JPH02177832A - Regeneration of limonium genus plant - Google Patents

Abstract

PURPOSE:To efficiently and surely multiply a seedling in a short time by differentiating an adventitious bud from the leaf blade section of a Limonium genus plant and subsequently culturing the differentiated adventitious bud. CONSTITUTION:For example, the leaf blade section of a Limonium genus plant is sterilized with 70% ethanol and further with a 1% sodium hypochlorite solution, etc., and the sterilized leaf blade section is cut into 1-3mm wide narrow pieces. The prepared tissue pieces are cultured in a solid medium prepared by adding gelane gum is an amount of 2.2g/l to a MS medium wherein the content of a saccharide is changed into 2%, thereby generating an adventitious bud. the adventitious bud is grown into a length of 5-10mm and transplanted in a solid medium comprising a MS medium and 0.1ml/l of NAA for generating the roots thereof to provide the objective seedling.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for regenerating plants of the genus Limonium and a method for propagating their seeds and seedlings, and more particularly relates to a method for mass propagating plants of the genus Limonium and their seeds and seedlings.

[Prior art]

As a method for regenerating plants of the genus Limonium and a method for mass propagating their seeds and seedlings, a shoot apex culture method is conventionally known in which the shoot apex of the plant is collected and cultured in an appropriate medium.

[Problem to be solved by the invention]

The above regenerative propagation method based on culturing the shoot top of a plant has the disadvantage that all the operating procedures involved in this method are complicated and that stable regeneration efficiency cannot be obtained.

[Failure to solve the problem]

As a result of extensive research into methods for regenerating plants of the genus Limonium and methods for propagating their seedlings, the present inventors have found that adventitious buds can be grown in Limonium plants and their seedlings by leaf piece culture or callus culture, which was completely unknown in the past. It was discovered that plants of the genus Limonium can be regenerated and propagated extremely efficiently by the differentiation method, and as a result of further research based on this new knowledge, the present invention was completed.

That is, the method for regenerating a Limonium genus plant of the present invention involves differentiating adventitious buds from leaf discs of Limonium genus (1, genus Imonium) plants, and then culturing the adventitious buds to obtain seedlings, or The present invention is characterized by inducing callus from tissue pieces of plants of the genus, culturing the callus to differentiate into adventitious buds, and culturing the adventitious buds to obtain seedlings. The method for propagating seeds and seedlings of plants of the genus Limonium of the invention involves differentiating adventitious buds from the leaf discs of plants of the genus Limonium, culturing the adventitious buds individually and rooting them to obtain seedlings, and further acclimating them to dirt. Shall I force you?
Alternatively, callus is induced from a tissue piece of a Limonium genus plant, the callus is then cultured to differentiate into a large number of adventitious buds, and the adventitious buds are individually cultured and rooted to obtain a seedling; , which is characterized by acclimatization.

The present invention will be explained in detail below.

As the plant of the genus Limonium used in the present invention, Limonium sinua
tum), Limonium swolowi (Limoni)
um suworowii), Limonium latifumerium (Limonium Iatif.

Iium), Limonium tartaricum (L i m
on i umtataricum), and Limonium sinuatum is particularly preferred.

As the tissue piece of the above-mentioned plant, for example, in the case of leaf culture, an explant obtained by cutting the leaf part into pieces is used. In addition, when culturing callus, various parts of the plant used as explants in normal tissue culture, such as leaves, stems,
Although those obtained from roots etc. can be used, leaves and stems are particularly suitable.

The tissue piece can be obtained, for example, as follows.

Seeds of the above-mentioned plants are sown in the soil normally used for cultivating Limonium plants, and tissues of the grown young plants, such as stems and leaves, are treated with 70% ethanol, sodium hypochlorite,
After sterilizing using a common sterilizing agent such as a surfactant, the tissue piece is washed with sterile water to obtain a sterile tissue piece.

This tissue piece is cut into pieces of about 1 to 5 mm to obtain tissue pieces for leaf culture or callus induction.

In addition, after sterilizing the seeds of the above-mentioned plants using a normal disinfectant such as 70% ethanol or sodium hypochlorite for about 10 to 40 minutes, the seeds after the sterilization treatment are thoroughly washed with sterilized water. , remove the disinfectant.

The obtained sterilized seeds are placed in a medium commonly used for plant tissue culture, such as a normal MS basic medium, with a solidifying agent such as agar or gellan gum, and plant hormones such as indole acetic acid (IAA) and naphthalene acetic acid (NAA). , 2.
Auxins such as 4-D, benzyladenine (BA),
The seeds are sown on a solid medium supplemented with at least one kind of plant hormone selected from cylindrical and kainin, such as kinetin and zeatin.

At this time, the content of plant hormones in the medium is 0.05 to 2.0 mg/42 in the case of auxins, preferably 0,
2 to 0.5 mg/about 12, in the case of cytokinins 0.05 to 5.0 mg/j2, preferably 0.1 to 2
．． It is about 0 mg/42.

In addition, the content of the solidifying agent is 0.7 to 1.5χ (in the case of agar)
W/V), in the case of gellan gum 0.2-0.3% (
W/V).

Next, the seeds are cultured under sterile conditions at about 20 to 30° C. and a photoperiod of 8 to 20 hours per day for about 8 weeks to germinate to obtain sterile seedlings.

Next, the above-mentioned young plants or their stems and leaves are cut into pieces of about 1 to 5 pieces to obtain tissue pieces for leaf piece culture or callus induction.

Leaf piece culture or callus induction using the tissue piece is performed as follows. That is, the length of the stem and leaf part is 1 to 5 mm.
For example, the concentration of sucrose is 10 to 2.56 A (W/V).
MS medium modified with anabolic agents such as dielan gum, auxins such as NAA and IAA, BA, kinetin,
Place the plate on a solid medium containing plant hormones such as cytokinins such as zeatin, and culture for about 20 to 50 days at a temperature of about 20 to 30°C and under a photoperiod of 8 to 20 hours to produce a large number of adventitious buds from the vein tissue of the leaf pieces. or induce callus from the leaf pieces.

In addition, the content of the assimilate in the above medium is 0.2 to 0.3χ
(W/V), and as plant hormones, it is desirable to contain 0.2 to 2.0 mg/p in the case of auxins, and 0.1 to 1.0 mg/p in the case of cytokinins.

In addition, when obtaining shoots by directly differentiating stems and leaves by culturing leaf pieces, cytokinins are added in a larger amount than auxins, specifically auxin at around 0.1 mg/p, and cytokinin at 0.2 to 0.2 mg/p. 1. It is desirable to contain about 0mg/12.

Also, if necessary, the obtained callus was mixed with the above-mentioned sucrose concentration of 1.0 to 2.5 x (hip/V)? Modified this,
Subculturing may be performed at monthly intervals on an MS solid medium containing plant hormones.

The callus obtained as described above was added to the modified MS solid medium as a plant hormone containing 0 cytokinins such as BA.
．． 1-2.0mg/E alone or in addition to IAA,
Auxins such as NAA 0.01-0.5mg/l
Placed on a medium supplemented with
A large number of adventitious buds of a Limonium genus plant were generated by culturing at 30°C, and the adventitious buds that had grown to about 5 or higher were transplanted to a solid medium in which only the inorganic salts of the MS medium were reduced to about Z-Z. The mixture is then cultured at 20 to 30°C under a 16 to 24 hour photoperiod for about one month or more to obtain shoots of plants of the genus Limonium.

In addition, when regenerating a complete plant from the shoot, for example, the MS solid medium or 0.01 to 0
．． The seedlings are transplanted to a medium containing auxins at a low concentration of about 1 mg/ffi, and adventitious roots are generated and grown. By further acclimatizing this plant under appropriate conditions, seeds and seedlings of Limonium plants can be obtained.

(Effect of the invention) According to the present invention, plants of the genus Limonium and their seeds and seedlings are
Adventitious buds that differentiate into many stems and leaves directly from the leaf disc,
Or, since it is propagated by adventitious buds differentiated from callus, it is possible to produce the desired plants or seedlings in a much shorter period of time, efficiently and reliably compared to the conventional method of obtaining plants by sowing seeds. The present invention is of great industrial significance.

The present invention will be specifically illustrated by examples below.

〔Example〕

Example 1 Limonium latifolium (Limonium I
Atifolium) seeds were sown in soil and allowed to germinate, and the leaves of the seedlings, which had been cultivated for about 40 days, were surface sterilized with 70% ethanol for several minutes, and then treated with 1% sodium hypochlorite and a few drops of Tween 20. Sterilize with the mixture for 15 minutes,
Thoroughly washed with sterile water. The sterilized leaves were shredded into 1-3 mm wide pieces and added to MS medium containing 2% saccharose at 0.2 mg/42 NAA and 0.5 mg/L B.
A, 2.2g/! The plants were placed on a solid medium containing gellan gum and cultured at 25° C. under a 16-hour photoperiod for about 40 days to generate a large number of adventitious buds. 1 piece of tissue (weight: approx. 0.
3 to 5 adventitious buds are obtained from 3 g).

Next, the adventitious buds that had grown to about 5 to 10 mm were taken out with sterile pins and added to MS medium at 0.1 mg/I NAA.
The seedlings were transplanted to a solid medium supplemented with 25° C. and completely rooted under 3000 ux to obtain seedlings. Furthermore, this seedling was transplanted to an MS solid medium that does not contain plant hormones, and after culturing for several times, the plant grew to a height of 3 to 5 cm. It was then transplanted to a sterilized Hermigulai 1 to 15 cm to the original height of 15 to 20 cm.
For days, cover with glassware and heat at 25°C 30001ux.
Seedlings of plants of the genus Limonium were obtained by acclimatization under conditions.

Example 2 Limonium sinuatum (Limonium sinuatum)
uatum) seeds for 3 minutes with 70% ethanol, then sterilized with 1% sodium hypochlorite and a few drops of Twee.
It was sterilized with a mixture of n20 for 20 minutes, and then thoroughly washed with sterilized water. The sterilized seeds were added to MS basic medium with 0.8χ
(W/V) Agar, 0.5mg/I! , BA, 0
．． 1mg#! IAA, 0.1. mg#! 2, 4-D
The seedlings were placed on a solid medium containing 0.5 mg/n Kinetin and cultured at 27° C. under a 116-hour photoperiod for about 35 days to obtain seedlings.

After removing the roots of the seedlings and cutting the stems and leaves into pieces several nanometers in size, 2.2 g of ##! Gellan gum, 1.0mg #2NA
A. Thin stems and leaves were placed on a solid medium supplemented with 0.5 mg/ρBA and cultured at 27° C. under a 116-hour photoperiod for about 40 days to obtain a yellow-green callus.

This callus was mixed with 0.5mg/IB8 and O, 1mg/p
, IAA were added to the MS solid medium. Adventitious buds were generated by culturing for about 5 to 6 weeks under the above conditions.

Approximately 25 adventitious buds were generated from 1 g of callus within the above culture period.

Next, adventitious buds that had grown to about 5 to 10 mm were taken out with tweezers, and NAA (0.1 mg/I
Transplanted into a medium supplemented with
When cultivated under these conditions, the plants were rooted and complete seedlings were obtained.

Furthermore, this young plant was taken out with a sterilized Vincent,
Transplanted to MS solid medium (does not contain plant hormones),
After culturing for several months, the plants that have grown to a height of 5 to 10 cm are transplanted to sterilized vermiculite and grown for the first time.
Cover with glassware and store at 25°C for 5 to 20 days.
Seedlings of plants of the genus Limonium were obtained by acclimatization under conditions of 0001 ux.

Claims (1)

[Scope of Claims] 1. A method for regenerating plants of the genus Limonium, which comprises differentiating adventitious buds from the leaf discs of plants of the genus Limonium, and then culturing the adventitious buds to obtain seedlings. . 2. A plant of the genus Limonium, which is characterized in that after differentiating adventitious buds from the leaf discs of the plant of the genus Limonium, the adventitious buds are individually cultured and rooted to obtain seedlings, which are further acclimatized. How to propagate seeds. 3. A plant of the genus Limonium, characterized in that callus is induced from a tissue piece of the plant of the genus Limonium, the callus is then cultured to differentiate into adventitious buds, and the adventitious buds are then cultured to obtain a seedling. How to play. 4. Inducing callus from a tissue piece of a Limonium genus plant, then culturing the callus to differentiate a large number of adventitious buds, and then culturing the adventitious buds individually and rooting them to obtain seedlings; Furthermore, a method for propagating seeds and seedlings of plants of the genus Limonium, which comprises acclimatizing them.