CN105557523A - Establishment method of kiwi fruit genetic transformation receptor system based on heat stress - Google Patents
Establishment method of kiwi fruit genetic transformation receptor system based on heat stress Download PDFInfo
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- CN105557523A CN105557523A CN201510951500.8A CN201510951500A CN105557523A CN 105557523 A CN105557523 A CN 105557523A CN 201510951500 A CN201510951500 A CN 201510951500A CN 105557523 A CN105557523 A CN 105557523A
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- heat stress
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to the technical field of plant tissue culture and gene engineering, and in particular relates to an establishment method of a kiwi fruit genetic transformation receptor system based on heat stress. The establishment method comprises the following steps: by utilizing Chinese gooseberry Funiu95-2 tissue culture seedling lamina subjected to heat stress treatment as an explant, processing the explant to obtain a callus tissue, and performing adventitious bud induction culture and rooting culture. The method is simple and feasible in operation, and is capable of greatly shortening the regeneration time of an original transformation receptor system; meanwhile, the differentiation ration of adventitious buds of the callus tissue is further improved, and the root development capability of adventitious buds is also greatly improved.
Description
Technical field
The present invention relates to Plant Tissue Breeding and gene engineering technology field, specifically, a kind of method for building up of the kiwi fruit genetic transformation system based on heat stress.
Background technology
Actinidia Actinidiaceae (Actinidiaceae) actinidia, due to they have that Vitamin C content is high, unique flavor, the advantage such as nutritious and receive much concern.Chinese gooseberry (Actinidiachinensis) is one of main cultispecies of kiwi fruit, its second of the three ten-day periods of the hot season, the pulp of ox 95-2 strain was red, special Chinese gooseberry germ plasm resource, but there is because of it deficiencies such as fruit is little, appearance poor, therefore await improveing and improve its fruit quality with meeting the market requirement.The cycle needed for breed improvement utilizing conventional breeding means to carry out kiwi fruit is grown, workload is large and efficiency is low, is difficult to the needs meeting production development, and technique for gene engineering is then for the Genetic improvement of kiwi fruit opens a new way.
Since the invention leaf disk method such as Horsch in 1985, engineered transformation relative simplicity.First successful Transformation Systems relies on the foundation of good Plant host systems, namely require that the explant for transforming efficiently, stably can regenerate clone, and the integration of foreign DNA can be accepted, certain susceptibility should be had to antibiotic for the transformant that screens or plant simultaneously.For the existing research of the genetic conversion system of Chinese gooseberry before present invention applicant, but this system still to there is the recovery time long, it is underdeveloped problem that indefinite bud is taken root.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide a kind of method for building up of the kiwi fruit genetic transformation system based on heat stress, described kiwi fruit genetic transformation system, it is long to solve the existing transformation system recovery time, and it is underdeveloped problem that indefinite bud is taken root.
Based on a method for building up for the kiwi fruit genetic transformation system of heat stress, comprising:
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process as explant, described explant is treated as callus and carries out Fiber differentiation and the culture of rootage of indefinite bud.
Each cell of plant contains whole hereditary information of these species, thus possesses the hereditary potency developing into whole plant.Under optimum conditions, any one cell can develop into a new individuality.Totipotency of plant cell is the theoretical foundation of Plant Tissue Breeding, and in its totipotent power and transformation system, the differentiation speed of callus is closely bound up.
The heat stress method that the application adopts is that inventor is serendipitous in an experiment.There is of short duration intensification because the temperature-adjusting device in culturing room breaks down, in cultivation subsequently, find the Calli Differentiation speed of this batch be significantly higher than before batch, and root system is more flourishing.Finally univers parameter is decided after inventor adjusts adjustment genetic transformation system.
About heat stress (heatstress), at present clear and definite mechanism is not also had to the impact of totipotency of plant cell, but Recent study finds, the totipotency having cell after stress processing in animal obtains the phenomenon [Nature510 of part recovery, 393 – 396 (19June2014)], find when researcher is by studying the muscle stem cell (sternzellen) of mouse, this kind of cell is be in a kind of inactive state under normal circumstances, is constantly carrying out DNA replication dna and differentiation unlike other cells.But once human body injured or need regeneration, these cells can be activated.And the totipotency of plant cell is inherently far more than zooblast, its totipotency may also can be subject to affecting, and then makes Calli Differentiation ability stronger, and this may have the molecular mechanism different from zooblast.
After heat stress process, the application achieves the recovery time and significantly shortens, and indefinite bud to take root be more flourishing effect.
Preferably, as above based on the method for building up of the kiwi fruit genetic transformation system of heat stress, the leaf age of the blade of described plantlet in vitro is 28 ~ 32d.
Preferably, as above based on the method for building up of the kiwi fruit genetic transformation system of heat stress, the concrete steps of described heat stress are:
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 18 ~ 22d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature is 23 ~ 27 DEG C.
The suitableeest cultivation temperature of Chinese gooseberry is 23 ~ 27 DEG C, just may cause the death of plant when environmental temperature is more than 40 DEG C.Morning 6:00 ~ 8:00 to carry out heat stress effect best, and the recovery time of plant can be shortened.The concrete operation method of heat stress process is, plantlet in vitro being transferred to room temperature is leave standstill 30 ~ 50min in the culture environment of 35 ~ 38 DEG C.
Preferably, as above based on the method for building up of the kiwi fruit genetic transformation system of heat stress, explant is treated as callus and the concrete operations carrying out the Fiber differentiation of indefinite bud comprise:
Remove blade tip and the leaf margin of blade, vertically be cut into the leaf dish of (0.4 ~ 0.6cm) × (0.4 ~ 0.6cm) as test explant along arteries and veins in blade, face of blade be upwards inoculated in callus and adventitious bud induction culture base and cultivate.
Preferably, described callus and adventitious bud induction culture base are MS+ (1.5 ~ 2.2) mg/LZT+ (0.5 ~ 0.7) mg/LNAA.
Preferred further, described callus and adventitious bud induction culture base are MS+ (1.5 ~ 2.2) mg/LZT+ (0.5 ~ 0.7) mg/LNAA.
Compound method is, in MS medium, add ZT and NAA by given amount.
Preferably, as above based on the method for building up of the kiwi fruit genetic transformation system of heat stress, the concrete operations of described culture of rootage comprise: be inoculated in root media by the healthy and strong indefinite bud growing to 2 ~ 3cm and cultivate.
Preferably, described root media is 1/2MS+ (2.3 ~ 2.6) mg/LNAA.
Compound method is, in MS medium, add NAA by given amount.
Preferred further, the condition of culture of described culture of rootage is:
Cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, incubation time is 20 ~ 30d.
Preferably, as above based on the method for building up of the kiwi fruit genetic transformation system of heat stress, MS in described callus and adventitious bud induction culture base and the 1/2MS in described root media is and obtains the part mineral salt improvement in basal MS medium, is modified to and is divided into: KNO
3be adjusted to 700 ~ 750mg/L, NH
4nO
3be adjusted to 600 ~ 650mg/L.
Table 1 modified MS medium mother liquor example (unit: mg)
Wherein, MS medium is Murashige and Skoog was tobacco cell Training Design in 1962, be characterized in mineral salt and ion concentration higher, more stable ionic equilibrium solution, its nitrate content is high, quantity and the ratio of its nutrient are suitable, but high salt ionic concentration is unfavorable for the differentiation of Chinese gooseberry volt ox 95-2 on the contrary.Therefore, the application is to the KNO in wherein macroelement
3and NH
4nO
3content is improved, and its concrete content is as shown in table 1.
Compared with prior art, beneficial effect of the present invention is:
(1) heat stress is simple to operate, and technical difficulty is low.
(2) induction time of callus and indefinite bud significantly reduces, and increases culture efficiency; Callus and indefinite bud differentiation rate can reach 96%.
(3) the culture of rootage incubation time of indefinite bud significantly reduces, and adds culture efficiency; Root biomass, point number, rhizome embedded depth all comparatively additive method progress are obvious, illustrate that the root system development that cultivation obtains is more flourishing.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment 1
A kind of method for building up of the kiwi fruit genetic transformation system based on heat stress
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 18d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature is 23 ~ 27 DEG C.
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process as explant, described explant is treated as callus and carries out Fiber differentiation and the culture of rootage of indefinite bud.
Embodiment 2
A kind of method for building up of the kiwi fruit genetic transformation system based on heat stress
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 22d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature is 23 ~ 27 DEG C.
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process 10d as explant, remove blade tip and the leaf margin of blade, vertically be cut into the leaf dish of 0.4cm × 0.4cm as test explant along arteries and veins in blade, face of blade be upwards inoculated in callus and adventitious bud induction culture base and cultivate.
Callus and adventitious bud induction culture base are MS+1.5mg/LZT+0.5mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, and incubation time is 25d.
The healthy and strong indefinite bud growing to 2 ~ 3cm is inoculated in root media and cultivates.
Root media is 1/2MS+2.6mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, and incubation time is 25d.
MS in above-mentioned callus and adventitious bud induction culture base and the 1/2MS in described root media is and obtains the part mineral salt improvement in basal MS medium, is modified to and is divided into: KNO
3be adjusted to 700mg/L, NH
4nO
3be adjusted to 600mg/L.
Embodiment 3
A kind of method for building up of the kiwi fruit genetic transformation system based on heat stress
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 18d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature is 23 ~ 27 DEG C.
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process 10d as explant, remove blade tip and the leaf margin of blade, vertically be cut into the leaf dish of 0.6cm × 0.6cm as test explant along arteries and veins in blade, face of blade be upwards inoculated in callus and adventitious bud induction culture base and cultivate.
Callus and adventitious bud induction culture base are MS+2.2mg/LZT+0.7mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, and incubation time is 27d.
The healthy and strong indefinite bud growing to 2 ~ 3cm is inoculated in root media and cultivates.
Root media is 1/2MS+2.3mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, and incubation time is 30d.
MS in above-mentioned callus and adventitious bud induction culture base and the 1/2MS in described root media is and obtains the part mineral salt improvement in basal MS medium, is modified to and is divided into: KNO
3be adjusted to 750mg/L, NH
4nO
3be adjusted to 650mg/L.
Embodiment 4
A kind of method for building up of the kiwi fruit genetic transformation system based on heat stress
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 20d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature is 23 ~ 27 DEG C.
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process 10d as explant, remove blade tip and the leaf margin of blade, vertically be cut into the leaf dish of 0.5cm × 0.5cm as test explant along arteries and veins in blade, face of blade be upwards inoculated in callus and adventitious bud induction culture base and cultivate.
Callus and adventitious bud induction culture base are MS+2.0mg/LZT+0.6mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 14h/d, and incubation time is 26d.
The healthy and strong indefinite bud growing to 2 ~ 3cm is inoculated in root media and cultivates.
Root media is 1/2MS+2.5mg/LNAA;
Condition of culture is: cultivation temperature 23 ~ 27 DEG C, light application time 14h/d, and incubation time is 27d.
MS in above-mentioned callus and adventitious bud induction culture base and the 1/2MS in described root media is and obtains the part mineral salt improvement in basal MS medium, is modified to and is divided into: KNO
3be adjusted to 725mg/L, NH
4nO
3be adjusted to 625mg/L.
With the adventitious bud induction culture base of the callus filtered out or root media for minimal medium, add different quality concentration (0,5,10,15,20,30,40,50mg/L) kanamycin carry out sensitivity tests, the best screening concentration of measurement result to be 20mg/L kanamycin concentration be Chinese gooseberry volt ox 95-2 blade genetic transformation.
For each embodiment effect of the application is described, experimental example 1 ~ 2 is set to explain.
Wherein, the cultural method of comparative example 1 is see Jiangsu's agriculture science, the foundation of 2013,41 (6): 46-48. Chinese gooseberry genetic transformation systems.Concrete cultural method repeats no more.
Comparative example 2 is consistent with the heat stress processing method of the embodiment of the present application 4, but follow-up cultural method is consistent with comparative example 1.
Experimental example 1
Table 2 heat stress is on the impact of Chinese kiwi fruit leaf sheet callus and Differentiation ration of adventitious buds
Note: explant number × 100% of healing rate=the induce explant number/inoculation of callus;
Explant number/callus number × 100% that Differentiation ration of adventitious buds=induction is sprouted;
Comparative example 1 and comparative example 2, in cultivation 30d " Invest, Then Investigate " Callus formation rate, cultivate 40d " Invest, Then Investigate " callus Differentiation ration of adventitious buds;
Experimental example 1 ~ 4, in cultivation 20d " Invest, Then Investigate " Callus formation rate, cultivates 25d " Invest, Then Investigate " callus Differentiation ration of adventitious buds.
As the table shows, although the incubation time of embodiment 1 ~ 4 is shorter than comparative example 1 ~ 2, but the indefinite bud number that callus Differentiation ration of adventitious buds and each leaf dish are formed all has significance to raise (p<0.05), and known by embodiment 2, after heat shock, Chinese gooseberry Callus of Leaf and the demand of indefinite bud to the suitableeest plant growth regulator also there occurs change.
Experimental example 2
Table 3 heat stress affects the root hydraulic conductivity of Chinese gooseberry indefinite bud
| Grouping | NAA(mg/L) | Weight of root system (g) | Embedded depth (cm) | Divide number (individual) |
| Embodiment 1 | 2.0 | 14.69 | 35.1 | 15.2 |
| Embodiment 2 | 1.5 | 12.53 | 38.5 | 15.8 |
| Embodiment 3 | 2.2 | 13.62 | 36.6 | 13.6 |
| Embodiment 4 | 2.0 | 15.79 | 42.7 | 16.1 |
| Comparative example 1 | 1.0 | 6.10 | 26.2 | 8.9 |
| Comparative example 2 | 1.0 | 7.56 | 24.2 | 9.2 |
As shown above, after heat stress process, the weight of root system (root biomass) of embodiment 1 ~ 4, root system divide number, maximum embedded depth is all significantly higher than comparative example 1 (p<0.01, versus comparative example 1), and than comparative example 2 also high (embedded depth and point number p<0.01, weight of root system p<0.05, versus comparative example 1), this illustrates that the root hydraulic conductivity of Chinese gooseberry indefinite bud has had and significantly raises, and also there occurs change to the optimum concentration of NAA.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1., based on a method for building up for the kiwi fruit genetic transformation system of heat stress, it is characterized in that, comprising:
Using the blade of the plantlet in vitro of the Chinese gooseberry volt ox 95-2 after heat stress process as explant, described explant is treated as callus and carries out Fiber differentiation and the culture of rootage of indefinite bud.
2. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 1, is characterized in that, the leaf age of the blade of described plantlet in vitro is 28 ~ 32d.
3. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 2, is characterized in that, the concrete steps of described heat stress are:
Get the plantlet in vitro of healthy and strong Chinese gooseberry volt ox 95-2, when its leaf age is 18 ~ 22d, when 6:00 ~ 8:00 in morning, by described plantlet in vitro in 35 ~ 38 DEG C of thermal treatment 30 ~ 50min, during 6:00 ~ 8:00 next day, the same terms reprocessing once;
Except heat stress process, remaining cultivation temperature of described plantlet in vitro is 23 ~ 27 DEG C.
4. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 1, is characterized in that, explant is treated as callus and the concrete operations carrying out the Fiber differentiation of indefinite bud comprise:
Remove blade tip and the leaf margin of blade, vertically be cut into the leaf dish of (0.4 ~ 0.6cm) × (0.4 ~ 0.6cm) as test explant along arteries and veins in blade, face of blade be upwards inoculated in callus and adventitious bud induction culture base and cultivate.
5. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 4, is characterized in that:
Described callus and adventitious bud induction culture base are MS+ (1.5 ~ 2.2) mg/LZT+ (0.5 ~ 0.7) mg/LNAA.
6. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 5, is characterized in that, explant is treated as callus and the condition of culture carrying out the Fiber differentiation of indefinite bud is:
Cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, incubation time is 25 ~ 27d.
7. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 1, it is characterized in that, the concrete operations of described culture of rootage comprise: be inoculated in root media by the healthy and strong indefinite bud growing to 2 ~ 3cm and cultivate.
8. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 7, is characterized in that:
Described root media is 1/2MS+ (2.3 ~ 2.6) mg/LNAA.
9. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 8, is characterized in that, the condition of culture of described culture of rootage is:
Cultivation temperature 23 ~ 27 DEG C, light application time 13 ~ 15h/d, incubation time is 25 ~ 30d.
10. the method for building up of the kiwi fruit genetic transformation system based on heat stress according to claim 5 or 9, it is characterized in that, MS in described callus and adventitious bud induction culture base and the 1/2MS in described root media is and obtains the part mineral salt improvement in basal MS medium, is modified to and is divided into: KNO
3be adjusted to 700 ~ 750mg/L, NH
4nO
3be adjusted to 600 ~ 650mg/L.
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| CN109392714A (en) * | 2018-11-22 | 2019-03-01 | 中国科学院武汉植物园 | A kind of tissue culture and rapid propagation method of Kiwi berry gold plum |
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| CN103548680A (en) * | 2013-10-30 | 2014-02-05 | 西南林业大学 | Tissue culture and rapid propagation method for actinidia chinensis |
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