CN105941147A - Betula papyrifera tissue culture seedling propagation method - Google Patents

Abstract

The invention provides a betula papyrifera tissue culture seedling propagation method, which comprises the following steps in order: (1) selecting betula papyrifera juvenile tender shoots cultivated for more than one year, and intercepting branches of stem-segments with auxiliary buds from terminal buds downward to serve as the explants, (2) disinfecting the explants, (3) cutting off both ends of the explants by 0.2-0.4 respectively, then introducing the explants into a medium to conduct culture; (4) longitudinally segmenting the sterile sprouts obtained in step (3) along the stem-segments into two, inoculating the segmented sprouts into a medium to conduct culture; (5) segmenting clustered shoots obtained in step (4) into individual shoots, and inoculating the individual shoots into a strong seedling medium to conduct culture; and (6) inoculating the individual shoots obtained in step (5) into a well configured rooting medium to perform rooting induction. The method provided by the invention has the advantages of simple operation, and good repeatability. The seedlings are robust, grow rapidly, and have uniform size and neat height. No callus is generated, and variation and trait segregation are low. The rooting effect is good, and the transplantation survival rate is more than 90%, therefore the method is economically feasible.

Description

Paper birch tissue culture of sprout mating system

Technical field

The present invention relates to the seedling breeding method of paper birch.

Background technology

Paper birch (BETULA PAPYRIFERA) is the woody arbor kind of Betula, main garden One of woods, economic forest plant, originate in North America.Present document relates to the introduction ornamental plant kind " Betula of paper birch Papyrifera ' Jacquemontii ' " and " Betula papyrifera ' Duraheat ' " Two commodity kinds.This kind wood quality is excellent, can make the material such as plywood, joiner, is one Important industrial cut stock seeds, bark can be pyrolyzed extraction tar, and the juice of tree can make beverage；Its Logical straight trunk, beautiful bark color and bark texture clearly, add the climatic adaptation that it is wide in range Property and soil suitability, and leaf pubescence is coated, along with sending out of afforestation in recent years and urban look Opening up, it is used as depositing dust plant action in urban look and becomes increasingly conspicuous, and nursery stock usage amount is increasing.

Traditional paper birch propagation method uses cuttage or seed broadcast mode to carry out seedling breeding, but cutting propagation is subject to Season limits, and only the ofest short duration among 1 year can carry out relevant operation the 5--9 month；In addition, skewer Slotting nursery also needs to intercept a large amount of branch from nursery stock, so can have a strong impact on the moulded of nursery stock, Serious will result directly in lopping nursery stock and cannot sell, lose commercial value.And use seed broadcast mode to educate Seedling, is limited by seed source again, and seedling raise period is confined to 2--5 month, and seed germination rate is subject to Seed activity and the restriction of weather conditions after planting, additionally seed is broadcast nursery and be there is also serious character Separate, be difficult to the business seedling that acquired character is consistent.

In a word, conventional seminal propagation speed is the slowest, seed selection of need to collecting seed, storage, accelerating germination sowing, educates The enforcement step of a series of longer cycles such as Seedling management, is limited by season simultaneously, and the sowing time limit is shorter； And the survival rate of seed is low, cause breeding coefficient low；During seminal propagation, there is trait segregation Probability is more, it is difficult to the batch seedling that acquired character is consistent.

Cutting propagation cuttage rooting is more difficult, and cutting time is of short duration, cutting medium proportioning kind and skewer Insert micro environment control bigger on the impact of rooting rate；And cutting propagation needs to intercept a large amount of branch, Have a strong impact on tree vigo(u)r and overall appreciation effect；In addition, plant virus can along with cutting propagation by Gradually accumulating, along with the increase of cottage propagation algebraically, trait depression is thought about it fairly obvious.

Summary of the invention

It is an object of the invention to provide a kind of paper birch tissue culture of sprout mating system, to overcome existing skill The defect that art exists.

The method of the present invention, comprises the steps:

(1) selection of outer implant:

Select the hot-house culture paper tender tip of birch juvenile form of more than a year, from terminal bud, intercept downwards about band 2～6 Individual, the preferably branch of 4 axillary bud stem sections, remove blade, cut single-unit stem segment with axillary bud as outer planting Body, every stem section carries an axillalry bud, long 1～2.5cm, the longest 1.5-2cm；

(2) outer implant sterilization:

Outer implant medical alcohol cotton rub being wiped, the ratio of being subsequently placed in is the dilution bleaching water (drift of 1:3～5 Plain boiled water: sterilized water volume ratio, lower same) middle concussion sterilization 10～30min, wash 1～3 with sterilized water Secondary；Described bleaching water is commercially available " white cat " board bleaching water.

(3) Primary culture:

Outer implant two ends after sterilization are excised 0.2～0.4CM, preferably 0.3cm respectively, and access is opened In dynamic culture medium, darkroom cultivation 36～60 hours, preferably 48 hours, then light application time 12～20 Hour, preferably 16h, dark 6～10 hours, preferably 8h, light intensity 2000～3000lux, preferably 2300lux, 20～30 degrees Celsius preferably 25 degrees Celsius cultivate 5～7 weeks, preferably 6 weeks, dormancy axillalry bud Sprout, it is thus achieved that aseptic rudiment, high about 2-3cm；

Described Primary culture base is WPM+6-BA 1.5mg/L+GA₃2.0mg/L+ sucrose 30g/L+ agar 6.8g/L, the pH of culture medium are 5.85；

Described WPM culture medium, 6-BA, GA₃, the medicine such as IAA, NAA be Sigma of the U.S. Selling finished product culture medium and plant growth regulator, AC (activated carbon), agar etc. are domestic procurement business Product, lower same.

(4) enrichment culture:

Aseptic rudiment step (3) obtained is longitudinally split along stem section, is divided into two, is inoculated in propagation In culture medium, light culture 36～60 hours, preferably 48 hours, 20～30 degrees Celsius preferably 25 Celsius Degree, remaining time illumination 12～20 hours, preferably 16h, dark 6～10 hours, preferably 8h, light Strong 2000～3000lux, preferably 2300lux, to cultivate 5～7 weeks, induction produces from sprouting 4, Bud clump highly reaches 2cm, and the rate of increase reaches 4 times；

Proliferated culture medium is WPM+6-BA2.0mg/L+IAA0.5mg/L+GA₃1.0mg/L+ adds sucrose 30g/L+ agar 6.8g/L, the pH of culture medium are 5.85；

(5) strong seedling culture

The Multiple Buds induced in increment culture medium step (4) obtained is divided into single bud, connects Kind in strong seedling culture base, light culture 36～60 hours, preferably 48 hours, cultivation temperature be 20～ 30 degrees Celsius, preferably 25 degrees Celsius, remaining time illumination 12～20 hours, preferably 16h, dark 6～ 10 hours, preferably 8h, light intensity 2000～3000lux, preferably 2300lux, cultivate 3～5 weeks, excellent Select 4 weeks；

Strong seedling culture base is that WPM+AC0.5g/L+ adds sucrose 30g/L+ agar 6.8g/L, and PH is 5.85；

(6) root culture

Step (5) is obtained single bud excision stem section base portion 0.1～0.3cm, preferably 0.2cm, cuts All blades within base portion 1cm, retain upper blade, are inoculated in the root media configured Carry out root induction, light culture 36～60 hours after inoculation, preferably 48 hours, then full exposure training Support.Cultivation temperature be temperature be 20～30 degrees Celsius, preferably 25 degrees Celsius, illumination 12～20 hours, Preferably 16h, dark 6～10 hours, preferably 8h, light intensity 2000～3000lux, preferably 2300lux, Cultivating 5～7 weeks, preferably 6 weeks, it is thus achieved that go out root, plant length up to 3cm, main root was more than 2, long Spend the seedling of about 3cm；

Root media is that 1/2WPM+IBA1.0mg/L+NAA0.5mg/L+AC1.0g/L+ adds sucrose 15g/L+ agar 6.8g/L, the pH of culture medium are 5.85.

The invention has the beneficial effects as follows:

Simple to operate, reproducible；Seedling is healthy and strong, growth is rapid, the thick 1.0mm of seedling stem, high 30mm Above, in the same size, highly ordered；Produce without callus, low variation, low trait segregation；Raw Root is effective, root system 24, and root system transplants survival rate height, transplants survival rate more than 90%, cost Saving, economically feasible.

Detailed description of the invention

Embodiment 1

The selection of the outer implant of 1.Duraheat:

In April in spring, select bright day gas, choose that robust growth in greenhouse, character be pure, nothing The hot-house culture of the pest and disease damage tender tip of Duraheat juvenile form of more than a year, from terminal bud, downward intercepting about carries The branch of 4 axillary bud stem sections, removes blade, cuts single-unit stem segment with axillary bud as outer implant, every stem section Carry an axillalry bud, long 1.5-2cm.

2.Duraheat outer implant sterilization:

It is divided into slight lignifying and non-degree of lignification individually to carry out disinfection according to degree of lignification stem section. On aseptic operating platform, by outer implant medical alcohol cotton wiped clean, ratio of pouring into immediately is 1:4 Bleaching water (commodity bleaching water: sterilized water volume ratio, lower with) in sterilization 20min, period is both needed to not Stop concussion, finally wash respectively with sterilized water 3 times, every time rinsing 5 minutes.

3. Primary culture:

In operative employee's station, the outer implant two ends after sterilization being excised 0.3cm respectively, access is opened In dynamic culture medium, Primary culture base is WPM+6-BA 1.5mg/L+GA₃2.0mg/L+ sucrose 30g/L+ Agar 6.8g/L, the pH of culture medium are 5.85.Culturing room's light culture is put into 48 hours, so after inoculation Rear light application time 16h, dark 8h, light intensity 2300lux., room temperature 25 degrees Celsius.Cultivate 6 weeks, Dormancy axillary bud sprouting, robust plant, aseptic seedling plant height about 2-3cm.

4. enrichment culture:

By longitudinally split along stem section for the dormancy axillalry bud of sprouting in Primary culture base, it be divided into two and be inoculated in In proliferated culture medium, proliferated culture medium is WPM+6-BA2.0mg/L+IAA0.5mg/L+GA₃1.0mg/L+ Adding sucrose 30g/L+ agar 6.8g/L, the pH of culture medium is 5.85.Light culture 48 hours, cultivates Temperature is 25 DEG C, remaining time illumination 16h, dark 8h, light intensity 2300lux.Cultivate 6 weeks and can lure Artificial deliviery life is from sprouting 4, and bud clump highly reaches 2cm, and the rate of increase reaches 4 times.

5. strong seedling culture

The bud of living again induced in increment culture medium is divided into single bud, is inoculated in strong seedling culture base In, strong seedling culture base is that WPM+AC0.5g/L+ adds sucrose 30g/L+ agar 6.8g/L, and pH value is 5.85.Light culture 48 hours, cultivation temperature is 25 DEG C, remaining time illumination 16h, dark 8h, light Strong 2300lux.Cultivate 4 weeks.

6. root culture

Single bud excision stem section base portion 0.2CM strong seedling culture in strong seedling culture base crossed, cuts base portion All blades within 1CM, retain upper blade, are inoculated in the root media configured and lure Leading and take root, root media is that 1/2WPM+IBA1.0mg/L+NAA0.5mg/L+AC1.0g/L+ adds Sucrose 15g/L+ agar 6.8g/L, the pH of culture medium are 5.85.Light culture 48 hours after inoculation, so Rear full exposure is cultivated.Cultivation temperature is 25 DEG C, light application time 16h, dark 8h, light intensity 2300lux. Cultivating 6 weeks and can induce root, plant length up to 3cm, healthy and strong, main root is more than 2, length about 3cm.

Using observational technique directly perceived, test, result shows, the method for the present invention has operation letter Single, reproducible；Seedling is healthy and strong, growth is rapid, the thick 1.0mm of seedling stem, high more than 30mm, In the same size, highly ordered；Produce without callus, low variation, low trait segregation；Rooting efficiency Good, root system 24, root system is transplanted survival rate height, is transplanted survival rate more than 90%.

Claims (7)

1. paper birch tissue culture of sprout mating system, it is characterised in that in turn include the following steps:

(1) selection of outer implant:

Select the hot-house culture tender tip of the juvenile form of more than 1 year, from terminal bud, intercept downwards stem segment with axillary bud Branch, as outer implant.

(2) outer implant carries out disinfection:

(3) Primary culture:

Outer implant two ends after sterilization are excised respectively 0.2～0.4CM, accesses in Primary culture base Cultivate；

(4) enrichment culture:

Aseptic rudiment step (3) obtained is longitudinally split along stem section, is divided into two, is inoculated in propagation Culture medium is cultivated；

(5) strong seedling culture:

The Multiple Buds induced in increment culture medium step (4) obtained is divided into single bud, connects Plant and cultivate in strong seedling culture base；

(6) root culture

Step (5) is obtained single bud excision stem section base portion 0.1～0.3cm, within cutting base portion 1cm All blades, retain upper blade, be inoculated in the root media configured and carry out root induction.

Method the most according to claim 1, it is characterised in that in step (1), from terminal bud Rise and intercept downwards the branch with 2～6 axillary bud stem sections, remove blade, cut single-unit stem segment with axillary bud and make For outer implant, every stem section carries an axillalry bud, length 1～2.5cm.

Method the most according to claim 1, it is characterised in that in step (2), by outer planting Body medical alcohol cotton rub is wiped, and is subsequently placed in bleaching water: sterilized water volume ratio is the dilution of 1:3～5 In bleaching water, concussion sterilization 10～30min, wash with sterilized water.

Method the most according to claim 1, it is characterised in that the cultural method of step (3) As follows；Darkroom is cultivated 36～60 hours, then light application time 12～20 hours, dark 6～10 little Time, light intensity 2000～3000lux, cultivate 5～7 weeks for 20～30 degrees Celsius, dormancy axillary bud sprouting, obtain Obtain aseptic seedling strain；

Described Primary culture base is WPM+6-BA 1.5mg/L+GA₃2.0mg/L+ sucrose 30g/L+ agar 6.8g/L, the pH of culture medium are 5.85.

Method the most according to claim 1, it is characterised in that the cultural method of step (4) As follows；Light culture 36～60 hours, 20～30 degrees Celsius, remaining time illumination 12～20 hours, Dark 6～10 hours, light intensity 2000～3000lux, to cultivate 5～7 weeks, induction produces from sprouting；

Proliferated culture medium is WPM+6-BA2.0mg/L+IAA0.5mg/L+GA₃1.0mg/L+ adds sucrose 30g/L+ agar 6.8g/L, the pH of culture medium are 5.85.

Method the most according to claim 1, it is characterised in that the cultural method of step (5) As follows: light culture 36～60 hours, cultivation temperature is 20～30 degrees Celsius, remaining time illumination 12～ 20 hours, dark 6～10 hours, light intensity 2000～3000lux, cultivate 3～5 weeks；

Strong seedling culture base is that WPM+AC0.5g/L+ adds sucrose 30g/L+ agar 6.8g/L, culture medium PH is 5.85.

Method the most according to claim 1, it is characterised in that the cultural method of step (6) As follows: light culture 36～60 hours after inoculation, then full exposure is cultivated.Cultivation temperature is 20～30 Degree Celsius, illumination 12～20 hours, dark 6～10 hours, light intensity 2000～3000lux, cultivate 5～7 weeks, it is thus achieved that go out root seedling；

Root media is that 1/2WPM+IBA1.0mg/L+NAA0.5mg/L+AC1.0g/L+ adds sucrose 15g/L+ agar 6.8g/L, the pH of culture medium are 5.85.