JPH0286772A - Method for isolating plant mesophyll protoplast - Google Patents

Abstract

PURPOSE:To obtain the subject protoplast in high activity in a stable number with an enzymic solution in a low concentration in simple operation by using a leaf of a plant in a vitrified state as a material and treating the leaf with the enzymic solution. CONSTITUTION:A plant is treated with an enzymic solution to isolate a plant protoplast. In the process, a leaf of a germ-free seedling prepared by aseptically growing a plant is cut out and dipped in a liquid culture medium containing an inorganic salt, vitamin B5, sucrose, phytohormone, etc., allowed to stand at 26 deg.C for 17hr in a dark place and vitrified. The resultant leaf without finely cutting is subsequently added to an enzymic solution containing an enzyme, such as cellulase, and allowed to stand at 30 deg.C for 5hr in a dark place to isolate the objective plant mesophyll protoplast.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] This invention relates to eleven methods for producing plant mesophyll protoplasts.

[Prior Art] Conventionally, plant mesophyll protoplasts have been isolated by cutting the leaves of plants grown in a greenhouse or under microbial conditions into small pieces of about 10 cm wide, or peeling off the epidermis on the underside, and then using cellulase, This is carried out by treatment with an enzyme solution containing enzymes such as hemicellulase or hemocutinase. It is also known that highly active protoplasts of some plants can be obtained by isolating protoplasts after treating leaves in a liquid medium under specific conditions.

[Disadvantages of the conventional technology] Although this method has achieved good results with some plants, it lacks reproducibility, and therefore it is extremely difficult to consistently obtain highly active mesophyll protoplasts. and no solution has been proposed.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for isolating protoplasts that can always provide stable and highly active mesophyll protoplasts.

[Hands-on research to solve the problem] As a result of intensive research, the inventors of the present application have discovered that leaves under certain physiological conditions can be used as a material in a method for isolating protoplasts by treatment with an enzyme solution. The present invention was completed based on the discovery that it is possible to reduce cell damage caused by protoplast isolation operations, suppress a decrease in protoplast activity, and always isolate a stable number of protoplasts Y.

That is, the present invention provides a method for isolating plant mesophyll protoplasts, which is characterized by using vitrified leaves as a material in isolating plant protoplasts by treating plants with an enzyme solution. The vitrified state is when the leaves and stems of plants or woody plants take on a translucent, glass-like appearance, as if swollen with water (LinOsey A Wither).
s, P, G, AlderSon.

(1986): Plant Ti5sue (:u
lture and its agriculture
al Application, Butterwor
ths).

[Effects of the Invention] The present invention provides a method capable of isolating a highly active and stable number of protoplasts. In addition, operations such as cutting the leaves into 1111-width pieces and peeling off the underside epidermis, which are performed in conventional isolation methods, can be omitted or simplified, and the concentration of the enzyme used can be low. The method of the present invention can be applied to the isolation of mesophyll protoplasts of any plant, and the obtained mesophyll protoplasts have a high survival rate in subsequent culture and are useful for cell fusion treatment and genetic manipulation.

[Detailed Description of the Invention] As described above, the isolation method of the present invention uses vitrified leaves as a material. This can be either naturally vitrified leaves or artificially vitrified leaves, but it is difficult to obtain vitrified leaves in a natural state, and it is desirable to produce artificially vitrified leaves.

As a method for producing artificially vitrified leaves, it is possible to obtain vitrified leaves by culturing the plant body under aseptic vitrification conditions, or to obtain vitrified leaves from normally growing plants. may be vitrified under suitable conditions in a liquid medium suitable for vitrifying.

To vitrify the plant - For boat fishing, increase the water potential in the culture medium, or use ammonia! It is said that this is caused by a medium rich in nitrogen, ethylene accumulation, etc. (Agricell Rep, 517° (1985)
)). According to the findings of the present inventors, agar 0.4 to 0
．． 7z, auxin 0.001 to Q, Q5pp+s and cytokinin 0.001 as plant hormones. A common method is to grow in a basal medium containing 0.1 to 0.10 ppm of ol.Also, to vitrify normal leaves in a liquid medium, add plant hormones to a 2- to 4-fold dilution of the basal medium. As auxin o, oos or 0.
10 pp- and cytokinin 0.05 to 0.20
ppm and culture in the dark for 5 to 24 hours. As the basic medium, MS medium or B-
5 media are common or are not limited to these.

In the method of this invention, the enzyme treatment for isolating protoplasts can be carried out in the same manner as in conventional methods. However, as mentioned above, the method of the present invention can be simplified by omitting the shredding of the mesophyll and peeling off the back epidermis, which were necessary in the conventional method, or by increasing the width of the shredding. Furthermore, the enzyme concentration used can be lower than in conventional methods. For example, Cellulase Onozuka R30, 1-2.5$, Macerozyme Onozuka I
t-100, 1-2.0%, Hectra-1' Knee X
It is possible to employ enzyme concentrations in the range of 1.0% for Y-230.1.

In addition, the temperature and time during enzyme treatment were the same as before.
Usually, the temperature is 27°C to 30°C for about 3 to 6 hours.

[Examples] Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Bachori, a plant belonging to the Lamiaceae family, was grown at 26°C in 12 hours of light (40
001ux), aseptically in the dark for 12 hours.
After growing for 2.5 months, sterile seedlings were obtained. Cut the sterile seedlings No. 3 and No. 4.1 and add inorganic salts, vitamin B5.3% sucrose and plant hormones as N A A O,01.
1) It was immersed in MS liquid medium (pl-15,7) containing 0.10 ppp of pl, B A and left to stand in the dark at 26°C for 17 hours to vitrify. After finishing the treatment, fresh leaves of approximately tg were added at 1.0% (W/V) without being shredded.
Cellulase Onozuka R3 (manufactured by Yakult Biochemical Co., Ltd.),
1. oX (W/Limacerozyme Onazuka R-10 (manufactured by Yakult Biochemical Co., Ltd.), 0.IOX (w/V) Hectolyase Y-23 (manufactured by Seishin Yakuhin Co., Ltd.) and 0.5M
It was added to 10 ml of an enzyme solution (pH 5.5) containing Amanil, and left standing in the dark at 30°C for 5 hours. When the state of isolation of protoplasts was observed under a WJ microscope, it was confirmed using a hemacytometer that there were 3.0XIO' protoplasts per gram of fresh type. In addition, the activity was detected under ultraviolet light after staining with FDA (Reference: Larkin, P, J.
, +978Purification and
mobility determination
ofplant protoplast: Plan
ta 128.2+3-216), and as a result, more than 80% of the obtained protoplasts emitted strong fluorescence indicating high activity.

Example 2 N. geranium, a plant of the family Pocoraceae, grows at 141°C at 27°C.
The seedlings were grown aseptically for about 2 months in the light for 1e (4DOO1ux) and in the dark for 10 hours to obtain sterile seedlings. The stem part including the growth point of sterile seedlings was treated with inorganic salts, vitamin H, 2% sucrose, and NAA O as a plant hormone, 01pp.
I11,;h i*chi:/0. Agar medium containing IOppm and purified agar 0.8% (W/V) (pH 5,7
) at 27°C for 14 hours in the light (40,001 ux) and in the dark for 10 hours. About 2 weeks later, the newly developed vitrified io, 5g (new a: #heavy) was shredded and 0. ! 4 (W/V) (! Ruraze Onozuka R
-10 (manufactured by Yakult Biochemical Co., Ltd.), 0.5X (W/V) Macerozyme Onazuka R-10 (manufactured by Yakult Biochemical Co., Ltd.)
, 0.02 $ (W/V) Hectolyase Y-23 (manufactured by Seishin Yakuhin) and 5 ml of enzyme solution (pl + 5.5) containing 0.5 M Amanil, and incubated at 27°C in the dark for 2 hours. I left it still. When the isolated state of protoplasts was observed under a microscope, it was confirmed that there were 5 x 10 6 protoplasts per gram of fresh type using a hemacytometer. In addition, when the protoplasts were stained with FDA and their activity was observed under ultraviolet light, more than 70% of the obtained protoplasts emitted strong fluorescence indicating high activity.

Comparative Example 1 The third and fourth leaves of the sterile patchouli seedlings used in Example 1 were treated with an enzyme solution in the same manner as in Example 1 without being vitrified. After treatment, the isolated state of protoplasts was examined under a microscope. As a result, no protoplasts were detected at all.

Comparative Example 2 Sterilization of Bachouri used in Example 1 [Gan no. 3 and 4]
The leaves were not subjected to vitrification treatment, but were cut into pieces with a width of about 31 mm and treated with an enzyme solution in the same manner as in Example 1. After treatment,
When the state of isolation of protoplasts was observed under a microscope, it was confirmed that there were 9,0XIO' protoplasts per gram of fresh type. However, only about 2% of the protoplasts emitted strong fluorescence when stained with FDA.

Comparative Example 3 The leaves of the sterile seedlings of N. geranium used in Example 2 were not vitrified or shredded, and were treated with Z, 0X (W/V) Cellular Zeonozuka RS,
1. O$(W/V)? (? l-2 Zyme Onozuka R-10, 0, 10% (W/V) ') TO') 7--
The mixture was added to 5 ml of an enzyme solution (pH 5.5) containing t'Y-23 and 0.511 mannitol, and allowed to stand at 27°C for 4 hours for enzyme solution treatment. After the treatment, the state of isolation of protoplasts was observed using a J2 microscope under a lAim microscope, and no protoplasts were observed.

Comparative Example 4 The leaves of the sterile seedlings of Nieugeranium used in Example 2 were not vitrified and were about 2H wide! B-cut I! 7ffi1g to 2.0% (W/V) Cellulase Off 1 Shikaro + o, +, oX (W/V) Macerozyme Onozuka R-10,0,10$ (W/V)')
)-') Enzyme solution containing 7-ze Y-23 and H++, 5M amanil (pf15.5) 5 In addition to o+1, 27
The mixture was allowed to stand at °C for 3 hours and treated with an enzyme solution.

After the treatment, the state of isolated protoplasts was observed under a microscope, and it was confirmed that there were 5 x 1O' protoplasts per gram of fresh gram weight. However, about 20% of the protoplasts emitted strong fluorescence when stained with FDA.

Claims (1)

[Claims] 1. A method for isolating plant mesophyll protoplasts, which comprises using vitrified leaves as a material for isolating plant protoplasts by treating plants with an enzyme solution.