JPH0327222A - Protoplast culture of patchouli and redifferentiation to plant body - Google Patents

Abstract

PURPOSE:To enable mass production of patchouli by culturing protoplasts of patchoui in a culture medium for protoplast cultivation having a specific ammonia nitrogen concentration. CONSTITUTION:Patchouli protoplasts are inoculated in a medium consisting of 200 to 800mu protoplast culture medium of less than 2mM ammonia nitrogen concentration containing 2 to 4mg/l of 2,4-D, 0.5 to 2.0mg/l of kinetin, 2 to 4mg/l of NAA, 2 to 6wt.% of sucrose and 0.4 to 0.6M of mannitol and, when needed, 400 to 1,500mul of solid medium and clutured at 25 to 27 deg.C under 200 to 700lux without addition of fresh medium to form colonies. Then, the colonies are inoculated in the basal medium containing less than 10mg/l of auxin and 5mg/l of cytokinin or only cytokinin and cultured at 25 to 27 deg.C under light for 1 to 2 months until they differentiate into plant bodies.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of Industry L] The present invention relates to a method for culturing Bro I-plasts of a plant of the Lamiaceae family, B. cucurbita, and a method for redifferentiating the obtained colonies into plants.

[Prior Art] Patchouli is a plant of the Lamiaceae family whose leaves contain a large amount of volatile components that are raw materials for perfume. I wonder if techniques such as cell theory will be applied to obtain something that has the desired effect on the volatile components. It's twisted. Regarding other plants, there have been many reports regarding Brotobulus 1 integration, protoplast culture methods, and plant regeneration from colonies, which are the basis of these techniques. However, because the culture conditions for protoplasts differ slightly depending on the plant family and species, no colony formation was observed even if the culture conditions for other plants were applied to B. japonicum. In addition, Brotobulus 1- of Drumstick 1 is a protoblast of other plants. If you try to culture Pro 1 to Plus 1- using the method commonly used for 1'8 cultivation, there is a tendency for the plants to change. There have been no reports regarding this. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for culturing protoplasts of Patchouli and a method for redifferentiating it into plants. [Means for Solving the Problems] The present inventors investigated various culture conditions in order to solve problem a above, and found that the ammonia nitrogen generally contained in the protoblast culture medium It was found that the formation of colonies from protoplasts was inhibited, and therefore, lowering the concentration of ammonia nitrogen in the medium was effective for colony formation. We also found that browning can be prevented by culturing protoblasts in combination with a solid medium and without adding medium until the colonies grow large. Furthermore, as a result of examining the conditions for redifferentiating the formed colonies into plants, they found effective conditions for redifferentiation and completed the present invention. That is, the present invention provides a method for culturing pachouli brotoplasts in a protoplast culture medium having an ammonia sM concentration of 21 M or less and forming colonies, a solid medium, and a suspension of protoplasts. To provide a method for culturing protoplasts of Patchouli, which is characterized in that it is used in combination with a turbid medium and no new medium is added from the start of culture to colony formation.
There are two more aspects of this invention. Patchouli protoblasts, characterized in that colonies or cultured cells derived from Patchouli protoblasts are cultured in a basal medium containing auxin 10g/I or less and cytokinin 5sg/I or more, or cytokinin only, and redifferentiated into plants. We provide a method for redifferentiation into plants. [Specific Description of the Invention] In the present invention, the formation of colonies of Patchouli is carried out by isolating protoplasts from suspension cultured cells of Patchouli, and then suspending the brotoblasts in a basic medium that does not contain ammonia nitrogen or contains ammonia nitrogen at a low concentration. It can be made by making it cloudy and culturing it. As the basic medium for protoplast culture, MS basic medium, 85 basic medium, Nitch and Nitch medium, etc. can be used, but it is preferable to use it after diluting it to about 1/2. In addition, it may contain other trace ingredients such as vitamins, plant hormones, sucrose, mannitol, etc. For example, 2.4-0 2 to 4 B/l in a basal medium that does not contain or contains ammonia nitrogen at a low concentration.
, kinetin 0.5-2.0 mg/l. NAA 2
~4 B/l. SHOP! It can be suspended and cultured in a medium containing 2-6% by weight of mannitol and 04-0.6M. In the present invention, protoplasts can be isolated by enzymatic treatment according to a conventional method. In the present invention, the ammonia nitrogen concentration is 2mM or less, preferably On+M. The basic media conventionally used for protoblast culture, such as B5 and MS, all contain ammonia nitrogen. However, Patchouli brotoblasts can form colonies by setting the ammonia nitrogen concentration in the medium to a low concentration, preferably OmM.

In order to prevent browning until the colony grows large, it is effective to layer a protoblast suspension medium on a medium solidified with agarose or agar, or to add a solid medium to the brotoblast suspension medium. be. The appropriate amount of the solid medium is about 400 μl to 1500 μl when culturing in a Petri dish with a diameter of 3.5 c+s, and the appropriate amount of the suspension is about 200 μl to 800 μl. Culture at 25°C to 27'C, 200 lux to T
This can be done with OO lux. In this case, do not add fresh medium until colonies have formed. Protoplasts tend to brown when fresh medium is added. In the present invention, the regeneration of plants from a patchouli colony is carried out at less than 10 mg/l of auxin. Preferably 0
．． 1-2.0 mg/l and cytokinin 5B71
The above can be carried out by using a basic agar medium containing only 10 to 15 mg/l of cytokinin, or this content. As Saiichi Kishin, I
AA fO-10 ag/11. N.A.A.
10-5mg/11. 2.4-D. IBA (
0.5 mg/11 etc. can be used. In addition, as zytokarinin, kinetin (5-20~g
/11. 8A flo~20mg/1.1 etc. can be used. Furthermore, glycine is a trace component.
Vitamins such as pyridoxine hydrochloride, nicotinic acid, thiamine hydrochloride, NAA. Contains plant hormones such as BA, etc. Cultivation can be carried out in the light for 25-276 CTl-2 months. E Example 1 Hereinafter, the present invention will be specifically explained with reference to Examples 7
However, the present invention is not limited to the following examples.

Example 1 2.4-0 3 mα/from leaves of Pachouli, a plant belonging to the Lamiaceae family.
l, MS agar plate containing 0.1 B/l kinetin,
Callus induction was performed in the dark, and subculture was performed every month in the same medium. This Calmata was placed in a liquid medium of similar composition and heated at 26°G. Rotary shaking culture was performed at 120 rpm. The sad S culture that was obtained. ll! Cells were passaged every 7 days. ! On the 3rd or 4th day after the 11th generation, the cultured cells were treated with 10Illl of cellulase per 1g of fresh cell weight.
Protoblasts were placed in an enzyme solution containing 23 0.3% and mannyl 1--0.3M and subjected to enzyme treatment for 4.5 hours at 27''C and 40''pm to obtain Bro 1-Plus!. Protoblasts. The medium for Ig cultivation was 1/2 times A Agency's 85 basic medium, however, the concentration of +NH.l.so. was set to OmlJ.
Mannitol 0.3M. C3WM 3%. 2.4-
0 3 mg/l. NAA O. 5B/l. BA0
．． 5 B/l. Glycine 2 B/ as a vitamin
l. A mixture containing pyridoxine hydrochloride lOIIIg/l, nicotinic acid 5B/1 and diamine hydrochloride 11) B/l was used. The obtained cotton protoblast was 1xlO'
The cells were suspended in the above medium to a concentration of /oal. 1 m of the same medium was placed in advance in a 35+ms diameter tube.
04% agarose fsIGMA. Type V
After firming up the Petri dish with 400 μl of brotoblast turbidity medium, the Petri dish was sealed with rose film]7. This is about 1-1. under 300 lux.
After static culture for 5 months, colony formation of approximately 27% of the cultured protoplasts was observed. On the other hand, F
When cultured in a medium containing (NH-1z5040.5 cmM), the colony formation rate decreased to approximately 0.1-0.5%, and fNH-1zS047.5
No ml4 or root colony formation was observed.

The obtained colonies were treated with NAA 1 mg/l, ROA 1
MS basic medium at 1/2 concentration containing 2 g/l (however, as vitamins: glycine 2 mg/1, pyridoxine hydrochloride 10 g/l, nicodic acid 4 Illg/l, thiamin hydrochloride 10 B/l, sucrose 3%) The plants were placed on an agar medium supplemented with the following, and cultured in the light at 26"C for about 2.5 months. Regeneration of the plants was observed at a frequency of about 5%.

[Effect of the invention]

Claims (3)

[Claims] (1) A method for culturing pachouli protoplasts, which comprises culturing pachyuli protoplasts in a protoplast culture medium having an ammonia nitrogen concentration of 2 mM or less to form colonies. (2) Using a solid medium and a protoplast suspension medium together,
2. The method for culturing pachouli protoplasts according to claim 1, characterized in that no new medium is added from the start of culture to colony formation. (3) Auxin 10mg/l or less and cytokinin 5
1. A method for regenerating a Patchouli plant, which comprises culturing a colony or cultured cells derived from Patchouli protoplasts in a basal medium containing mg/1 or more or only cytokinin and redifferentiating them into a plant.