CN101502238B - A Micropropagation Method of Populus alba in Novosibirsk - Google Patents

Abstract

The present invention provides a micropropagation method of populus hybride, and relates to a micropropagation method. The invention settles the problems of long breeding cycle, low efficiency of propagation and high cost. The method comprises the following steps: 1. sterilizing the explant; 2. cutting the explant to stem segment and then executing induction culture; 3. transferring the explant and proliferative sterilized bud in a secondary culture medium for secondarily culturing to micron-branch; 4. cutting each micro-branch from the stem segment separately, and then executing invigoration culture; and 5. cutting the invigorated micro-branch to stem segment, then executing enrichment culture, and sticking into a rooting substrate for executing rooting culture for finishing the micropropagation of populus hybride. The micropropagation method of populus hybride according to the invention has the advantages of short micropropagation period, high efficiency, excellent genetic stability and low cost. The propagation efficiency can obtain 30 times of prior art. Furthermore the rooting rate and survival rate of seedling propagation equally can obtain 100%.

Description

A kind of micro-propagation method of Populus hybride

Technical field

The present invention relates to a kind of micro-propagation method.

Background technology

Populus hybride (P.bachofenii * P.pyramidalis ROZ) originates in Russia; it is the high-quality quick growing species of trees of cultivating high and cold, high temperature that form, extremely anti-through Russian several generations scientist's painstaking efforts; and have good sight and stronger salt tolerant alkali and a drought-resistance ability; the utilization of can becoming a useful person in the general more than ten years; it is the good raw material of making paper pulp and plywood; also be to be used for ecological construction, one of important species of aspects such as environmental protection simultaneously.

Populus hybride was introduced China in 2004 by Russian Siberia, during introduction a limited number of spray stem ends, the nursery stock that obtains through cottage propagation can safe overwintering in Heihe Area, not having any freeze injury takes place, the northern China that is through with city does not have the resist cold history of fast growth poplar kind of high latitude coldly, has very big development space.Heihe Area has been planned in a short time (before 2010) and has been cultivated 1,000,000 strains of high-quality fast growth poplar nursery stock, be used for the construction of base of high-yield and high-efficiency forest plantation, but because the tender stem limited amount of introducing, and the cycle of cottage propagation Populus hybride is long, at least need 6 months, proliferate efficiency is low, the cost height, can not satisfy the needs in market far away, this makes Populus hybride be greatly limited in China domestic the popularization cultivation and the utilization of resources.At present, have and utilize poplar leaf to induce the propagation technique that produces indefinite bud, but exist the offspring who produces to make a variation greatly the problem of genetic stability difference.

Summary of the invention

The objective of the invention is for solve the Populus hybride breeding cycle long, proliferate efficiency is low, cost is high and the problem of genetic stability difference, and the micro-propagation method of a kind of Populus hybride that provides.

The micro-propagation method of Populus hybride is realized according to the following steps: the Populus hybride explant after, will cleaning is 75% alcohol solution dipping 60s again with another part mass concentration after on the superclean bench being 75% alcohol solution dipping 60s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 12～15min with mass concentration again, use aseptic water washing then 3～5 times; Two, the explant after will sterilizing cuts into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, be inoculated in then in the inducing culture, and be that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to the long length of aseptic blastogenesis be 0.5～1cm; Three, will transfer in subculture medium together with the aseptic bud of hyperplasia through the explant that step 2 is handled and cultivated and hyperplasia goes out aseptic bud, be that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to the long length of aseptic blastogenesis be 2～5cm, be little branch (unrooted test-tube plantlet); Four, each little branch being cut off separately on the stem section, be inoculated in then and increase in the strong medium, is that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to little branch growth length be 4～6cm; Five, will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, being inoculated in piecemeal then and being cultured to little branch growth length in the proliferated culture medium is 4～6cm, and cuttage is carried out promptly finishing the little numerous of Populus hybride after the culture of rootage in the matrix of taking root again; Wherein inducing culture is to be minimal medium with improvement MS solid culture medium in the step 2, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.5～1.0mg/L and mass concentration and be 2.5～3.0% sucrose, the pH value is 5.6～6.0; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step 3, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.1～0.3mg/L and mass concentration and be 2.0～2.5% sucrose, the pH value is 5.6～6.0; Increasing strong medium in the step 4 and be with the solid medium of MS is minimal medium, and also to comprise mass concentration be 2.0～2.5% sucrose, and the pH value is 5.6～6.0; Proliferated culture medium is to be minimal medium with the solid medium of improvement MS in the step 5, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.1～0.3mg/L and mass concentration and be 2.0～2.5% sucrose, the pH value is 5.6～6.0; The matrix of taking root in the step 5 is made up of 25% turfy soil, 30% vermiculite and 45% perlite by volume, and the water content of substrate of taking root is 50%～60%.

Populus hybride is little numerous among the present invention, and the breeding cycle is short, the efficient height, and its proliferate efficiency can reach 30 times, and utilizes original terminal bud on the maternal plant or axillalry bud directly to carry out the propagation of bud, the descendant inheritting good stability that is produced; Little branch behind the enrichment culture can directly be taken root into complete regeneration plant without any need for chemical reagent with plant hormone outside test tube, take root and tame simultaneously and carry out, saving of work and time, cost is low, and the rooting rate and the survival rate of transplanting seedlings all can reach 100%, realized Populus hybride fast, the efficient target of breeding and large-scale promotion plantation.

Embodiment

Embodiment one: the micro-propagation method of present embodiment Populus hybride is realized according to the following steps: the Populus hybride explant after, will cleaning is 75% alcohol solution dipping 60s again with another part mass concentration after on the superclean bench being 75% alcohol solution dipping 60s with mass concentration, use aseptic water washing then 3～5 times, be 0.1% mercuric chloride solution sterilization, 12～15min with mass concentration again, use aseptic water washing then 3～5 times; Two, the explant after will sterilizing cuts into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, be inoculated in then in the inducing culture, and be that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to the long length of aseptic blastogenesis be 0.5～1cm; Three, will transfer in subculture medium together with the aseptic bud of hyperplasia through the explant that step 2 is handled and cultivated and hyperplasia goes out aseptic bud, be that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to the long length of aseptic blastogenesis be 2～5cm, be little branch (unrooted test-tube plantlet); Four, each little branch being cut off separately on the stem section, be inoculated in then and increase in the strong medium, is that 25～28 ℃, humidity are 70%～80%, intensity of illumination is 40～60 μ molcm in temperature ^-2S ^-1, illumination every day 16～18h condition under to be cultured to little branch growth length be 4～6cm; Five, will increase little branch after strong and cut into that to have 1 terminal bud or 1～2 axillalry bud and length be the stem section of 1～2cm, being inoculated in piecemeal then and being cultured to little branch growth length in the proliferated culture medium is 4～6cm, and cuttage is carried out promptly finishing the little numerous of Populus hybride after the culture of rootage in the matrix of taking root again; Wherein inducing culture is to be minimal medium with improvement MS solid culture medium in the step 2, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.5～1.0mg/L and mass concentration and be 2.5～3.0% sucrose, the pH value is 5.6～6.0; Subculture medium is to be minimal medium with the solid medium of improvement MS in the step 3, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.1～0.3mg/L and mass concentration and be 2.0～2.5% sucrose, the pH value is 5.6～6.0; Increasing strong medium in the step 4 and be with the solid medium of MS is minimal medium, and also to comprise mass concentration be 2.0～2.5% sucrose, and the pH value is 5.6～6.0; Proliferated culture medium is to be minimal medium with the solid medium of improvement MS in the step 5, and also comprise the naa (NAA) of 6-benzylaminopurine (6-BA), 0.05～0.1mg/L of 0.1～0.3mg/L and mass concentration and be 2.0～2.5% sucrose, the pH value is 5.6～6.0; The matrix of taking root in the step 5 is made up of 25% turfy soil, 30% vermiculite and 45% perlite by volume, and the water content of substrate of taking root is 50%～60%.

The healthy individual plant that present embodiment selected speed from the Populus hybride treelet gives birth to, cold-resistant, resistance, sight do very well is as the maternal plant of gathering explant.

Present embodiment is gathered the full young tender branch of sleeping bud as explant.

Cleaning in the present embodiment step 1 is to clean explant (can add a washing agent in the water) with clear water, and the dust and the assorted bacterium of scrubbing the branch surface gently with the fine, soft fur brush during cleaning are noted not damaging sleeping bud, then branch are washed 0.5～1h under running water.

The stem section is inoculated in inducing culture in the present embodiment step 2, is that the mode that keeps stem section morphology upper end to make progress is vertically inserted the stem section in the inducing culture, and the degree of depth is 0.5cm.

To increase in the present embodiment step 5 and be inoculated in proliferated culture medium piecemeal after little branch after strong cuts into the stem section, be that the stem section with the tool terminal bud vertically is inserted in the proliferated culture medium, and the degree of depth is 0.5cm; The stem section of tool axillalry bud is lain in the enrichment culture primary surface.

In the present embodiment step 5 per about 30 days with the culture in the enrichment culture (not cutting) switching once, be transferred on the fresh proliferated culture medium, aseptic bud is grown to little branch, general switching once can realize new little formation, the propagation multiple can reach 30.

Step 4 and step 5 are capable of circulation in the present embodiment carries out, until producing little the quantity that can satisfy the demands.

The culture of rootage room temperature is 25～28 ℃ in the present embodiment step 5, and humidity is 70%～80%, illumination every day 16～18h, and intensity of illumination is 40～60 μ molcm ^-2S ^-1

Culture of rootage can be cultivated with domestication and be carried out simultaneously in the present embodiment step 5, be with around little branch take root the matrix compacting and on take root stromal surface and little branches and leaves face water spray slightly, be covered with preservative film then and cultivating indoor cultivation; Water 2 time in the mode of spraying every day, takes off film 2～5min ventilation every day sooner or later, removes preservative film after 20 days, and little branch can be taken root when cultivating 7～10 days, and rooting rate reaches 100%.

In the present embodiment after little numerous the finishing of Populus hybride, can carry out nursery stock transplanting and land for growing field crops field planting, little that is after taking root is continued to cultivate 30 days, be transplanted to then (culture matrix in the little alms bowl is the mixture of turfy soil and sandy soil) in the little alms bowl of plastics, be put in greenhouse or the booth and cultivate, note keeping good ventilation in greenhouse and the booth, the survival rate of transplanting seedlings reaches 100%; Can supply big Tanaka's production field planting when treating 15～40 centimetres of heights of seedling.

Embodiment two: aseptic water washing 3 times are used in not being both of present embodiment and embodiment one in the step 1, are 0.1% mercuric chloride solution sterilization 13min again with mass concentration, use aseptic water washing then 3 times.Other step and parameter are identical with embodiment one.

Mercuric chloride solution can add a Tween-20 in the present embodiment.

Embodiment three: the explant that is not both in the step 2 of present embodiment and embodiment two cuts into that to have 1 terminal bud or 2 axillalry buds and length be the stem section of 1.5cm.Other step and parameter are identical with embodiment two.

Embodiment four: present embodiment and embodiment three not to be both in the step 2 in temperature be that 25 ℃, humidity are 75%, intensity of illumination is 50 μ molcm ^-2S ^-1, illumination every day 17h condition under to be cultured to the long length of aseptic blastogenesis be 1cm.Other step and parameter are identical with embodiment three.

Embodiment five: present embodiment and embodiment four not to be both in the step 3 in temperature be that 27 ℃, humidity are 70%, intensity of illumination is 55 μ molcm ^-2S ^-1, illumination every day 17h condition under to be cultured to the long length of aseptic blastogenesis be 4cm.Other step and parameter are identical with embodiment four.

Embodiment six: present embodiment and embodiment five not to be both in the step 4 in temperature be that 27 ℃, humidity are 80%, intensity of illumination is 50 μ molcm ^-2S ^-1, illumination every day 17h condition under to be cultured to little branch growth length be 5cm.Other step and parameter are identical with embodiment five.

Embodiment seven: not being both of present embodiment and embodiment six will increase little branch after strong and cut into that to have 1 terminal bud or 2 axillalry buds and length be the stem section of 1.5cm in the step 5, being inoculated in piecemeal then and being cultured to little branch growth length in the proliferated culture medium is 5cm.Other step and parameter are identical with embodiment six.

Claims (7)

1. a micropropagation method of Populus alba Novosibirsk is characterized in that the poplar alba Novosibirsk micropropagation method is realized by the following steps: one, the Populus alba Novosibirsk explants after cleaning are cleaned with quality Soak in 75% ethanol solution for 60s, then soak in another 75% ethanol solution for 60s, rinse with sterile water for 3 to 5 times, and then disinfect with 0.1% mercuric chloride solution 12 to 15 minutes, and then rinsed with sterile water for 3 to 5 times; 2. Cut the sterilized explants into stem segments with 1 terminal bud or 1 to 2 axillary buds and a length of 1 to 2 cm, and then Inoculate in the induction medium, cultivate until sterile under the conditions of temperature 25-28°C, humidity 70%-80%, light intensity 40-60μmol·cm ^-2 ·s ^-1 , light 16-18h per day The growth length of the buds is 0.5-1cm; 3. Transfer the explants that have been treated and cultivated in step 2 and have grown sterile buds together with the grown sterile buds to the subculture medium at a temperature of 25-28°C. , the humidity is 70% to 80%, the light intensity is 40 to 60 μmol·cm ^-2 ·s ^-1 , and the conditions of 16 to 18 hours of light per day are cultivated until the growth length of the sterile buds is 2 to 5 cm, which is the microbranch; 4. 1. Separate each branchlet from the stem segment, and then inoculate it in the growth medium at a temperature of 25-28°C, a humidity of 70%-80%, and a light intensity of 40-60 μmol·cm ^-2 . s ^-1 . Cultivate under the condition of 16-18 hours of light every day until the growth length of the micro-branches is 4-6 cm; 5. Cut the strengthened micro-branches into 1 terminal bud or 1-2 axillary buds with a length of 4-6 cm. Stem segments of 1 to 2 cm are then inoculated segment by segment in the proliferation medium and cultivated until the growth length of the microbranches is 4 to 6 cm, and then cuttages are placed in the rooting matrix for rooting culture to complete the micropropagation of Populus alba Novosibirsk; wherein step 2 The medium induction medium is based on the improved MS solid medium, and also contains 0.5-1.0mg/L 6-benzylaminopurine, 0.05-0.1mg/L naphthalene acetic acid and a mass concentration of 2.5-3.0% sucrose, the pH value is 5.6-6.0; the subculture medium in step 3 is based on the improved MS solid medium, and also contains 0.1-0.3mg/L 6-benzylaminopurine, 0.05-0.1mg Naacetic acid per L and sucrose with a mass concentration of 2.0 to 2.5% have a pH value of 5.6 to 6.0; in step 4, the enhancement medium is based on MS solid medium, and also contains a mass concentration of 2.0 to 2.5 % sucrose, the pH value is 5.6～6.0; the proliferation medium in step 5 is based on the improved MS solid medium, and also contains 0.1～0.3mg/L of 6-benzylaminopurine, 0.05～0.1mg The naphthalene acetic acid of /L and mass concentration are the sucrose of 2.0～2.5%, and pH value is 5.6～6.0; The volume ratio is composed of 25% peat soil, 30% vermiculite and 45% perlite, and the water content of the rooting matrix is 50% to 60%. 2. the micropropagation method of a kind of Novosibirsk alba poplar according to claim 1 is characterized in that in step 1, rinse 3 times with sterile water, then use mass concentration to be 0.1% mercuric chloride solution disinfection 13min, Then rinse 3 times with sterile water. 3. the micropropagation method of a kind of Novosibirsk Alba poplar according to claim 2, is characterized in that in step 2, explant is cut into the stem section that has 1 terminal bud or 2 axillary buds, and length is 1.5cm. 4. The micropropagation method of Populus alba Novosibirsk according to claim 3, characterized in that in step 2, the temperature is 25°C, the humidity is 75%, and the light intensity is 50 μmol·cm ^-2 ·s ^-1 , Under the condition of 17h of light every day, culture until the growth length of sterile buds is 1cm. 5. A method for micropropagation of Populus alba Novosibirsk according to claim 4, characterized in that in step 3, the temperature is 27°C, the humidity is 70%, and the light intensity is 55 μmol·cm ^-2 ·s ^-1 , Under the condition of 17h of light every day, culture until the growth length of sterile buds is 4cm. The micropropagation method of Populus alba Novosibirsk according to claim 5, characterized in that in step 4, the temperature is 27°C, the humidity is 80%, the light intensity is 50μmol·cm ^-2 ·s ^-1 , Under the condition of 17 hours of light every day, cultivate until the growth length of the micro-twigs is 5 cm. 7. the micropropagation method of a kind of Novosibirsk alba poplar according to claim 6 is characterized in that in step 5, the micro-branches after strengthening are cut to have 1 terminal bud or 2 axillary buds, and the length is 1.5 cm stem segments, and then inoculated segment by segment in the proliferation medium and cultured until the growth length of the microbranches was 5 cm. the