CN101011035A - Seashore paspalum young spike isolated culture strain-reproducing technique - Google Patents

Abstract

The invention relates to a method for cultivating the barnyard grass in vitro. The invention uses the spike in blooming period as the explant, to be cultivated in the callus cultivate medium which contains 2.omg/L of 2, 4-fenclofenac, and 0.05mg/L of 6-benzyl adenine to obtain yellow dry particle callus whose number is more than 40% higher than the induce method; then cultivates in the cultivate medium whose agar-solidifier content is improved one time to generate, while the callus is particle structure, via three generations, the green differentiation can reach 98%. The invention can be used to cultivate barnyard grass, or the like.

Description

Seashore paspalum young spike isolated culture strain-reproducing technique

Technical field

Seashore paspalum young spike isolated culture strain-reproducing technique of the present invention relates to a kind of culture technique that is applicable to the acceptor material of micropropagation, somatic mutants screening and genetic transformation, belongs to method for plant tissue culture.

Technical background

Puffin barnyard grass (Paspalum vaginatum Sw.) has higher salt tolerant, drought-resistant, waterlogging and mar proof, as turfgrass, widely plant with the subtropical zone in the torrid zone, the world, be the up-and-coming youngster (Xie Xinming that after warm season dogstail such as Bermuda grass, Korea lawn grass and centipede grass, rises, Lu Xiaoliang. the good characteristic of puffin barnyard grass germ plasm resource and value [J] thereof, Agricultural University Of South China's journal, 2004,25 (supplementary issue II): 64～67).The puffin barnyard grass mainly is distributed in provinces such as Taiwan, Yunnan, Guangdong and Hainan in China.The introduction and Experiment result shows for many years, and the puffin barnyard grass is at Hangzhou and two places, Nanjing energy safe overwintering, but the green phase is shorter partially than local Bermuda grass and Korea lawn grass kind, and fungal disease is expansion trend.Triploid puffin barnyard grass is because of obstacles such as pollen abortion, selfing are not affine, and the propagation by division of crawling by stem (Malaysian China, Zhao Nanxian. Paspalum cytology and reproductive biology research [J], the subtropical plant science, 2003,32 (3): 5～9), work brings difficulty to conventional breeding.

Reaching its maturity of plant soma screening mutant and transgenic technology opened up cell and molecular biology breeding new way for improveing turfgrass.So far, somatic mutants screening and genetic transformation do not appear in the newspapers on the puffin barnyard grass as yet, and its main cause is to be difficult to obtain granular embryo callus.In Plant Tissue Breeding, translucent, moistening, sticking shape embryo callus is lost the ability of green seedling differentiation gradually in the successive transfer culture process, especially depress (physics and chemistry mutagen) in the selection of artificial screening and cultivate the thorough ability of losing plant regeneration in back by agroinfection with being total to, somatic mutants screens and the progress of agrobacterium-mediated transformation genetic transformation work thereby influence.

Up to now, both at home and abroad 1 piece of report (Cardona C A is only arranged about puffin barnyard grass tissue culture plant regeneration and somaclonal variation, Duncan R R.In Vitro culture, somaclonal variation, and transformation strategies with paspalum turf ecotypes.In:MBSticklen and MP Kenna (ed) Turfgrass biotechnology-cell and moleculargenetic approaches to turfgrass improvement[M] .Ann Arbor Press, MI, 1997,229～236).Cardona and Duncan are material with the puffin barnyard grass, have obtained the variant that internode shortens from the regeneration plant of callus.

Summary of the invention

The technical assignment of the technical problem to be solved in the present invention and proposition is to overcome translucent that existing puffin barnyard grass cultured in vitro technological guide produces, moistening, sticking shape embryo callus is lost the ability of green seedling differentiation in the successive transfer culture process, the defective that should not be used for somatic mutants screening and agrobacterium-mediated transformation genetic transformation, a kind of seashore paspalum young spike isolated culture strain-reproducing technique is provided, with the young fringe by selecting the puffin barnyard grass for use as explant material, the osmotic pressure of regulation and control exogenous plant hormones and medium, obtain a high proportion of graininess embryo callus, set up the technical system of high-frequency plant regeneration.For this reason, the present invention is by the following technical solutions:

Seashore paspalum young spike isolated culture strain-reproducing technique is characterized in that:

1) material

The young fringe of selecting for use the puffin barnyard grass to be in grain husk flower potted flower period is an explant material;

2) medium

Minimal medium is by MS macroelement, MS trace element and B ₅Vitamin is formed, and adds sucrose 30g/L;

Callus inducing medium and callus subculture medium: additional hormone 2,4 dichlorophenoxyacetic acid 1.0～5.0mg/L and 6-benzylaminopurine 0.01～0.2mg/L;

Callus differential medium: additional hormone 6-benzylaminopurine 1.0～3.0mg/L;

In callus inducing medium and callus differential medium, with the agar strip of 8g/L as curing agent; In the callus subculture medium, with the agar strip of 12～16g/L as curing agent;

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

3) cultural method

Get and be in the grain husk flower potted flower young fringe in period, on superclean bench, wrap in the leaf sheath and the stem of young fringe outside with the thorough wiping of 70% alcohol, strip out young fringe, be cut into the long section piece of 2～3mm, be seeded on the callus inducing medium, under scattered light, cultivate, induce from young fringe about 15d to produce light yellow, dry, granular callus; Selecting light yellow, dry, granular callus transfers to the callus subculture medium and carries out successive transfer culture, transfer to again on the callus differential medium through the graininess callus behind continuous 3 successive transfer culture, under illumination condition, cultivate, differentiate green seedling about 15d; Culturing room's temperature is 26 ± 2 ℃ in the cultured in vitro process.

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

Under isolated culture condition, be in the grain husk flower potted flower young fringe in period by selecting for use, promptly the long young fringe of 1～2cm is an explant material, regulates and control the proportioning of exogenous hormone in the callus inducing medium, has improved the induction frequency of puffin barnyard grass graininess embryo callus.Increase the consumption of the curing agent-agar strip in the callus subculture medium, graininess callus structure remains unchanged in callus successive transfer culture process, helps keeping high-frequency plant regeneration ability.The seashore paspalum young spike isolated culture strain-reproducing technique system that the present invention sets up, suitable acceptor material as somatic mutants screening and agrobacterium-mediated transformation genetic transformation is for carrying out puffin barnyard grass cell and molecular biology breeding research work provides basic condition.

In callus inducing medium, additional 2,4 dichlorophenoxyacetic acid 2.0mg/L and 6-benzylaminopurine 0.05mg/L, the quantity light yellow, dry, the graininess callus of acquisition accounts for the callus sum more than 40% of inducing generation.On the callus subculture medium that improves 1 times of agar strip consumption, the nutty structure of callus remains unchanged, and forwards that green seedling differentiation rate reaches 98% on the callus differential medium to.

Light yellow, dry, the graininess callus that use the inventive method to obtain carry out sieving psychrotolerant somatic mutants work, have obtained cell engineering plant in batches.

Description of drawings

Fig. 1 children fringe evoked callus.

Fig. 2 graininess callus.

Fig. 3 callus breaks up green seedling.

Fig. 4 somatic cell screening mutant engineering plant that resists cold.

Embodiment

1. material and method

1.1 test material

The puffin barnyard grass kind of introducing from the U.S. " Adalay " (Paspalum vaginatum Sw.cv.Adalay) is provided by lake, Guanlan, Shenzhen golf course, is test material.

1.2 medium

Minimal medium is by the MS macroelement, MS trace element (Murashige T, Skoog F.Arevised medium from rapid growth and bioassays with tobacco tissuecultures[J] .Physiol plant, 1962,15:473～497) and B5 vitamin (GamborgOL, Miller R A, Ojima K.Nutrient requirements of suspension culturesof 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;

Callus inducing medium and callus subculture medium add hormone: 2,4-dichlorphenoxyacetic acid (being called for short 2,4-D, Shanghai chemical reagent four factories) 2.0mg/L, 6-benzylaminopurine (being called for short BAP, the full bio tech ltd of Shanghai snow) 0.05mg/L;

The callus differential medium adds hormone: 6-benzylaminopurine (being called for short BAP, the full bio tech ltd of Shanghai snow) 2.0mg/L;

In callus inducing medium and callus differential medium, the consumption of agar strip (board, Quanzhou City, Fujian spring Hong Kongnization factory) with fair wind is 8g/L; In the callus subculture medium, the consumption of agar strip is 16g/L;

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

1.3 cultural method

Get the puffin barnyard grass and be in the grain husk flower potted flower young fringe in period, children's spike length degree is 1～2cm, wraps in the leaf sheath and the stem of young fringe outside on superclean bench with the thorough wiping of 70% alcohol, strips out young fringe then, be cut into the long section piece of 2～3mm, be seeded on the callus inducing medium.Add up induction frequency light yellow, dry, the graininess callus behind the 25d.

Select light yellow, dry, graininess callus and carry out successive transfer culture on the callus subculture medium, 20d was 1 cycle, and being commissioned to train through continuous 3 forwards on the callus differential medium the green seedling of differentiation about 15d, the green seedling differentiation frequency of statistics behind the 25d to after supporting.

Culturing room's temperature is 26 ± 2 ℃.Callus induction and successive transfer culture carry out under scattered light, and the callus differentiation is carried out under illumination condition.

2. result

2.1 2,4-D is to the influence of callus induction rate

Getting the long young fringe of 1.0～2.0cm is inoculation material, 2, and 4-D concentration is respectively 2.0mg/L and 4.0mg/L, and the induction frequency of callus all reaches more than 90% (table 1).

Table 12,4-D is to the influence of callus induction rate

2,4-D concentration (mg/litre)	Inoculate the hop count of young fringe	The young fringe hop count of evoked callus	Callus induction rate (%)
				2.0	136	131	96.32
4.0	130	119	91.54

2.2 the young fringe puberty is to the influence of callus induction rate

The young fringe puberty of table 2 is to the influence of callus induction rate

Children's spike length degree (centimetre)	Inoculate the hop count of young fringe	The young fringe hop count of evoked callus	Callus induction rate (%)
				1.0～1.5	98	98	100.
1.5～2.0	112	94	83.93
				2.0～2.5	104	62	59.62

2, when 4-D concentration is 2.0mg/L, inoculate the long young fringe of 1.0～1.5cm, 1.5～2.0cm and 2.0～2.5cm respectively, the induction frequency of callus obviously successively decreases successively, and when the length of young fringe surpassed 2.0cm, the inductivity of callus was lower than 60% (table 2).Result of the test shows, puffin barnyard grass children fringe cultured in vitro evoked callus, and the best period of drawing materials is the long young fringe of 1.0～2.0cm.

2.3 BAP is to the influence of graininess callus induction

Add the BAP of low concentration in callus inducing medium, the total induction frequency of callus adds 2 with independent, and the effect of 4-D is suitable substantially, all more than 90%; But wherein the ratio of graininess callus then is significantly improved, and rises to (table 3) more than 42% from 26%.Result of the test shows, adds 2 in callus inducing medium, and 4-D 2.0mg/L and BAP 0.05mg/L can keep high-frequency callus induction frequency, can improve the ratio of graininess callus again greatly.

Table 3 BAP is to the influence of graininess callus induction

Hormone kind and concentration (milligram)	The callus sum	Graininess callus number	Graininess callus number/callus sum (%)
				2，4-D 2.0	143	38	26.57
2，4-D 2.0；BAP 0.05	105	45	42.86

2.4 the plant regeneration ability of graininess callus

Select the graininess callus from callus inducing medium and carry out the shoot proliferation cultivation, when the agar strip consumption was 8g/L, part graininess callus changed translucent, moistening, granular structure into; When the agar strip consumption was 16g/L, light yellow, dry, nutty structure callus was remaining unchanged in shape, transferred to after 3 generations on the callus differential medium through continuous successive transfer culture, and green seedling differentiation rate reaches 98% (table 4).

Table 4 graininess callus is to the influence of green seedling differentiation rate

Graininess callus sum	Green seedling callus number	Green seedling differentiation rate (%)
			100	98	98

3. conclusion

This research is in the grain husk flower potted flower young fringe in period with the puffin barnyard grass, children's spike length degree is 1～2cm, be explant material, callus inducing medium additional 2,4-D 2.0mg/L and BAP 0.05mg/L induce light yellow, dry, the graininess callus of generation to account for more than 40% of callus induction sum.The graininess callus remains nutty structure on the medium of 1 times of the consumption that improves curing agent-agar strip, reach 98% through its green seedling differentiation rate behind the continuous 3 generation successive transfer culture.Set up the seashore paspalum young spike isolated culture strain-reproducing technique system, the graininess embryo callus is applicable to somatic mutants screening and agrobacterium-mediated transformation genetic transformation, for carrying out puffin barnyard grass cell and molecular biology breeding research work provides basic condition.

More than with a concrete example technical scheme of the present invention is described in detail, but be not to be only limited to above-mentioned concrete implementation detail; Implement technical scheme of the present invention with other data in the data area of technical solution of the present invention, reached same technique effect, do not repeat them here.

Claims (1)

1, seashore paspalum young spike isolated culture strain-reproducing technique is characterized in that:

1) material

The young fringe of selecting for use the puffin barnyard grass to be in grain husk flower potted flower period is an explant material;

2) medium

Minimal medium is by MS macroelement, MS trace element and B ₅Vitamin is formed, and adds sucrose 30g/L;

Callus inducing medium and callus subculture medium: additional hormone 2,4 dichlorophenoxyacetic acid 1.0～5.0mg/L and 6-benzylaminopurine 0.01～0.2mg/L;

Callus differential medium: additional hormone 6-benzylaminopurine 1.0～3.0mg/L;

In callus inducing medium and callus differential medium, with the agar strip of 8g/L as curing agent; In the callus subculture medium, with the agar strip of 12～16g/L as curing agent;

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

3) cultural method

Get and be in the grain husk flower potted flower young fringe in period, on superclean bench, wrap in the leaf sheath and the stem of young fringe outside with the thorough wiping of 70% alcohol, strip out young fringe, be cut into the long section piece of 2～3mm, be seeded on the callus inducing medium, under scattered light, cultivate, induce from young fringe about 15d to produce light yellow, dry, granular callus; Selecting light yellow, dry, granular callus transfers to the callus subculture medium and carries out successive transfer culture, transfer to again on the callus differential medium through the graininess callus behind continuous 3 successive transfer culture, under illumination condition, cultivate, differentiate green seedling about 15d; Culturing room's temperature is 26 ± 2 ℃ in the cultured in vitro process.

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