CN103988776B - A kind of Nantong little side's persimmon tissue culture and rapid propagation method - Google Patents
A kind of Nantong little side's persimmon tissue culture and rapid propagation method Download PDFInfo
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Abstract
The present invention relates to a kind of Nantong little side's persimmon tissue culture and rapid propagation method, belong to crops fast breeding technique field.Have studied sleeping bud sterilization method, determine the medium component being suitable for little side's persimmon early and middle portion shoot proliferation, analyze the different basic element of cell division, impact that growth hormone grows plantlet in vitro.Peel the sleeping bud taken from the little side's persimmon annotinous branch of Nantong off outer scale, sterilize with 75% alcohol, 10% hydrogen peroxide respectively after 2 hours with running water, the sleeping bud of disinfecting is inoculated in minimal medium, is transferred in subculture multiplication medium after sprouting.This method solves Nantong little side persimmon substantially just for the problem that brownization is serious, just for survival rate to 80%.Nantong little side's persimmon shoot proliferation problem is solved by adding appropriate gibberellin.Facilitate fast breeding and the production popularization of the little side's persimmon in dwarf form persimmon kind Nantong.
Description
Technical field
The invention belongs to field of tissue culture, relate to a kind of Nantong little side's persimmon tissue culture and rapid propagation method.
Background technology
Persimmon (DiospyroskakiL.f.) belongs to Ebenaceae (Ebenaceae), Diospyros (Diospyros)), persimmon contains the nutriments such as significant quantities of fat, protein, carbohydrate, and the mineral element such as calcium, phosphorus, iron and multivitamin nutritious, both can eat raw, dried persimmon, persimmon vinegar, persimmon wine etc. can be processed into again; Persimmon leaf can do tea, and persimmon paint can be used by fuel feeding umbrella.Simultaneously persimmon tree performance is graceful, and leaf, fruit are beautiful in colour, are a kind of good ornamental tree species.
Persimmon is tall and big deciduous tree, usual height of tree 7-9 rice.For a long time, many fruit tree workers make great efforts the persimmon germ plasm resource seeking to have Dwarfing Gene, the persimmon germ plasm resource of dwarf form persimmon kind Nantong little Fang Shishi China preciousness one by one, this kind tree low body is little, early fruiting character is good, and high yield is stable, and fruit look gorgeous is, seedless, of fine quality, can naturally take away the puckery taste, be the excellent persimmon kind of Cultivation With Dwarf Varieties And Close Spacing.Think producing far-reaching shadow to China's cultivation of persimmon from now on, breeding and development thereof.But the more difficult transplanting of traditional persimmon propagation technique, after survival rate and grafting, survival rate is all lower.
Summary of the invention
The object of the invention is for survival rate after the more difficult transplanting survival rate of traditional persimmon propagation technique and grafting low, a kind of Nantong little side's persimmon tissue culture and rapid propagation method is provided.
A group training tissue culture method for fast propagation for the little side's persimmon in Nantong, comprises the following steps:
(1) disinfect
Get the sleeping bud on the little side's persimmon annotinous branch of Nantong, peel off outside 2-3 sheet scale, rinse 2 hours under flowing water; Sleeping bud is relayed on superclean bench, with 75% alcohol surface disinfection 30s (second) aseptic water washing 3 times afterwards; Afterwards sleeping bud is put into 10% hydrogen peroxide and soaks 20min (minute), must not stop between soak period to rock and make hydrogen peroxide produce a large amount of bubble, finally use aseptic water washing 3 times, be put into be lined with aseptic blotting paper culture dish in;
Sleeping bud through sterilization is inoculated on the MS medium not adding growth hormone and the basic element of cell division containing 500mg/LPVP (polyethylene adjoins pyrrolidone) and cultivates;
(2) shoot proliferation is cultivated
The little side's persimmon in Nantong sprout on MS medium growth sleeping bud be inoculated in MS (1/2N) medium, the basic element of cell division of interpolation is ZT2mg/L, and squamous subculture 2-3 is reduced to 1mg/L for rear zeatin concentration; Archusia is NAA0.05mg/L; PVP500mg/L; Sucrose 30g/L; Agar 6.0g/L;
Plantlet in vitro squamous subculture 4-5 uses (1/2N) MS+ZT1.0mg/L+IAA0.05-0.3mg/L+GA for rear (seedling growing way is healthy and strong, highly about about 1cm)
30.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L and (1/2N) MS+ZT1.0mg/L+IAA0.05-0.3mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L medium alternate culture, the plantlet in vitro axillalry bud of last culture is cut and inserts medium and carry out propagation and expand numerous, often kind of medium shoot proliferation cultivation about 25-30 days.
Beneficial effect
The present invention is directed to the traditional propagation method of persimmon is that after stock grafting transplanting survival rate and grafting, survival rate is all low compared with other fruit trees, on the basis of forefathers' research, establishes the group culturation rapid propagating technology of the little side's persimmon in dwarf form tree kind Nantong first.
This method solves Nantong little side persimmon substantially just for the problem that brownization is serious, just for survival rate to 80%.By adding appropriate gibberellin and screening specific basal medium and variety classes, the basic element of cell division of concentration, growth hormone, solve Nantong little side's persimmon shoot proliferation problem.Facilitate fast breeding and the production popularization of the little side's persimmon in dwarf form persimmon kind Nantong.The research of Nantong little side's persimmon fast breeding technique, provides possibility for obtaining a large amount of nursery stock fast, facilitates dwarf form persimmon kind industry in the popularization of China and fast development.Meanwhile, be established as physiological Study and the molecule aspect of little side's persimmon persimmon plantlet in vitro regenerating system are studied it and are downgraded mechanism proterties etc. and lay a good foundation.
Accompanying drawing explanation
Fig. 1: the impact that different disinfectant is cultivated sleeping bud
Fig. 2: Nantong little side's persimmon plantlet in vitro picture
Embodiment
The materials and methods that the best sterilization method of 1 explant is determined
Nantong little side persimmon sleeping bud cuts from annotinous branch the outside 2-3 sheet scale peelling off bud, and in suds, wash bud section 2-3 time, running water is disinfection on super-clean bench after 2 hours.
Disinfecting time and step are: with 75% alcohol surface sterilizing 30s, carry out follow-up sterilization respectively by following three kinds of methods: (1) is containing 10%H
2o
2soak 10min, 20min respectively in solution, in immersion process otherwise time shake, make decomposing hydrogen dioxide solution produce a large amount of oxygen and play bactericidal action.Finally use aseptic water washing 5 times; (2) containing 0.1% tween 1 5%NaCIO (antiformin) solution in soak 10min, in immersion process however time shake, finally use aseptic water washing 5 times; (3) containing the 0.1%HgCI:(mercuric chloride of 0.1% tween 1) soak 10min in solution, in immersion process however time shake, finally use aseptic water washing 5 times.Being inoculated in by the sleeping bud of sterilizing through distinct methods does not add on the MS medium of growth hormone and the basic element of cell division containing 500mg/LPVP (polyethylene adjoins pyrrolidone), and each process 60 bottles, added up survival rate after 2 weeks.
As Fig. 1, although low by the serious survival rate of sleeping bud Disinfection Effect low brownization of good pollution rate of clorox and mercuric chloride sterilization, be respectively 54%, 40%.Hydrogen peroxide is 68% and 80% to the carry out disinfection survival rate of 10min, 20min of sleeping bud respectively, and explant is without browning, and survival rate is high.The active oxygen that during sterilization, decomposing hydrogen dioxide solution produces simultaneously has facilitation to the sprouting of little side's persimmon sleeping bud, growth.So adopt alcohol surface sterilizing 30s during little side's persimmon sleeping bud inoculation pre-treatment; hydrogen peroxide dipping sterilization 20min (need not stop to rock); finally use the method survival rate of aseptic water washing 5 times high, and efficiently solve sleeping bud just for the problem of brownization death.
2. the determination of best shoot proliferation culture method
2.1 materials and methods
Sucrose concentration in test used medium is 3%, and agar concentration is 0.6%, PVP (polyethylene adjoins pyrrolidone) 0.5%, and pH value is 5.7-5.8; Medium is through 121 DEG C of (0.105-0.12MPa) high-temperature heat sterilization 15min, and condition of culture is: cultivation temperature about 25 DEG C, light application time 16h/d, is to cultivate under the incandescent lamp of 1500-2000lx in intensity of illumination.Often all cultivate 25-30 days for plantlet in vitro.
The sprout sleeping bud of growth of the little side's persimmon in Nantong is inoculated in I subculture medium, and squamous subculture 2-3 uses No. II (zeatin content reduces by half) subculture medium instead and cultivates after generation; Described I squamous subculture based formulas is MS (1/2N)+zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; II squamous subculture based formulas is MS (1/2N)+zeatin 1mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L.
2.2 different minimal mediums are to Nantong little side's persimmon plantlet in vitro proliferative effect
Sprout growth the Nantong little side persimmon plantlet in vitro of sleeping bud on MS (1/2N) medium containing ZT1.0mg/L+NAA0.05mg/L after squamous subculture 2 generation, be seeded in respectively in MS, 1/2MS, MS (1/2N) medium containing ZT1.0mg/L+NAA0.05mg/L, compare the impact of minimal medium on the numerous propagation of Nantong little side persimmon tissue culture sprout quick.
As shown in table 1, the plantlet in vitro plant height adopting improved culture medium (1/2N) MS to cultivate and other two kinds of medium culture deposit significant difference, and growing way is good, and blade is emerald green greatly, and base portion callus cell is little.Between the plantlet in vitro height of 1/2MS and MS medium culture, difference is not remarkable, and plantlet in vitro is rosette-stape growth substantially, and stem is very short, and plantlet in vitro growing way is weak, and blade is yellow green part brownization, and it is numerous that base portion callus is unfavorable for that greatly subculture expands.Illustrate that persimmon explant growth needs the mineral element of low concentration, especially the nitrogen of low concentration.
The different minimal medium of table 1. is to Nantong little side's persimmon plantlet in vitro proliferative effect
Note: in result, lowercase alphabet shows significance level P=0.05, as follows
2.3 variable concentrations gibberellin affect Nantong little side's persimmon shoot proliferation
By tissue culture plant inoculation to 1.0mg/LZT, 0.1mg/LIAA, 0.0,0.05,0.1GA
3mS (1/2N) medium in grew for 2 generations after, statistics plantlet in vitro growing state, compare gibberellin plantlet in vitro growth in effect.
Gibberellin can promote cell division, also can show and help promotion cell elongation, is conducive to the leaf expansion of plantlet in vitro, stem extends and collateral generation.Add 0.05 in the medium respectively, 0.1mg/L gibberellin cultivate do not add gibberellin with contrast after one month compared with all there is significant difference between plant height and axillalry bud number, along with gibberellin concentration increase plant height and axillalry bud number increase all to some extent, number of blade change is little, stem obviously extends, and effectively improves the numerous rate of increase of expansion of little side's persimmon.But the plantlet in vitro growing way under gibberellin effect obviously dies down, narrow along with concentration increase blade diminishes gradually, when concentration is greater than 0.1mg/L, blade is yellow green willow leaf shape, and plant becomes short brownization.Be sprouting and the Stem nematode of the gibberellin inducement plant axillalry bud of 0.05mg/L by concentration, the proliferation times that strong seedling culture effectively can improve the little side's persimmon in Nantong is carried out again with the medium not adding gibberellin, the emerald green growing way of plant leaf is vigorous, and be all significantly higher than in plant height, axillalry bud number and the number of blade containing gibberellin concentration 0.00,0.05, the plantlet in vitro cultivated separately of the medium of 0.1mg/L.
Illustrate that adding the propagation that gibberellin is conducive to persimmon plantlet in vitro when persimmon subculture expands numerous.Available ZT1.0mg/L+IAA0.1mg/L+GA0.05mg/L and ZT1.0mg/L+IAA0.1mg/L medium alternate culture carries out the Fast-propagation of plantlet in vitro.
Table 2: variable concentrations GA
3(gibberellin) affects Nantong little side's persimmon shoot proliferation
The impact that the basic element of cell division of 2.4 variety classeses, concentration grows plantlet in vitro
Be chosen at (1/2N) MS+ZT1.0mg/L+IAA0.05-0.3mg/L+GA
3the plant height of 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L and (1/2N) MS+ZT1.0mg/L+IAA0.05-0.3mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L medium alternate culture about 3cm plantlet in vitro for carry out variety classes, concentration the basic element of cell division on the experiment of the impact that plantlet in vitro grows.
The axillalry bud of plantlet in vitro is cut in (1/2N) MS+NAA0.05mg/L medium of access respectively containing ZT (0.5,1.0,3.0mg/L), 6-BA (0.5,1.0,3.0mg/L), TDZ (0.1,0.5,1.0mg/L), each process 25 bottles, adds up growing height and the growing state of plantlet in vitro after one month.
Table 3 result shows, and the growing state adding different types of basic element of cell division plantlet in vitro is obviously different.Add the plantlet in vitro growing way grown in the medium of ZT prosperous, blade is emerald green, does not have browning, and seedling base portion callus is less, and axillalry bud number is more, and cauline leaf ratio is moderate, is applicable to expanding numerous propagation.Add the plantlet in vitro growing way grown in the medium of 6-BA and TDZ more weak, plant is short and small, and base portion callus is large and partial blade has browning, and the shoot proliferation not being suitable for the little side's persimmon in Nantong is cultivated.The growing way of Integrated comparative seedling, highly, to find to add in medium concentration be the subculture growth that the ZT of 1-3mg/L is more suitable for the little side's persimmon in Nantong to callus size.
The impact that the basic element of cell division of table 3. variety classes, concentration grows plantlet in vitro
Note: English alphabet represents different disposal difference, capitalization represents difference in Duncan analysis and sees significance level (0.01), and lowercase is that significance level (0.05) is as follows.
The impact that the archusia of 2.5 variety classeses, concentration grows plantlet in vitro
The plantlet in vitro being chosen at height about the 10mm of subculture in (1/2N) MS+ZT1.0mg/L+0.1mg/LIAA medium carries out squamous subculture process in mid-term.
The axillalry bud of plantlet in vitro is cut access respectively containing concentration be 0.05,0.1, in (1/2N) MS+ZT1.0mg/L medium of IAA, IBA, NAA of 0.3mg/L, control group CK does not add archusia, each process 25 bottles, adds up growing height and the growing state of plantlet in vitro for 30 days afterwards.
Test finds that the height impact of different types of archusia on little side's persimmon plantlet in vitro is not obvious.From the growing state of seedling, the seedling growth grown in the medium of interpolation IAA and IBA is better than the seedling of adding in NAA.The growing way of adding the archusia seedling of low concentration is better, and excessive concentration can stimulate base portion calli induction, yellow leaf brownization, medium brown stain.Integrated comparative finds that the IBA plantlet in vitro growing way of adding IAA or 0.05mg/L of 0.05-0.3mg/L in medium is better, and base portion callus is little, is substantially more suitable for the growth of Nantong little side's persimmon plantlet in vitro without browning.
The impact that the archusia of table 4. variety classes, concentration grows plantlet in vitro
Embodiment 1
Nantong little side's persimmon sleeping bud cuts from annotinous branch 3, the outside scale peelling off bud, washs bud 2 times in suds, and running water forwards disinfection on superclean bench to afterwards to alleviate explant surface microorganism pollution level in 2 hours.With 75% alcohol surface sterilizing 30s, aseptic water washing 3 times; Containing 10%H
2o
2soak 20min in solution, in immersion process otherwise time shake, make decomposing hydrogen dioxide solution produce a large amount of oxygen and play bactericidal action; Finally use aseptic water washing 3 times.Being inoculated in by sleeping bud after sterilization does not add on the MS medium of growth hormone and the basic element of cell division containing 500mg/LPVP (polyethylene adjoins pyrrolidone), cultivates 2 weeks.
The sleeping bud of growth of sprouting is inoculated in I subculture medium: on MS (1/2N)+zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L, subculture grew for 2 generations, plantlet in vitro growing way is healthy and strong, after height of seedling about 1cm, the medium (II subculture medium) changing MS (1/2N)+zeatin 1mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L into that reduced by half by the zeatin content in medium cultivated for 2 generations.
Growing way stalwartness is chosen, the plantlet in vitro of highly about about 1cm, with (1/2N) MS+ZT1.0mg/L+IAA0.1mg/L+GA after above-mentioned medium squamous subculture Dual culture 4 generation
30.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L and (1/2N) MS+ZT1.0mg/L+IAA0.1mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L medium alternate culture, the plantlet in vitro axillalry bud of last culture is cut insert medium carry out propagation expansion numerous, often kind of medium shoot proliferation cultivates 30 days.
Claims (1)
1. a tissue culture and rapid propagation method for the little side's persimmon in Nantong, is characterized in that:
(1) disinfect
Get the sleeping bud on the little side's persimmon annotinous branch of Nantong, peel off outside 2-3 sheet scale, running water 2-3 hour;
Sleeping bud is relayed on superclean bench, with aseptic water washing after 75% alcohol surface disinfection 30s 3 times; Afterwards sleeping bud is put into 10% hydrogen peroxide and soak 20min, need between soak period not stop to rock to make hydrogen peroxide produce a large amount of bubble, finally use aseptic water washing 3 times, be put in the culture dish being lined with aseptic blotting paper;
(2) to sprout cultivation: the sleeping bud through sterilization is inoculated in do not add growth hormone and the basic element of cell division containing 500mg/LPVP MS medium on cultivate 2 weeks; Cultivation temperature is 25 ~ 28 DEG C, light application time 16 ~ 18h/d, and intensity of illumination is 1500-2000lx;
(3) shoot proliferation is cultivated
Sleeping bud previous step cultivated 2 weeks is inoculated in I subculture medium, and squamous subculture 2-3 uses II subculture medium instead and cultivates 2-3 generation after generation; Described I squamous subculture based formulas is 1/2NMS+ zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; II squamous subculture based formulas is 1/2NMS+ zeatin 1mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; Cultivation temperature is 25 ~ 28 DEG C, light application time 16 ~ 18h/d, and intensity of illumination is 1500-2000lx; In above-mentioned per generation, all cultivates 25-30 days;
Plantlet in vitro squamous subculture 4-5 is for rear III medium and IV medium alternate culture, during alternate culture, the plantlet in vitro axillalry bud of last culture is cut insertion medium and carry out the numerous cultivation of propagation expansion, often kind of medium shoot proliferation cultivates 25-30 days, cultivation temperature is 25 ~ 28 DEG C, light application time 16 ~ 18h/d, intensity of illumination is 1500-2000lx; III culture medium prescription is 1/2NMS+ zeatin 1.0mg/L+ indole-3-acetic acid 0.05-0.3mg/L+ gibberellin GA
30.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L, or 1/2NMS+ zeatin 1.0mg/L+IBA0.05mg/L+ gibberellin GA
30.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; IV culture medium prescription is 1/2NMS+ zeatin 1.0mg/L+ indole-3-acetic acid 0.05-0.3mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L, or 1/2NMS+ zeatin 1.0mg/L+IBA0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L.
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| CN104255499B (en) * | 2014-09-29 | 2016-06-29 | 中国计量学院 | A kind of method of Radix seu Ramulus Diospyroris rhombifoliae tissue culture and rapid proliferation |
| CN104686364A (en) * | 2015-03-31 | 2015-06-10 | 桂林得坤生物科技股份有限公司 | Culture medium for persimmon tree tissue culture |
| CN104663464A (en) * | 2015-03-31 | 2015-06-03 | 桂林得坤生物科技股份有限公司 | Culture medium for persimmon tree tissue culture |
| CN109168687A (en) * | 2018-09-25 | 2019-01-11 | 上海市农业科学院 | A method of vegetative propagation is carried out using loquat stock |
| CN110583485A (en) * | 2019-10-18 | 2019-12-20 | 浙江大学 | Method for inducing and rapidly propagating axillary buds of persimmon |
| CN115053811B (en) * | 2022-06-30 | 2023-06-20 | 中南林业科技大学 | Method for culturing oil persimmon aseptic seedlings |
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| CN102037882A (en) * | 2009-10-09 | 2011-05-04 | 付秀兰 | Novel sweet persimmon cultivation technology |
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| RU2490871C1 (en) * | 2012-04-06 | 2013-08-27 | Общество с ограниченной ответственностью "Агро-консалт" | Method of surface sterilisation of explants and apical buds of pine strawberry, grape, persimmon of variety "korolek" in vitro |
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