CN107258536A - A kind of eucalyptus breeding tail eucalyptus camaldulensis DH191 7 tissue culture and rapid propagation method - Google Patents
A kind of eucalyptus breeding tail eucalyptus camaldulensis DH191 7 tissue culture and rapid propagation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a kind of eucalyptus breeding tail eucalyptus camaldulensis DH191 7 tissue culture and rapid propagation method, this method uses the clone select trees of DH191 7, sprouts of stump is carried out to it, outside shade is used as using stem section of the rudiment bar with resting bud, in-vitro inducing sterile bud, without callus approach, lateral bud is directly germinated by stem section, sterile bud obtains regeneration plant after the links such as shoot proliferation culture, culture of rootage and transplantation of seedlings of taking root.The present invention is using eucalyptus breeding as breeding object, with the direct seedling of bud organ in-vitro inducing sterile bud mode, regeneration plant can completely keep the excellent inhereditary feature of select tree, be not susceptible to variation, and this method operation difficulty is low, production cost is low, nursery stock goes out root rate height, and transplanting survival rate is high, as a result stablizes, it is repeated good, can be with large-scale industrialized nursery.
Description
Technical field
The present invention relates to eucalyptus panting technical field, more particularly to a kind of eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture
Quick-breeding method.
Background technology
Eucalyptus originates in Australia, is Myrtaceae（Mytaceae) eucalyptus belongs to（Eucalyptus), cup fruit tree category
（Angophora）, umbrella room category（Corymbia）The general designation of 3 category seeds.Have 1039 kinds, subspecies and mutation, wherein eucalyptus
Belong to up to 945 kinds of seeds, geographical distribution span is big, and performance difference is also big.Country's introducing and planting, equal table more than 100 in the world
Reveal strong adaptability, grow the merits such as fast, yield is high, the period of felling in turn is short.Eucalyptus Wood can be used for car and boat, building, mine, people
Plate is made, and is important pulp and paper industry raw material, is to cultivate one of optimal seeds of short-cycle industrial material.At present
As one of three universally acknowledged big artificial forest seeds.
Nineteen eighty-two, middle Australia's technological cooperation --- the east gate eucalyptus demonstration forest project implementation, the state-owned east gate forest farm in Guangxi is big from Australia
Seven countries such as Leah, Indonesia, have introduced 174 eucalypt species/introduces a collections, introduction and cultivation, survey by science
After fixed, analysis, determine this 6 seeds of Eucalyptus urophylla, alpine ash, eucalyptus camaldulensis, garden angle eucalyptus, Eucalyptus pellita, Eucalyptus cloeziana with being adapted to In South China
Area grows.Then, artificialpollination is carried out to the Eucalyptus urophylla of successful introduction, alpine ash, eucalyptus camaldulensis, garden angle eucalyptus, passes through crossbreeding
Hereditary basis is improved, and obtains the hybrid generation that a large amount of genetic gain height, strong stress resistance, increment are protruded, and is built up
The eucalyptus germ plasm resource gene pool of Fia.Present east gate forest farm is national eucalyptus Seed multiplication base, Eucalyptus In Guangxi prevalent variety cultivation
Center, national characteristic seedling base.
Because eucalyptus is the perennial woody plant of cross-pollination, easy natural hybrization, after the filial generation of generation is bred, character
Occur serious separation, be difficult to keep its good characteristic with sexual reproduction method, using vegetative propagations such as traditional cuttage, graftings
Method breeding nursery stock, usually because select tree material source is not enough, mating period and seedling raising environment are restricted, seedling cost height etc.
Factor, constrains the speed of popularization and application.Eucalyptus breeding is bred using group culturation rapid propagating technology, the heredity of select tree can be kept special
Property, the purpose quickly bred can be reached again, be to produce the widely used method of eucalyptus choiceness.
1989, E.urophylla×E.grandis hybrid generation bred success, and scale popularization and afforestation through tissue culture.
Eucalyptus successful introduction and the popularizing planting after crossbreeding and improvement, the development to China Forest serve prominent effect.
According to statistics, by 2013, Chinese 4,400,000 hectares of eucalyptus plantation area, the 2.2% of Jin Zhan China forest gross area, but provide
Timber accounts for the 25% of national timber yield, because the ability of eucalyptus plantation production timber is 4-10 times of natural shaw, institute
To have eucalyptus plantation, also be considerably reduced the pressure cut down to wildwood, therefore, eucalyptus provides to alleviate China's timber
The shortage and imbalance between supply and demand in source, to safeguard that China's ecological safety aspect has played prominent effect.
Due to the tail alpine ash of present popularizing planting, cold resistance is limited, and Bioclimatic analysis is smaller, afforest ground area expansion by
Restriction, in order to obtain the timber required for more production and construction, there is two approach, one is to cultivate cold-resistant eucalypt species, facilitates eucalyptus
Tree northwards develop, to expand afforestation area, two be to breed high-yield variety, improve instant forest unit area timber yield, with up to
To producing more timber on limited soil.
The eucalyptus breeding tail eucalyptus camaldulensis DH191-7 of east gate forest farm seed selection, has two excellent product of fast growing and cold resistance concurrently
Matter, it is the filial generation hybridized by Eucalyptus urophylla and eucalyptus camaldulensis by artificialpollination, from F1In generation, selects excellent acquisition select tree, through pierre
Into clone.Eucalyptus urophylla originates in Indonesia, with preferable fast growing, and growth is fast, and yield is high, but cold resistance
Poor, eucalyptus camaldulensis originates in Australia, and it has good adaptability to poor environments such as cold sweltering heats, there is wider life to temperature
Amplitude of adaptation is managed, its winter resistance is more than alpine ash, so, using Eucalyptus urophylla as female parent, alpine ash is male parent, the tail eucalyptus camaldulensis of selection cross
DH191-7, yielding ability and winter resistance are all than more prominent, in clone contrast tests, and DH191-7 performances are good, and 5.5 years raw
DH191-7 Stock growths reach 198.06m3/hm2, average annual Stock growth reach 36.01m3/hm2；Examination is promoted in regionality afforestation
In testing, DH191-7 growths are rapid, and winter resistance is good, and in the northern regional testing in osmanthus, 4.1 years raw stand average breast diameter 12.9cm are put down
The equal height of tree 17.2m, Stock growth 178.0m3/hm2, the average annual m of Stock growth 43.43/hm2, in the southern regional testing in osmanthus, 4
Year raw stand average breast diameter 13.2cm, mean height 17.1m, Stock growth 137.5m3/hm2, average annual Stock growth 34.4
m3/hm2, in the regional testing of Guangdong, 4.1 years raw stand average breast diameter 10.3cm, mean stand height 15.2m, Stock growth
149.4m3/hm2, average annual Stock growth 36.4m3/hm2, in addition, DH191-7 is dry-shaped straight, volume recovery is high, and wood quality is good,
Texture is straight, is detected through Wood Properties Within, and it is that a relatively good solid wood utilizes type commerical tree species to show tail eucalyptus camaldulensis DH191-7.Because
Above-mentioned good characteristic, the Guangxi forest variety certification committee was in 2013, after expert appraisal, authorized Guangxi high quality tree species.
Quick this eucalyptus breeding of popularization DH191-7 on a large scale is realized, is maximally effective way using group culturation rapid propagating technology
Footpath.Tissue culture factory was set up in east gate forest farm in 1992, had successfully bred more than 100 eucalyptus choiceness so far, cultivated tissue-cultured seedling
Nearly 1,000,000,000 plants.Since by the way of bud organ in-vitro inducing sterile bud, non-callus Plantlet formation way, so tissue-cultured seedling can
The good characteristic of complete holding select tree, is not susceptible to forest form is neat after variation, afforestation, and increment is high, is ground using same method
Study carefully DH191-7 group culturation rapid propagating technology, achieve success.
The domestic research to eucalyptus camaldulensis tissue culture at present is very more, but studies then less for tail eucalyptus camaldulensis tissue culture, 2007
Zhang Yuanhua etc. is to east gate forest farm D2、D6、D5Tissue-culturing rapid propagation has been carried out to succeed；Chen Jian in 2006 is bravely waited to tail eucalyptus camaldulensis carry out group
Training is numerous soon to succeed, but without specific clone title, originates also indefinite；Zhang Lianshui in 2006 etc. is to tail eucalyptus camaldulensis
DH201 tissue-culturing rapid propagations succeed, but DH201 is the hybrid generation of alpine ash and garden angle eucalyptus, not tail eucalyptus camaldulensis.
The content of the invention
It is an object of the invention to the deficiency for above technology a large amount of inhereditary features are quickly obtained there is provided one kind unanimously,
The regeneration plant of elite stand merit can be completely kept, as a result the regeneration plant obtained using this method is stablized, repeatability is good
Tail eucalyptus camaldulensis DH191-7 tissue culture regeneration method.
To realize the purpose of the present invention, adopt the following technical scheme that：
A kind of eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, is the cuttage bred using the former strains of tail eucalyptus camaldulensis DH191-7
Fine individual plant in seedling clone stands, sprouts of stump is carried out to it, using the rudiment bar at nearly rhizome, is taken with resting bud
Semi-lignified stem section makees explant, in-vitro inducing sterile bud, by the shoot proliferation culture of sterile bud, culture of rootage and taking root
Transplantation of seedlings, obtains regeneration plant.
The step of this method, is as follows：
（1）From east gate forest farm Eucalyptus clone contrast experiment determine woods in, by the METHOD FOR CONTINUOUS DETERMINATION diameter of a cross-section of a tree trunk 1.3 meters above the ground, the height of tree, Wood Properties Within with
And stem shape indix, through calculating accumulated, current annual increment, comprehensive analysis contrast after, select choiceness DH191-7,
Then in DH191-7 clones, selection, on-the-ground assessment, select DH191-7 fine individual plants again.
（2）DH191-7 fine individual plants are cuted down at 10-15cm height from the ground, promote rudiment, when rudiment bar grows to 10-
During 15cm, clip rudiment bar, be soaked in water base portion, takes back laboratory standby.
（3）Rudiment bar prunes away blade, rinses 30min with running water flowing water, then washed with saturation washing powder solution
10min, is cleaned up with pure water.
（4）On superclean bench, rudiment bar is cut off into tender tip section and base portion aging part, long 2-3cm is cut, remains with
Surface inactivation material is done in the segment of 1-2 resting bud.
（5）Material to be inactivated is placed in the treated aseptic bottle of autoclaving, with the alcoholic solution that volume fraction is 75%
15-17s is soaked, sterile water wash 3-5 times washes away residual alcohol, then with mass fraction be 0.1%HgCL2Solution soaks, and fits
HgCL is removed after working as stirring, 7-9min2Solution, with sterile water wash material 5-6 times, washes away into special collecting tank and remains in material
Expect the HgCL on surface2Solution.
（6）Cleaned stem section is gripped with the tweezers after sterilizing, is placed on the inoculation dish of sterilizing, is blotted with aseptic filter paper
It is attached to after material surface moisture, cuts the two ends of petiole and stem section, is inoculated on sterile bud inducing culture, every bottle of inoculation one
Branch explant, full light culture 8-12d, 26 ± 2 DEG C of temperature, the axillary bud not medium well that starts to sprout is long, is transferred under illumination condition and cultivates
15-20d, light application time 12h/d, intensity of illumination 1500-2000lux, 26 ± 2 DEG C of cultivation temperature.
（7）The sterile bud without pollution is chosen, is transferred in subculture multiplication medium and cultivates 18-23 days after cutting；Cultivate bar
Part：26 ± 2 DEG C of temperature, intensity of illumination 2000-2500lux.
（8）Sterile bud culture terminates, and chooses the long 2-3cm of sprout, and has the subculture sprout of 3-4 section, is cut into long 1.5-
Cultivated on 1.8cm simple bud, insertion root media, every bottle is inoculated with 25 plants, condition of culture：28-30 DEG C of temperature, intensity of illumination
1200-1500lux；After culture 6-10 days, treat that otch is emerged short or root point, when going out root rate and reaching 60%, shifting bottle to temperature 28-
35 DEG C, continue under intensity of illumination 3000-5000lux natural light to cultivate 15-20 days；Under growth root bottle seedling is up to 3.0-4.5cm, leaf
4-6 pairs of piece, well developed root system, culture terminates when master root is obvious.
（9）By height of seedling 3.0-4.5cm, the bottle seedling of taking root of well developed root system washes away agar, transplants what is extremely sterilized with KMnO4
Yellow soil and vermiculite are done on the nutrition cup or Light media seedling-raising cup of matrix, and the ratio of yellow soil and vermiculite is 5：1, cultivate 60-80
My god, obtain height of seedling 20-25cm, ground diameter 0.3-0.4cm regeneration plant.
In above-mentioned eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, described breeding DH191-7 clones are
Refer to, the eucalyptus camaldulensis introduced a fine variety from Australia, the Eucalyptus urophylla introduced a fine variety from Indonesia carries out provenance test in east gate forest farm, selected
Excellent suitable non-hibernating eggs source and family, select fine individual plant in family, and using Eucalyptus urophylla as female parent, eucalyptus camaldulensis is male parent, artificial by AIP
Controlled pollination techniques, obtain Hybrid F1 generation, are analyzed by being determined to 6 years raw F1 generation family trials, in F1 generation family trial
Superior families DH191 is selected, select tree is selected in DH191 familys, wherein No. 7 select tree claims tail eucalyptus camaldulensis DH191-7, DH191-
7 by cutting propagation, obtains reproductive population --- DH191-7 ortets, the material of this tissue-culturing rapid propagation research institute, just
It is taken from the fine individual plant in DH191-7 ortets.
In above-mentioned eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, step（6）In sterile bud inducement cultivation
Base（Initial culture base）Constitute and be：Improve MS+N6Benzyladenine（6-BA) 1.0mg/L+ methyl α-naphthyl acetates（NAA）0.2-0.4mg/L+
The mg/L+ cysteine 5.0g/L+ sucrose 30g/L+ agar of 2.0 mg/L+ riboflavin (vitamin B2) of vitamin C (Vc) 7.0
4.0g/L, pH 6.0-6.2.
In above-mentioned eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, step（7）In subculture increment culture medium
Constitute and be：Improve MS+N6Benzyladenine（6-BA) 0.3-0.5mg/L+ methyl α-naphthyl acetates (NAA) 0.1-0.15mg/L+ riboflavin (dimension
Raw element B2) 7.0 mg/L+ vitamin Cs (Vc) 2.0mg/L+ sucrose 30g/L+ agar 4.0g/L, pH 5.8-6.0.
In above-mentioned eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, step（8）In root media composition
For：1/2 improvement MS+ABT10.6mg/L+IBA0.1mg/L+ sucrose 15g/L+ agar 4.2g/L, pH 6.0-6.4.
The modified MS medium refers to：In MS（murashige and skoog.1962）On the basis of culture medium, adjust
Whole a great number of elements, trace element, the concentration of organic matter, and add part former base basal culture medium without nutriment, be modulated into
It is exclusively used in specific Object of Development DH191-7 culture medium.
ABT in root media of the present invention1Can by reinforcing, the regulation and control content of plant endogenous hormones, important enzyme work
Property, promote the morphogenesis of the synthesis of biomolecule, induction plant adventitious root or adventitious bud, adjust plant metabolism action intensity,
So as to improve the survival rate of tissue-cultured seedling；IBA can promote plant main root to grow, and improve germination percentage, survival rate.It is of the invention other
Cultivate that based compound can chemically dictionary or the textbooks of arviculture be to its title and effect, the present invention uses this
A little compounds are combined obtained culture medium, and tail eucalyptus camaldulensis DH191-7 is adapted to the most, are just final by test of many times screening
Determine.
Beneficial effects of the present invention are：
1. the object that the present invention is studied is the eucalyptus breeding authorized through Guangxi Zhuang Autonomous Region forest variety certification committee, have
Two fine qualities of fast-growing character and winter resistance, have important economic benefit and social benefit after successfully promoting.
2. the present invention uses the clonal fine individual plant rudiments of tail eucalyptus camaldulensis DH191-7 as outside shade, in tissue-culturing rapid propagation side
In method, tissue culture sterile bud acquisition pattern is by bud organ（Stem section with resting bud）In-vitro inducing Multiple Buds, without callus group
Knit and do not pass through Dedifferentiation in approach, sterile bud Induction Process, the tissue-cultured seedling stabilization characteristics of genetics of cultivation can be protected completely
The hereditary capacity of elite stand is held, variation is not susceptible to.
3. the tail eucalyptus camaldulensis DH191-7 of invention tissue culture and rapid propagation method operation difficulty is low, production cost is low, rooting rate is up to
More than 96%, the tissue culture shoot survival percent of production is high, strong adaptability, can be promoted the use of interior on a large scale, using the present invention's
DH191-7 tissue culture and rapid propagation method, a large amount of neat and consistents, the regeneration plant of stabilization characteristics of genetics, examination can be obtained in a short time
Test result stable, repeatability is good, can be applied to large-scale commercial nursery, nursery stock can keep the excellent heredity of select tree
Shape, is adapted to large area region plantation.
Brief description of the drawings
Fig. 1 is tail eucalyptus camaldulensis DH191-7 breeding certificate；
Fig. 2 is the fine individual plant in tail eucalyptus camaldulensis DH191-7 clone stands；
Fig. 3 is the rudiment after fine individual plant in tail eucalyptus camaldulensis DH191-7 is cuted down；
Fig. 4 is tail eucalyptus camaldulensis DH191-7 select trees rudiment bar as outside shade, the sterile bud of induction germinating；
Fig. 5 is tail eucalyptus camaldulensis DH191-7 shoot proliferation seedlings；
Fig. 6 is the bottle seedling of taking root of tail eucalyptus camaldulensis DH191-7 sterile buds induction；
Fig. 7 is seedling growing state after tail eucalyptus camaldulensis DH191-7 tissue-culture container seedlings are transplanted；
Fig. 8 is tail eucalyptus camaldulensis DH191-7 plantlet in vitro regeneration plant, will go out the seedling of garden afforestation.
Fig. 9 is the standing forest after 4 years raw DH191-7 tissue-cultured seedling afforestation of tail eucalyptus camaldulensis.
Embodiment
Embodiment below to the present invention, case study on implementation are described in detail, so that advantages of the present invention and spy
Levying can be easier to be readily appreciated by one skilled in the art.
Embodiment 1
The specific method that eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue-culturing rapid propagations obtain regeneration plant is as follows：
（1）In eucalyptus breeding tail eucalyptus camaldulensis DH191-7 first generation clone stands, by determining the diameter of a cross-section of a tree trunk 1.3 meters above the ground, the height of tree, Wood Properties Within
And stem shape indix, fine individual plant is selected after calculating analysis, object is bred as tissue-culturing rapid propagation, it is determined that breeding object and being
The first generation plant division of DH191-7 ortets.
（2）DH191-7 fine individual plants are cuted down at 10-15cm height from the ground, promote rudiment, when rudiment bar grows to 10-
During 15cm, clip rudiment bar, be soaked in water base portion, takes back laboratory standby.
（3）Rudiment bar prunes away blade, rinses 30min with running water flowing water, then washed with saturation washing powder solution
10min, is cleaned up with pure water.
（4）On superclean bench, rudiment bar is cut off into tender tip section and base portion aging part, semi-lignified stem section is stayed, cuts
Grow up 2-3cm, and pasteurization material is done in the segment for remaining with 1-2 resting bud.
（5）Material to be sterilized is placed in the treated aseptic bottle of autoclaving, 15-17s is soaked with 75% alcoholic solution,
Sterile water wash 3-5 times, washes away residual alcohol, then use 0.1%HgCL2Solution is soaked and suitably stirred, and HgCL is removed after 7-9min2
Solution is into special collecting tank, with sterile water wash material 5-6 times.
（6）Cleaned stem section is gripped with the tweezers after sterilizing, is placed on the inoculation dish of sterilizing, with the filter paper sterilized
Blot after surface moisture, cut the two ends of petiole and stem section, be inoculated on sterile bud inducing culture, one explant of every bottle of inoculation
Body, full light culture is after 8-12 days, and axillary bud starts to sprout, and is transferred under illumination condition and cultivates, illumination 12h/d, intensity of illumination 1500-
2000lux, 26 ± 2 DEG C of cultivation temperature, under the conditions of cultivate 15-20 days.Sterile bud inducing culture（Initial culture base）Constitute and be：
Improve MS+N6Benzyladenine（6-BA) 1.0mg/L+ methyl α-naphthyl acetates（NAA）The mg/L+ of 0.2-0.4mg/L+ vitamin Cs (Vc) 2.0
Riboflavin (vitamin B2) 7.0 mg/L+ cysteine 5.0g/L+ sucrose 30g/L+ agar 4.0g/L, pH 6.0-6.2.
（7）Choose without the sterile bud polluted, be transferred to after cutting in subculture multiplication medium, in 26 ± 2 DEG C of temperature, illumination
Under the conditions of intensity 2000-2500lux, cultivate 18-23 days, with promote clump bud breed and growth, on proliferated culture medium repeatedly after
It is commissioned to train foster, sterile sprout quantity is on the increase, and is increased by geometric progression, 4-4.5 times of growth coefficient；Subculture increment culture medium composition
For：Improve MS+N6Benzyladenine（6-BA) 0.3-0.5mg/L+ methyl α-naphthyl acetates (NAA) 0.1-0.15mg/L+ riboflavin (vitamins
B2) 7.0 mg/L+ vitamin Cs (Vc) 2.0mg/L+ sucrose 30g/L+ agar 4.0g/L, pH 5.8-6.0.
（8）The long 2-3cm of sprout is chosen, and has the subculture sprout of 3-4 section, long 1.5-1.8cm simple bud, insertion life is cut into
Cultivated on root culture medium, every bottle is inoculated with 25 plants, and 28-30 DEG C of cultivation temperature, intensity of illumination 1200-1500lux is cultivated 6-10 days,
When otch is emerged short or root point, when going out root rate and reaching 60%, bottle is moved to 28-35 DEG C of temperature, intensity of illumination 3000-5000lux's
Cultivated 15-20 days under natural light, height of seedling reaches 3.0-4.5cm, 4-6 pairs of blade, well developed root system, master root is obvious.Root media
Constitute and be：1/2 improvement MS+ABT10.6mg/L+IBA0.1mg/L+ sucrose 15g/L+ agar 4.2g/L, pH 6.0-6.4.
（9）As height of seedling 3.0-4.5cm, the bottle seedling of taking root taken root of well developed root system washes away agar, transplants to using KMnO4Disappear
The yellow soil and vermiculite that poison is crossed are done on the nutrition cup or Light media seedling-raising cup of matrix, and the ratio of yellow soil and vermiculite is 5：1, culture
60-80 days, obtain height of seedling 20-25cm, ground diameter 0.3-0.4cm regeneration plant.
Embodiment 2
（1）In the block tail eucalyptus camaldulensis DH191-7 of east gate forest farm 25 first generation clone stands, by determining the diameter of a cross-section of a tree trunk 1.3 meters above the ground, the height of tree, branch
The indexs such as lower high and form, select fine individual plant after calculating analysis, object are bred as tissue-culturing rapid propagation, it is determined that breeding object and being
The first generation plant division of DH191-7 ortets.
（2）DH191-7 fine individual plants are cuted down rush at 15cm height from the ground and sprouted by selection in spring, the morning without rain,
After 25 days, when rudiment bar grows to 10-15cm, the rudiment bar at the continuous sunny morning of more than 3 days, the nearly rhizome of clip is selected,
Be soaked in water base portion, takes back laboratory standby；
（3）Rudiment bar prunes away blade, stays petiole, rinses 30min with running water flowing water, then washed with saturation washing powder solution
10min, is cleaned up with pure water；On superclean bench, rudiment bar is cut off into tender tip section and base portion aging part, clip is long
2-3cm, the semi-lignified stem section with 1-2 resting bud completes the preliminary treatment of explant as Surface inactivation material.
（4）Material to be sterilized is placed in the treated aseptic bottle of autoclaving, 15-17s is soaked with 75% alcoholic solution,
Alcohol is removed, it is quick to use sterile water wash 3-5 times, residual alcohol is washed away, then use 0.1%HgCL2Solution soaks and suitably stirred,
HgCL is removed after 8min2Solution is into special collecting tank, with sterile water wash material 5-6 times；Go out on the surface for completing propagating materials
Processing living.
（5）Cleaned stem section is gripped with the tweezers after sterilizing, is placed on the inoculation dish of sterilizing, with the filter paper sterilized
Blot after surface moisture, cut the two ends of petiole and stem section, be inoculated on sterile bud inducing culture, one explant of every bottle of inoculation
Body, full light culture is after 10 days, and axillary bud starts to sprout, and is transferred under illumination condition and cultivates, light application time 12h/d, intensity of illumination 1500-
2000lux, 26 ± 2 DEG C of cultivation temperature, under the conditions of cultivate 18 days, the expansion of the axillary bud of germinating is changed into green by white or blush
Color, forms the clearly demarcated budlet of cauline leaf.
（6）Choose without the sterile bud polluted, be transferred to after cutting in subculture multiplication medium, in 26 ± 2 DEG C of temperature, illumination
Under the conditions of intensity 2000-2500lux, cultivate 18-23 days, with promote clump bud breed and growth, on proliferated culture medium repeatedly after
It is commissioned to train foster, sterile sprout quantity is on the increase, and is increased by geometric progression, 4.0 times of growth coefficient；
（7）The long 2-3cm of sprout is chosen, and has the subculture sprout of 3-4 section, long 1.5-1.8cm simple bud is cut into, inserts training of taking root
Support and cultivated on base, every bottle is inoculated with 25 plants, and 28-30 DEG C of cultivation temperature, intensity of illumination 1200-1500lux is cultivated 6-10 days, when cutting
Mouth is emerged short or root point, when going out root rate and reaching 60%, moves bottle to 28-35 DEG C of temperature, intensity of illumination 3000-5000lux nature
Cultivated 20 days under light.
（9）Rooted seedling is constantly grown tall, and leaf area expands, and as height of seedling 3.0-4.5cm, development forms the bottle of taking root of complete root system
Miao Shi, washes away agar, transplants to using KMnO4The yellow soil and vermiculite sterilized does the nutrition cup or Light media seedling-raising cup of matrix
On, the ratio of yellow soil and vermiculite is 5：1, cultivate 60-80 days, during which carry out imposing N, P, K fertilizer or compound containing N, P, K
Fertilizer, and necessary pest control measure is done, obtain height of seedling 20-25cm, ground diameter 0.3-0.4cm regeneration plant.
Claims (6)
1. a kind of eucalyptus breeding tail eucalyptus camaldulensis DH191-7 tissue culture and rapid propagation method, it is characterised in that：Using Guangxi Zhuang Autonomous Region woods
The eucalyptus breeding tail eucalyptus camaldulensis DH191-7 ortets of wooden variety certification committee authorization, are cuted down after rush sprouts, latent with rudiment bar band
The stem section of bud is lied prostrate as outside shade, in-vitro inducing sterile bud directly germinates lateral bud by stem section, sterile bud through shoot proliferation culture,
After culture of rootage and transplantation of seedlings link of taking root, regeneration plant is obtained.
2. eucalyptus breeding tail eucalyptus camaldulensis DH191-7 according to claim 1 tissue culture and rapid propagation method, it is characterised in that：It is described
Method is comprised the following steps that：
（1）In eucalyptus breeding tail eucalyptus camaldulensis DH191-7 clonal test woodss, the former strain of select tree is selected；
（2）The former strain of DH191-7 select trees is cuted down at 10-15cm height from the ground, promotees rudiment, when rudiment bar length to 10-15cm
When, clip rudiment bar, be soaked in water base portion, takes back laboratory standby；
（3）Rudiment bar prunes away blade, and 30min is rinsed with running water flowing water, then washs 10min with saturation washing powder solution, uses
Pure water is cleaned up；
（4）On superclean bench, rudiment bar is cut off into tender tip section and base portion aging part, long 2-3cm is cut, remains with 1-2
Surface inactivation material is done in the segment of individual resting bud；
（5）Material to be inactivated is placed in the treated aseptic bottle of autoclaving, soaked with volume fraction for 75% alcoholic solution
15-17s, sterile water wash 3-5 times washes away residual alcohol, then with mass fraction is 0.1%HgCL2Solution soaks, and suitably stirs
Mix, HgCL is removed after 7-9min2Solution, with sterile water wash material 5-6 times, washes away into special collecting tank and remains in material list
The HgCL in face2Solution；
（6）Cleaned stem section is gripped with the tweezers after sterilizing, is placed on the inoculation dish of sterilizing, attachment is blotted with aseptic filter paper
After material surface moisture, the two ends of petiole and stem section are cut, are inoculated on sterile bud inducing culture, every bottle of inoculation one is outer
Implant, full light culture 8-12d, 26 ± 2 DEG C of temperature, the axillary bud not medium well that starts to sprout is long, is transferred under illumination condition and cultivates 15-
20d, light application time 12h/d, intensity of illumination 1500-2000lux, 26 ± 2 DEG C of cultivation temperature；
（7）The sterile bud without pollution is chosen, is transferred in subculture multiplication medium and cultivates 18-23 days after cutting；Condition of culture：
26 ± 2 DEG C of temperature, intensity of illumination 2000-2500lux；
（8）The long 2-3cm of sprout is chosen, and has the subculture sprout of 3-4 section, long 1.5-1.8cm simple bud is cut into, inserts training of taking root
Support and cultivated on base, every bottle is inoculated with 25 plants, condition of culture：28-30 DEG C of temperature, intensity of illumination 1200-1500lux；Culture 6-10 days
Afterwards, treat that otch is emerged short or root point, when going out root rate and reaching 60%, move bottle to 28-35 DEG C of temperature, intensity of illumination 3000-
Continue to cultivate 15-20 days under 5000lux natural light；Under growth root bottle seedling is up to 3.0-4.5cm, 4-6 pairs of blade, well developed root system,
Culture terminates when master root is obvious；
（9）The bottle seedling of taking root of well developed root system is chosen, the agar of root adhesion is washed away, transplanted to using KMnO4The yellow soil that sterilized and
Vermiculite is done on the nutrition cup or Light media seedling-raising cup of matrix, is cultivated 60-80d, is obtained height of seedling 20-25cm, ground diameter 0.3-0.4cm's
Regeneration plant；Yellow soil and the vermiculite ratio is 5:1.
3. the tissue culture and rapid propagation method of the eucalyptus breeding tail eucalyptus camaldulensis DH191-7 according to claim any one of 1-2, its feature exists
In：Described eucalyptus breeding tail eucalyptus camaldulensis DH191-7 is cultivated by east gate forest farm, is entrusted through Guangxi Zhuang Autonomous Region forest variety certification
Eucalyptus urophylla × eucalyptus camaldulensis that member can authorize（E.urophylla ×E.camaldulensis）What the fine individual plant of hybrid filial generation bred
Clone, character is：Fast growing and cold resistance.
4. eucalyptus breeding tail eucalyptus camaldulensis DH191-7 according to claim 2 tissue culture and rapid propagation method, it is characterised in that：It is described
Step（6）In sterile bud inducing culture composition be：Improve MS+N6Benzyladenine（6-BA) 1.0mg/L+ methyl α-naphthyl acetates
（NAA）Mg/L+ riboflavin (the vitamin Bs of 0.2-0.4mg/L+ vitamin Cs (Vc) 2.02) 7.0 mg/L+ cysteines 5.0g/L+
Sucrose 30g/L+ agar 4.0g/L, pH 6.0-6.2.
5. eucalyptus breeding tail eucalyptus camaldulensis DH191-7 according to claim 2 tissue culture and rapid propagation method, it is characterised in that：It is described
Step（7）In subculture multiplication medium composition be：Improve MS+N6Benzyladenine（6-BA) 0.3-0.5mg/L+ methyl α-naphthyl acetates
(NAA) the mg/L+ vitamin C 2.0mg/L+ sucrose 30g/L+ agar of 0.1-0.15mg/L+ riboflavin (vitamin B2) 7.0
4.0g/L, pH 5.8-6.0.
6. eucalyptus breeding tail eucalyptus camaldulensis DH191-7 according to claim 2 tissue culture and rapid propagation method, it is characterised in that：It is described
Step（8）In root media be：1/2 improvement MS+ABT10.6mg/L+IBA 0.1mg/L+ sucrose 15g/L+ agar
4.2mg/L, pH 6.0-6.4.
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