CN104067942B - A kind of method of apple rootstock MM116 tissue-culturing rapid propagation - Google Patents
A kind of method of apple rootstock MM116 tissue-culturing rapid propagation Download PDFInfo
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- CN104067942B CN104067942B CN201410333345.9A CN201410333345A CN104067942B CN 104067942 B CN104067942 B CN 104067942B CN 201410333345 A CN201410333345 A CN 201410333345A CN 104067942 B CN104067942 B CN 104067942B
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Abstract
The invention discloses a kind of method of apple rootstock MM116 tissue-culturing rapid propagation, the method of this apple rootstock MM116 tissue-culturing rapid propagation is the medium Initial culture of MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L, the squamous subculture adopting MS+6-BA0.6mg/L+IBA0.1mg/L process, the culture of rootage of employing root media 1/2MS+IBA0.9mg/L, the acclimatization and transplants with pure sawdust by adopting medium; Achieve apple rootstock MM116 to breed fast, not only ensure that higher survival rate, and remain the good characteristic of stock, for the development of Apple Industry provides method and access easily with expanding further.
Description
Technical field
The invention belongs to apple nursery plant cultivation technology field, particularly relate to a kind of method of apple rootstock MM116 tissue-culturing rapid propagation.
Background technology
Apple Industry occupies very important status in Chinese agriculture economy, and Chinese apple cultivated area 225.3 ten thousand hectares in 2013, output is 3,170 ten thousand tons, accounts for 1/2 of Gross World Product.Compared with apple advanced production country of the world, also there is larger gap in the apple production of China, main manifestations is that unit area output is low, fruit quality is poor, economic benefit is not high, possession share on international fresh fruit market is few, extremely unbecoming with the status of apple production big country of China, wherein apple nursery stock is of poor quality is one of major reason causing this situation.At present, China apple production pattern by traditional poor efficiency closing orchard pattern progressively change the intensive High-efficient Production cultivation mode of modernization short anvil dense planting into.Nursery stock is the basis of apple production cultivation, and the quality of nursery stock directly affects the growth of apple tree, blooms, result and resistance, resistance against diseases and life-span.After nursery stock is subject to infecting of virus, tree body normal cell proliferation, will be damaged, and serious words can affect the normal physiological mechanism such as the growth potential of fruit tree, fruit quality and output, and this becomes the hidden danger of Chinese apple industry development.One period is the critical period that China's apple updates on a large scale from now on, and how providing support from nursery stock aspect and ensureing just becomes a problem being worth paying much attention to.At present, China downgrades apple nursery stock market, and supply falls short of demand, and the key of restriction Nursery Stock Industry development is, lacks the excellent dwarfing rootstock of wide adaptability on the one hand, not yet makes a breakthrough on the other hand to existing dwarfing rootstock raising technology.MM116 is half vigorous stock utilizing M9 and MM106 parents to cultivate by Dong Mao woods experiment station of Britain, it has the early fruiting character of M9 and the good resistance of MM106 concurrently, the present invention sets up a kind of method of apple rootstock MM116 tissue-culturing rapid propagation, virus-free nursery stock is downgraded for production high-quality, significant, development prospect is wide.
Summary of the invention
The object of the embodiment of the present invention is a kind of method providing apple rootstock MM116 tissue-culturing rapid propagation, is intended to, for the cultivation of China's apple nursery stock provides a kind of method of tissue-culturing rapid propagation, update on a large scale to accelerate China's apple.
The embodiment of the present invention is achieved in that a kind of method of apple rootstock MM116 tissue-culturing rapid propagation, and the method for this apple rootstock MM116 tissue-culturing rapid propagation comprises the following steps:
Step one, with apple rootstock MM116 for material, the healthy and strong elite stand without damage by disease and insect of growth selection, 1 year raw branch is got March, carry out water planting in the greenhouse placing 25 DEG C, every premium on currency contains 30g sucrose, changes a water and cut old clip every 3d, when bud pumping on the annotinous branch of water planting is 1.5 ~ 2.0cm, clip tender shoots, removes and launches blade, under running water, rinse 6 ~ 8h, with 75% ethanol postincubation 8s, 0.1% mercuric chloride process 7min;
Step 2, after disinfection, 3min is cleaned with supersonic wave cleaning machine, use aseptic water washing again 3 ~ 4 times, be inoculated into by the tender shoots handled well in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP (polyvinylpyrrolidone) 500mg/L and cultivate, often liter of medium adds 30g sucrose, 8g agar, then be placed on illumination 16h/d, illumination 2000lux, cultivate under 26 DEG C of conditions;
Step 3, by the MM116 aseptic explant inoculation MS+6-BA0.6mg/L+IBA0.1mg/L agar 8g/L obtained, carries out squamous subculture in the medium of illumination 2000lux, often liter of additional 30g sucrose, 8g agar;
Step 4, select the robust growth that squamous subculture obtains, be highly greater than 1.5cm, growth time is bud of 20 days, is inoculated in 1/2MS+IBA0.9mg/L (sucrose 30g/L, agar 8g/L) root media and carries out culture of rootage;
Step 5, after plantlet in vitro root length reaches 2cm, move on to outdoor to shelter from heat or light hardening about 10 ~ 12 days, then opened by culture vessel bottleneck, carry out uncork hardening after 2 ~ 4 days under natural daylight, seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary for root is cleaned up, finally rinse 2 ~ 3 times with clear water, in the nutritive cube be then transplanted to pure sawdust.
Further, in step 5, the way to manage of transplanted seedling is: seedling of taking root after transplanting manages under shade net, within first week, adopts concentration to be that 0.1% carbendazim solution is sprayed, and the sprinkling irrigation of second week use every day water once, keeps matrix moistening ventilative.Offspring pumping to be generated is extensible shade net after going out sprouting.
The method of apple rootstock MM116 tissue-culturing rapid propagation provided by the invention, be the medium Initial culture of MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L by employing medium, survival rate is 41.38%, is conducive to the foundation of MM116 plantlet in vitro; Adopt the squamous subculture of MS+6-BA0.6mg/L+IBA0.1mg/L process, expand numerous coefficient up to 5.46; Adopt the culture of rootage of root media 1/2MS+IBA0.9mg/L and rooting rate carries out acclimatization and transplants survival rate up to 90% up to 91.0% with pure sawdust; That repeats carries out squamous subculture to MM116 kind, and culture of rootage and transplanting can achieve the tissue-culturing rapid propagation of the virus-free nursery stock of apple rootstock MM116 in the short period of time.Not only ensure that higher survival rate by tissue culture technique, and remain the good characteristic of stock, for the development of Apple Industry provides method and access easily with expanding further.
Accompanying drawing explanation
Fig. 1 is the method flow diagram of the apple rootstock MM116 tissue-culturing rapid propagation that the embodiment of the present invention provides.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the method for the apple rootstock MM116 tissue-culturing rapid propagation of the embodiment of the present invention comprises the following steps:
S101: with apple rootstock MM116 for material, the healthy and strong elite stand without damage by disease and insect of growth selection, gets 1 year raw branch March, carries out water planting in the greenhouse placing 25 DEG C, every premium on currency contains 30g sucrose, change a water every 3d and cut old clip, when the bud pumping on the annotinous branch of water planting is 1.5 ~ 2.0cm, clip tender shoots, remove and launch blade, 6 ~ 8h is rinsed under running water, with 75% ethanol postincubation 8s, 0.1% mercuric chloride process 7min;
S102: after disinfection, 3min is cleaned with supersonic wave cleaning machine, use aseptic water washing again 3 ~ 4 times, the tender shoots handled well is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP (polyvinylpyrrolidone) 500mg/L and cultivates, often liter of medium adds 30g sucrose, 8g agar, is then placed on illumination 16h/d, illumination 2000lux, cultivates under 26 DEG C of conditions;
S103: by the MM116 aseptic explant inoculation best medium process M14 obtained, i.e. MS+6-BA0.6mg/L+IBA0.1mg/L agar 8g/L, carries out squamous subculture in the medium of illumination 2000lux, often liter of additional 30g sucrose, 8g agar;
S104: select the robust growth that squamous subculture obtains, be highly greater than 1.5cm, growth time is bud of 20 days, is inoculated in 1/2MS+IBA0.9mg/L (sucrose 30g/L, agar 8g/L) root media and carries out culture of rootage;
S105: after plantlet in vitro root length reaches 2cm, move on to outdoor to shelter from heat or light hardening about 10 ~ 12 days, then culture vessel bottleneck is opened, uncork hardening is carried out after 2 ~ 4 days under natural daylight, seedling of taking root takes out from medium, and putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary for root is cleaned up, finally rinse 2 ~ 3 times with clear water, in the nutritive cube be then transplanted to pure sawdust.
In step S105, the way to manage of transplanted seedling is: seedling of taking root after transplanting manages under shade net, within first week, adopts concentration to be that 0.1% carbendazim solution is sprayed, and the sprinkling irrigation of second week use every day water once, keeps matrix moistening ventilative.Offspring pumping to be generated is extensible shade net after going out sprouting.
Concrete steps of the present invention are as follows:
Step one, Initial culture
Apple rootstock MM116 is material, the healthy and strong elite stand without damage by disease and insect of growth selection, 1 year raw branch is got March, water planting (every premium on currency contains 30g sucrose) is carried out in the greenhouse placing 25 DEG C, change a water every 3d and cut old clip, when bud pumping on the annotinous branch of water planting is 1.5 ~ 2.0cm, clip tender shoots, remove and launch blade, 6 ~ 8h is rinsed under running water, with 75% ethanol postincubation 8s, 0.1% mercuric chloride process 7min, after disinfection, 3min is cleaned with supersonic wave cleaning machine, use aseptic water washing again 3 ~ 4 times, the tender shoots handled well is inoculated into MS+6-BA0.5mg/L+IBA0.1mg/L+ variable concentrations process (0, 0.3, 0.5, 0.7, cultivate in the medium of polyvinylpyrrolidone (PVP) 1.0g/L), often liter of medium adds 30g sucrose, 8g agar, each triangle bottle graft 3 buds, often process 15 bottles, then illumination 16h/d is placed on, illumination 2000lux, cultivate under 26 DEG C of conditions, pollution rate is investigated respectively after 14d, melting brown rate, survival rate, show that the Initial culture of MM116 be optimum medium is MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L (sucrose 30g/L, agar 8g/L),
Step 2, squamous subculture
The aseptic explant of acquisition is inoculated into containing the capable squamous subculture of variable concentrations 6-BA, often liter of additional 30g sucrose, 8g agar, every bottle of 3 strains, each process 10 bottles, repeat 3 times, the best medium of MM116 is, i.e. MS+6-BA0.6mg/L+IBA0.1mg/L (agar 8g/L, illumination 2000lux);
Step 3, culture of rootage
Select the robust growth that squamous subculture obtains, highly be greater than the plant of 1.5cm, growth time is bud of 20 days, be inoculated in 1/2MS+ variable concentrations IBA process (0,0.8,0.9,1.0,1.1mg/L) culture of rootage in carry out culture of rootage, every bottle graft kind 3, each process 10 bottles, repeats 3 times, obtaining the optimum root media of MM116 is 1/2MS+IBA0.9mg/L (sucrose 30g/L, agar 8g/L);
Step 4, acclimatization and transplants
After plantlet in vitro root length reaches 2cm, move on to outdoor to shelter from heat or light hardening about 10 ~ 12 days, then culture vessel bottleneck is opened, uncork hardening is carried out after 2 ~ 4 days under natural daylight, seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary for root is cleaned up, finally rinse 2 ~ 3 times with clear water, then different substrates process (the pure sawdust that the capacity that is transplanted to is identical, sawdust+matrix, pure matrix, matrix+perlite) nutritive cube in, the way to manage of transplanted seedling is: seedling of taking root manages under shade net, within first week, concentration is adopted to be that 0.1% carbendazim solution is sprayed, the sprinkling irrigation of second week use every day water once, keep matrix moistening ventilative.Added up acclimatization and transplants survival rate afterwards in 14 days, draw by the acclimatization and transplants survival rate of pure sawdust best.
By following experiment and data, principle of the present invention is described further:
1, reference table 1 variable concentrations PVP process is on the impact of explant growth;
Table 1 variable concentrations PVP process is on the impact of explant growth
Note: represent difference not significantly (P>0.05) with columns same letter, different letter representation significant difference (P<0.05).Data are Mean ± SE (mean value ± standard error); Lower same.
2, the process of different 6-BA concentration is on the impact of MM116 squamous subculture:
The different 6-BA concentration of table 2 is on the impact of MM116 squamous subculture
| Minimal medium | 6-BA(mg/L) | IBA(mg/L) | Expand numerous coefficient |
| MS | 0 | 0.1 | 1.24±0.07c |
| MS | 0.3 | 0.1 | 1.2±0.08c |
| MS | 0.4 | 0.1 | 2.29±0.32b |
| MS | 0.5 | 0.1 | 3.94±0.12ab |
| MS | 0.6 | 0.1 | 5.46±0.31a |
3, the different I BA concentration process MM116 impact of taking root:
The impact that table 3 different I BA concentration process MM116 is taken root
4 different hardening matrix are on the impact of seedling growth of taking root:
The impact that the different cultivation matrix process of table 4 grows transplanted seedling
| Matrix treatments | MM116 transplanting survival rate |
| Sawdust | 90.00±5.32a |
| Sawdust+matrix | 82.50±2.33b |
| Matrix | 72.50±3.42c |
| Matrix+perlite | 77.50±.233bc |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. a method for apple rootstock MM116 tissue-culturing rapid propagation, is characterized in that, apple rootstock MM116 carries out tissue-culturing rapid propagation under tissue culture technology, and the concrete grammar of this apple rootstock MM116 tissue-culturing rapid propagation comprises the following steps:
Step one, with apple rootstock MM116 for material, the healthy and strong elite stand without damage by disease and insect of growth selection, 1 year raw branch is got March, carry out water planting in the greenhouse placing 25 DEG C, water planting is that every premium on currency contains 30g sucrose, changes a water and cut old clip every 3d, when bud pumping on the annotinous branch of water planting is 1.5 ~ 2.0cm, clip tender shoots, removes and launches blade, under running water, rinse 6 ~ 8h, with 75% ethanol postincubation 8s, 0.1% mercuric chloride process 7min; And then clean 3min with supersonic wave cleaning machine cleaning, use aseptic water washing again 3 ~ 4 times, the tender shoots handled well is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L and cultivates, often liter of medium adds 30g sucrose, 8g agar, then be placed on illumination 16h/d, intensity of illumination 2000lux, cultivate under 26 DEG C of conditions;
Step 2, tissue culture plant inoculation step one obtained, to MS+6-BA0.6mg/L+IBA0.1mg/L, carries out squamous subculture in the medium of intensity of illumination 2000lux, often liter of additional 30g sucrose, 8g agar;
Step 3, the robust growth selecting squamous subculture to obtain, is highly greater than the plant of 1.5cm, selects growth time on this plant to be that the bud of 20 days is inoculated in 1/2MS+IBA0.9mg/L, sucrose 30g/L, carries out culture of rootage in agar 8g/L root media;
Step 4, in step 3 culture of rootage after plantlet in vitro root length reaches 2cm, move on to outdoor to shelter from heat or light hardening 10 ~ 12 days, then opened by culture vessel bottleneck, carry out uncork hardening after 2 ~ 4 days under natural daylight, seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary for root is cleaned up, finally rinse 2 ~ 3 times with clear water, be then transplanted to in the pure sawdust nutritive cube that is matrix;
In step 4, the way to manage of transplanted seedling is: seedling of taking root after transplanting manages under shade net, within first week, adopts concentration to be that 0.1% carbendazim solution is sprayed, and the sprinkling irrigation of second week use every day water once, keeps matrix moistening ventilative; Namely offspring pumping to be generated shifts out shade net after going out sprouting.
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| CN104686355A (en) * | 2015-03-04 | 2015-06-10 | 鲁东大学 | Method for increasing callus rate and sprouting ratio of leaves of aseptic apple seedlings |
| CN104737920B (en) * | 2015-04-26 | 2017-03-08 | 芜湖东源新农村开发股份有限公司 | A kind of tissue culture fast seedling-cultivating method of apple rootstock M9 |
| CN106613934A (en) * | 2015-10-28 | 2017-05-10 | 北京市农林科学院 | Method for rapidly propagating apple rootstock SH6 |
| CN106613933A (en) * | 2015-10-28 | 2017-05-10 | 北京市农林科学院 | Method for inducing rooting of apple dwarf rootstock tissue culture seedling |
| CN105900836B (en) * | 2016-04-22 | 2018-07-31 | 山东省果树研究所 | The culture medium and cultural method of adventitious bud are generated for apple cold-resistant dwarfing rootstock excised leaf |
| CN106134997A (en) * | 2016-07-06 | 2016-11-23 | 中国农业科学院郑州果树研究所 | The group training fast seedling-cultivating method of apple rootstock M26 |
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| CN107278893A (en) * | 2017-07-01 | 2017-10-24 | 西北农林科技大学 | A kind of apple rootstock SH40 rapid propagation methods |
| CN107371880A (en) * | 2017-07-28 | 2017-11-24 | 济南浩隆生物科技有限公司 | A kind of apple rootstock tissue culturing fast seedling-cultivating method |
| CN108157182B (en) * | 2018-02-06 | 2021-05-14 | 山东省烟台市农业科学研究院 | Culture method for reducing browning rate of apple explants |
| CN111616052A (en) * | 2020-05-30 | 2020-09-04 | 西北农林科技大学 | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei |
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| CN101926287B (en) * | 2010-09-06 | 2012-05-30 | 中国农业大学 | Method for culturing tissue of 'Zhongzhen No.1' |
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