CN101422135A - Ciliate desert-grass regeneration method via green spherical body and special culture medium thereof - Google Patents
Ciliate desert-grass regeneration method via green spherical body and special culture medium thereof Download PDFInfo
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Abstract
The invention discloses a method for culturing the green spheroids of a pteris vittata and a special medium thereof. In the method, the rhizomes of ferns are used as explants and are cultured by a plant tissue medium to obtain the green spheroids; the plant tissue medium is a medium obtained by adding cane sugar with a final concentration of 18 to 22g/L, agar with a final concentration of 4 to 6g/L, GA with a final concentration of 0.3 to 0.7g/L and 6-BA with a final concentration of 0.4 to 0.6g/L into a 1/2MS-MS medium; wherein, the final concentration refers to the concentration in the plant tissue medium. Experiments show that the induction rate of the green spheroids obtained by the medium and the culturing method can achieve more than 70 percent. The differentiation rate of the green spheroids to carry out bud initiation can achieve 98 percent. Moreover, the method has the characteristics of short time, low cost and simple operation.
Description
Technical field
The present invention relates to method and the special culture media thereof of a kind of ciliate desert-grass through the green spheroids regeneration plant.
Background technology
Arsenic contamination has become a global main Environmental Problems (Meharg, 2004) in soil and the water.There is at present the tens of millions of people just suffering the threat (Smith et al., 1992) of chronic toxicity effect of soil, underground water and the various foods of arsenic contamination in the world.Pteridophyte ciliate desert-grass (Pteris vittata L) is in the news as first and has the plant of the super enrichment function of arsenic, can be applied to the phytoremediation (Ma et al., 2001) of As polluted soil.Yet up to the present, ciliate desert-grass is to the accumulation and the detoxifcation mechanism and unclear of arsenic, its major reason is that on the one hand the ciliate desert-grass sporophyte is the big and poky plant of a kind of genome, be difficult to it be studied, be that on the other hand the tissue culture plant regeneration of ciliate desert-grass and the system of genetic transformation do not set up as yet with genetic methods such as mutation inductions.
Can improve plant trait effectively and provide experimental system by setting up plant efficient tissue culture regenerating system and biological heredity method for transformation for studying the molecular mechanism that these proterties take place.For pteridophyte, more existing at present methods by the spore aftergrowth, studies show that, wherein used raw material (as having the leatherleaf of spore or the spore of separation) (Pierik et al., 1986), different surface disinfectant (as clorox, calcium hypochlorite, mercury chloride or hydrogen peroxide) (Fay, 1994), the physical state of mineral nutrition and illumination, pH, medium and plant growth regulator etc. all can influence the gametophytic growth of pteridophyte (Swami and Raghavan, 1980; Sheffield and Bell, 1987; Fern á ndez et al., 1997a, b, 1999).Most possessor utilizes the ciliate desert-grass spore to carry out quickly tissue culture and breeds to provide enough seedlings to be used for reparation (Chen Tongbin, 2007 that arsenic in soil pollutes; Breznovits and Mohay, 1987), but up to the present still do not set up a plant regeneration system that effectively is used for the ciliate desert-grass genetic transformation.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating the pteridophyte green spheroids.
The method of cultivation provided by the present invention pteridophyte green spheroids is that the root-like stock with pteridophyte is an explant, cultivates with plant tissue culture media, obtains green spheroids; Described plant tissue culture media is by add the medium that 6-BA that sucrose that final concentration is 18-22g/L, agar that final concentration is 4-6g/L, GA that final concentration is 0.3-0.7mg/L and final concentration be 0.4-0.6mg/L obtains in the 1/2MS-MS medium; Described final concentration is the concentration in described plant tissue culture media.
Described plant tissue culture media is preferably by add the medium that 6-BA that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L, GA that final concentration is 0.5mg/L and final concentration be 0.5mg/L obtains in the 1/2MS medium.
The pH value of described plant tissue culture media is 5.8-6.0.
Described culture condition is: temperature is that 23-27 ℃, photoperiod are that 14-16h illumination/10-8h dark, intensity of illumination are 2500-2300lux; Described temperature is preferably 25 ℃, and the described photoperiod is preferably 15h illumination/9h dark, and described intensity of illumination is preferably 2400lux.
Wherein, described root-like stock specifically can obtain by tissue culture.
The method of described tissue culture can be spore inoculating with described pteridophyte in the spore germination medium, cultivates and obtains described root-like stock.
Described spore germination medium is by add the medium that sucrose that final concentration is 10-40g/L, agar that final concentration is 3-7g/L obtain in the 1/8MS-MS medium; Described final concentration is the concentration in described spore germination medium; Described spore germination medium is preferably by add the medium that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L obtain in the 1/2MS medium.
Above-mentioned pteridophyte specifically can be ciliate desert-grass (Pteris vittata L).
Another object of the present invention provides a kind of plant tissue culture media.
Plant tissue culture media provided by the present invention is by add the medium that 6-BA that sucrose that final concentration is 18-22g/L, agar that final concentration is 4-6g/L, GA that final concentration is 0.3-0.7mg/L and final concentration be 0.4-0.6mg/L obtains in the 1/2MS-MS medium; Described final concentration is the concentration in described plant tissue culture media; Described plant tissue culture media is preferably in the 1/2MS medium and adds the medium that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L, GA that final concentration is 0.5mg/L, 6-BA that final concentration is 0.5mg/L obtain.
Described plant tissue culture media is adapted to inducing of pteridophyte green spheroids.
Another object of the present invention provides a kind of method of cultivating ciliate desert-grass, and promptly pteridophyte is through the method for green spheroids regeneration plant.
The method of cultivation ciliate desert-grass provided by the present invention is to cultivate with above-mentioned arbitrary described method to obtain the ciliate desert-grass green spheroids, more described green spheroids is cultivated, and obtains the ciliate desert-grass regeneration plant.Wherein, described green spheroids being carried out cultured method is that green spheroids is progressively carried out inducing of bud and root.
The present invention has set up a cover and has been explant, induces and obtain green spheroids, induce the cultivating system of green spheroids differentiation regeneration sporophyte again with ciliate desert-grass aseptic sporophyte root-like stock.Experiment showed, with medium of the present invention and cultural method and induce the inductivity that obtains green spheroids can reach more than 70% that the differentiation rate that green spheroids is carried out the bud differentiation can reach 98%; And the inventive method has the advantages that the time is short, cost is low, simple to operate.Therefore, can breed fast in a large number with the inventive method and to obtain green spheroids, for various Study on Genetic Transformation operations (as agrobacterium-mediated transformation, particle bombardment etc.) provide desirable material, for the molecular mechanism of studying ciliate desert-grass arsenic enrichment and detoxifcation provides desirable research system.The inventive method and medium are suitable in the tissue culture of ciliate desert-grass and also can be applicable in the breeding fast.
Description of drawings
Fig. 1 is the ciliate desert-grass green spheroids.
Fig. 2 is the ciliate desert-grass sporophyte with root and bud by green spheroids differentiation regeneration.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.Used material reagent all can be bought from market company like no specified otherwise.
Use MS, 1/2MS, 1,4MS, 1/8MS medium among the following embodiment; Wherein, among 1/2MS, 1/4MS, the 1/8MS concentration of each material be in the MS medium corresponding each material concentration 1/2,1/4,1/8, the pH of medium is transferred to 5.8-6.0 with Na0H or HCl.
The composition of table 1, MS medium and concentration (not containing sucrose and agar)
The plant regeneration of embodiment 1, ciliate desert-grass
One, the plant regeneration I of ciliate desert-grass
1, ciliate desert-grass spore germination and gametophyte, sporophyte are cultivated
From the greenhouse, gather ripe ciliate desert-grass (Pteris vittata L) spore 0.1g, in the centrifuge tube of 1.5ml, adopt 0.3%NaClO sterilization 10min, then 13, the centrifugal 5min of 000g/min, centrifugal back is collected the bottom spore and is adopted aseptic water washing 4 times, be suspended in the 0.5ml sterile water, be seeded in following spore germination and gametophyte, sporophyte medium then, 25 ℃, photoperiod be 15h illumination/9h dark, intensity of illumination is to cultivate under the condition of 2400lux.
Spore germination and gametophyte, sporophyte growth medium are composed as follows: 1/2 MS+20g/L sucrose+5g/L agar; The pH value is 5.9.
3 repetition are established in experiment, and average 18d spore begins to sprout, and cultivates the average every bottle of 6.15 ± 0.07g of gametophytic fresh weight (every bottle graft kind 0.1ml spore suspension) that produces behind the 45d, on average cultivate 70d after formation have rhizomatic sporophyte seedling.
2, green spheroids (Green globular bodies, inducing GGB)
Get the aseptic sporophyte root-like stock of the ciliate desert-grass that obtains in the step 1 as growing body outward;
Aseptic ciliate desert-grass sporophyte root-like stock is placed in the inducing culture, 25 ℃, photoperiod be 15h illumination/9 hour dark, intensity of illumination is to cultivate under the condition of 2400lux.Cultivate 50 altogether and grown body outward.
Inducing culture is composed as follows: 1/2 MS+20g/L sucrose+5g/L agar+0.5mg/L GA+0.5mg/L6-BA; The pH value is 5.9.
3 repetitions are established in experiment, cultivate after 20 days, and position, root-like stock top produces single green spheroids gradually, and green spheroids is the green hair of consolidation shape graininess (Fig. 1), and its structure is different with callus, and similar to the protocorm of orchid.Green spheroids constantly increases or constantly produces new green spheroids and a plurality of together intensive afterwards, and after 50 days, the green spheroids inductivity reaches maximum substantially, and average inductivity is 76%.Average inductivity=generation green spheroids is grown number/total outer body number X100% that grows of body outward.
2, bud induces
After cultivating ciliate desert-grass and producing green spheroids, green spheroids is transferred on the differential medium, 25 ℃, photoperiod be 15h illumination/9h dark, intensity of illumination is to cultivate under the condition of 2400lux.
Differential medium is composed as follows: 1/2 MS+20g/L sucrose+5g/L agar; The pH value is 5.9.
3 repetitions are established in experiment, and green spheroids begins to take place the differentiation of bud behind the average 30d, produces green point, and green subsequently point is upwards grown and produced blade, forms the unrooted seedling.Average differentiation rate is 98%.The number of the green spheroids of the number of the green spheroids of differentiation rate=generation bud/total.
3, root induces
From green spheroids induce form the unrooted seedling after, seedling is transferred in the root media, 25 ℃, photoperiod be 15h illumination/9h dark, intensity of illumination is to cultivate under the condition of 2400lux.
Root media is composed as follows: 1/2 MS+20g/L sucrose+5g/L agar; The pH value is 5.1.
3 repetitions are established in experiment, and the result shows, continues to cultivate 1～2 month, and along with the growth of sporophyte acrial part, under ground portion grows adventive root (Fig. 2) gradually.
Two, the plant regeneration II of ciliate desert-grass
1, ciliate desert-grass spore germination and gametophyte, sporophyte are cultivated
From the greenhouse, gather ripe ciliate desert-grass spore 0.1g, in the centrifuge tube of 1.5ml, adopt 0.3%NaCl0 sterilization 10min, then 13, the centrifugal 5min of 000g/min, centrifugal back is collected the bottom spore and is adopted aseptic water washing 4 times, be suspended in the 0.5ml sterile water, be seeded in then on the 1/8MS minimal medium of additional saccharose 10g/L, 23 ℃, photoperiod be 14h illumination/10h dark, intensity of illumination is to cultivate under the condition of 2300lux.
The spore germination medium is composed as follows: 1/8MS+10g/L sucrose+3g/L agar; The pH value is 5.8.
3 repetition are established in experiment, and average 30d spore begins to sprout, and cultivates average every bottle 4.35 ± 0.12 of the gametophytic fresh weight (every bottle graft kind 0.1ml spore suspension) that produces behind the 45d, on average cultivate 70d after formation have rhizomatic sporophyte seedling.
2, green spheroids induces
Get the aseptic sporophyte root-like stock of ciliate desert-grass as growing body outward;
Aseptic ciliate desert-grass sporophyte root-like stock is placed in the inducing culture, 23 ℃, photoperiod be 14h illumination/10 hour dark, intensity of illumination is to cultivate under the condition of 2300lux.Cultivate 50 altogether and grown body outward.
Inducing culture is composed as follows: 1/2 MS+18g/L sucrose+4g/L agar+0.3mg/L GA+0.4mg/L6-BA; The pH value is 5.8.
3 repetitions are established in experiment, cultivate after 20 days, and position, root-like stock top produces single green spheroids gradually, and green spheroids is the green hair of consolidation shape graininess, and its structure is different with callus, and similar to the protocorm of orchid.Green spheroids constantly increases or constantly produces new green spheroids and a plurality of together intensive afterwards, and after 50 days, the green spheroids inductivity reaches maximum substantially, and average inductivity is 46%.Average inductivity=generation green spheroids is grown number/total outer body number X100% that grows of body outward.
2, bud induces
After cultivating ciliate desert-grass and producing green spheroids, green spheroids is transferred on the differential medium, 23 ℃, photoperiod be 14h illumination/10h dark, intensity of illumination is to cultivate under the condition of 2300lux.
Differential medium is composed as follows: 1/2 MS+18g/L sucrose+4g/L agar; The pH value is 5.8.
3 repetitions are established in experiment, and green spheroids begins to take place the differentiation of bud behind the average 30d, produces green point, and green subsequently point is upwards grown and produced blade, forms the unrooted seedling.Average differentiation rate is 98%.The number of the green spheroids of the number of the green spheroids of differentiation rate=generation bud/total.
3, root induces
From green spheroids induce form the unrooted seedling after, seedling is transferred in the root media, 23 ℃, photoperiod be 14h illumination/10h dark, intensity of illumination is to cultivate under the condition of 2300lux.
Root media is composed as follows: 1/2 MS+18g/L sucrose+4g/L agar; The pH value is 5.0.
3 repetitions are established in experiment, and the result shows, continues to cultivate 1～2 month, and along with the growth of sporophyte acrial part, under ground portion grows adventive root gradually.
Three, the plant regeneration III of ciliate desert-grass
1, ciliate desert-grass spore germination and gametophyte, sporophyte are cultivated
From the greenhouse, gather ripe ciliate desert-grass spore 0.1g, in the centrifuge tube of 1.5ml, adopt 0.3%NaCl0 sterilization 10min, then 13, the centrifugal 5min of 000g/min, centrifugal back is collected the bottom spore and is adopted aseptic water washing 4 times, be suspended in the 0.5ml sterile water, be seeded in then on the MS minimal medium of additional saccharose 40g/L, 27 ℃, photoperiod be 16h illumination/8h dark, intensity of illumination is to cultivate under the condition of 2500lux.
The spore germination medium is composed as follows: MS+40g/L sucrose+7g/L agar; The pH value is 6.0.
3 repetition are established in experiment, and average 30d spore begins to sprout, and cultivates average every bottle 4.75 ± 0.15 of the gametophytic fresh weight (every bottle graft kind 0.1ml spore suspension) that produces behind the 45d, on average cultivate 55d after formation have rhizomatic sporophyte seedling.
2, green spheroids induces
Get the aseptic sporophyte root-like stock of ciliate desert-grass as growing body outward;
Aseptic ciliate desert-grass sporophyte root-like stock is placed in the inducing culture, 27 ℃, photoperiod be 16h illumination/8 hour dark, intensity of illumination is to cultivate under the condition of 2500lux.Cultivate 50 altogether and grown body outward.
Inducing culture is composed as follows: MS+22g/L sucrose+6g/L agar+0.7mg/L GA+0.6mg/L6-BA; The pH value is 6.0.
3 repetitions are established in experiment, cultivate after 20 days, and position, root-like stock top produces single green spheroids gradually, and green spheroids is the green hair of consolidation shape graininess, and its structure is different with callus, and similar to the protocorm of orchid.Green spheroids constantly increases or constantly produces new green spheroids and a plurality of together intensive afterwards, and after 50 days, the green spheroids inductivity reaches maximum substantially, and average inductivity is 63%.Average inductivity=generation green spheroids is grown number/total outer body number X100% that grows of body outward.
2, bud induces
After cultivating ciliate desert-grass and producing green spheroids, green spheroids is transferred on the differential medium, 27 ℃, photoperiod be 16h illumination/8h dark, intensity of illumination is to cultivate under the condition of 2500lux.
Differential medium is composed as follows: MS+22g/L sucrose+6g/L agar; The pH value is 6.0.
3 repetitions are established in experiment, and green spheroids begins to take place the differentiation of bud behind the average 30d, produces green point, and green subsequently point is upwards grown and produced blade, forms the unrooted seedling.Average differentiation rate is 80%.The number of the green spheroids of the number of the green spheroids of differentiation rate=generation bud/total.
3, root induces
From green spheroids induce form the unrooted seedling after, seedling is transferred in the root media, 27 ℃, photoperiod be 16h illumination/8h dark, intensity of illumination is to cultivate under the condition of 2500lux.
Root media is composed as follows: MS+22g/L sucrose+6g/L agar; The pH value is 5.0.
3 repetitions are established in experiment, and the result shows, continues to cultivate 1～2 month, and along with the growth of sporophyte acrial part, under ground portion grows adventive root gradually.
Claims (10)
1, a kind of method of cultivating the pteridophyte green spheroids is that the root-like stock with pteridophyte is an explant, cultivates with plant tissue culture media, obtains green spheroids; Described plant tissue culture media is by add the medium that 6-BA that sucrose that final concentration is 18-22g/L, agar that final concentration is 4-6g/L, GA that final concentration is 0.3-0.7mg/L and final concentration be 0.4-0.6mg/L obtains in the 1/2MS-MS medium; Described final concentration is the concentration in described plant tissue culture media.
2, method according to claim 1 is characterized in that: described plant tissue culture media is by add the medium that 6-BA that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L, GA that final concentration is 0.5mg/L and final concentration be 0.5mg/L obtains in the 1/2MS medium.
3, method according to claim 1 and 2 is characterized in that: described culture condition is: temperature is that 23-27 ℃, photoperiod are that 14-16h illumination/10-8h dark, intensity of illumination are 2500-2300lux; Described temperature is preferably 25 ℃, and the described photoperiod is preferably 15h illumination/9h dark, and described intensity of illumination is preferably 2400lux.
4, according to claim 1,2 or 3 described methods, it is characterized in that: described root-like stock obtains by tissue culture.
5, method according to claim 4 is characterized in that: the method for described tissue culture for the spore inoculating of described pteridophyte in spore germination and gametophyte, sporophyte growth medium, cultivate and obtain described root-like stock;
Described spore germination and gametophyte, sporophyte growth medium are by add the medium that sucrose that final concentration is 10-40g/L, agar that final concentration is 3-7g/L obtain in the 1/8MS-MS medium; Described final concentration is the concentration in described spore germination and gametophyte, sporophyte growth medium.
6, method according to claim 5 is characterized in that: described spore germination and gametophyte, sporophyte growth medium are by add the medium that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L obtain in the 1/2MS medium.
7, according to arbitrary described method among the claim 1-6, it is characterized in that: described pteridophyte is ciliate desert-grass (Pteris vittataL).
8, a kind of plant tissue culture media is by add the medium that 6-BA that sucrose that final concentration is 18-22g/L, agar that final concentration is 4-6g/L, GA that final concentration is 0.3-0.7mg/L and final concentration be 0.4-0.6mg/L obtains in the 1/2MS-MS medium; Described final concentration is the concentration in described plant tissue culture media.
9, plant tissue culture media according to claim 8 is characterized in that: described plant tissue culture media is to add the medium that sucrose that final concentration is 20g/L, agar that final concentration is 5g/L, GA that final concentration is 0.5mg/L, 6-BA that final concentration is 0.5mg/L obtain in the 1/2MS medium.
10, a kind of method of cultivating ciliate desert-grass is to cultivate with arbitrary described method among the claim 1-7 to obtain the ciliate desert-grass green spheroids, more described green spheroids is cultivated, and obtains ciliate desert-grass.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101828528A (en) * | 2010-06-11 | 2010-09-15 | 江苏省农业科学院 | Tissue culture and quick propagation method suitable for pteridophyte scarecrow |
| CN104206280A (en) * | 2014-09-21 | 2014-12-17 | 云南省农业科学院花卉研究所 | Tissue culture and rapid propagation method of hemionitis arifolia seedlings by virtue of green spherical body manner |
| CN104206279A (en) * | 2014-09-21 | 2014-12-17 | 云南集创园艺科技有限公司 | Induction medium for green spherical bodies of hemionitis arifolia |
| CN104782484A (en) * | 2015-04-13 | 2015-07-22 | 云南省农业科学院生物技术与种质资源研究所 | Method for obtaining tissue culture plantlets fast by utilizing homogenate of stems and leaves of hydrilla verticillata |
| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN108094206A (en) * | 2017-12-25 | 2018-06-01 | 绵阳师范学院 | A kind of cultural method of cystoathyrium chinense sporinite breeding |
| CN108575714A (en) * | 2018-05-17 | 2018-09-28 | 湖南省农业环境生态研究所 | A kind of ciliate desert-grass spore germination and intensive seedling production method |
| CN115623987A (en) * | 2022-12-01 | 2023-01-20 | 贵州师范大学 | Method for inducing green spheroid approach plant tissue culture by cyathea spores |
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| CN100586274C (en) * | 2005-10-27 | 2010-02-03 | 中国科学院地理科学与资源研究所 | Method for quickly tissue culture reproducing using ciliate desert-grass spore |
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| CN101828528A (en) * | 2010-06-11 | 2010-09-15 | 江苏省农业科学院 | Tissue culture and quick propagation method suitable for pteridophyte scarecrow |
| CN104206280A (en) * | 2014-09-21 | 2014-12-17 | 云南省农业科学院花卉研究所 | Tissue culture and rapid propagation method of hemionitis arifolia seedlings by virtue of green spherical body manner |
| CN104206279A (en) * | 2014-09-21 | 2014-12-17 | 云南集创园艺科技有限公司 | Induction medium for green spherical bodies of hemionitis arifolia |
| CN104782484A (en) * | 2015-04-13 | 2015-07-22 | 云南省农业科学院生物技术与种质资源研究所 | Method for obtaining tissue culture plantlets fast by utilizing homogenate of stems and leaves of hydrilla verticillata |
| CN104938336A (en) * | 2015-06-11 | 2015-09-30 | 仲恺农业工程学院 | Rapid propagation method for promoting germination of aquilaria sinensis isolated bud |
| CN108094206A (en) * | 2017-12-25 | 2018-06-01 | 绵阳师范学院 | A kind of cultural method of cystoathyrium chinense sporinite breeding |
| CN108575714A (en) * | 2018-05-17 | 2018-09-28 | 湖南省农业环境生态研究所 | A kind of ciliate desert-grass spore germination and intensive seedling production method |
| CN108575714B (en) * | 2018-05-17 | 2023-01-06 | 湖南省农业环境生态研究所 | Centipede spore germination and intensive seedling raising method |
| CN115623987A (en) * | 2022-12-01 | 2023-01-20 | 贵州师范大学 | Method for inducing green spheroid approach plant tissue culture by cyathea spores |
| CN115623987B (en) * | 2022-12-01 | 2023-07-21 | 贵州师范大学 | Cyathea spores induced green spheroid plant tissue culture method |
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