CN103070078A - Rapid propagation method for performing tissue culture by using taro stem tip - Google Patents
Rapid propagation method for performing tissue culture by using taro stem tip Download PDFInfo
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- CN103070078A CN103070078A CN2013100478235A CN201310047823A CN103070078A CN 103070078 A CN103070078 A CN 103070078A CN 2013100478235 A CN2013100478235 A CN 2013100478235A CN 201310047823 A CN201310047823 A CN 201310047823A CN 103070078 A CN103070078 A CN 103070078A
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Abstract
The invention discloses a rapid propagation method for performing tissue culture by using a taro stem tip, belonging to the technical field of biology. The method comprises the following steps of: selecting an aseptic taro stem tip as an explant; and performing a series of operating steps such as bud induction, subculture multiplication, rooting culture and the like to form a complete test-tube plantlet. The method comprises treatment methods for culture medium components at each abovementioned stage and induction culture. A seedling with two or more leaves can grow by performing primary culture on the taro stem tip for about 20 days, so that the process of primary culture is accelerated greatly; the growth coefficient is 4.3 on average after one-time secondary culture, secondary culture can be performed for five times generally to culture 1,470 (4.35) seedlings, and two batches can be cultured every year to form 2,940 tissue culture seed tuber seedlings, so that large-scale commercial production of taros is facilitated; and the method is also easy for obtaining regeneration plants with high genetic stability and low aberration rates.
Description
Technical field
The present invention's " a kind of quick-breeding method that utilizes the taro stem apex to organize cultivation " is to be cultivated by taro stem apex in vitro tissue to obtain test-tube plantlet, belongs to biological technical field.
Background technology
Jiangsu Province has more local characteristic taro kind, has preferably nutrition and health care quality, and contains multivitamin, both can work as grain, can do vegetables again, is suitable to people of all ages invigorant, and processing prospect is wide, the well-known and market that enjoys a good market both at home and abroad.At present, the production of Jiangsu Province local characteristic taro normally adopts the masses to breed from the mode of reserving seed for planting, and kind sexual involution problem is comparatively outstanding, and disease resistance and resistance go down.And, domestic to its group culturation rapid propagating technology aspect research seldom, therefore carry out Jiangsu Province's local characteristic taro tissue culture regeneration system technical research, not only be conducive to the rejuvenating of kind, more plant seedling industrialized production and breeding of new variety technical support and deposit are provided.At present, the taro Study on tissue culture all has report both at home and abroad, but is research contents mainly with the stem apex evoked callus, is unfavorable for keeping the stability of kind.The present invention utilizes the stem apex isolated condition to obtain test-tube plantlet, is easy to keep Different Varieties, can obviously improve reproduction coefficient and reproduction speed simultaneously, has accelerated fast numerous process, has kept the good characteristic of kind, has effectively protected the germ plasm resource with local characteristic.
Summary of the invention
Technical problem
The objective of the invention is to find and to improve taro reproduction coefficient and reproduction speed, the method that can keep again this variety stability, the method has the reproduction coefficient height, reproduction speed is fast, reproductive efficiency is high, seedling virus is few, the advantages such as the good aberration rate of genetic stability is low, can remedy the shortcoming that existing modes of reproduction cost is high, coefficient is low, callus induction group training seedling is loaded down with trivial details, the time is long, aberration rate is high, thereby provide a kind of new method for the taro seeding propagation.
Technical scheme
1, a kind of quick-breeding method that utilizes the taro stem apex to organize cultivation may further comprise the steps:
(1) preparation of explant: choose intact, without the taro of the spot that rots, the K through 0.2%
2MnO
4Sterilization fitly is arranged in the Turnover Box behind the 5min, covers 2/3 place of whole taro piece with sand, is placed into and treats its sprouting in the illumination box.
(2) sterilization of explant: when bud grows into 1-3cm, cut stem apex, divest the outer layer 2-3 blade of stem apex, about circulating water flushing 10min, at super-clean bench with 75% alcohol-pickled 30s, with the 84 thimerosals 10-15min that sterilizes, rinsed with sterile water 5-7 time.
(3) spore induction differentiation: explant is seeded in interpolation TDZ concentration after sterilization be in the minimal medium of 0.5mg/L, and 20d can grow up to two or two seedling that blade is above.
(4) shoot proliferation: it is long time 2.0cm to sprout the indefinite bud that on the inducing culture, and being seeded in interpolation TDZ concentration is in the minimal medium of 1.0mg/L, general continuous subculture 3-5 time.
(5) culture of rootage: with more than high 2.0 cm, downcut with the healthy and strong seedling of 2 leaves, change on the root media and cultivate, the culture of rootage based component is 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 0.5mg/L; Cultivate and to form complete group training seedling about 30d.
(6) condition of culture: culturing room's temperature is 25 ± 2 ℃, intensity of illumination 1200-1500lx, illumination every day 12h.Minimal medium is to add agar 7 g/L among the MS, adds sucrose 30g/L.
Wherein the MS medium is the M0222.0050 medium.
Beneficial effect
The method that the present invention utilizes tissue to cultivate for the first time solves the fast numerous problem of taro kind taro with Jiangsu Local characteristic.
The present invention utilizes taro stem apex in vitro to obtain test-tube plantlet, is conducive to realize taro sapling multiplication batch production production.
The present invention has the reproduction coefficient height, reproduction speed is fast, reproductive efficiency is high, the characteristics that seedling virus less, genetic stability is good.The present invention utilize the taro stem apex just culture can grow up to two or two seedling that blade is above about 20 days, greatly improved the process of first culture, subculture is cultivated the one-step growth coefficient and is on average reached 4.3, generally continuous subculture 5 times is cultivated 1470 young plants (4.3
5=1470), can cultivate 2 batches in 1 year, the formation group is cultivated taro 2940 strains, is conducive to the taro large-scale commercial production; The method also has and is easy to obtain the low regeneration plant of the good aberration rate of genetic stability.
Description of drawings
Accompanying drawing 1 is culture just;
Accompanying drawing 2 shoot proliferations;
Accompanying drawing 3 culture of rootage;
4 groups of trainings of accompanying drawing seedling.
Embodiment
Further specify the present invention below by obtaining test-tube plantlet based on the significant agricultural product Jingjiang of Chinese geography Xiangsha dasheen stem apex in vitro.
1 materials and methods
1.1 test material
Adopt potted plant taro, selecting stem apex is explant.
1.2 method
1.2.1 the processing of sterilizable material
Choose intact, without the taro of the spot that rots, the K through 0.2%
2MnO
4Sterilization fitly is arranged in the Turnover Box behind the 5min, covers 2/3 place of whole taro piece with sand, is placed into and treats its sprouting in the illumination box.Treat to cut stem apex when bud grows into 1-3cm, divest outer layer 2-3 blade, about circulating water flushing 10min, at super-clean bench with 75% alcohol-pickled 30s, process sterilization with 3 time periods of two kinds of disinfectants respectively, every kind of processing is inoculated respectively 12 stem apexs in minimal medium, observes bactericidal effect.Minimal medium is to add agar 7g/L and sucrose 30g/L among the MS.MS is M0222.0050 medium (Dutch Duchefa Biochemie company).
1. use that 84 thimerosals (Jiangsu Ai Tefu aerosol Co., Ltd) soak respectively 10,15,20min, relatively its pollution rate;
2. with 5% NaClO soak respectively 5,10,15min, relatively its pollution rate.
1.2.2 bud is induced differentiation
Explant is seeded in 6-BA and NAA combination and the TDZ(Thidiazuron that adds variable concentrations after sterilization) and the IBA(indolebutyric acid) in the minimal medium of combination, the definite suitable medium of differentiation rate, bud height and the number of blade etc. of bud relatively behind the inoculation 30d.Hormone concentration arranges 7 processing, and each is processed and respectively inoculates 15 buds.
1. minimal medium+6-BA 0.5mg/L+NAA 0.2mg/L;
2. minimal medium+6-BA 1.0mg/L+NAA 0.2mg/L;
3. minimal medium+6-BA 2.0mg/L+NAA 0.2mg/L;
4. minimal medium+6-BA 4.0mg/L+NAA 0.2mg/L;
5. minimal medium+TDZ 0.5mg/L;
6. minimal medium+TDZ 1.0mg/L;
7. minimal medium+TDZ 1.0mg/L+IBA 0.4mg/L.
1.2.3 shoot proliferation
Sprout on the inducing culture in the long minimal medium that is seeded in the 6-BA that adds variable concentrations and NAA combination and TDZ after certain degree of the sprout that and observe its propagation multiple.Hormone concentration arranges 3 processing, and each is processed and respectively inoculates 20 simple buds.
1. minimal medium+6-BA 2.0mg/L+NAA 0.2mg/L;
2. minimal medium+TDZ 0.5mg/L;
3. minimal medium+TDZ 1.0mg/L.
1.2.4 culture of rootage
With more than high 2.0 cm, with the healthy and strong seedling cutting-out of 2 leaves, change on the root media and cultivate.The fate of on average taking root of investigation different disposal, the number of taking root of 30d, rooting rate, total leaf number, root system average length and Root morphology.Root media arranges 4 processing, and each is processed and respectively inoculates 15 seedlings.
1. 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 0.5mg/L;
2. 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 1.0mg/L;
3. 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 2.0mg/L;
4. 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 4.0mg/L.
1.2.5 medium sterilization and condition of culture
Sodium hydroxide solution with concentration 0.1mol/L before the autoclaving is adjusted Medium's PH Value to 5.8, keeps 121 ℃ of sterilization 20min.Culturing room's temperature is 25 ± 2 ℃, intensity of illumination 1200-1500lx, illumination every day 12h/d.
2 results and analysis
2.1 different thimerosals and sterilization time are on the impact of explant sterilization effect
As can be seen from Table 1, explant is best with 84 thimerosals sterilization 10-15min effect, and the sterilization success rate reaches more than 83.3%.The NaClO success rate of sterilizing is well below 84 thimerosals sterilization success rate.Therefore, the sterilizing methods of explant is: cut stem apex when bud grows into 1-3cm, divest the outer layer 2-3 blade of stem apex, about circulating water flushing 10min, at super-clean bench with 75% alcohol-pickled 30s, with 84 thimerosals sterilization 10-15min, rinsed with sterile water 5-7 time.
The different thimerosals of table 1 and sterilization time are on the impact of explant sterilization effect
2.2 bud is induced differentiation
Can find out that from table 2 select minimal medium+TDZ 0.5mg/L and minimal medium+TDZ 1.0mg/L medium that the Xiangsha dasheen explant is induced cultivation, spore induction rate and bud growing way are better than other several medium.Wherein the highest with minimal medium+TDZ 0.5mg/L medium inductivity, inductivity is 80%, and the bud plant height of cultivating after 30 days is 2.3cm, and launching the number of blade is 3.Therefore, the method for spore induction differentiation is: explant is seeded in to add in the minimal medium that TDZ concentration is 0.5mg/L after sterilization and cultivates.
Table 2 different culture media is on the impact of explant induction
2.3 shoot proliferation
Can be found out that by table 3 inducing adventitious buds proliferation optimum medium is minimal medium+TDZ 1.0mg/L, differentiation speed is fast, 4.3 times of propagation multiple average out to.Therefore the subculture method of cultivating is: sprout on the inducing culture indefinite bud length that to about the 2.0cm time, be seeded in to add in the minimal medium that TDZ concentration is 1.0mg/L and cultivate.General continuous subculture is no more than 5 times.
Table 3 hormon concentration is on the impact of explant propagation
2.4 culture of rootage
Can be found out that by table 4 impact that hormon concentration is taken root on Xiangsha dasheen is larger.Hormone concentration and media is 6-BA 0.1mg/L+NAA 0.5mg/L, and rooting efficiency is best, and the fate of on average taking root is short, and rooting rate reaches the root length of 100%, 30d, and blade amt and height of seedling are the highest, and root system is long and thick.Along with NAA concentration increases, root length, the number of blade and height of seedling have decline in various degree, and the root system chap that shortens gradually finally is the shank shape.Therefore the method for culture of rootage is: with more than high 2.0 cm, with the healthy and strong seedling cutting-out of 2 leaves, change on the root media and cultivate.The culture of rootage based component is 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 0.5mg/L; Cultivate and to form complete group training seedling about 30d.
The impact of table 4 hormon concentration on taking root
The present invention utilize the taro stem apex just culture can grow up to two or two seedling that blade is above about 20 days, greatly improved the process of first culture, subculture is cultivated the one-step growth coefficient and is on average reached 4.3, generally continuous subculture 5 times is cultivated 1470 young plants (4.3
5=1470), can cultivate 2 batches in 1 year, the formation group is cultivated taro 2940 strains, is conducive to the taro large-scale commercial production; The method also has and is easy to obtain the low regeneration plant of the good aberration rate of genetic stability.
Claims (2)
1. quick-breeding method that utilizes the taro stem apex to organize cultivation may further comprise the steps:
(1) preparation of explant: choose intact, without the taro of the spot that rots, through the K of mass ratio 0.2%
2MnO
4Sterilization 5min covers 2/3 place of whole taro piece with sand, is placed into and treats its sprouting in the illumination box;
(2) sterilization of explant: when bud grows into 1-3cm, cut stem apex, divest the outer layer 2-3 blade of stem apex, circulating water flushing 10min, at super-clean bench with 75% alcohol-pickled 30s, with the 84 thimerosals 10-15min that sterilizes, rinsed with sterile water 5-7 time;
(3) spore induction differentiation: explant is seeded in interpolation TDZ concentration after sterilization be in the minimal medium of 0.5mg/L, and 20d namely grows up to two or two seedling that blade is above;
(4) shoot proliferation: it is long time 2.0cm to sprout the indefinite bud that on the inducing culture, and being seeded in interpolation TDZ concentration is in the minimal medium of 1.0mg/L, general continuous subculture 3-5 time;
(5) culture of rootage: with more than high 2.0 cm, downcut with the healthy and strong seedling of 2 leaves, change on the root media and cultivate, the culture of rootage based component is 1/2MS+ agar 7g/L+ sucrose 30g/L+ active carbon 0.2g/L+6-BA 0.1mg/L+NAA 0.5mg/L;
The above-mentioned steps condition of culture: culturing room's temperature is 25 ± 2 ℃, and intensity of illumination 1200-1500lx, illumination every day 12h, minimal medium add agar 7g/L, sucrose 30g/L among the MS.
2. a kind of quick-breeding method that utilizes the taro stem apex to organize cultivation according to claim 1, wherein MS is the M0222.0050 medium.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103931504A (en) * | 2014-05-13 | 2014-07-23 | 江苏省农业科学院 | Rapid purification and rejuvenation method for local varieties of taros |
| CN104026022A (en) * | 2014-06-30 | 2014-09-10 | 镇江瑞繁农艺有限公司 | Alocasia macrorrhizos test-tube plantlet multiplication method |
| CN106688886A (en) * | 2016-12-06 | 2017-05-24 | 广西壮族自治区农业科学院 | Method for explant disinfection culture in taro stem tip tissue culture and plant |
| CN111919747A (en) * | 2020-07-27 | 2020-11-13 | 衡阳市蔬菜研究所 | Method for improving inductivity and rooting rate based on detoxified areca taro culture |
| CN116686707A (en) * | 2023-06-12 | 2023-09-05 | 江苏省农业科学院 | Pretreatment method for plant stem tip tissue culture |
| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103931504A (en) * | 2014-05-13 | 2014-07-23 | 江苏省农业科学院 | Rapid purification and rejuvenation method for local varieties of taros |
| CN103931504B (en) * | 2014-05-13 | 2016-04-27 | 江苏省农业科学院 | The method of a kind of taro landrace Fast Purification rejuvenation |
| CN104026022A (en) * | 2014-06-30 | 2014-09-10 | 镇江瑞繁农艺有限公司 | Alocasia macrorrhizos test-tube plantlet multiplication method |
| CN104026022B (en) * | 2014-06-30 | 2016-06-15 | 镇江瑞繁农艺有限公司 | A kind of Rhizoma alcasiae odorae test tube seedling proliferation method |
| CN106688886A (en) * | 2016-12-06 | 2017-05-24 | 广西壮族自治区农业科学院 | Method for explant disinfection culture in taro stem tip tissue culture and plant |
| CN111919747A (en) * | 2020-07-27 | 2020-11-13 | 衡阳市蔬菜研究所 | Method for improving inductivity and rooting rate based on detoxified areca taro culture |
| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
| CN116686707A (en) * | 2023-06-12 | 2023-09-05 | 江苏省农业科学院 | Pretreatment method for plant stem tip tissue culture |
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Application publication date: 20130501 |