CN101347100B - Method for plant tissue culture by propagation with root - Google Patents
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Abstract
本发明公开了一种带根增殖的植物组织培养方法，该方法不同于常规的组织培养方法，常规的组织培养方法是把经过灭菌处理的外植体按启动培养、增殖培养、壮苗培养和生根培养的程序进行，缺点是增殖缓慢，生根率较低，而且根长较小。而本发明是把经过启动培养的外植体，在增殖培养和壮苗培养一定时间后就进行生根培养，然后进行带根增殖。这样的带根增殖，由于根主动可以进行吸收营养，因而增殖和壮苗过程营养吸收要远远优于常规植物组织培养方法。本发明所提供的新方法从技术、经济角度来讲其效果均非常明显。The invention discloses a method for plant tissue culture with root proliferation. The method is different from the conventional tissue culture method. The conventional tissue culture method is to start culture, proliferate culture and strong seedling culture of sterilized explants. It is carried out with the procedure of rooting culture, and the disadvantage is that the proliferation is slow, the rooting rate is low, and the root length is small. And the present invention just carries out the rooting culture after the explant of starting culture is cultured in proliferation and strong seedlings for a certain period of time, and then carries out the proliferation with roots. Such rooted proliferation, because the roots can actively absorb nutrients, the nutrient absorption in the process of proliferation and seedling growth is far superior to conventional plant tissue culture methods. The effect of the new method provided by the invention is very obvious in terms of technology and economy.
Description
Technical field
The present invention relates to a kind of method of Plant Tissue Breeding, particularly a kind of new method of the Plant Tissue Breeding explant propagation with root propagation.
Background technology
Along with the deep development of China's reform and opening-up, Party and government further pay attention to scientific research, and China constantly has the strain of new breeding and rare plant to occur at aspects such as forest plants, commodity trees, ornamental plantss.Will be these plant breedings and the rapid expanding propagation of rare strain, satisfy and produce needs, tissue culture is as one of asexual fast numerous important means of plant breeding, China has set up the group training and has bred the base from country to local and many companies in recent years, and many plant breedings and rare strain are here bred rapidly.So method for tissue culture is constantly studied and improved is very necessary, further satisfies and produce actual needs.
Conventional tissue culture procedures is: under aseptic condition, explant through sterilization treatment carries out the bud inducing culture on the bud inducing culture, and then transfer on proliferated culture medium, carry out enrichment culture, after propagation quantity reaches production requirement, transfer again and on the strong seedling culture base, carry out strong seedling culture, at last the no offspring under cutting on the explant of strong seedling culture is carried out culture of rootage on root media.In the tissue culture procedures of routine, enrichment culture is not produce under the situation of root at explant on the proliferated culture medium, breeds through subculture repeatedly, therefore, it is the osmosis of leaning on cell wall that the incubation explant absorbs nutrition, and the nutrient absorption effect is very limited.
Summary of the invention
Deficiency at above-mentioned prior art exists the objective of the invention is to, and a kind of method for plant tissue culture with root propagation is provided, and this method can improve tissue culture growth coefficient and bud seedling growing height effectively, and unrooted seedling rooting rate.This has important function to raising tissue culture benefit, reduction tissue culture cost.
To achieve these goals, the present invention takes following technical solution:
A kind of method for plant tissue culture with root propagation is characterized in that, specifically comprises the following steps:
1) selection group training plant explants is carried out sterilization treatment;
2), be inoculated in and induce sprouting on the inducing culture through the plant explants of sterilization treatment;
3) induce and sprout to certain hour, the enrichment culture on proliferated culture medium of transferring, the bud quantum count on proliferated culture medium is increased to specified quantity, is transferred on the strong seedling culture base again and cultivates, the bud seedling of strong seedling culture is long to specified altitude, promptly forms bud clump shape explant;
4) bud clump shape explant is transferred on root media, carry out culture of rootage, after bud clump shape explant bears root, transfer again and on proliferated culture medium, be with the root enrichment culture;
5) behind band root enrichment culture, when bud is bred to some, and bud height of seedling degree reaches when necessarily requiring, and carries out the cutting of unrooted seedling from bud clump body explant;
6) cutting no offspring down carries out culture of rootage according to the culture of rootage method of routine, bears whole plant of formation behind the root, can practice transplantation of seedlings;
7) the band root after the cutting of no offspring and the remainder of a small amount of bud is arranged continues band root enrichment culture on proliferated culture medium, treat that propagation reaches specified degree after, do not have the offspring cutting once more, the band root portion proceeds to cultivate propagation, successively repeatedly.
Principle of the present invention is, explant through the bud inducing culture, on propagation and strong seedling culture base, cultivate certain hour, when bud of breeding on the explant reaches specified quantity and bud height more than 2 centimetres the time, the switching of the explant of bud clump shape is cultivated on root media, after explant sends out roots, the explant of band root is transferred to again is with root propagation in the proliferated culture medium.Root absorbs nutrient component in the medium and belongs to active absorption, and root length in medium constantly increases, and lateral root is constantly a large amount of to be formed, and Root Distribution is in the various piece of medium.So band root explant is far superior to not be with the explant of root to the absorption of nutriment and other compositions.And propagation and strong sprout two processes be easy to be combined into a process.Its growth coefficient and aseptic seedling plant height are all greater than conventional enrichment procedure.
Band radical bud clump shape explant is cultivated certain hour in proliferated culture medium after, when the bud height of seedling degree of propagation reaches requirement, can from the cutting of gemmule clump shape explant down no offspring carry out culture of rootage, bear whole plant of formation behind the root, can practice transplantation of seedlings; The band root portion explant that will be left is then still cultivated in proliferated culture medium, carries out the enrichment culture of a new round, by the explant of this band root portion of transferring, can constantly carry out good explant propagation, can continue the no offspring of cutting and carry out culture of rootage.
In the inventive method, the no offspring that cuts from the band radical bud clump shape explant through enrichment culture carries out culture of rootage, the no offspring that its rooting rate and root are grown up and cultivated in routine.
Embodiment
The present invention is described in further detail below in conjunction with embodiment that the inventor provides.
The applicant is an example with long pretty pawpaw, tetraploid locust, raspberry etc., illustrates that the Plant Tissue Breeding new method of band root propagation of the present invention is compared the technique effect that brings with conventional enrichment procedure.
Embodiment 1: the tissue culture of long pretty pawpaw:
(Chaeomeles speciosa (sweet) Nakai C.Lagenearia (changjun) is the rose family (Rosaceae) to long pretty pawpaw, Chaenomeles (Chaenomeles lindi) plant.
1) material source and sterilization: pick up from grew in the 3 years germinating branch of pretty pawpaw plant of Shaanxi Yang Ling silver company of heap of stone nursery one strain.
With just adopting the germinating branch that comes, cut blade, after the germinating branch fully washes with running water,, then the germinating branch is cut into stem section about 10 centimetres again with liquid detergent water flushing three times.
On superclean bench,, take out the back and immerse 0.1% HgCl 30s in the ethanol of stem section immersion 75%
26min in the solution handles back aseptic water washing three times, again the stem section is cut into a plurality of segments, every segment band one bud.
2) segment of process sterilization treatment is seeded on the bud inducing culture and induces sprouting, and the prescription of bud inducing culture is: MS+6-BA (0.5mg/L)+IBA (0.05mg/L)+GA3 (0.5mg/L).
3) induce sprout 30d after, be transferred to enrichment culture on the proliferated culture medium again, the prescription of proliferated culture medium is: MS+6-BA (0.5mg/L)+IBA (0.01mg/L)+GA3 (0.01mg/L) bud is bred to more than 5-6, be transferred to again on the strong seedling culture base and cultivate, the prescription of strong seedling culture base is: MS+IBA (0.1mg/L)+GA3 (0.5mg/L)+0.1% active carbon, long through the bud seedling of strong seedling culture to certain altitude, become bud clump shape explant.
4) the part of some quantity bud clump shape explants of the cultivating culture of rootage on root media of transferring, the prescription of root media is: 1/2MS+NAA (3.0mg/L)+IBA (1.0mg/L); Another part continues to carry out proliferation and subculture and cultivates in contrast through cutting on proliferated culture medium.
5) in the aseptic seedling with root enrichment procedure and conventional enrichment procedure comparative trial, it is some respectively to select the similar seedling of size, writes down its plant height and bud number, puts into proliferated culture medium then, carries out enrichment culture.Do 3 repetitions, each repeats 20 bottles.The method of bud height of seedling degree and bud seedling number statistical is, every bottle of plant height is with the high computational mean value greater than the 1cm bud, and bud seedling number is only added up the bud that is higher than 0.5cm.Growth coefficient is the ratio of the every bottle of average bud number in propagation back with the preceding every bottle of average bud number of propagation.Observe the value-added coefficient and the plant height situation of two groups of aseptic seedling after one month.The value-added coefficient result is as shown in table 1:
The different enrichment procedures of table 1 are to the influence of aseptic seedling bud growth coefficient
Value-added coefficient to result of the test carries out statistical hypothesis test T=9.2018, and t
0.01(4)=4.604, the processing differences is extremely remarkable.Pass through band root enrichment procedure as can be seen from Table 1, average growth coefficient 3.5, and the commonsense method growth coefficient only is 2.0, illustrates with band root enrichment procedure very obvious to the proliferation function of bud.Plant height situation result such as table 2.
The different enrichment procedures of table 2 are to the influence of aseptic seedling plant height
Plant height to every group of aseptic seedling carries out variance analysis, the results are shown in Table 3:
The different enrichment procedures of table 3 are to the variance analysis of aseptic seedling plant height
| Source of variation | Sum of squares | Degree of freedom | All square | The F value | The p value |
| Between processing | 1.2150 | 1 | 1.2150 | 24.3000 | 0.0079 |
| In the processing | 0.2000 | 4 | 0.0500 | ||
| Total variation | 1.4150 | 5 |
Utilize the DPS6.55 statistical analysis software to carry out variance analysis (F=24.3000, p＜0.0079) to result of the test, it is extremely remarkable to handle differences.By as can be seen, be with total on average the increasing of aseptic seedling after root is bred to be 2.2cm.And total on average the increasing of the aseptic seedling after the commonsense method propagation only is 1.3cm, so band root enrichment procedure effect is significantly better than the enrichment procedure of routine.
6) from being with root propagation and not having offspring, cultivate and on root media, carry out culture of rootage with the simple bud of plant height more than 4 centimetres and two buds under the bud clump body cutting of root propagation.3 repetitions of every processing, 20 bottles of every repetitions.Experimental result such as table 4:
Table 4 band root enrichment procedure with not with the comparison sheet of root enrichment procedure unrooted seedling rooting rate
Band root enrichment procedure with not with the experimental result data of root enrichment procedure unrooted seedling rooting rate, analytical control by statistics, 1 bud seedling: U=1.7593, the one-sided quantile U of α=0.05
0.05=1.645, because U〉U
0.05So the rooting rate of being with root to increase method increases the rooting rate of method significantly greater than routine; 2 bud seedling: U=5.3049, the one-sided quantile U of α=0.01
0.01=1.96 because U〉U
0.01So the band root increases the extremely significant rooting rate that increases method greater than routine of rooting rate of method.Illustrate that the band root is bred and not with the unrooted seedling rooting under the cutting on the explant of root propagation, the former is significantly greater than the latter.The rooting efficiency that the no offspring of band root enrichment procedure is described is better than significantly is not with the root enrichment procedure not have offspring.
7) the bud clump shape explant of band root propagation, residue band root portion aseptic seedling behind the no offspring of cutting has the different seedling attitudes of simple bud, two bud, three buds and the shape of growing thickly, the aseptic seedling of these different seedling attitudes is transferred to continuation cultivation on the proliferated culture medium, after one month, observing its growth and propagation situation finds, the equal well-grown of the aseptic seedling of each seedling attitude, propagation and growing ability are stronger.
More than each incubation condition of culture be: the medium sucrose concentration is 30g/L, and agar concentration is 6g/L, and PH is transferred to 5.6-5.8.Cultivation temperature is about 25 ℃, and intensity of illumination is 1200-25001x, illumination every day 16h, and relative moisture is 60-80%.
Embodiment 2: tetraploid locust K
2Tissue culture:
Tetraploid locust K
2(Gigas type locust (2)) is Papilionaceae (Papilionaceae) robinia (Robinia pseudoacacia L) plant:
1) material source and sterilization: pick up from and draw tetraploid locust K from Beijing Forestry University
2The branch of annual grafting.
With just adopting the branch that comes, cut blade, after branch fully washes with running water,, then branch is cut into stem section about 10 centimetres again with liquid detergent water flushing three times.
On superclean bench,, take out the back and immerse 0.1% HgCl 30-60s in the ethanol of stem section immersion 75%
26min in the solution handles back aseptic water washing three times, again the stem section is cut into a plurality of segments, every segment band one bud.
2) segment of process sterilization treatment is seeded on the bud inducing culture and induces sprouting, and the prescription of bud inducing culture is: MS+6-BA (1.0mg/L)+GA3 (0.5mg/L).
3) induce sprout 30d after, be transferred on propagation and the strong seedling culture base again and breed and strong seedling culture, the prescription of propagation and strong seedling culture base is: MS+6-BA (1.0mg/L)+NAA (0.5mg/L+IBA (0.5mg/L) (the existing proliferation function of this medium has the effect in strong sprout again).Bud is bred to more than 5-6, and the bud seedling is long to certain altitude, becomes bud clump shape explant.
4) the part of some quantity bud clump shape explants of the cultivating culture of rootage on root media of transferring, the prescription of root media is: 1/2MS+6-BA (0.5mg/L)+IBA (0.5mg/L)+IAA (0.5mg/L); Another part continues to carry out proliferation and subculture on propagation strong seedling culture base and cultivates in contrast through cutting.
5) in the aseptic seedling with root enrichment procedure and conventional enrichment procedure comparative trial, it is some respectively to select the similar seedling of size, writes down its plant height and bud number, puts into propagation strong seedling culture base then, cultivates.Do 3 repetitions, each repeats 20 bottles.The method of bud height of seedling degree and bud seedling number statistical is, every bottle of plant height is with the high computational mean value greater than the 1cm bud, and bud seedling number is only added up the bud that is higher than 0.5cm.Growth coefficient is the ratio of the every bottle of average bud number in propagation back with the preceding every bottle of average bud number of propagation.Observe the value-added coefficient and the plant height situation of two groups of aseptic seedling after one month.The value-added coefficient result is as shown in table 5:
The different enrichment procedures of table 5 are to the influence of aseptic seedling bud growth coefficient
Value-added coefficient to result of the test carries out statistical hypothesis test T=7.5969, and t
0.01(4)=4.604, difference is extremely remarkable.The growth coefficient that band root propagation is described is better than conventional propagation extremely significantly, and is very obvious to the proliferation function of bud with band root enrichment procedure.
Plant height situation result such as table 6.
The different enrichment procedures of table 6 are to the influence of aseptic seedling plant height
The effect data that increases to the aseptic seedling of result of the test carries out statistical hypothesis test T=5.1180, and t
0.01(4)=4.604, difference is extremely remarkable.Illustrate that the aseptic seedling of being with root propagation increases effect and is better than conventional propagation extremely significantly, total on average the increasing of aseptic seedling after the band root is bred is 2.1cm.And total on average the increasing of the aseptic seedling after the commonsense method propagation only is 1.3cm, so band root enrichment procedure effect is significantly better than the enrichment procedure of routine.
6) from being with root propagation and not having offspring, cultivate and on root media, carry out culture of rootage with the simple bud of plant height more than 3 centimetres under the bud clump body cutting of root propagation.3 repetitions of every processing, 20 bottles of every repetitions.Experimental result such as table 7:
Table 7 band root enrichment procedure with not with the comparison sheet of root enrichment procedure unrooted seedling rooting rate
Band root enrichment procedure with not with the experimental result data of root enrichment procedure unrooted seedling rooting rate, analytical control by statistics, U=3.2826, U
0.01=2.576. band root propagation is not with extremely significant with difference between the unrooted seedling rooting rate under the cutting on the explant of root propagation, and the rooting efficiency that the no offspring of being with the root enrichment procedure is described is better than significantly is not with the root enrichment procedure not have offspring.
7) the bud clump shape explant of band root propagation, residue band root portion aseptic seedling behind the no offspring of cutting has the different seedling attitudes of simple bud, two bud, three buds and the shape of growing thickly, the aseptic seedling of these different seedling attitudes is transferred to continuation cultivation on the proliferated culture medium, after one month, observing its growth and propagation situation finds, the equal well-grown of the aseptic seedling of each seedling attitude, propagation and growing ability are stronger.
More than each incubation condition of culture be: the medium sucrose concentration is 30g/L (root media is 20g/L), and agar concentration is 6g/L, and PH is transferred to 5.6-5.8.Cultivation temperature is about 25 ℃, and intensity of illumination is 1200-25001x, illumination every day 16h, and relative moisture is 60-80%.
Embodiment 3: the tissue culture of raspberry:
Raspberry (Rubus idaeus) is the rose family (Rosaceae) rubus (Rubus) raspberry subgenus (Ideobatus) plant.
1) material source and sterilization: pick up from and draw from the branch of Chinese forest-science academy kind for 1 year living nursery stock of figure Latin America.
With just adopting the branch that comes, cut blade, after branch fully washes with running water,, then branch is cut into stem section about 5 centimetres again with liquid detergent flushing three times.
On superclean bench,, take out the back and immerse 0.1% HgCl 30s in the ethanol of stem section immersion 75%
25min in the solution handles back aseptic water washing three times, again the stem section is cut into a plurality of segments, every segment band one bud.
2) segment of process sterilization treatment is seeded on the bud inducing culture and induces sprouting, and the prescription of bud inducing culture is: MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA3 (0.5mg/L).
3) induce sprout 30d after, be transferred to again and carry out enrichment culture on the proliferated culture medium, the prescription of proliferated culture medium is: MS+6-BA (1.0mg/L)+IBA (0.1mg/L). bud is bred to more than 5-6, be transferred to again on the strong seedling culture base and cultivate, the prescription of strong seedling culture base is: MS+6-BA (1.0mg/L)+IBA (0.1mg/L)+GA3 (3.0mg/L), long through the bud seedling of strong seedling culture to certain altitude, become bud clump shape explant.
4) the part of some quantity bud clump shape explants of the cultivating culture of rootage on root media of transferring, the prescription of root media is: 1/2MS+IBA (0.1mg/L); Another part continues to carry out proliferation and subculture and cultivates in contrast through cutting on proliferated culture medium.
5) in the aseptic seedling with root enrichment procedure and conventional enrichment procedure comparative trial, it is some respectively to select the similar seedling of size, writes down its plant height and bud number, puts into proliferated culture medium then, cultivates.Do 3 repetitions, each repeats 20 bottles.The method of bud height of seedling degree and bud seedling number statistical is, every bottle of plant height is with the high computational mean value greater than the 1cm bud, and bud seedling number is only added up the bud that is higher than 0.5cm.Growth coefficient is the ratio of the every bottle of average bud number in propagation back with the preceding every bottle of average bud number of propagation.Observe the value-added coefficient and the plant height situation of two groups of aseptic seedling after one month.The value-added coefficient result is as shown in table 8:
The different enrichment procedures of table 8 are to the influence of aseptic seedling bud growth coefficient
Value-added coefficient to result of the test carries out statistical hypothesis test T=10.4103, and t
0.01(4)=4.604, difference is extremely remarkable.The growth coefficient that band root propagation is described is better than conventional propagation extremely significantly, and is very obvious to the proliferation function of bud with band root enrichment procedure.
Plant height situation result such as table 9.
The different enrichment procedures of table 9 are to the influence of aseptic seedling plant height
The effect data that increases to the aseptic seedling of result of the test carries out statistical hypothesis test T=8.5732, and t
0.01(4)=4.604, difference is extremely remarkable.Illustrate that the aseptic seedling of being with root propagation increases effect and is better than conventional propagation extremely significantly, total on average the increasing of aseptic seedling after the band root is bred is 2.0cm.And total on average the increasing of the aseptic seedling after the commonsense method propagation only is 1.3cm, so band root enrichment procedure effect is significantly better than the enrichment procedure of routine.
6) from being with root propagation and not having offspring, cultivate and on root media, carry out culture of rootage with the simple bud of plant height more than 2 centimetres under the bud clump body cutting of root propagation.3 repetitions of every processing, 20 bottles of every repetitions.Experimental result such as table 10:
Table 10 band root enrichment procedure with not with the comparison sheet of root enrichment procedure unrooted seedling rooting rate
Band root enrichment procedure with not with the experimental result data of root enrichment procedure unrooted seedling rooting rate, analytical control by statistics, U=2.6968, U
0.01=2.576. band root propagation is not with extremely significant with difference between the unrooted seedling rooting rate under the cutting on the explant of root propagation, and the rooting efficiency that the no offspring of being with the root enrichment procedure is described is better than significantly is not with the root enrichment procedure not have offspring.
7) the bud clump shape explant of band root propagation, residue band root portion aseptic seedling behind the no offspring of cutting has the different seedling attitudes of simple bud, two bud, three buds and the shape of growing thickly, the aseptic seedling of these different seedling attitudes is transferred to continuation cultivation on the proliferated culture medium, after one month, observing its growth and propagation situation finds, the equal well-grown of the aseptic seedling of each seedling attitude, propagation and growing ability are stronger.
More than each incubation condition of culture be: the medium sucrose concentration is 30g/L, and agar concentration is 6g/L, and PH is transferred to 5.6-5.8.Cultivation temperature is about 25 ℃, and intensity of illumination is 1200-25001x, illumination every day 16h, and relative moisture is 60-80%.
Need to prove that the present invention is the result that applicant's long-term experiment is explored, and is not limited to the foregoing description, can expand on the tissue culture of asexual other the numerous plant soon of plant breeding.According to method of the present invention, behind enrichment culture and strong seedling culture certain hour, just carry out culture of rootage, be with root propagation then.Such band root propagation because root initiatively absorbs nutrition, thereby propagation and strong sprout the process nutrient absorption to be far superior to the conventional plant method for tissue culture.All very obvious from technology, its effect of economic angle.
Claims (2)
1. the method for plant tissue culture with root propagation is characterized in that, specifically comprises the following steps:
1) selection group training plant explants is carried out sterilization treatment;
2), be inoculated in and induce sprouting on the inducing culture through the plant explants of sterilization treatment;
3) induce sprouting after 30 days, the enrichment culture on proliferated culture medium of transferring, when the bud quantum count on the proliferated culture medium is increased to more than 5～6, bud height of seedling degree reaches desired height, be transferred to again on the strong seedling culture base and cultivate, the bud seedling length of strong seedling culture promptly forms bud clump shape explant to more than the 2cm;
4) bud clump shape explant is transferred on root media, carry out culture of rootage, after bud clump shape explant bears root, transfer again and on proliferated culture medium, be with the root enrichment culture;
5) behind band root enrichment culture, when bud is bred to more than 9.7, and bud height of seedling degree reaches 3cm when above, carries out the cutting of unrooted seedling from bud clump shape explant;
6) cutting no offspring down carries out culture of rootage according to the culture of rootage method of routine, bears whole plant of formation behind the root, can practice transplantation of seedlings;
7) the band root after the cutting of no offspring and the remainder of a small amount of bud is arranged, on proliferated culture medium, continue band root enrichment culture, wait to breed the bud propagation quantity and bud height of seedling degree that reaches step 5) after, do not have offspring cutting once more, the band root portion proceeds to cultivate propagation, successively repeatedly.
2. the method for plant tissue culture of band root propagation as claimed in claim 1 is characterized in that, described sterilization treatment is with 30s in the ethanol of plant explants immersion 75%, takes out the back and immerses 0.1% HgCl
26min in the solution handles the back and gets final product for three times with aseptic water washing.
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| CN105918126B (en) * | 2016-05-19 | 2018-12-11 | 绍兴文理学院 | The rapid propagation in vitro method of Rubus chingii virus-elimination seedlings |
| CN110915652A (en) * | 2019-11-18 | 2020-03-27 | 三亚耀众农业发展有限公司 | Culture method and culture medium for non-transgenic pawpaw seedlings |
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| Title |
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| 孟强,董丽芬,邵崇斌.长俊木瓜组培苗叶片数及外源激素对生根影响的研究.西北林学院学报.2007,22(1),67-69. * |
| 郭有燕,陈明绪,董丽芬,贾彩霞,李胜奇.长俊木瓜芽增殖最适茎长的确定及壮苗培养研究.西北林学院学报.2007,22(6),72-73. * |
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