After centrifugation, protoplasts need to be carefully collected from the interface using a pipette. The collection process requires skill, is prone to impurities, and is longer than usual. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing protoplasts from kale leaves and an enzymatic hydrolysate to address the current problems of limited germplasm resources and urgent need for quality improvement in kale breeding. The method is simple, has a short processing flow, low cost, and yields a large number of kale protoplasts with high survival rate.

[0005] The technical solution of this invention:

[0006] A method for preparing protoplasts from kale leaves, the specific method including the following steps:

[0007] Step 1: Use an enzymatic hydrolysate to hydrolyze the leaves of sterile kale seedlings to obtain crude kale protoplast fluid. The enzymatic hydrolysate consists of an enzyme solution and an SCW solution with a volume ratio of 3-7:5.

[0008] Step 2: Add mannitol-CPW solution to the crude protoplast solution of kale from Step 1, centrifuge and collect the precipitate, repeat three times, then wash once with protoplast culture medium to obtain pure protoplast solution, centrifuge and collect the precipitate, which is the precipitate containing the kale protoplasts.

[0009] Furthermore, in step one, the enzyme solution comprises 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.8; the SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.6-5.8.

[0010] Furthermore, in step one, the enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 5:5. The enzyme solution consists of 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80mM CaCl2, and the pH of the enzyme solution is 5.8. The SCW solution consists of 0.5M sorbitol, 10mM CaCl2, and 5mM MES, and the pH of the SCW solution is 5.8.

[0011] Furthermore, in step two, the mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution includes 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, and 1480 mg / L CaCl2·2H2O, with a pH of 5.8.

[0012] An enzymatic hydrolysate used in a method for preparing protoplasts from kale leaves.

[0013] Includes enzymatic hydrolysate and mannitol-CPW solution.

[0014] The enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 3-7:5. The enzyme solution includes 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.8. The SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.4-5.8.

[0015] The mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution contains 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, and 1480 mg / L CaCl2·2H2O, with a pH of 5.8.

[0016] Furthermore, the enzymatic hydrolysate consisted of an enzyme solution and an SCW solution in a 5:5 volume ratio. The enzyme solution contained 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80mM CaCl2, with a pH of 5.8. The SCW solution contained 0.5M sorbitol, 10mM CaCl2, and 5mM MES, with a pH of 5.8.

[0017] This invention establishes a highly efficient technique for isolating kale protoplasts. It utilizes cellulase, pectinase, mannitol, and CaCl2 as enzymatic hydrolysates, combined with an SCW solution containing sorbitol, CaCl2, and MES. Centrifugation and precipitation are then performed using a mannitol-CPW solution, resulting in a high number of extracted kale protoplasts, reaching 3.08 × 10^6. 6 The high survival rate (approximately 90%) provides a theoretical basis for the subsequent construction of protoplast fusion culture and transient transformation systems for kale, and offers a new approach for kale genetic breeding. Compared with existing technologies, the SCW solution (mass-to-weight ratio separation solution) formulation in the enzymatic hydrolysate of this invention uses fewer reagents, is easier to prepare, and has lower costs; the centrifugal precipitation method is simple, rapid, gentle, and highly efficient; no pretreatment is required before enzymatic hydrolysis of kale leaves, simplifying the experimental process. Attached Figure Description

[0018] Figure 1 The images show optical microscope comparisons of protoplasts from kale leaves in Examples 1, 2, and 3 of this invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a method for preparing protoplasts from kale leaves, the specific method comprising the following steps:

[0021] Step 1: Use an enzymatic hydrolysate to hydrolyze the leaves of sterile kale seedlings to obtain crude kale protoplast fluid. The enzymatic hydrolysate consists of an enzyme solution and an SCW solution with a volume ratio of 3-7:5.

[0022] Step 2: Add mannitol-CPW solution to the crude protoplast solution of kale from Step 1, centrifuge and collect the precipitate, repeat three times, then wash once with protoplast culture medium to obtain pure protoplast solution, centrifuge and collect the precipitate, which is the precipitate containing the kale protoplasts.

[0023] Furthermore, in step one, the enzyme solution comprises 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.8; the SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.6-5.8.

[0024] Furthermore, in step one, the enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 5:5. The enzyme solution consists of 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80mM CaCl2, and the pH of the enzyme solution is 5.8. The SCW solution consists of 0.5M sorbitol, 10mM CaCl2, and 5mM MES, and the pH of the SCW solution is 5.8.

[0025] Furthermore, in step two, the mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution includes 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, and 1480 mg / L CaCl2·2H2O, with a pH of 5.8.

[0026] This invention also provides an enzymatic hydrolysate for the preparation of protoplasts from kale leaves.

[0027] Includes enzymatic hydrolysate and mannitol-CPW solution.

[0028] The enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 3-7:5. The enzyme solution includes 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.8. The SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.4-5.8.

[0029] The mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution contains 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, and 1480 mg / L CaCl2·2H2O, with a pH of 5.8.

[0030] Furthermore, the enzymatic hydrolysate consisted of an enzyme solution and an SCW solution in a 5:5 volume ratio. The enzyme solution contained 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80 mM CaCl2, with a pH of 5.8. The SCW solution contained 0.5 M sorbitol, 10 mM CaCl2, and 5 mM MES, with a pH of 5.8.

[0031] Example 1.

[0032] This embodiment provides a method for preparing kale protoplasts, including the following steps:

[0033] (1) Kale seeds were inoculated onto MS medium without any hormones for tissue culture, and the culture conditions were 25℃ and 16h / d light culture to obtain sterile seedlings;

[0034] (2) Take leaves from the aseptic kale seedlings obtained in step (1) at about four weeks of age, cut them into strips of about 1 mm with a blade, and place them at the bottom of a 50 mL centrifuge tube. Soak the strips in 10-15 mL of the prepared enzymatic hydrolysate. The enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 5:5. The enzyme solution consists of 1.5% cellulase, 0.1% pectinase, 0.2 M mannitol, and 80 mM CaCl2, and the pH of the enzyme solution is 5.8. The SCW solution consists of 0.5 M sorbitol, 10 mM CaCl2, and 5 mM MES, and the pH of the SCW solution is 5.8. Place the above system on a shaker at 25°C and 30 rpm for 4 hours for enzymatic hydrolysis.

[0035] (3) Use the enzymatic hydrolysate of kale leaves obtained in step (2) to filter out undigested leaf tissue or impurities by filtration through a 300-mesh nylon mesh to obtain crude protoplast fluid.

[0036] (4) The crude protoplast solution obtained in step (3) is added to mannitol-CPW solution (wherein the mannitol-CPW solution includes 0.4M mannitol, and the CPW solution includes 27.2mg / L H2PO4, 101mg KNO3, 246mg / L MgSO4·7H2O, 0.16mg / L KI, 0.025 mg / L CuSO4·5H2O, 1480 mg / L CaCl2·2H2O, and pH is 5.8) and mixed in a 50ml centrifuge tube. The mixture is centrifuged at 900rpm / min for 5min, and the precipitate is collected. This process is repeated three times. Afterward, the mixture is washed once with protoplast culture medium to obtain a pure protoplast solution. The precipitate is then collected by centrifugation, and the precipitate is the solution containing the kale protoplasts. The optical microscope image of the kale protoplasts is shown below. Figure 1 As shown in Figure A, the highest number of protoplasts extracted from kale was 3.08 × 10^ 6 The number of protoplasts extracted was approximately 90% per gram.

[0037] Comparative Example 2

[0038] The comparative example uses the same method and steps as Example 1, except that the enzymatic hydrolysate used in step (2) consists of 0.5% cellulase, 0.2% pectinase, 0.2M mannitol and 80mM CaCl2, and the pH of the enzyme solution is 5.8; the SCW solution consists of 0.5M sorbitol, 10mM CaCl2 and 5mM MES, and the pH of the SCW solution is 5.8. The system is placed on a shaker at 25°C and the hydrolysis is carried out at 30 rpm for 8 hours.

[0039] In this embodiment, the number of protoplasts prepared from 1g of kale was the minimum, reaching only 1.98×10^ 6 The number of protoplasts per gram is relatively small, and the protoplasts are mostly intact and fragmented. Figure 1 As shown in B.

[0040] Comparative Example 3

[0041] The comparative example uses the same methods and steps as Example 1, except that the enzymatic hydrolysate used in step (2) consists of 1% cellulase, 0.3% pectinase, 0.2M mannitol and 80mM CaCl2, and the pH of the enzyme solution is 5.8; the SCW solution consists of 0.5M sorbitol, 10mM CaCl2 and 5mM MES, and the pH of the SCW solution is 5.8. The system is placed on a shaker at 25°C and rotated at 30 rpm for 8 hours.

[0042] In this embodiment, the number of protoplasts prepared from 1g of kale was relatively small, reaching only 2.31×10^ 6 The number of protoplasts per gram was not very high, and most of them were deformed into elliptical shapes, such as... Figure 1 As shown in C.

[0043] In addition to the preferred embodiments described above, there are other embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection claimed by the present invention.

Claims

1. A method for preparing protoplasts from kale leaves, characterized in that, The specific method includes the following steps: Step 1: Use an enzymatic hydrolysate to hydrolyze the leaves of sterile kale seedlings to obtain crude kale protoplast fluid. The enzymatic hydrolysate consists of an enzyme solution and an SCW solution with a volume ratio of 3-7:

5. Step 2: Add mannitol-CPW solution to the crude protoplast solution of kale from Step 1, centrifuge and collect the precipitate, repeating the process three times. Then wash once with protoplast culture medium to obtain a pure protoplast solution. Centrifuge and collect the precipitate, which contains kale protoplasts.

2. The method for preparing protoplasts from kale leaves according to claim 1, characterized in that, The enzyme solution in step one comprises 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.

8. The SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.4-5.

8.

3. The method for preparing protoplasts from kale leaves according to claim 2, characterized in that, In step one, the enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 5:

5. The enzyme solution consists of 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80mM CaCl2, and the pH of the enzyme solution is 5.

8. The SCW solution consists of 0.5M sorbitol, 10mM CaCl2, and 5mM MES, and the pH of the SCW solution is 5.

8.

4. The method for preparing protoplasts from kale leaves according to claim 1, characterized in that, In step two, the mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution includes 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O and 1480 mg / L CaCl2·2H2O, with a pH of 5.

8.

5. The enzymatic hydrolysate used in the method for preparing protoplasts from kale leaves according to claims 1-4, characterized in that, Includes enzymatic hydrolysate and mannitol-CPW solution. The enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a volume ratio of 3-7:

5. The enzyme solution includes 0.5-1.5% cellulase, 0.1-0.3% pectinase, 0-0.4M mannitol, and 70-80mM CaCl2, with a pH of 5.

8. The SCW solution consists of 0.3-0.8M sorbitol, 8-15mM CaCl2, and 2-7mM MES, with a pH of 5.4-5.

8. The mannitol-CPW solution consists of 0.4M mannitol and CPW solution. The CPW solution contains 27.2 mg / L H2PO4, 101 mg KNO3, 246 mg / L MgSO4·7H2O, 0.16 mg / L KI, 0.025 mg / L CuSO4·5H2O, and 1480 mg / L CaCl2·2H2O, with a pH of 5.

8.

6. The enzymatic hydrolysate according to claim 5, characterized in that, The enzymatic hydrolysate consists of an enzyme solution and an SCW solution in a 5:5 volume ratio. The enzyme solution comprises 1.5% cellulase, 0.1% pectinase, 0.2M mannitol, and 80mM CaCl2, with a pH of 5.

8. The SCW solution comprises 0.5M sorbitol, 10mM CaCl2, and 5mM MES, with a pH of 5.8.

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