CN105660417A - Yangmai 14 mature embryo callus successive induced culture medium - Google Patents

Abstract

The invention relates to a Yangmai 14 mature embryo callus successive induced culture medium. The culture medium is composed of MS+700 mg/L of L-proline, 1000 mg/L of casein hydrolysate, 2 mg/L of 2, 4-D, 30 g/L of sucrose and 2.6 g/L of phytagel, and PH of the culture medium is 5.8. By using the culture medium, culture time of Yangmai 14 mature embryo callus can be prolonged, high regeneration rate of the callus can be maintained, and stability of wheat conversion is facilitated.

Description

Raise wheat 14 mature embryo callus subculture inducing culture

Technical field

The invention belongs to Crop Genetic Breeding and field of plant tissue culture, be specifically related to the external subculture callus induction of mature embryo.

Background technology

1. the breeding objective of Semen Tritici aestivi

Semen Tritici aestivi (Triticumaestivum) is one of world's staple food crop, and its yield and quality is related to social and economic activities. Development along with population increase and national economy, it is necessary to more grain meets the life requirement of people. But soil erosion causes farmland constantly to be reduced and global warming makes grain yield in recent years increase and inconspicuous. According to Correlative data analysis, analyzing global yield of wheat in recent years, it has been found that yield of wheat is unstable, and amplification is less, the volume increase of Semen Tritici aestivi has become as one of problem of people's concern. In the face of so big challenge, any method that can improve grain yield is all worth utilizing. For cereal crops breed improvement, people mainly take the mode of traditional breeding method and transgenic. Both essential distinctions are: the former promotes the variation of parental gene group by emasculation hybridization, and the later stage, with merit for index, utilizes field test, screening to obtain the wheat breed of required merit. Utilizing transgenic technology cause plant gene directed variation and produce merit, compared to conventional hybridization breeding, transgenic technology can break reproduction isolation, expands the scope of plant gene variation further. Plant tissue culture is the key of wheat transgenic technology, and traditional breeding method carries out under field conditions (factors), so cycle length, climate environmental effect are big. Transgenic technology utilizes tissue culture, and whole process can carry out at incubator and greenhouse, is prevented effectively from the impact of environmental factors. Transgenic technology specifically includes that gene clone, genes of interest Function Identification, genes of interest are inserted Plant Genome and obtained breed improvement commercially valuable plant by Physiological Appraisal. Plant Genome inserts the method for exogenous gene particle bombardment and Agrobacterium-mediated Transformation method and pollen tube passage method etc.

2. Wheat Transformation method

Semen Tritici aestivi is allohexaploid plant, genome is big, gene regulation is difficult, the callus ubiquity wound healing embryo that the outer implant of Semen Tritici aestivi is induced simultaneously is poor, differentiation rate is low, convert the problems such as cell regeneration difficulty, is the universally acknowledged raise crop being most difficult to convert. Scientist realizes conversion to Semen Tritici aestivi by various methods, but current topmost Wheat Transformation technology is particle bombardment and agriculture bacillus mediated. The first in the world strain transgenic wheat is that Vasil passed through particle bombardment by gus, bar channel genes wheat breed Pavon in 1992.Within 1997, Cheng utilizes agrobacterium-mediated transformation that GUS and npt II has been proceeded to Semen Tritici aestivi first. Except particle gun and agrobacterium-mediated transformation, pollen tube passage method, microinjection, Laser microbeam puncture, PEG method and electric shocking method etc. are also affected by increasing concern.

3. WHEAT CALLUS induction

In 1987 Wheat Tissue cultivated according to christy and form callus classification, 3 classes can be classified as: embryo callus (E), non embryogenic callus (NE) and the smooth wound healing of compacting (SC) of embryo callus can be changed into. E type wound healing color is white or faint yellow, glossy, nodositas, one touch easily be broken into graininess, cell ball shape; NE type wound healing is frangible, and color is brown or Lycoperdon polymorphum Vitt; SC type wound healing is mainly characterized by smooth compacting, and has very strong Reproductive activity, can be converted into E type wound healing. The E type wound healing of mature embryo is mainly from SC type Transformation of Callus, and the embryo callus of wheat immature embryo directly can go out E class wound healing from embryonal induction. It is vigorous that embryoid directly can be divided into whole plant, vitality by individual cells, is greatly shortened the seedling cycle.

4. affect callus induction factor

4.1 genotype different cultivars can affect generation embryo callus subculture ability owing to genotype is different, and this all can find in the tissue culture procedures of the plants such as Semen Tritici aestivi, Oryza sativa L. and Semen Maydis. Bright phoenix etc. 2000 to the Liao Dynasty 10,7742,7757, after new gram of drought 9 and Roblin Semen Tritici aestivi subculture, statistics generates embryo callus ratio, and distant 10 Semen Tritici aestivis are after inoculation 414 days, and the embryo callus of 78% generates, and new gram of drought 9 callus brownization.

4.2 outer implant types snow prunus mume (sieb.) sieb.et zucc.s perhaps etc. study its impact on callus induction with the different parts of Picea koraiensis Nakai immature embryo for material in 2009. Found that wound healing induction is had a great impact by the outer implant of different parts, the frequency of embryonic callus induction best result on full embryo and plumular axis top does not reach 81.27% and 75.96%.

4.3 medium component culture medium are not only plant cell In vitro culture and provide nutrient substance, provide exogenous inducing factors and pressure for the dedifferentiation of cell, again differentiation yet. Plant dedifferentiation is limited mainly by the effect of auxin and the basic element of cell division period, so for the generation of callus, the adjustment of hormone is one of key. He Shenglian etc. are with Henan wheat 49, Henan wheat 18 and Lankao 906 for material, the impact that wheat immature embryo embryo callus is generated by the different allogenic material of research, it has been found that along with increasing of 2,4-D concentration, embryo callus subculture ratio and green some rate are continuously increased, but decline therewith again after reaching finite concentration. The Semen Tritici aestivi of 3 kinds is when 2,4-D is 2mg/L, and embryo callus ratio all reaches the highest. Except hormone, the impact with embryo callus subculture has been also affected by the concern of people by the nutrient substance of culture medium such as aminoacid or trace element etc. Gao Wujun etc. add 6 kinds of Organic substances respectively at the minimal medium of Semen Maydis, it has been found that the L-PROLINE of 500mg/L caseinhydrolysate and 700mg/L significantly improves the ratio of Semen Maydis embryo callus subculture. Yan Huajun etc. study it is again seen that embryo callus is remarkably promoted effect by caseinhydrolysate, L-PROLINE, inositol etc. with boat wheat No. 2.

5. orthogonal test

Orthogonal test is to arrange multifactorial experiment by global design orthogonal test table, through Integrated comparative, statistical analysis, affects size and best of breed by less the effects difference factor.

Being mainly characterized by of orthogonal experiment: for multifactorial experimental design, it is possible to the level choosing experimental factor representative compares, effectively reduces the number of times of test, but does not reduce the feasibility of experiment. Finally select the level that each factor is optimum and best of breed between different factor each level. On the other hand, by the analysis of orthogonal test, it is possible to find key factor and mutual relation that may be present between each factor

Nineteen forty-two orthogonal test starts to receive publicity, and all obtains in every field afterwards and is widely applied. Domestic to use the mathematical method such as orthogonal experiment to carry out Vitro Plant culture studies the earliest be Institute of Zoology heredity room, Guangdong Province, and increasing researcher starts with this method and carries out the improvement of culture medium afterwards. Within 2010, Kong Weiping adopts 5 kinds of additional factors of orthogonal test research and the concentration impact on corn mature embryo callus, by the statistics to various combination healing rate, finally determines the best of breed of corn mature embryo induction. Tang Wenzhongs in 2012 etc. utilize four factor three levels, study minimal medium kind, IBA, NAA and AC impact on papaya seedling rooting.

Summary of the invention

It is an object of the invention to provide one and raise wheat 14 mature embryo callus subculture inducing culture, this culture medium is conducive to raising wheat 14 mature embryo subculture more than about 40 days, still maintains the callus of higher regeneration capacity. (acceptance amendment)

The embryo callus that wheat mature embryo long-term subculture obtains, has higher regeneration capacity, is the good material of genetic transformation. In order to when not affecting the regeneration capacity raising wheat 14 mature embryo wound healing, obtaining more embryo callus further and extend the holding time of embryo callus, the present invention is filtered out most beneficial for the inducing culture raising wheat 14 mature embryo long term subculture by orthogonal. With L-PROLINE, caseinhydrolysate and 2,4-D for influence factor, being respectively provided with 3 concentration, configure the culture medium of 9 kinds of various combinations altogether, cut and raise wheat 14 mature embryo and be respectively placed in 9 kinds of culture medium, experiment repeats 3 times. After subculture 5 times, all calluss of every kind of culture medium shift on same division culture medium respectively, add up green some rate of wound healing and situation of emerging. Final with green some rate for standard, analyze the contribution rate of each factor and each factor best of breed.

The wheat 14 mature embryo callus subculture inducing culture of raising that the present invention is determined by orthogonal test is MS+700mg/LL-proline+1000mg/L casein hydrolysate+2mg/L2,4-D+30g/L sucrose+2.6g/L plant gel, PH5.8. What this culture medium was induced raises wheat 14 mature embryo callus when long-term subculture, and callus still keeps light yellow, and callus increases very fast; After long-term subculture, the wheat 14 mature embryo callus regeneration capacity of raising of other culture medium induction is suppressed, and does not all emerge, and the wheat 14 mature embryo callus regeneration rate of raising of best medium is up to 6.7%.

Accompanying drawing explanation

Fig. 1 is 9 kinds of inducing culture callus induction regeneration figure.

Note: in figure, 1 to 9 represents the callus raising wheat 14 that 9 kinds of culture medium culturings go out respectively and is placed under same division culture medium, same growth conditions, regeneration seedling and form the situation of green some rate. (9 kinds of culture medium combinations that in 9 kinds of culture medium correspondence embodiments 1, orthogonal test obtains, concrete formula changes in Table 3)

Detailed description of the invention

Embodiment 1:

1. experiment material

1.1 experimental cultivars: preserve 1 year raise wheat 14 seed (buying in gold soil, Jiangsu Zhong Ye company limited)

1.2 culture medium: callus induction consists of MS minimal medium (concrete composition is in Table 1), 30g/L sucrose, 2.6g/L plant gel, add variable concentrations 2,4-D, L-PROLINE and casein hydrolysate (table 2) respectively.Division culture medium is made up of MS minimal medium, 30g/L sucrose, 2.6g/L plant gel, 0.5mg/L kinetins, 2mg/L zeatin. All culture medium PH are 5.8

Table 1.MS culture medium mother solution

2. experimental technique

2.1 set experimental factor level in Table 2. By orthogonal design assistant V3.1, design L9 (3³) Three factors-levels orthogonal design table is in Table 3, and the inducing culture of 9 kinds of combinations is configured according to orthogonal test table.

2.2 take laboratory preserves the seed raising wheat 14 of a year, selects the wheat seed of full seed, is placed in the triangular flask that bacterium of having gone out is clean. After 75% ethanol sterilizing 5min, soak 30min with 0.1% mercuric chloride, finally rinse 3～5 times with aquesterilisa. With the rataria of dissecting knife allocation diameter 1mm size on superclean bench, scultellum is inoculated on 9 kinds of calli induction medias upward, and every kind of culture medium inoculated 30, each process is repeated 3 times, changes once same inducing culture every 10 days, altogether subculture 5 times.

2.3 all wheat 14 mature embryo callus of raising all transfer to same division culture medium respectively after subculture 5 times, and 25 ± 1 DEG C of illumination cultivation add up emergence rate after one month.

The factor of table 2 orthogonal test and level

3. result and analysis

Data process formula:

Callus is affected by the interpolation of 3 three kinds of materials of table and variable concentrations

What R value represented each factor affects size, with raise wheat 14 mature embryo-derived callus differentiation phase again occur green some rate for index, the more big representative impact of R value is more big, so the relation that affects of three kinds of compositions is 2,4-D > L-PROLINE > casein hydrolysates. Finding out optimum combination according to K value size: K value is more big, effect is more good, is A3B3C2 that is the 9th kind culture medium according to statistics optimum combination: MS+700mg/LL-proline+1000mg/L casein hydrolysate+2mg/L2,4-D+30g/L sucrose+2.6g/L plant gel.

Embodiment 2:

The different culture media of the acquisition of the subculture 5 times processed according to example 1 raise wheat 14 mature embryo callus, transfer on same division culture medium respectively, (25 ± 1) DEG C illumination cultivation observes growth of cereal crop seedlings condition after one month.

Drawing according to the situation of emerging: after subculture 5 times, other culture medium are raised wheat 14 mature embryo callus and are subject to subculture impact, all do not sprout; Optimal medium (MS+700mg/LL-proline+1000mg/L casein hydrolysate+the 2mg/L2 that example 1 obtains, 4-D+30g/L sucrose+2.6g/L plant gel, that PH5.8) cultivates raises wheat 14 mature embryo-derived callus, and after subculture 5 times, emergence rate is 6.7%, such as Fig. 1.

Claims (1)

1. raise wheat 14 mature embryo callus subculture inducing culture for one kind, it is characterised in that composition is: MS+700mg/LL-proline+1000mg/L casein hydrolysate+2mg/L2,4-D+30g/L sucrose+2.6g/L plant gel; The PH5.8 of culture medium.