CN104067942A - Apple stock MM116 tissue culture rapid propagation method - Google Patents
Apple stock MM116 tissue culture rapid propagation method Download PDFInfo
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Abstract
The invention discloses an apple stock MM116 tissue culture rapid propagation method. The method comprises the steps as follows: firstly, conducting primary culture through adopting a culture medium containing MS+6-BA 0.5 mg/L+IBA 0.1 mg+PVP 500mg/L; secondly, conducting subculture by using MS+6-BA 0.6 mg/L+IBA 0.1 mg/L; thirdly, adopting a rooting culture medium of 1/2 MS+IBA 0.9 mg/L for rooting culture; fourthly, adopting pure saw dust for acclimatization and transplant. Through the method, the apple stock MM116 tissue culture rapid propagation is realized, a higher survival rate is guaranteed, the excellent characteristics of a stock is maintained, and a convenient method and approach are brought to the development and further expansion of the apple industry.
Description
Technical field
The invention belongs to apple nursery stock culture technique field, relate in particular 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, 225.3 ten thousand hectares of Chinese apple cultivated areas in 2013, and 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 yield per unit area 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, the apple production pattern of China by traditional poor efficiency closing orchard pattern progressively change the intensive High-efficient Production cultivation mode of the short anvil dense planting of modernization into.Nursery stock is the basis of apple production cultivation, the quality of nursery stock directly affect apple tree growth, bloom, result and resistance, resistance against diseases and life-span.After nursery stock is subject to infecting of virus, the normal cell proliferation of tree body, will be damaged, and serious words can affect the normal physiological mechanism such as growth potential, fruit quality and the output of fruit tree, and this becomes the hidden danger of Chinese apple industry development.Be the extensive critical period of updating of China's apple one period from now on, how to provide support from nursery stock aspect and to ensure that just becoming one is worth the problem of 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 good dwarfing rootstock of wide adaptability on the one hand, on the other hand existing dwarfing rootstock raising technology is not yet made a breakthrough.MM116 utilizes M9 and MM106 parent to hybridize by Dong Mao woods experiment station of Britain to cultivate half vigorous stock forming, it has the good resistance of early fruiting character and the MM106 of M9 concurrently, the present invention sets up a kind of method of apple rootstock MM116 tissue-culturing rapid propagation, downgrade virus-free nursery stock for production high-quality, significant, development prospect is wide.
Summary of the invention
The object of the embodiment of the present invention is to provide a kind of method of apple rootstock MM116 tissue-culturing rapid propagation, is intended to provide for the cultivation of China's apple nursery stock a kind of method of tissue-culturing rapid propagation, updates 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 1, taking apple rootstock MM116 as material, selects the elite stand of robust growth without damage by disease and insect, get 1 year raw branch March, place in the greenhouse of 25 DEG C and carry out water planting, every premium on currency contains 30g sucrose, changes a water and cuts old clip every 3d, in the time that the bud pumping on the annotinous branch of water planting is 1.5～2.0cm, clip tender shoots, removes and launches blade, under running water, rinses 6～8h, process 8s with 75% alcohol, 0.1% mercuric chloride is processed 7min;
Step 2, after disinfection, clean 3min with supersonic wave cleaning machine, use again aseptic water washing 3～4 times, the tender shoots of handling well is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP (polyvinylpyrrolidone) 500mg/L and cultivates, every liter of additional 30g sucrose of medium, 8g agar, then be placed on illumination 16h/d, illumination 2000lux, under 26 DEG C of conditions, cultivate;
Step 3, by the MM116 aseptic explant inoculation MS+6-BA0.6mg/L+IBA0.1mg/L agar 8g/L obtaining, carries out subculture cultivation, every liter of additional 30g sucrose, 8g agar in the medium of illumination 2000lux;
Step 4, selects subculture to cultivate the robust growth obtaining, and is highly greater than 1.5cm, and 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, when group training shoot root length reaches after 2cm, move on to the outdoor hardening of sheltering from heat or light about 10～12 days, then culture vessel bottleneck is opened, carried out uncork hardening after 2～4 days under natural daylight, the seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary root is cleaned up, finally rinse 2～3 times with clear water, in the nutritive cube being then transplanted to pure sawdust.
Further, in step 5, the way to manage of transplanted seedling is: the seedling of taking root after transplanting manages under shade net, and first week adopts concentration is that 0.1% carbendazim solution is sprayed, and second week water every day is sprayed 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, by adopting the just culture of medium that medium is MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L, survival rate is 41.38%, is conducive to the foundation of MM116 group training seedling; Adopt the subculture that MS+6-BA0.6mg/L+IBA0.1mg/L processes to cultivate, expand numerous coefficient up to 5.46; Adopt culture of rootage and the rooting rate of root media 1/2MS+IBA0.9mg/L to carry out acclimatization and transplants survival rate up to 90% up to the pure sawdust of 91.0% use; That repeats carries out subculture cultivation to MM116 kind, and culture of rootage and transplanting can realize the tissue-culturing rapid propagation of the virus-free nursery stock of apple rootstock MM116 in the short period of time.Not only having ensured higher survival rate by tissue culture technique, and retained the good characteristic of stock, is development and further method and the approach easily of providing that expand of Apple Industry.
Brief description of the drawings
Fig. 1 is the method flow diagram of the apple rootstock MM116 tissue-culturing rapid propagation that provides of the embodiment of the present invention.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, 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, is 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: taking apple rootstock MM116 as material, select robust growth without the elite stand of damage by disease and insect, get 1 year raw branch March, place in the greenhouse of 25 DEG C and carry out water planting, every premium on currency contains 30g sucrose, change a water and cut old clip every 3d, in the time that the bud pumping on the annotinous branch of water planting is 1.5～2.0cm, clip tender shoots, remove and launch blade, under running water, rinse 6～8h, process 8s with 75% alcohol, 0.1% mercuric chloride is processed 7min;
S102: after disinfection, clean 3min with supersonic wave cleaning machine, use again aseptic water washing 3～4 times, the tender shoots of handling well is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP (polyvinylpyrrolidone) 500mg/L and cultivates, every liter of additional 30g sucrose of medium, 8g agar, is then placed on illumination 16h/d, illumination 2000lux, under 26 DEG C of conditions, cultivates;
S103: the MM116 aseptic explant inoculation best medium obtaining is processed to M14, i.e. MS+6-BA0.6mg/L+IBA0.1mg/L agar 8g/L, carries out subculture cultivation in the medium of illumination 2000lux, every liter of additional 30g sucrose, 8g agar;
S104: select subculture to cultivate the robust growth obtaining, 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: when group training shoot root length reaches after 2cm, move on to the outdoor hardening of sheltering from heat or light about 10～12 days, then culture vessel bottleneck is opened, under natural daylight, carry out uncork hardening after 2～4 days, the seedling of taking root takes out from medium, and putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary root is cleaned up, finally rinse 2～3 times with clear water, in the nutritive cube being then transplanted to pure sawdust.
In step S105, the way to manage of transplanted seedling is: the seedling of taking root after transplanting manages under shade net, and first week adopts concentration is that 0.1% carbendazim solution is sprayed, and second week water every day is sprayed 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 1, first culture
Apple rootstock MM116 is material, select the elite stand of robust growth without damage by disease and insect, get 1 year raw branch March, place in the greenhouse of 25 DEG C and carry out water planting (every premium on currency contains 30g sucrose), change a water and cut old clip every 3d, in the time that the bud pumping on the annotinous branch of water planting is 1.5～2.0cm, clip tender shoots, remove and launch blade, under running water, rinse 6～8h, with 75% alcohol processing 8s, 0.1% mercuric chloride is processed 7min, after disinfection, clean 3min with supersonic wave cleaning machine, use again aseptic water washing 3～4 times, the tender shoots of handling well is inoculated into MS+6-BA0.5mg/L+IBA0.1mg/L+ variable concentrations processing (0, 0.3, 0.5, 0.7, in the medium of polyvinylpyrrolidone (PVP) 1.0g/L), cultivate, every liter of additional 30g sucrose of medium, 8g agar, 3 buds of each triangle bottle graft, 15 bottles of every processing, then be placed on illumination 16h/d, illumination 2000lux, under 26 DEG C of conditions, cultivate, after 14d, investigate respectively pollution rate, brown rate, survival rate, the first culture that draws MM116 is that optimum medium is MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L (sucrose 30g/L, agar 8g/L),
Step 2, subculture is cultivated
The aseptic explant of acquisition is inoculated into and contains the capable subculture cultivation of variable concentrations 6-BA, every liter of additional 30g sucrose, 8g agar, every bottle of 3 strains, 10 bottles of each processing, repeat 3 times, and the best medium of MM116 is, be MS+6-BA0.6mg/L+IBA0.1mg/L (agar 8g/L, illumination 2000lux);
Step 3, culture of rootage
Select subculture to cultivate the robust growth obtaining, highly be greater than the plant of 1.5cm, growth time is bud of 20 days, be inoculated in the culture of rootage that 1/2MS+ variable concentrations IBA processes (0,0.8,0.9,1.0,1.1mg/L) and carry out culture of rootage, 3 of every bottle graft kinds, 10 bottles of each processing, repeat 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
When group training shoot root length reaches after 2cm, move on to the outdoor hardening of sheltering from heat or light about 10～12 days, then culture vessel bottleneck is opened, under natural daylight, carry out uncork hardening after 2～4 days, the seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary root is cleaned up, finally rinse 2～3 times with clear water, then different substrates processing (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: the seedling of taking root manages under shade net, first week adopts concentration is that 0.1% carbendazim solution is sprayed, second week water every day is sprayed once, keep matrix moistening ventilative.After 14 days, add up acclimatization and transplants survival rate, 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 processes the impact on explant growth;
Table 1 variable concentrations PVP processes the impact on explant growth
Note: represent not remarkable (P>0.05) of difference, different letter representation significant differences (P<0.05) with columns same letter.Data are Mean ± SE (mean value ± standard error); Lower same.
2, different 6-BA concentration is processed the impact that MM116 subculture is cultivated:
The impact that the different 6-BA concentration of table 2 is cultivated MM116 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, different I BA concentration is processed the impact that MM116 is taken root:
Table 3 different I BA concentration is processed the impact that MM116 is taken root
The impact of 4 different hardening matrix on the seedling growth of taking root:
The different cultivation matrixes of table 4 are processed the impact on transplanted seedling growth
| 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 of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
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 method concrete grammar of this apple rootstock MM116 tissue-culturing rapid propagation comprises the following steps:
Step 1, taking apple rootstock MM116 as material, clean 3min with supersonic wave cleaning machine, use again aseptic water washing 3～4 times, the tender shoots of handling well is inoculated in the medium of MS+6-BA0.5mg/L+IBA0.1mg+PVP500mg/L and cultivates, every liter of additional 30g sucrose of medium, 8g agar, then be placed on illumination 16h/d, illumination 2000lux, under 26 DEG C of conditions, cultivate;
Step 2, is inoculated into MS+6-BA0.6mg/L+IBA0.1mg/L by the aseptic explant of acquisition, carries out subculture cultivation in the medium of illumination 2000lux, i.e. every liter of additional 30g sucrose, 8g agar;
Step 3, selects subculture to cultivate the robust growth obtaining, and is highly greater than the plant of 1.5cm, and growth time is bud of 20 days, is inoculated in 1/2MS+IBA0.9mg/L sucrose 30g/L, in agar 8g/L root media, carries out culture of rootage.
2. the method for apple rootstock MM116 tissue-culturing rapid propagation as claimed in claim 1, it is characterized in that, in step 1 taking apple rootstock MM116 as material, select the elite stand of robust growth without damage by disease and insect, get 1 year raw branch March, place in the greenhouse of 25 DEG C and carry out water planting, water planting is that every premium on currency contains 30g sucrose, change a water and cut old clip every 3d, in the time that the bud pumping on the annotinous branch of water planting is 1.5～2.0cm, clip tender shoots, remove and launch blade, under running water, rinse 6～8h, process 8s with 75% alcohol, 0.1% mercuric chloride is processed 7min; And then clean with supersonic wave cleaning machine.
3. the method for apple rootstock MM116 tissue-culturing rapid propagation as claimed in claim 1, it is characterized in that, in step 3 culture of rootage, training shoot root length when group reaches after 2cm, move on to the outdoor hardening 10～12 days of sheltering from heat or light, then culture vessel bottleneck is opened, under natural daylight, carry out uncork hardening after 2～4 days, the seedling of taking root takes out from medium, putting into concentration is that 0.1% carbendazim solution soaks 5min, and medium unnecessary root is cleaned up, finally rinse 2～3 times with clear water, in the nutritive cube being then transplanted to pure sawdust.
4. the method for apple rootstock MM116 tissue-culturing rapid propagation as claimed in claim 3, it is characterized in that, in step 5, the way to manage of transplanted seedling is: the seedling of taking root after transplanting manages under shade net, first week adopts concentration is that 0.1% carbendazim solution is sprayed, second week water every day is sprayed once, keeps matrix moistening ventilative.Offspring pumping to be generated shifts out shade net after going out sprouting.
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| CN105900836A (en) * | 2016-04-22 | 2016-08-31 | 山东省果树研究所 | Culture medium and culture method used for generating adventitious buds of cold-resistant dwarf apple rootstock in-vitro leaves |
| 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 |
| 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 |
| CN106879470A (en) * | 2017-03-30 | 2017-06-23 | 天水市果树研究所 | A kind of method of apple rootstock tissue culture Initial culture prevent-browning |
| CN106937597A (en) * | 2017-04-12 | 2017-07-11 | 山东农业大学 | A kind of tissue-culturing rapid propagation method suitable for high flavonoids apple new lines |
| CN107258538A (en) * | 2017-07-01 | 2017-10-20 | 西北农林科技大学 | A kind of apple rootstock SH38 rapid propagation methods |
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| 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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| 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 |
| CN104737920A (en) * | 2015-04-26 | 2015-07-01 | 芜湖东源新农村开发股份有限公司 | Tissue culture fast breeding method of apple rootstock M9 |
| 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 |
| 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 |
| CN106613934A (en) * | 2015-10-28 | 2017-05-10 | 北京市农林科学院 | Method for rapidly propagating apple rootstock SH6 |
| CN105900836A (en) * | 2016-04-22 | 2016-08-31 | 山东省果树研究所 | Culture medium and culture method used for generating adventitious buds of cold-resistant dwarf apple rootstock in-vitro leaves |
| 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 |
| CN106879470A (en) * | 2017-03-30 | 2017-06-23 | 天水市果树研究所 | A kind of method of apple rootstock tissue culture Initial culture prevent-browning |
| CN106937597A (en) * | 2017-04-12 | 2017-07-11 | 山东农业大学 | A kind of tissue-culturing rapid propagation method suitable for high flavonoids apple new lines |
| CN106937597B (en) * | 2017-04-12 | 2018-11-27 | 山东农业大学 | A kind of tissue-culturing rapid propagation method suitable for high flavonoids apple new lines |
| CN107258538A (en) * | 2017-07-01 | 2017-10-20 | 西北农林科技大学 | A kind of apple rootstock SH38 rapid propagation methods |
| 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 |
| CN108157182A (en) * | 2018-02-06 | 2018-06-15 | 山东省烟台市农业科学研究院 | A kind of cultural method for reducing apple Explant browning rate |
| 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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