CN102138522B

CN102138522B - Method for regenerating plants under high frequency by inducing adventitious buds with leaves of davallia bullata

Info

Publication number: CN102138522B
Application number: CN2010106123543A
Authority: CN
Inventors: 叶晓青; 佘建明; 邓衍明; 汤日圣; 童红玉
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2010-12-30
Filing date: 2010-12-30
Publication date: 2012-08-22
Anticipated expiration: 2030-12-30
Also published as: CN102138522A

Abstract

The invention provides a method for regenerating plants under high frequency by inducing adventitious buds with leaves of davallia bullata, belonging to the technical field of plant culture methods. The existing technology for regenerating plants by inducing adventitious buds with pteridophytes is still on the laboratory research stage and is limited to rhynioceae and nephrolepidaceae. The method is characterized in that the leaves of davalliaceae davallia bullata aseptic seedlings are taken as the explant materials; the adventitious buds are induced and generated on the culture media to which 2.0mg/L of phenylurea cytokinins (CPPU) and 0.1mg/L of indolebutyric acid (IBA) are added; the induction frequency of the adventitious buds reaches 95%; multiplication of the adventitious buds is carried on the culture media to which 0.5mg/L of CPPU and 0.1mg/L of IBA are added; the multiplication coefficient of each generation of the adventitious buds is 5; and the seedling rate of the adventitious buds on the culture media to which 0.2mg/L of IBA is added reaches 100% and the adventitious buds grow to become complete plantlets with roots, stems and leaves. The method is suitable for propagating plantlets of davallia bullata and provides conditions for developing screening of cell mutants and gene transformation study.

Description

Wolf tail fern blade evoking adventive bud high-frequency plant regeneration method

Technical field

Wolf tail fern blade evoking adventive bud high-frequency plant regeneration method of the present invention relates to a kind of regeneration of pteridophyte somatic cell isolated culture plant strain and test-tube plantlet micro-propagation technique of being applicable to, belongs to the plant cultivation method technical field.

Background technology

Up to now, in 12000 kinds of pteridophytes, that successfully carries out tissue culture only accounts for a few part; Though belonging to planting to have organized, part trains successfully; But exist the cycle long, and the problem that the rate of increase is not high (Han Jing, Zhao Li. pteridophyte breeding progress [J]. the Anhui agricultural science; 2005,33 (7): 1261～1263).Compare as explant with spore, utilizing root-like stock stem apex on the sporophyte, tender leaf etc. to organize training as explant has into advantages such as seedling speed is fast.Thereby, the pteridophyte group training research that with the sporophyte is explant be one of emphasis of fern group training research (Wang Shengpei is etc. pteridophyte Study on tissue culture progress [J] for Jiang Shengjun, Song Xiqiang. gardening journal, 2002,29 (supplementary issues): 651～656).

Wolf tail fern (Davallia bullata) belongs to pteridophyte wall fern order (Polypodiales) Davalliaceae (Davalliaceae).Li Wenan etc. are explant material with the blade that wolf tail fern has spore; From spore germination, induce produce that phyllome has root to growing up to, the plantlet of stem, leaf reaches (Li Wenan more than a year; Wang Yuqin. the cultured in vitro [J] of wolf tail fern. Plant Physiology Communications, 1990 (3): 44).It is explant that Li Hua grant to wait the blade with Platycerium bifurcatum, 0.1～1.0mg/L BAP help inducing of indefinite bud and breed (Li Hua grants, Chen Zhihong. the tissue culture of Platycerium bifurcatum [J]. Plant Physiology Communications, 1988 (2): 58).In tuber fern platymiscium root-like stock point is cultivated; Loescher thinks evoking adventive bud generation needing no foreign hormone; But KT can promote the formation and the propagation of indefinite bud; BAP then plays inhibitory action (Loescher W H and Albrecht C N.Development in vitro of Nephrolepis exaltala cv.Bostoniensis runner tissues [J] .Physiol Plant, 1979,47:250～254).Camloh points out that BAP can promote Platycerium bifurcatum stem end blade to form indefinite bud (Camloh M; Gogala N and Rode J.Plant regeneration from leaf explants of the fern Platycerium bifureatum in vitro [J] .Scientia Horticulture; 1994,56:257～266).

Summary of the invention

The technical problem that technical problem the present invention will solve and the technical assignment of proposition are: the blade with wolf tail fern is an explant material; Through selecting for use phenyl ureas basic element of cell division CPPU to induce and breeding indefinite bud; Improve the planting percent of indefinite bud, set up high-frequency plant regeneration technique system.

Technical scheme

Wolf tail fern blade evoking adventive bud high-frequency plant regeneration method comprises:

1) material

Selecting the blade of wolf tail fern aseptic seedling for use is material;

2) medium

Minimal medium is made up of with B5 vitamin MS macroelement, MS trace element, adds sucrose 30g/L, and the agar strip of 8g/L is as curing agent;

Adventitious bud induction culture base: additional phenyl ureas basic element of cell division CPPU 2.0mg/L and IBA 0.1mg/L;

Adventitious bud proliferation medium: additional phenyl ureas basic element of cell division CPPU 0.5mg/L and IBA 0.1mg/L;

Cheng Miao and root media: the plain IBA 0.2mg/L of appositional growth, active carbon 0.1%;

Above-mentioned various medium is adjusted pH value to 5.8 with sodium hydroxide and hydrochloric acid before autoclaving;

3) method

The blade of clip wolf tail fern aseptic seedling is seeded in step 2 on superclean bench) on the said adventitious bud induction culture base, induce from blade about 20d and produce indefinite bud; Indefinite bud forwards step 2 to) carry out shoot proliferation on the said adventitious bud proliferation medium and cultivate, 30d is an one-period; Indefinite bud is transferred to step 2) on said Cheng Miao and the root media, develop into have root, the complete plantlet of stem, leaf;

Indefinite bud induce and propagation, Cheng Miao and culture of rootage are all carried out under illumination condition, intensity of illumination is 1500lx, light application time is 12h/d; Culturing room's temperature is 26 ℃ ± 2 ℃.

Beneficial effect the present invention compared with prior art has following advantage and good effect:

The present invention selects for use phenyl ureas basic element of cell division CPPU to induce the leaf bud shape indefinite bud ratio that produces normal condition to reach about 95% from wolf tail fern blade, and the planting percent of leaf bud reaches 100%; BAP induces and breeds the thallus indefinite bud of abnormal condition, and planting percent is merely 6%; KT induces the leaf bud shape indefinite bud that produces the higher proportion normal condition, but the indefinite bud of propagation presents vitrifying and soft rotten phenomenon, and planting percent is 40%.

Chinese scholars selects for use BAP and KT to induce and breed indefinite bud from blade on nephrolepidaceae and Rhynioceae pteridophyte, and plant regeneration technique still rests on conceptual phase.The present invention sets up the wolf tail fern blade evoking adventive bud high-frequency plant regeneration technique of Davalliaceae first, reaches degree of being practical, can offer reference for carrying out pteridophyte micropropagation, somatic mutants screening and transgenosis work.

Description of drawings

The indefinite bud that Fig. 1 CPPU induces.

The indefinite bud that Fig. 2 BAP induces and breeds.

The indefinite bud that Fig. 3 CPPU induces and breeds.

The complete plantlet of Fig. 4.

Embodiment

1. material and method

1.1 test material

Blade with wolf tail fern (Davallia bullata) aseptic seedling (wolf tail fern seedling is available from Huambo flowers market, Nanjing) of pteridophyte wall fern order (Polypodiales) Davalliaceae (Davalliaceae) is an explant.

1.2 medium

Minimal medium is by MS macroelement, MS trace element (Murashige T; Skoog F.A revised medium from rapid growth and bioassays with tobacco tissue cultures [J] .Physiol plant; 1962,15:473～497) and B5 vitamin (Gamborg O L, Miller R A; Ojima K.Nutrient requirements of suspension cultures of soybeanroot cells [J] .Exp.Cell Res.; 1968.50:151～158) form, be a kind of medium commonly used in the Plant Tissue Breeding, sucrose 30g/L; Agar strip (board, Quanzhou City, Fujian spring Hong Kongnization factory) with fair wind 8g/L;

The adventitious bud induction culture base: the 6-benzylaminopurine of additional 2.0mg/L (is called for short BAP respectively; The full bio tech ltd of Shanghai snow); Kinetin (is called for short KT; Nanjing Sheng Xing Bioisystech Co., Ltd) and the phenyl ureas basic element of cell division (be called for short CPPU, Nanjing Sheng Xing Bioisystech Co., Ltd);

The adventitious bud proliferation medium: the 6-benzylaminopurine of additional 0.5mg/L (is called for short BAP respectively; The full bio tech ltd of Shanghai snow); Kinetin (is called for short KT; Nanjing Sheng Xing Bioisystech Co., Ltd) and the phenyl ureas basic element of cell division (be called for short CPPU, Nanjing Sheng Xing Bioisystech Co., Ltd);

Cheng Miao and root media: additional 0.2mg/L growth hormone indolebutyric acid (being called for short IBA, source, Shanghai consor thing Science and Technology Ltd.), 0.1% active carbon (Nanjing assistant research fellow Bioisystech Co., Ltd);

Medium is adjusted pH value to 5.8 with sodium hydroxide and hydrochloric acid before autoclaving.

1.3 method

Blade with wolf tail fern aseptic seedling is a material, is inducing the generation indefinite bud on the adventitious bud induction culture base about 20d; The blade that has indefinite bud is transferred on the adventitious bud proliferation medium, and 30d is an one-period, the indefinite bud shoot proliferation; Indefinite bud is transferred on seedling and the root media, forms about 25d to have the complete regenerated plant of root, stem, leaf; Isolated culture condition: culturing room's temperature is 26 ± 2 ℃, and intensity of illumination is 1500 lx, and light application time is 16h/d.

2. result

2.1 the basic element of cell division is to the influence of blade evoking adventive bud

The blade inoculation of wolf tail fern aseptic seedling is on the inducing culture of additional KT 2.0mg/L, IBA 0.1mg/L and CPPU 2.0mg/L, IBA 0.1mg/L; Induce the generation indefinite bud about 20d, form intensive thallus bud about 30d and follow macroscopic leaf bud shape indefinite bud (Fig. 1); And on the medium that adds BAP 2.0mg/L, IBA 0.1mg/L, only induce to produce intensive thallus bud, do not see leaf bud shape indefinite bud.

Table 1 basic element of cell division is to the influence of blade evoking adventive bud

Table 1 result shows: on the adventitious bud induction culture base of additional 2.0mg/L KT, CPPU or BAP; The induction frequency of indefinite bud is all more than 90%; But induce the state of the indefinite bud of generation that notable difference is arranged, KT and CPPU help inducing the leaf bud shape indefinite bud that produces normal condition.

2.2 the basic element of cell division is to the influence of adventitious bud proliferation

On the adventitious bud proliferation medium of additional CPPU 0.5mg/L, IBA 0.1mg/L; All transfer to form the leaf bud shape indefinite bud (Fig. 2) of normal condition early stage from the thallus bud on interpolation KT or the CPPU medium; And in earlier stage from the thallus indefinite bud of this maintenance of the thallus bastem abnormal condition on the BAP medium, only other thallus bud forms leaf bud shape indefinite bud.

Forward the adventitious bud proliferation medium of additional KT 0.5mg/L, IBA 0.1mg/L on from the indefinite bud that adds on the KT medium early stage, and most buds transfer to form the leaf bud shape indefinite bud of normal condition, but still have the part bud to keep thalliform.Forward the adventitious bud proliferation medium of additional BAP 0.5mg/L, IBA0.1mg/L on from the indefinite bud on the BAP medium early stage, and bud all keeps intensive thalliform (Fig. 3).

0.5mg/L KT, CPPU or BAP are suitable basically to the proliferate efficiency of indefinite bud, per generation growth coefficient be about 5.

Table 2 basic element of cell division is to the influence of indefinite bud Cheng Miao

Observe in the test and forward enrichment culture on the medium that adds CPPU 0.5mg/L, IBA 0.1mg/L to from the indefinite bud that adds on the KT medium early stage, the indefinite bud more than 50% presents vitrifying and soft rotten phenomenon, thereby causes planting percent to be merely 40%; And forward the medium that add CPPU 0.5mg/L, IBA 0.1mg/L on enrichment culture from the indefinite bud on the additional CPPU medium early stage; The indefinite bud robust growth; Planting percent is up to 100% (table 2) on the medium of additional IBA0.2mg/L; Induce to generate the light/dark balance root system, rooting rate reaches 98%, forms the complete plantlet (Fig. 4) with root, stem, leaf.This shows the inducing, breeding and become seedling of the suitable wolf tail fern blade indefinite bud of phenyl ureas basic element of cell division CPPU.

3. conclusion

CPPU is a kind of phenyl ureas basic element of cell division; Can promote various plants evoking adventive bud in cultured in vitro generation (Luo Zhengrong. new plant growth regulator CPPU and the application on fruit tree and vegetables [J] thereof. Plant Physiology Communications; 1993,29 (1): 297～299).This test is an explant with the blade of pteridophyte Davalliaceae wolf tail fern; Phenyl ureas basic element of cell division CPPU induces and the effect of breeding indefinite bud obviously is superior to KT and BAP; The ratio of the leaf bud shape indefinite bud of normal condition reaches more than 95%; Indefinite bud growth coefficient of per generation is 5, and the planting percent of indefinite bud is set up high-frequency plant regeneration technique system up to 100%.

Claims

1. wolf tail fern blade evoking adventive bud high-frequency plant regeneration method is characterized in that:

1) material

Selecting the blade of wolf tail fern aseptic seedling for use is material;

2) medium

Minimal medium is by MS macroelement, MS trace element and B ₅Vitamin is formed, and adds sucrose 30g/L, and the agar strip of 8g/L is as curing agent;

Cheng Miao and root media: the plain IBA0.2mg/L of appositional growth, quality of activated carbon is than 0.1%;

3) method

The blade of clip wolf tail fern aseptic seedling is seeded in step 2 on superclean bench) on the said adventitious bud induction culture base, 20d induces from blade and produces indefinite bud; Indefinite bud forwards step 2 to) carry out shoot proliferation on the said adventitious bud proliferation medium and cultivate, 30d is an one-period; Indefinite bud is transferred to step 2) on said Cheng Miao and the root media, develop into have root, the complete plantlet of stem, leaf;

Indefinite bud induce and propagation, Cheng Miao and culture of rootage are all carried out under illumination condition, intensity of illumination is 1500lx, light application time is 16h/d; Culturing room's temperature is 26 ℃ ± 2 ℃.