AU2020101602A4 - A Method for Rapidly Preparing Stevia Leaf Protoplasts - Google Patents
A Method for Rapidly Preparing Stevia Leaf Protoplasts Download PDFInfo
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- AU2020101602A4 AU2020101602A4 AU2020101602A AU2020101602A AU2020101602A4 AU 2020101602 A4 AU2020101602 A4 AU 2020101602A4 AU 2020101602 A AU2020101602 A AU 2020101602A AU 2020101602 A AU2020101602 A AU 2020101602A AU 2020101602 A4 AU2020101602 A4 AU 2020101602A4
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- Prior art keywords
- stevia
- solution
- protoplasts
- leaves
- mannitol
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
- A01H6/1488—Stevia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Physiology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for rapidly preparing stevia leaf protoplasts. Choosing
tissue culture of young leaves of stevia as material. The most suitable conditions for isolation
of leaf protoplasts of stevia were screened by controlling the factors such as the time of
plasmolysis, the ratio and concentration of Onozuka R-10, hemicellulase, macrozyme R-10,
and the concentration of mannitol in the enzymolysis solution, the pH value of the enzymatic
hydrolysis solution, and the time of enzymatic hydrolysis. The method can obtain more free
protoplasts and has high activity, which is a simple and quick method for preparing stevia
leaf protoplasts.
-1/3
Figure 1
Description
-1/3
Figure 1
PATENTS ACT 1990
A Method for Rapidly Preparing Stevia Leaf Protoplasts
The invention is described in the following statement: -
The invention relates to a method for rapidly preparing the protoplast of the homogenized
branch, belonging to the field of biotechnology.
Stevia (Stevia rebaudianaBertoni), as an ideal sweetener and functional health care
product, has been the focus of research and development in the field of food and
medicine. In our stevia research group, systematic research has been done on stevia
variety selection, polyploid mutagenesis, tissue culture and rapid propagation, seed
quality and high-yield cultivation. At present, there is no report about the protoplast
preparation of stevia. The isolation and preparation of the protoplasts of stevia were the
basis for the studies of protoplast fusion, protoplast culture, plant regeneration, gene
transient expression and protein subcellular localization and the like.
Object of the invention: The object of the present invention is to provide a method for
rapidly preparing stevia leaf protoplasts.
Technical solution : In that invention, the most suitable factor for separating the
protoplast of stevia are screened by adjusting the factors such as the separation time of
the plasmolysis wall, the enzyme type and the ratio, the concentration of mannitol in the
enzymolysis solution, the pH of the enzymic solution, and the enzymatic hydrolysis time, etc. The purpose of the present invention is to provide a method for rapidly preparing protoplasts of stevia leaves, which comprises the following embodiments:
(1) Sample selection: The young leaves of stevia were selected.
(2) The leaves were cut into 0.5-1mm strips with a blade and placed in a petri dish
containing a mass-wall separation solution (CPW + 13% Mannitol) for mass separation.
(3) The leaf strip which is subject to the plasmolysis treatment is transferred into a petri
dish containing the enzymolysis solution and is subjected to enzymatic hydrolysis under
dark conditions on a shake bed for a period of time. After no complete leaf strip is in the
enzymatic hydrolysis solution and the colour is turned green, put the solution flaking
under a microscope to observe, and a large number of round protoplasts appeared in the
field of vision.
According to the above method, it is characterized in that the material is preferably a
young leaf on a stevia tissue culture seedling.
According to the above method, it is characterized in that the hydrolysate combination in
step (3) was CPW + Mannitol + 0.1% MES + 0.5%, Onzuka R-10 + 0.4%, Hemicellulase
+ 0.4%, and macrozyme R-10.
According to the above method, it is characterized in that the concentration of mannitol
in the enzymatic hydrolysate in step (3) is 9%.
According to the above method, it is characterized in that the pH of the enzymatic
hydrolysis solution in step (3) is 6.2.
According to the above method, it is characterized in that the rotation speed of the
shaking table in step (3) is 50 rpm.
According to the above method, it is characterized in that the enzymolysis time in step (3)
is 4 h.
Advantageous effects: The method for rapidly preparing the protoplasts of stevia leaf is
simple and practical, and the separation material is easy to obtain. The method of
enzymatic hydrolysis is simple and can get a large number of protoplats with high
activity. This technology will lay a foundation for cell research, gene transient expression
and protein subcellular localization, protoplast culture and plant regeneration of stevia
leaves.
FIG. 1 Schematic diagram of leaf protoplasts of tissue culture seedlings of stevia.
FIG. 2 Schematic diagram of tissue culture stevia leaves protoplasts stained by FDA in
the same field of view under white light.
FIG. 3 Schematic diagram of FDA-stained protoplasts of tissue culture stevia seedlings in
the same field of view under excitation blue light.
In order to deepen the understanding of the present invention, the invention will be
further described in detail below with reference to the following examples, which are
only for explaining the invention and are not intended to limit the scope of the invention.
Materials: Young and tender leaves of tissue culture seedlings of stevia.
In the examples, the formulation of the CPW solution is:
Components Concentration (g-L-1)
KH 2PO 4 27.2
KNO 3 101.0
CaCl2-2H20 1480.0
MgSO4-7H20 246.0
KI 0.16
CuSO 4 -5H20 0.025
Protoplast separation:
(1) Take the tender leaves from tissue culture stevia seedlings for later use.
(2) Cutting the blade into thin strips of 0.5-1 mm, and placing the strips in a mass-wall
separation liquid for 4 hours.
(3) Transferring the stevia leaf strips subjected to the plasmolysis treatment to the
enzymolysis solution at a temperature of 28C at a rotation speed of 50 rpm for 4 hours
in the dark; The cell protoplast washing solution was composed of CPW + 9% mannitol+
0.1% MES + 0.5% Onozuka R-10 + 0.4% hemicellulase and 0.4% Macerozyme R-10.
The pH was 6.2.
(4) Detecting the protoplasts isolated in the step (3): Taking 1OPL protoplast solution
and adding IpL 0.1% FDA stain solution, mixing well; Blood cell counting plate was
used to make flaking and then observed under fluorescence microscope. The number of
protoplasts was observed under white light, and then switched to excitation light to
observe the number of protoplasts with green fluorescence. The protoplast yield
(protoplast yield = total protoplast number in 25 squares / 0.1 x 1000 x 10 cells / g - FW)
was 1.85 x 107 / g - FW, and the survival rate was 93.37%.
The above embodiments are only for illustrating the technical idea and features of the
present invention, and the purpose thereof is to enable a person skilled in the art to
understand the contents of the invention and carry out the same, and not to limit the scope
of protection thereof. Any equivalent variation or modification made in accordance with
the spirit of the present invention is intended to be within the scope of the invention.
Claims (7)
1. A method for rapidly preparing protoplasts of stevia leaves, is characterized in that the
method comprises the steps of:
(1) Sample screening: The young and tender leaves of stevia were selected.
(2) The selected young leaves were cut into thin strips of 0.5-1mm with a blade and
placed in a culture dish containing a mass-wall separation solution (CPW + 13%
Mannitol) for mass separation; and
(3) Transferring the leaf strips subjected to the plasmolysis treatment to a petri dish
containing an enzymolysis solution, subjecting the leaves to an enzymatic hydrolysis
under dark conditions on a shaking table (50 rpm), and observing the solution under a
microscope after the solution turns green and then a large number of round protoplasts
will appear in the field of vision.
2. The method of claim 1, the method is characterized in that the material is a young leaf
of a tissue culture stevia seedling.
3. The method of claim 1, the method is characterized in that the hydrolysate combination
in step (3) was CPW + Mannitol + 0.1% MES + 0.5% Onzuka R-10 + 0.4%
Hemicellulase + 0.4% macrozyme R-10.
4. The method of claim 1, the process is characterized in that the concentration of
mannitol in the enzymatic hydrolysate in step (3) is 9%.
5. The method of claim 1, the process is characterized in that the pH of the enzymatic
hydrolysis solution in step (3) is 6.2.
6. The method of claim 1, the process is characterized in that the rotation speed of the
shaking table in step (3) is 50 rpm.
7. The method of claim 1, the process is characterized in that the enzymolysis time in step
(3) is 4 h.
-1/3-
Figure 1
-2/3-
Figure 2
-3/3-
Figure 3
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020101602A AU2020101602A4 (en) | 2020-07-31 | 2020-07-31 | A Method for Rapidly Preparing Stevia Leaf Protoplasts |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020101602A AU2020101602A4 (en) | 2020-07-31 | 2020-07-31 | A Method for Rapidly Preparing Stevia Leaf Protoplasts |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458036A (en) * | 2020-12-10 | 2021-03-09 | 上海交通大学 | Preparation and instantaneous transformation method of eggplant protoplast |
| CN113249296A (en) * | 2021-06-30 | 2021-08-13 | 安徽农业大学 | Method for separating and purifying fresh tissue protoplast of tea tree |
| CN113897330A (en) * | 2021-11-10 | 2022-01-07 | 北京林业大学 | Enzymolysis method for quickly removing cell walls of poplar or eucalyptus and application |
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
-
2020
- 2020-07-31 AU AU2020101602A patent/AU2020101602A4/en not_active Ceased
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458036A (en) * | 2020-12-10 | 2021-03-09 | 上海交通大学 | Preparation and instantaneous transformation method of eggplant protoplast |
| CN113249296A (en) * | 2021-06-30 | 2021-08-13 | 安徽农业大学 | Method for separating and purifying fresh tissue protoplast of tea tree |
| CN113249296B (en) * | 2021-06-30 | 2024-01-30 | 安徽农业大学 | Separation and purification method of fresh tissue protoplast of tea tree |
| CN113897330A (en) * | 2021-11-10 | 2022-01-07 | 北京林业大学 | Enzymolysis method for quickly removing cell walls of poplar or eucalyptus and application |
| CN114214305A (en) * | 2022-01-18 | 2022-03-22 | 中国科学院东北地理与农业生态研究所 | Enzymolysis liquid for preparing Lonicera caerulea protoplast and preparation method and application thereof |
| CN114214305B (en) * | 2022-01-18 | 2023-05-09 | 中国科学院东北地理与农业生态研究所 | Enzymatic hydrolysate for preparing lonicera caerulea protoplast and preparation method and application thereof |
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