CN106342682A - Tissue culture method of quercus rubra L. - Google Patents
Tissue culture method of quercus rubra L. Download PDFInfo
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- CN106342682A CN106342682A CN201610717246.XA CN201610717246A CN106342682A CN 106342682 A CN106342682 A CN 106342682A CN 201610717246 A CN201610717246 A CN 201610717246A CN 106342682 A CN106342682 A CN 106342682A
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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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Abstract
The invention provides a tissue culture method of quercus rubra L.. The tissue culture method comprises the following steps: cutting out semi-lignified stem buds of a quercus rubra L. individual plant for pretreatment, then grafting the semi-lignified stem buds to an induction medium, and obtaining sterile bud seedlings; cutting the obtained sterile bud seedlings into stem fragments having apical buds, grafting the stem fragments to a proliferation medium, and obtaining clustering bud seedlings; selecting strong clustering bud seedlings, cutting and separating by taking one single bud seedling as a unit, cutting single bud seedlings after cutting into stem fragments having apical buds, then grafting the stem fragments to a rooting medium, and obtaining regenerated plants; carrying out seedling hardening on the regenerated plants in a seedling hardening room in an opening way, washing a medium attached to roots, transplanting the regenerated plants in a culture medium, irrigating thoroughly, covering an opening of a container by using a plastic thin film, removing the covered plastic thin film after new growing leaves unfold completely, and completing tissue culture of the quercus rubra L.. The tissue culture method of the quercus rubra L., provided by the invention, is scientific and reasonable in steps, and the tissue culture method adopted for carrying out tissue culture on the quercus rubra L. has the advantages of simpleness, high efficiency, low cost and high seedling yield.
Description
Technical field
The present invention relates to tissue culture technology, the tissue culture method of the red oak in more particularly, to a kind of North America.
Background technology
The red oak in North America (scientific name: quercus rubra l.) tree crown is well-balanced, treelet oval, with the age of tree increase gradually
Become round, leaf-shaped is beautiful, bright in luster, blade alternate, and leaf 7 to 11 splits.Autumn leaf colours gradually become red, sufficient
Illumination Autumn leaf colours can be made more bright-coloured, twig be in green or rufous, nut brown.The North America red oak speed of growth is relatively
Hurry up, drought-enduring, cold-resistant, barren-resistant, fire-resistance calamity, more resistance to shade, light is shone, and sprout tillers ability is strong, is resistant to -29 DEG C of low temperature.Resistance to environmental pollution,
Adaptable to the soil of different pH values.The red oak in North America originates in eastern United States, and Yangtze River in China middle and lower reaches are also distributed, and are
Excellent city ornamental tree species.In street, park, campus and court as tree of sheltering from heat or light, it is widely used in urban afforestation, has simultaneously
The ecological value, is resumed, is particularly suitable for commerial growing with can be used for.
Due to carrying out having some limitations property of seed propagation using seed, cuttage and grafting and breeding technology are given birth in China at present
Root survival rate is relatively low, therefore strengthens its vegetative research, sets up vegetative propagation technique system, improves the red oak Seedling in China North America
The production capacity of wood, can be widely used in city trees and shrubs providing technical guarantee for the red oak in North America.China is red for North America at present
The research of oak tissue culture, the storage rate only 66% of sterilizable material, stem eye inductivity only up to 53%, that is, the red oak tissue culture in existing North America exist
Breeding is slow, and growth coefficient is low, the low shortcoming of planting percent.
Content of the invention
It is an object of the invention to, for the problems referred to above, a kind of tissue culture method of the red oak in North America is proposed, the method is easy to be high
Effect, low cost, inductivity is high, and growth coefficient is high, and survival rate is high.
For achieving the above object, the technical solution used in the present invention is: a kind of tissue culture method of the red oak in North America, including following
Step:
Step one, the semi-lignified stem eye cutting North America red oak (basket oak) individual plants carry out pretreatment, then inoculate
To containing 0.01～0.1mg/l basic element of cell division, 0.01～0.1mg/l auxin, 20～40g/l sucrose and 5～7g/l agar
On inducing culture, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination 12～24h, obtain aseptic sprout within 20～30 days；
Step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 1.5～3.0cm, be inoculated into containing 0.1～
The 1.5mg/l basic element of cell division, the proliferated culture medium of 0.01～0.1mg/l auxin, 20～40g/l sucrose and 5～7g/l agar
On, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination
Under conditions of 12～24h, every 10～30 days replacing fresh cultures, culture 1～3 month, obtains crowd shoots；
Step 3, select stalwartness crowd shoots, cut off in units of single sprout, the single sprout after cutting off
Cut into the stem section with terminal bud that length is 2.0～3.0cm, then stem section is inoculated into containing 0.1～0.5mg/l (two kinds of lifes
Long element combination, concentration is 0.1-0.5mg/l) on the root media of auxin, 20～40g/l sucrose and 5～7g/l agar,
Temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination 12～
Cultivate 10～30 days under the conditions of 24h, obtain regeneration plant；
Step 4, plant height is reached the regeneration plant of 4cm, well developed root system, mounted blade in the indoor uncovered seedling exercising 3d of seedling exercising, so
The culture medium of afterwash root system attachment, then is transplanted in cultivation matrix and pours permeable, temperature be 20～30 DEG C, humidity be 60%
～80%, intensity of illumination is 40～100 μm of ol m-2·s-1, held with covered rearing with plastic film under conditions of daily illumination 12～18h
Device mouth cultivate to the young leaves growing fully deployed after, remove the plastic sheeting of covering, obtain North America red oak (basket oak) nursery stock, that is,
Complete the micro-propagation method of the red oak in North America (basket oak).
Further, pretreatment described in step one comprises the following steps: (Dalian Area) 5-6 month, the fine day morning, choosing life
Length is healthy and strong, tree-like graceful, the excellent female parent of no disease and pests harm, gathers annotinous branch, is cut into the band axillary bud stem being about 1.5-2.5cm
Section (preferably 2cm), after neutral detergent rinsing, flowing water rinses more than 2h, after to be placed in workbench standby.In superclean bench
On, first with 70% Ethanol Treatment 10-30s, process 1-5min with 0.1% mercuric chloride afterwards, then use rinsed with sterile water 5 times..
Further, step one, the semi-lignified stem eye cutting North America red oak (basket oak) individual plants carry out pretreatment,
It is then seeded into the basic element of cell division containing 0.02-0.05mg/l, 0.01-0.04mg/l auxin, 25～35g/l sucrose and 5～7g/
On the inducing culture of l agar, temperature be 20～30 DEG C, humidity be 50%～70%, intensity of illumination be 40～80 μm of ol
m-2·s-1, daily illumination 15～20h, obtain aseptic sprout within 20～30 days.Further, inducing culture described in step one
For ms culture medium, 1/2ms culture medium, dkw culture medium, wpm culture medium or ql culture medium, described inducing culture ph value for 5～
6, the described basic element of cell division is benzyladenine, zeatin, kinetins or forchlorfenuron, and described auxin is naphthalene acetic acid or indole
Butanoic acid.
Further, step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 2.0～3.0cm, be inoculated into
The basic element of cell division containing 1.1-1.5mg/l, the propagation of 0.01-0.04mg/l auxin, 25～35g/l sucrose and 5～7g/l agar
In culture medium, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 50～100 μm of ol m-2·s-1, every
Under conditions of its illumination 12～24h, every 10～30 days replacing fresh cultures, culture 2～3 months, obtains crowd shoots.
Further, proliferated culture medium described in step 2 is that ms culture medium, dkw culture medium, wpm culture medium or ql cultivate
Base, described proliferated culture medium ph value is 5～6, and the described basic element of cell division is benzyladenine, zeatin, kinetins or chlorine pyrrole benzene
Urea, described auxin is naphthalene acetic acid or indolebutyric acid.
Further, step 3, select stalwartness crowd shoots, cut off in units of single sprout, after cutting off
Single sprout cut into the stem section with terminal bud that length is 2.0～3.0cm, then stem section is inoculated into containing 0.1-0.3 (two
The combination of kind of auxin, concentration is 0.1-0.3) root media of mg/l auxin, 20-30g/l sucrose and 5～7g/l agar
On, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination
Cultivate 10～30 days under the conditions of 12～24h, obtain regeneration plant.
Further, root media described in step 3 is that ms culture medium, dkw culture medium or wpm cultivate for auxin
Base, described root media ph value is 5～6, and described auxin is naphthalene acetic acid and/or indolebutyric acid.
Further, step 4, plant height is reached 4cm, well developed root system, mounted blade regeneration plant uncovered in seedling exercising interior
Seedling exercising 3d, then cleans the culture medium of root system attachment, then is transplanted in cultivation matrix and pours permeable, temperature be 20～30 DEG C,
Humidity is 60%～80%, intensity of illumination is 40～100 μm of ol m-2·s-1, use plastics under conditions of daily illumination 12～18h
Thin film cover vessel port cultivate to the young leaves growing fully deployed after, remove the plastic sheeting of covering, (natural pond is given birth to obtain the red oak in North America
Oak) nursery stock, that is, complete the micro-propagation method of the red oak in North America (basket oak).
Further, cultivation matrix described in step 4 comprises volume than following each component: 3 parts of turfy soil, perlite
1-3 part.It is preferably 3 parts of turfy soil, 2 parts of perlite.
The tissue culture method step science of the red oak in North America of the present invention, rationally, has the advantage that compared with prior art
1st, pretreatment is easy, and pollution rate is low, and the storage rate of sterilizable material is high, up to 80%.
2nd, the inductivity of Initial culture base is high, up to more than 90%.
3rd, the growth rate of proliferated culture medium is fast, Seedling growth neat and consistent, growth coefficient are high, up to 7.0.
4th, Seedling robust growth, without strong sprout, rooting culture of can directly taking root.
Specific embodiment
The present invention is further described with reference to embodiments:
Embodiment 1
The present embodiment provides a kind of tissue culture method of the red oak in North America, comprises the following steps:
Step one, the semi-lignified stem eye cutting North America red oak (basket oak) individual plants carry out pretreatment, then inoculate
To on the basic element of cell division containing 0.05mg/l, the inducing culture of 0.05mg/l auxin, 30g/l sucrose and 6g/l agar, in temperature
Spend for 25 DEG C, humidity be 60%, intensity of illumination be 60 μm of ol m-2·s-1, daily illumination 18h, obtain aseptic sprout within 25 days；
Step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 1.5～3.0cm, be inoculated into containing 0.5mg/l
On the basic element of cell division, the proliferated culture medium of 0.05mg/l auxin, 30g/l sucrose and 6g/l agar, it is 25 DEG C, humidity in temperature
It is 60 μm of ol m for 60%, intensity of illumination-2·s-1, daily illumination 18h, every 20 days change fresh culture under conditions of cultivate
2 months, obtain crowd shoots；
Step 3, select stalwartness crowd shoots, cut off in units of single sprout, the single sprout after cutting off
Cut into the stem section with terminal bud that length is 2.0～3.0cm, then stem section is inoculated into containing 0.3mg/l auxin, 30g/l
On the root media of sucrose and 6g/l agar, temperature be 25 DEG C, humidity be 60%, intensity of illumination be 60 μm of ol m-2·s-1, cultivate 20 days under the conditions of daily illumination 20h, obtain regeneration plant；
Step 4, plant height is reached the regeneration plant of 4cm, well developed root system, mounted blade in the indoor uncovered seedling exercising 3d of seedling exercising, so
The culture medium of afterwash root system attachment, then is transplanted in cultivation matrix and pours permeable, temperature be 25 DEG C, humidity be 80%, light
It is 80 μm of ol m according to intensity-2·s-1, cultivated to grow with covered rearing with plastic film vessel port under conditions of daily illumination 15h new
After leaf is fully deployed, remove the plastic sheeting of covering, obtain North America red oak (basket oak) robust growth, stem is straight, growing way one
The nursery stock causing, that is, complete the micro-propagation method of the red oak in North America (basket oak).
Above-mentioned pretreatment taking Dalian Area as a example, comprises the following steps: 5-6 month, in the fine day morning, selects robust growth, tree
Shape is graceful, the excellent female parent of no disease and pests harm, gathers annotinous branch, is cut into the stem segment with axillary bud of long 1.5-2.5cm, is washed with neutrality
After washing agent rinsing, flowing water rinses more than 2h, after to be placed in workbench standby；On superclean bench, first with 70% Ethanol Treatment 10-
30s, processes 1-5min with 0.1% mercuric chloride afterwards, then uses rinsed with sterile water 5 times.
Wherein in step one, inducing culture is ms culture medium, and ph value is 5, and the basic element of cell division is benzyladenine, raw
Long element is naphthalene acetic acid；In step 2, proliferated culture medium is ms culture medium, and ph value is 5, and the basic element of cell division is benzyladenine, raw
Long element is naphthalene acetic acid；In step 3, root media is ms culture medium for auxin, and ph value is 5, auxin be naphthalene acetic acid with
Indolebutyric acid；In step 4, cultivation matrix presses the perlite that volume parts are than the turfy soil for 3 parts, 2 parts.
The present embodiment methods described can make storage rate reach more than 80%, the stem eye inductivity of Initial culture base up to 90% with
On；Propagation is fast, and growth coefficient is high, neat and consistent, and planting percent is high.
Embodiment 2
The present embodiment provides a kind of tissue culture method of the red oak in North America, comprises the following steps:
Step one, the semi-lignified stem eye cutting North America red oak (basket oak) individual plants carry out pretreatment, then inoculate
To on the basic element of cell division containing 0.02mg/l, the inducing culture of 0.08mg/l auxin, 20g/l sucrose and 7g/l agar, in temperature
Spend for 20 DEG C, humidity be 80%, intensity of illumination be 20 μm of ol m-2·s-1, daily illumination obtain aseptic sprout within 12,30 days；
Step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 1.5～3.0cm, be inoculated into containing 0.1mg/l
On the basic element of cell division, the proliferated culture medium of 0.01mg/l auxin, 40g/l sucrose and 7g/l agar, it is 30 DEG C, humidity in temperature
It is 20 μm of ol m for 80%, intensity of illumination-2·s-1, daily illumination 20h, every 10 days change fresh culture under conditions of cultivate
3 months, obtain crowd shoots；
Step 3, select stalwartness crowd shoots, cut off in units of single sprout, the single sprout after cutting off
Cut into the stem section with terminal bud that length is 2.0～3.0cm, then stem section is inoculated into containing 0.3mg/l auxin, 30g/l
On the root media of sucrose and 5g/l agar, temperature be 30 DEG C, humidity be 40%, intensity of illumination be 100 μm of ol m-2·
s-1, cultivate 10 days under the conditions of daily illumination 24h, obtain regeneration plant；
Step 4, plant height is reached the regeneration plant of 4cm, well developed root system, mounted blade in the indoor uncovered seedling exercising 3d of seedling exercising, so
The culture medium of afterwash root system attachment, then is transplanted in cultivation matrix and pours permeable, temperature be 30 DEG C, humidity be 60%, light
It is 80 μm of ol m according to intensity-2·s-1, cultivated to grow with covered rearing with plastic film vessel port under conditions of daily illumination 18h new
After leaf is fully deployed, remove the plastic sheeting of covering, obtain North America red oak (basket oak) nursery stock, that is, (natural pond is given birth to complete the red oak in North America
Oak) micro-propagation method.
Above-mentioned pretreatment taking Dalian Area as a example, comprises the following steps: 5-6 month, in the fine day morning, selects robust growth, tree
Shape is graceful, the excellent female parent of no disease and pests harm, gathers annotinous branch, is cut into the stem segment with axillary bud of long 1.5-2.5cm, is washed with neutrality
After washing agent rinsing, flowing water rinses more than 2h, after to be placed in workbench standby；On superclean bench, first with 70% Ethanol Treatment 10-
30s, processes 1-5min with 0.1% mercuric chloride afterwards, then uses rinsed with sterile water 5 times.
Wherein in step one, inducing culture is dkw culture medium, and ph value is 6, and the basic element of cell division is zeatin, auxin
For indolebutyric acid；In step 2, proliferated culture medium is wpm culture medium, and ph value is 5, and the basic element of cell division is zeatin, auxin
For indolebutyric acid；In step 3, root media is dkw culture medium for auxin, and ph value is 5, and auxin is indolebutyric acid；
In step 4, cultivation matrix presses the perlite that volume parts are than the turfy soil for 3 parts, 2 parts.
The present embodiment methods described can make storage rate reach more than 80%, the stem eye inductivity of Initial culture base up to 90% with
On；Propagation is fast, and growth coefficient is high, neat and consistent, and planting percent is high.
Embodiment 3
The present embodiment provides a kind of tissue culture method of the red oak in North America, comprises the following steps:
Step one, the semi-lignified stem eye cutting North America red oak (basket oak) individual plants carry out pretreatment, then inoculate
To on the basic element of cell division containing 0.08mg/l, the inducing culture of 0.04mg/l auxin, 30g/l sucrose and 7g/l agar, in temperature
Spend for 30 DEG C, humidity be 40%, intensity of illumination be 60 μm of ol m-2·s-1, daily illumination 24h, obtain aseptic sprout within 30 days；
Step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 1.5～3.0cm, be inoculated into containing 0.5mg/l
On the basic element of cell division, the proliferated culture medium of 0.05mg/l auxin, 20g/l sucrose and 7g/l agar, it is 20 DEG C, humidity in temperature
It is 30 μm of ol m for 70%, intensity of illumination-2·s-1, daily illumination 12h, every 10 days change fresh culture under conditions of cultivate
2 months, obtain crowd shoots；
Step 3, select stalwartness crowd shoots, cut off in units of single sprout, the single sprout after cutting off
Cut into the stem section with terminal bud that length is 2.0～3.0cm, then stem section is inoculated into containing 0.4mg/l auxin, 30g/l
On the root media of sucrose and 7g/l agar, temperature be 20 DEG C, humidity be 50%, intensity of illumination be 50 μm of ol m-2·s-1, cultivate 20 days under the conditions of daily illumination 12h, obtain regeneration plant；
Step 4, plant height is reached the regeneration plant of 4cm, well developed root system, mounted blade in the indoor uncovered seedling exercising 3d of seedling exercising, so
The culture medium of afterwash root system attachment, then is transplanted in cultivation matrix and pours permeable, temperature be 30 DEG C, humidity be 80%, light
It is 40 μm of ol m according to intensity-2·s-1, cultivated to grow with covered rearing with plastic film vessel port under conditions of daily illumination 18h new
After leaf is fully deployed, remove the plastic sheeting of covering, obtain North America red oak (basket oak) nursery stock, that is, (natural pond is given birth to complete the red oak in North America
Oak) micro-propagation method.
Above-mentioned pretreatment taking Dalian Area as a example, comprises the following steps: 5-6 month, in the fine day morning, selects robust growth, tree
Shape is graceful, the excellent female parent of no disease and pests harm, gathers annotinous branch, is cut into the stem segment with axillary bud of long 1.5-2.5cm, is washed with neutrality
After washing agent rinsing, flowing water rinses more than 2h, after to be placed in workbench standby；On superclean bench, first with 70% Ethanol Treatment 10-
30s, processes 1-5min with 0.1% mercuric chloride afterwards, then uses rinsed with sterile water 5 times.
Wherein in step one, inducing culture is ql culture medium, and ph value is 6, and the basic element of cell division is forchlorfenuron, growth
Element is naphthalene acetic acid；In step 2, proliferated culture medium is ql culture medium, and ph value is 5, and the basic element of cell division is forchlorfenuron, auxin
For naphthalene acetic acid；In step 3, root media is wpm culture medium for auxin, and ph value is 6, and auxin is naphthalene acetic acid；Step
In four, cultivation matrix presses the perlite that volume parts are than the turfy soil for 3 parts, 2 parts.
The present embodiment methods described can make storage rate reach more than 80%, the stem eye inductivity of Initial culture base up to 90% with
On；Propagation is fast, and growth coefficient is high, neat and consistent, and planting percent is high.
Finally it is noted that various embodiments above, only in order to technical scheme to be described, is not intended to limit；To the greatest extent
Pipe has been described in detail to the present invention with reference to foregoing embodiments, it will be understood by those within the art that: its according to
So the technical scheme described in foregoing embodiments can be modified, or wherein some or all of technical characteristic is entered
Row equivalent；And these modifications or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a kind of tissue culture method of the red oak in North America is it is characterised in that comprise the following steps:
Step one, the semi-lignified stem eye cutting North America red oak individual plants carry out pretreatment, be then seeded into containing 0.01～
The 0.1mg/l basic element of cell division, the inducing culture of 0.01～0.1mg/l auxin, 20～40g/l sucrose and 5～7g/l agar
On, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination
12～24h, obtain aseptic sprout within 20～30 days；
Step 2, the aseptic sprout obtaining is cut into the stem section with terminal bud of 1.5～3.0cm, be inoculated into containing 0.1～1.5mg/
On the l basic element of cell division, the proliferated culture medium of 0.01～0.1mg/l auxin, 20～40g/l sucrose and 5～7g/l agar, in temperature
Spend for 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, daily illumination 12～24h,
Cultivate 1～3 month under conditions of changing within every 10～30 days fresh culture, obtain crowd shoots；
Step 3, select the crowd shoots of stalwartness, cut off in units of single sprout, the single sprout cutting after cutting off
Become length be 2.0～3.0cm the stem section with terminal bud, then by stem section be inoculated into containing 0.1～0.5mg/l auxin, 20～
On the root media of 40g/l sucrose and 5～7g/l agar, temperature be 20～30 DEG C, humidity be 40%～80%, illumination strong
Spend for 20～100 μm of ol m-2·s-1, cultivate 10～30 days under the conditions of daily illumination 12～24h, obtain regeneration plant；
Step 4, plant height is reached the regeneration plant of 4cm, well developed root system, mounted blade in seedling exercising indoor uncovered seedling exercising 3d, Ran Houxi
The culture medium of net root system attachment, then be transplanted in cultivation matrix and pour permeable, temperature be 20～30 DEG C, humidity be 60%～
80%th, intensity of illumination is 40～100 μm of ol m-2·s-1, use covered rearing with plastic film container under conditions of daily illumination 12～18h
Mouth cultivate to the young leaves growing fully deployed after, remove the plastic sheeting of covering, complete the tissue culture of the red oak in North America.
2. according to claim 1 the red oak in North America tissue culture method it is characterised in that pretreatment described in step one include following
Step: 5-6 month, the fine day morning, select robust growth, tree-like graceful, the excellent female parent of no disease and pests harm, gather annotinous branch,
Be cut into the stem segment with axillary bud of long 1.5-2.5cm, after neutral detergent rinsing, flowing water rinses more than 2h, after to be placed in workbench standby
With；On superclean bench, first with 70% Ethanol Treatment 10-30s, process 1-5min with 0.1% mercuric chloride afterwards, then use sterilized water
Rinsing 5 times.
3. according to claim 1 the tissue culture method of the red oak in North America it is characterised in that step one, cutting North America red oak plant
Individual semi-lignified stem eye carries out pretreatment, is then seeded into the basic element of cell division containing 0.02-0.05mg/l, 0.01-0.04mg/
On the inducing culture of l auxin, 25～35g/l sucrose and 5～7g/l agar, temperature be 20～30 DEG C, humidity be 50%
～70%, intensity of illumination is 40～80 μm of ol m-2·s-1, daily illumination 15～20h, obtain aseptic sprout within 20～30 days.
4. the tissue culture method of the red oak in North America according to claim 1 or 3 is it is characterised in that inducing culture described in step one
Base is ms culture medium, 1/2ms culture medium, dkw culture medium, wpm culture medium or ql culture medium, and described inducing culture ph value is 5
～6, the described basic element of cell division is benzyladenine, zeatin, kinetins or forchlorfenuron, and described auxin is naphthalene acetic acid or Yin
Diindyl butanoic acid.
5. according to claim 1 the red oak in North America tissue culture method it is characterised in that step 2, will obtain aseptic sprout
Cut into the stem section with terminal bud of 2.0～3.0cm, be inoculated into the basic element of cell division containing 1.1-1.5mg/l, 0.01-0.04mg/l life
On the proliferated culture medium of long element, 20-30g/l sucrose and 5～7g/l agar, temperature be 20～30 DEG C, humidity be 40%～
80%th, intensity of illumination is 50～100 μm of ol m-2·s-1, daily illumination 12～24h, every 10～30 days change fresh culture
Under conditions of cultivate 2～3 months, obtain crowd shoots.
6. the tissue culture method of the red oak in North America according to claim 1 or 5 is it is characterised in that enrichment culture described in step 2
Base is ms culture medium, dkw culture medium, wpm culture medium or ql culture medium, and described proliferated culture medium ph value is 5～6, described cell
Mitogen is benzyladenine, zeatin, kinetins or forchlorfenuron, and described auxin is naphthalene acetic acid or indolebutyric acid.
7. according to claim 1 the red oak in North America tissue culture method it is characterised in that step 3, select stalwartness Multiple Buds
Seedling, is cut off in units of single sprout, and the single sprout after cutting off cuts into the band terminal bud that length is 2.0～3.0cm
Stem section, then stem section is inoculated into taking root containing 0.1-0.3mg/l auxin, 30～40g/l sucrose and 5～7g/l agar
In culture medium, temperature be 20～30 DEG C, humidity be 40%～80%, intensity of illumination be 20～100 μm of ol m-2·s-1, every
Cultivate 10～30 days under the conditions of its illumination 12～24h, obtain regeneration plant.
8. the tissue culture method of the red oak in North America according to claim 1 or 7 is it is characterised in that root culture described in step 3
Base is ms culture medium, dkw culture medium or wpm culture medium for auxin, and described root media ph value is 5～6, described auxin
For naphthalene acetic acid and/or indolebutyric acid.
9. according to claim 1 the tissue culture method of the red oak in North America it is characterised in that step 4, plant height is reached 4cm, root system
Flourishing, mounted blade regeneration plant, in the indoor uncovered seedling exercising 3d of seedling exercising, is then cleaned the culture medium of root system attachment, then is transplanted to
In cultivation matrix and pour permeable, temperature be 20～30 DEG C, humidity be 60%～80%, intensity of illumination be 40～100 μm of ol
m-2·s-1, cultivated with covered rearing with plastic film vessel port under conditions of daily illumination 12～18h fully deployed to the young leaves growing
Afterwards, remove the plastic sheeting of covering, obtain North America red oak nursery stock.
10. the tissue culture method of the red oak in North America according to claim 1 or 9 is it is characterised in that cultivation matrix described in step 4
Comprise volume than following each component: 3 parts of turfy soil, perlite 1-3 part.
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| Title |
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| 丁彤: ""北美红栎无性繁殖体系的研究"", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 赵丹等: ""麻栎的组织培养与快速繁殖"", 《中国农学通报》 * |
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| CN110402818A (en) * | 2019-08-20 | 2019-11-05 | 黄冈师范学院 | A kind of method of excellent kind of Chinese chestnut Mature Embryo Tissue culture rapid seedling cultivation |
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