CN103988776A - Nantong xiaofang persimmon tissue culture rapid propagation method - Google Patents
Nantong xiaofang persimmon tissue culture rapid propagation method Download PDFInfo
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Abstract
The present invention relates to a Nantong xiaofang persimmon tissue culture rapid propagation method, and belongs to the technical field of crop rapid propagation. According to the present invention, a method for disinfection of dormant buds is researched, culture medium components for early stage and middle stage subculture proliferation of xiaofang persimmon are determined, and influence of different cytokinins and different auxins on tissue culture seedling growth is analyzed; outer layer scales of dormant buds taken from Nantong xiaofang persimmon annual branch are peeled, are rinsed for 2 h by using running water, and are disinfected respectively by using 75% alcohol and 10% hydrogen peroxide, and the disinfected dormant buds are inoculated in a base culture medium, and are transferred to a subculture proliferation culture medium after germination; with the method, the serious browning problem of the Nantong xiaofang persimmon initial generation is basically solved, and the initial generation survival rate achieves 80%; the proper amount of gibberellin is added so as to solve the subculture proliferation problem of the Nantong xiaofang persimmon; and the rapid propagation and production promotion work of the dwarf type persimmon variety Nantong xiaofang persimmon is promoted.
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 (Diospyros kaki L.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, can be processed into again dried persimmon, persimmon vinegar, persimmon wine etc.; Persimmon leaf can do tea, and persimmon paint can be used by fuel feeding umbrella.Persimmon tree performance grace simultaneously, leaf, really beautiful in colour, is a kind of good ornamental tree species.
Persimmon is tall and big deciduous tree, conventionally height of tree 7-9 rice.For a long time, many fruit tree workers make great efforts to seek to have the persimmon germ plasm resource of Dwarfing Gene, dwarf form persimmon kind is the persimmon germ plasm resource of 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 good persimmon kind of Cultivation With Dwarf Varieties And Close Spacing.By being produced to far-reaching shadow, China's cultivation, breeding and development thereof of persimmon from now on think.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 lowly, 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, under flowing water, rinse 2 hours; Sleeping bud is relayed on superclean bench, with aseptic water washing after 75% alcohol surface disinfection 30s (second) 3 times; Afterwards sleeping bud is put into 10% hydrogen peroxide soak 20min (minute), between soak period, must not stop to rock and make hydrogen peroxide produce a large amount of bubbles, finally use aseptic water washing 3 times, be put in the culture dish that is lined with aseptic blotting paper;
To inoculate on the MS medium that contains 500mg/L PVP (polyethylene adjoins pyrrolidone) and do not add growth hormone and the basic element of cell division and cultivate through the sleeping bud of sterilization;
(2) shoot proliferation is cultivated
Sprout on the MS medium sleeping bud of growth of the little side's persimmon in Nantong is inoculated in MS (1/2N) medium, and the basic element of cell division of interpolation is ZT2mg/L, and subculture is cultivated 2-3 generation rear zeatin concentration and is reduced to 1mg/L; Archusia is NAA0.05mg/L; PVP500mg/L; Sucrose 30g/L; Agar 6.0g/L;
Organize training seedling subculture cultivation 4-5 generation rear (seedling growing way stalwartness, highly about 1cm left and right) and use (1/2N) MS+ZT1.0mg/L+IAA0.05-0.3mg/L+GA
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, by the group training seedling axillalry bud of last culture cut insert medium breed expand numerous, every 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, has set up first the group culturation rapid propagating technology of the little side's persimmon in dwarf form tree kind Nantong.
This method has solved the just serious problem of brownization of generation of the little side's persimmon in Nantong substantially, just reaches 80% for survival rate.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.Fast breeding and the production popularization of the little side's persimmon in dwarf form persimmon kind Nantong are promoted.The research of Nantong little side's persimmon fast breeding technique, provides possibility for obtaining fast a large amount of nursery stocks, has promoted popularization and the fast development of dwarf form persimmon kind industry in China.Meanwhile, be established as physiological Study and the molecule aspect of little side's persimmon persimmon group training seedling regenerating system are studied it and are downgraded mechanism proterties etc. and lay a good foundation.
Brief description of the drawings
Fig. 1: the impact that different disinfectants are cultivated sleeping bud
Fig. 2: Nantong little side's persimmon group training seedling picture
Embodiment
The definite materials and methods of the best sterilization method of 1 explant
Nantong little side's persimmon sleeping bud cuts from annotinous branch the outside 2-3 sheet scale of peelling off bud, washs bud section 2-3 time in suds, and flowing water rinses 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: (1) is containing 10%H by following three kinds of methods
2o
2in solution, soak respectively 10min, 20min, in immersion process otherwise time shake makes decomposing hydrogen dioxide solution produce a large amount of oxygen and plays bactericidal action.Finally use aseptic water washing 5 times; (2) in the 5%NaCIO that contains 0.1% tween 1 (antiformin) solution, soak 10min, in immersion process otherwise time shake is finally used aseptic water washing 5 times; (3) at the 0.1%HgCI:(mercuric chloride that contains 0.1% tween 1) soak 10min in solution, in immersion process however time shake, finally use aseptic water washing 5 times.To inoculate in containing 500mg/LPVP (polyethylene adjoins pyrrolidone) and not add on the MS medium of growth hormone and the basic element of cell division through the sleeping bud of distinct methods sterilization, 60 bottles of each processing, statistics 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 respectively 68% and 80% to the carry out disinfection survival rate of 10min, 20min of sleeping bud, and explant is without browning, and survival rate is high.Sprouting, the growth of the active oxygen that while sterilization, decomposing hydrogen dioxide solution produces simultaneously to little side's persimmon sleeping bud has facilitation.So adopt alcohol surface sterilizing 30s when 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 the just dead problem of brownization of generation of sleeping bud.
2. determining 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: 25 DEG C of left and right of cultivation temperature, light application time 16h/d, cultivates under the incandescent lamp that is 1500-2000lx in intensity of illumination.Every Dai Zupei seedling is all cultivated 25-30 days.
The sprout sleeping bud of growth of the little side's persimmon in Nantong is inoculated in being inoculated in I subculture medium, and subculture is cultivated and used No. II (zeatin content reduces by half) subculture medium instead after 2-3 generation and cultivate; Described I subculture medium formula is MS (1/2N)+zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; II subculture medium formula 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 on the impact of Nantong little side's persimmon group training seedling proliferation
The sleeping bud of growth of sprouting is cultivated the Nantong little side's persimmon group training seedling after 2 generations containing subculture on the MS of ZT1.0mg/L+NAA0.05mg/L (1/2N) medium, be seeded in respectively in the MS, the 1/2MS that contain ZT1.0mg/L+NAA0.05mg/L, MS (1/2N) medium the relatively impact of minimal medium on the numerous propagation of Nantong little side's persimmon tissue culture sprout quick.
As shown in table 1, adopt the group of improved culture medium (1/2N) MS cultivation to train the significant difference of depositing of seedling plant height and other two kinds of medium culture, and growing way is good, blade is emerald green greatly, and base portion callus cell is little.Between the group training height of seedling degree of 1/2MS and MS medium culture, difference is not remarkable, and group training seedling is rosette-stape growth substantially, and stem is very short, and a little less than group training seedling growing way, blade is brownization of yellow green part, 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, the especially nitrogen of low concentration.
The different minimal mediums of table 1. are on the impact of Nantong little side's persimmon group training seedling proliferation
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
Tissue culture plant inoculation is arrived to 1.0mg/L ZT, 0.1mg/L IAA, 0.0,0.05,0.1GA
3mS (1/2N) medium in grow after 2 generations, the growing state of statistics group training seedling, the relatively effect of gibberellin in the growth of group training seedling.
Gibberellin can promote cell division, also can show and help promotion cell elongation, and the blade that is conducive to group training seedling expands, stem extends and collateral generation.In medium, add respectively 0.05,0.1mg/L gibberellin cultivates after one month and not add gibberellin and compare between plant height and axillalry bud number and all have significant difference with contrasting, along with gibberellin concentration increases plant height and all increases to some extent of axillalry bud number, the number of blade changes little, stem obviously extends, and has effectively improved the numerous rate of increase of expansion of little side's persimmon.But the training of the group under gibberellin effect seedling growing way obviously dies down, and diminishes and narrows gradually along with concentration increases blade, and in the time that concentration is greater than 0.1mg/L, blade is yellow green willow leaf shape, and plant becomes short brownization.The sprouting of the gibberellin inducement plant axillalry bud that is 0.05mg/L by concentration and stem growth, carry out strong seedling culture and can effectively improve again the propagation multiple of the little side's persimmon in Nantong with the medium that does not add gibberellin, the emerald green growing way of plant leaf is vigorous, and the group training seedling that is all being significantly higher than the medium that contains gibberellin concentration 0.00,0.05,0.1mg/L aspect plant height, axillalry bud number and the number of blade and cultivates separately.
Illustrate that the propagation that adds gibberellin to be conducive to persimmon group training seedling expands numerous in the time of persimmon subculture.Available ZT1.0mg/L+IAA0.1mg/L+GA0.05mg/L and ZT1.0mg/L+IAA0.1mg/L medium alternate culture are organized the Fast-propagation of training seedling.
Table 2: variable concentrations GA
3(gibberellin) affects Nantong little side's persimmon shoot proliferation
2.4 the impact of the basic element of cell division of variety classes, concentration on the growth of group training seedling
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 is the experiment of the impact on the growth of group training seedling for the basic element of cell division that carries out variety classes, concentration at the group training seedling of about 3cm.
The axillalry bud of group training seedling is cut in (1/2N) MS+NAA0.05mg/L medium that access respectively contains 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), 25 bottles of each processing, growing height and the growing state of statistics group training seedling after one month.
Table 3 result shows, adds the obviously difference of growing state of different types of basic element of cell division group training seedling.Add the group training seedling growing way of growing in the medium of ZT prosperous, blade is emerald green, there is no 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 group training seedling growing way of growing in the medium of 6-BA and TDZ a little less than, plant is short and small, the large and partial blade of base portion callus has browning, the shoot proliferation that is not suitable for the little side's persimmon in Nantong is cultivated.The growing way of comprehensive relatively 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 of the basic element of cell division of table 3. variety classes, concentration on the growth of group training seedling
Note: English alphabet represents different disposal difference, capitalization represents that in Duncan analysis, difference is shown in significance level (0.01), lowercase is that significance level (0.05) is as follows.
2.5 the impact of the archusia of variety classes, concentration on the growth of group training seedling
The group training seedling that is chosen at the height 10mm left and right of subculture in (1/2N) MS+ZT1.0mg/L+0.1mg/LIAA medium carries out subculture cultivation in mid-term to be processed.
By the axillalry bud of group training seedling cut that access respectively contains that concentration is 0.05,0.1, in (1/2N) MS+ZT1.0mg/L medium of IAA, the IBA of 0.3mg/L, NAA, control group CK does not add archusia, 25 bottles of each processing, 30 days growing height and the growing states of statistics group training seedling afterwards.
Test finds that different types of archusia is not obvious to the effect of altitude of little side's persimmon group training seedling.From the growing state of seedling, the seedling growth of growing in the medium of interpolation IAA and IBA is better than the seedling of adding in NAA.The growing way of the archusia seedling of interpolation low concentration is better, and excessive concentration can stimulate the formation of base portion callus, brownization of yellow leaf, medium brown stain.Comprehensive relatively find to add the IAA of 0.05-0.3mg/L in medium or the IBA of 0.05mg/L group training seedling growing way is better, base portion callus is little, the growth that substantially be more suitable for Nantong little side's persimmon group and train seedling without browning.
The impact of the archusia of table 4. variety classes, concentration on the growth of group training seedling
Embodiment 1
Nantong little side's persimmon sleeping bud cuts from annotinous branch 3 of the outsides scale of peelling off bud, washs bud 2 times in suds, and flowing water rinses 2 hours to alleviate explant surface microorganism pollution level, forwards afterwards disinfection on superclean bench to.With 75% alcohol surface sterilizing 30s, aseptic water washing 3 times; Containing 10%H
2o
2in solution, soak 20min, in immersion process otherwise time shake makes decomposing hydrogen dioxide solution produce a large amount of oxygen and plays bactericidal action; Finally use aseptic water washing 3 times.Sleeping bud after sterilization is inoculated in containing 500mg/LPVP (polyethylene adjoins pyrrolidone) and do not added on the MS medium of growth hormone and the basic element of cell division, cultivate 2 weeks.
The sleeping bud of the growth of sprouting is inoculated to the upper subculture of subculture medium: MS (1/2N)+zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L in I grew for 2 generations, group training seedling growing way stalwartness, behind height of seedling 1cm left and right, the zeatin content in medium is reduced by half and changes medium (II subculture medium) 2 generations of cultivation of MS (1/2N)+zeatin 1mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L into.
Above-mentioned medium subculture is chosen growing way stalwartness after cultivating and cultivating altogether for 4 generations, and the group training seedling of highly about 1cm left and right, with (1/2N) MS+ZT1.0mg/L+IAA0.1mg/L+GA
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, by the group training seedling axillalry bud of last culture cut insert medium breed expand numerous, every kind of medium shoot proliferation cultivation 30 days.
Claims (1)
1. the group of the little side's persimmon in a Nantong training tissue culture method for fast propagation, 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, flowing water rinses 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 soaked 20min, between soak period, must not stop to rock and make hydrogen peroxide produce a large amount of bubbles, finally use aseptic water washing 3 times, be put in the culture dish that is lined with aseptic blotting paper;
(2) cultivation of sprouting: cultivate 2 weeks inoculating through the sleeping bud of sterilization on the MS medium that contains 500mg/L PVP and do not add growth hormone and the basic element of cell division; 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
The sleeping bud that previous step is cultivated 2 weeks is inoculated in I subculture medium, and subculture is used II subculture medium cultivation 2-3 generation instead after cultivating 2-3 generation; Described I subculture medium formula is MS (1/2N)+zeatin 2mg/L+ methyl α-naphthyl acetate 0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L; II subculture medium formula is MS (1/2N)+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, is all cultivated 25-30 days.
It is rear with III medium and IV medium alternate culture that group training seedling subculture is cultivated 4-5 generation, when alternate culture, the group training seedling axillalry bud of last culture is cut to insertion medium and breed the numerous cultivation of expansion, every kind of medium shoot proliferation is cultivated 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 (1/2N) MS+ 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 (1/2N) MS+ 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 (1/2N) MS+ zeatin 1.0mg/L+ indole-3-acetic acid 0.05-0.3mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L, or (1/2N) MS+ zeatin 1.0mg/L+IBA0.05mg/L+PVP500mg/L+ sucrose 30g/L+ agar 6.0g/L.
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Cited By (6)
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| CN104255499A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Diospyros rhombifolia tissue culture and immediate propagation method |
| 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 |
| 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 |
| 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 |
| CN115053811A (en) * | 2022-06-30 | 2022-09-16 | 中南林业科技大学 | Method for culturing aseptic seedlings of persimmon |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104255499A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Diospyros rhombifolia tissue culture and immediate propagation method |
| 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 |
| 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 |
| 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 |
| 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 |
| CN115053811A (en) * | 2022-06-30 | 2022-09-16 | 中南林业科技大学 | Method for culturing aseptic seedlings of persimmon |
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