CN103155866B - Malus zumi tissue culture rapid propagation seedling raising method - Google Patents
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Abstract
The invention discloses a malus zumi tissue culture rapid propagation seedling raising method which comprises the following steps that (1) raw materials are processed: a terminal bud or a lateral bud of a field annual seedling malus zumi sapling is sheared; (2) primary culture is carried out: the terminal bud or the lateral bud is inoculated onto an induction medium to be cultured, the induction medium is made by the fact that raw materials are added into an MS minimal medium of one liter, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and 6-benayl aminopurine of 0.2-0.3mg; (3) enrichment culture is carried out: the terminal bud or the lateral bud is transferred into an enrichment medium to be cultured, the enrichment medium is made by the fact that raw materials are added into an MS enrichment medium of one liter, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and 6-benayl aminopurine 0.6-0.8mg; (4) rooting culture is carried out: the terminal bud or the lateral bud is placed into a root medium to be cultured, the root medium is made by the fact that raw materials are added into 1/2 MS minimal medium, and the raw materials and proportions of the raw materials are white granulated sugar of 10g, agar powder of 4.5g, and indolebutyric acid of 0.1-0.2mg; and (5) hardening-seedling and transplantation are carried out.
Description
Technical field
The invention belongs to field of plant tissue culture technique, specifically a kind of method by plant tissue culture technique breeding Mains Zumi.
Background technology
Mains Zumi originates in Japanese Mountainous Area of North, it is the wild Natural hybrid of Siberian crabapple and toringo, there is stronger saline-alkali tolerance, cold resistance, Ye Shi China scientific worker goes through the time of 13 years, screens anti-saline and alkaline new varieties of breeding out, through observing discovery for many years, Mains Zumi robust growth, tree crown circle are attractive in appearance, disease-free, be the desirable green tree species of varieties in saline-alkali areas, also can be used as stock grafting apple in addition, the area heavier in saline land is applied.
Plant tissue culture technology is the totipotency utilizing plant cell, namely each cell forming plant corpus has the potential ability budding into a whole plant, take the individual cells on plant corpus, cell mass, a part for meristematic tissue or organ, cultivated by the medium manually preparing Different Nutrition composition and hormone and regulate and control it and grow, make these cells, tissues etc. form thousands of plantlets and keep whole merits of maternal plant, this method can obtain a large amount of plantlet in vitro at short notice, and anniversary large-scale production can be carried out under manual control condition.Utilize plant tissue culture technology to carry out the factorial praluction of fruit tree flowers good seed, become the foundation engineering of modern horticultural agricultural development.
In the prior art, Mains Zumi provenance lacks, cuttage difficulty is taken root, and easily genetic variation occurs, is difficult to realize the large-scale industrialized production of high-quality.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Mains Zumi tissue-culturing rapid propagation seedling-cultivating method.
Technical scheme of the present invention is: a kind of Mains Zumi tissue-culturing rapid propagation seedling-cultivating method, it comprises the following steps: (1) raw-material process clip land for growing field crops annual Mains Zumi treelet terminal bud or lateral bud, stand-by as explant after using alcohol, mercuric chloride solution, hydrogen peroxide surface sterilization sterilizing respectively; (2) described explant is seeded on inducing culture and cultivates by Initial culture, this inducing culture for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2 ~ 0.3mg in 1 liter of MS minimal medium, condition of culture is: temperature 23 ± 2 DEG C, intensity of illumination 2000 ~ 2500Lx, light application time 10 ~ 13 hours/day, humidity 40% ~ 50%; (3) the bud seedling after grow thickly stem apex or the propagation of Initial culture is transferred in proliferated culture medium and cultivates by Multiplying culture, this proliferated culture medium for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.6 ~ 0.8mg indolebutyric acid 0.1 ~ 0.2mg in 1 liter of MS minimal medium, condition of culture is: temperature 23 ± 2 DEG C, intensity of illumination 2500 ~ 3000Lx, light application time 10 ~ 13 hours/day, humidity 40% ~ 50%; (4) 1.5 ~ 3cm stem apex of Multiplying culture is put into root media and is cultivated by culture of rootage, this root media for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.1 ~ 0.2mg in 1/2MS minimal medium, condition of culture is: temperature 23 ± 2 DEG C, humidity 40 ~ 50%, intensity of illumination 2500 ~ 3000Lx, light application time 24 hours/day; (5) acclimatization and transplants comprises following link: in closing that bottle is taken exercise, uncork is taken exercise, be transplanted to vermiculite matrix, being transplanted to the Nutrition Soil that is made up of soil, husky, decomposed manure.
Preferably, described inducing culture for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2mg in 1 liter of MS minimal medium; Described proliferated culture medium for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g6-benayl aminopurine 0.8mg indolebutyric acid 0.2mg in 1 liter of MS minimal medium; Described root media for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.2mg in 1/2MS minimal medium.
For medium composition and the relation of the stage culture effect such as proportioning, condition of culture and Initial culture, Multiplying culture, culture of rootage thereof or impact, inventor has carried out experimental study, in order to set forth the present invention further, experimental data and result of study are described below:
1, the different culture media impact that bud survived and grows
Young shoot after the sterilizing of stalwartness is inoculated in different minimal medium MS, 1/2MS and F by this test, on the variable concentrations of same hormone 6-benzyl aminopurine (6-BA), checks the survival rate of bud after 1 week.
Experiment conclusion: different minimal mediums all exists certain facilitation to the sprouting of bud, all can the sprouting of evoking adventive bud, but show obvious otherness, its inductivity of MS, 1/2MS is higher, inoculates and within about 5 days, start to sprout, grow in the stem apex performance of MS better, blade is dark green, and on 1/2MS medium, stem apex growth potential is more weak, and on L medium, bud is sprouted slower, one Zhou Houcai induces part callus, forms a small amount of indefinite bud.In MS medium, when 6-BA concentration is 0.2 ~ 0.3 time, bud not only survival rate is high, and robust growth, and sprout neat, with the increase of concentration, callus degree increases the weight of, and seedling abnormal rate increases, and when concentration is more than 1.0, only forms bulk tissue, sprouts less.
The impact that the same hormone different culture media of table 1 survives bud and grows
2, hormone is on the impact of Shoot propagation
1) 6-benzyl aminopurine (6-BA) is on the impact of Shoot propagation
The 6-BA adding variable concentrations in MS minimal medium carries out Shoot propagation cultivation, and experimental result is in table 2.
Table 2 6-BA is on the impact of Shoot propagation
Experiment conclusion: when 6-BA concentration is lower, bud robust growth, leaf look dark green, along with the increase propagation quantity of concentration strengthens, but 1.0 and above time, pumping bud is grown thickly thin and delicate, cannot be used for breeding next time, and callus produces in a large number, is unfavorable for taking root; Bud robust growth when concentration is too low, but germination negligible amounts, can not meet fast-propagation.So 0.6 ~ 0.8mg is the suitable concentration of 6-BA for Shoot propagation, 0.8mg/l is optium concentration.
2) 6-benzyl aminopurine (6-BA) and indolebutyric acid (IBA) combinationally use the impact on Shoot propagation
6-BA and IBA adding variable concentrations in MS minimal medium carries out Shoot propagation cultivation, and experimental result is in table 3.
Table 3 6-BA and IBA is on the impact of Shoot propagation
Experiment conclusion: 6-BA0.6mg/l+IBA0.2mg/l or 6-BA0.8mg/l+IBA0.2mg/l is healthy and strong with the use of the bud seedling of, pumping, growth is neat, and be more conducive to follow-on expansion numerous and take root, growth coefficient is up to 6.80 and 7.68.Find in test that 6-BA is that Mains Zumi stem apex Multiplying culture is necessary, it can break apical dominance, promotes axillary bud sprouting.
3, medium, hormone and condition of culture are to the facilitation of taking root
The propagation seedling cut-grafting of long 1.5-3cm, robust growth in the IBA of different minimal medium MS, 1/2MS and variable concentrations, is checked situation of taking root after 20d by this test.
1) different culture media and variable concentrations IBA are on the impact of bud seedling rooting
Experiment conclusion: the test-tube plantlet of different culture media and variable concentrations IBA process, its rooting rate and the amount of taking root have larger difference.Situation of taking root on 1/2MS minimal medium is generally higher than MS minimal medium; In two kinds of different minimal mediums, when IBA concentration is 0.2mg/l, rooting rate is all high, and along with concentration increases, rooting rate and root system number decline on the contrary, show, when IBA concentration is higher than 0.2mg/l, can suppress taking root of test-tube plantlet.In all process, 1/2MS+IBA0.2mg/l rooting rate is the highest, is 100%, and number of on average taking root is 4.48.
Table 4 different culture media and variable concentrations IBA are on the impact of bud seedling rooting.Note: on average take root and count the mean of stock number of making a living.
2) different light is on the impact of bud seedling rooting
Table 5 different illumination conditions is on the impact of bud seedling rooting
| Cultivate under culturing room's full exposure | Cultivate under culturing room's natural lighting | |
| Rooting rate (%) | 97.4 | 81.4 |
| Number of taking root (bar) | 4.43 | 3.61 |
Experiment conclusion: the test-tube plantlet of access 1/2MS+IBA0.2mg/l root media, group training room full exposure (2500 ~ 3000lx) cultivation in 24 hours through about 10 days effectively can improve rooting rate and number of taking root, average rooting rate reaches 97.4%, and number of on average taking root reaches 4.43.Under Indoor Natural illumination (2000 ~ 5000lx), rooting of vitro seedling rate also reaches 81.4%, number of on average taking root reaches 3.61, but the root produced under Indoor Natural optical condition is more sturdy, quality is crisp, and the root produced under the illumination cultivation condition of group training room is thinner, pliability is good, and not easily damaged during transplanting, planting survival rate is high.
3) different cultivation temperature is on the impact of bud seedling rooting
Test-tube plantlet in 1/2MS+IBA0.2mg/l root media, under not temperature condition, cultivates 15 days its rooting rates of " Invest, Then Investigate " and number of taking root under 24 hours full exposure (2500 ~ 3000lx) conditions in group training room.
The impact that table 6 different temperatures grows bud seedling rooting
Experiment conclusion: the culture of rootage of different cultivation temperature on test-tube plantlet has significant impact, cultivate 15d in same medium after, under 25 DEG C of conditions, rooting rate is the highest, and the new root number sent is maximum; Next is 30 DEG C, 35 DEG C, and under 20 DEG C of conditions, rooting rate is minimum.Show that temperature is at about 25 DEG C, be conducive to growth and the elongation of new root, and lower temperature is unfavorable for root growth.
4, different relative moisture, different substrates are on the impact of test-tube seedling transplanting
In greenhouse by solar heat, the impact that different relative moisture, different substrates survive test-tube seedling transplanting.
The impact that table 7 different humidity, different substrates survive test-tube seedling transplanting
Experiment conclusion: find out from three process table, damp condition very large situation make decision transplant success or failure, under greenhouse natural humidity condition, the transplanting survival rate of three kinds of different substrates is only about 10%, and the survival rate of three kinds of matrix all reaches more than 70% under Small plastic shed manually sprays damp condition.In three kinds of matrix, the transplanting survival rate of vermiculite is all high, is issued to more than 98% at the artificial spray condition of Small plastic shed.
The invention has the beneficial effects as follows, the present invention adopts light to cultivate the preparation carrying out plantlet in vitro, nurturing staff and hestening rooting, and at natural daylight lower refining seedling, transplant the method for tissue culture synchronously carried out, group training step have employed proliferated culture medium and the root media of applicable Mains Zumi Fast-propagation, propagation and rooting efficiency good, the rooting rate of Mains Zumi is made to reach 100%, and root system is neatly sturdy, plantlet in vitro robust growth simultaneously, leaf look dark green, transplanting survival rate can reach about 95%, for factorial praluction is laid a good foundation, the inventive method is simple and easy to do, production efficiency is high, can be used for the Fast-propagation of Mains Zumi.
Embodiment
Embodiment 1
A kind of Mains Zumi tissue-culturing rapid propagation seedling-cultivating method, it comprises the following steps:
(1) raw-material process
Raw-material process: the annual treelet terminal bud of the vigorous excellent Mains Zumi of clip grown in field or lateral bud, by running water flow wash 20 minutes, then by bud on superclean bench with 75% alcohol surface sterilization 30 seconds, 0.1% mercuric chloride sterilizing 5 minutes, 15% hydrogen peroxide process 10 minutes, aseptic water washing 4 times, blots with aseptic filter paper, stand-by as explant.
(2) plantlet in vitro Initial culture
Explant after sterilization is seeded on inducing culture and carries out differentiation-inducing cultivation, and this inducing culture for adding following raw material and proportioning is made in 1 liter of MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
6-benzyl aminopurine 0.2mg
The Initial culture base configured is divided in blake bottle, often bottled 25 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.1MPa, being inserted by explant material has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 2000Lx, temperature is 21 DEG C, humidity is 40% cultivates, and irradiates 10 hours every day; Newly connecing material was transferred in new medium every 3 days, repeated 2 times, when medium no longer brown stain time, bud is proceeded in growth medium and grows.
(3) plantlet in vitro Multiplying culture
The stem section of growing thickly of Fiber differentiation be transferred in proliferated culture medium and carry out Fast-propagation, this proliferated culture medium for adding following raw material and proportioning is made in 1 liter of MS minimal medium.
The proliferated culture medium configured is divided in blake bottle, often bottled 25 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.1MPa, being inserted by induced bud has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 2500Lx, temperature is 21 DEG C, humidity is 40% cultivates, and irradiates 10 hours every day, illumination cultivation 20 angel seedling and bud proliferation; In order to expanding propagation quantity, be repeatedly transferred to by the bud seedling after propagation in the proliferated culture medium of new configuration, squamous subculture condition is identical with first generation condition of culture, by production task, breed 8-20 for time propagation bud seedling is carried out culture of rootage.
(4) plantlet in vitro culture of rootage
The stem apex of the 1.5cm of squamous subculture robust growth is put into root media and carries out culture of rootage, this root media for adding following raw material and proportioning is made in 1/2MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
Indolebutyric acid 0.1mg
Root media is divided in blake bottle, 25 milliliters every bottle, seal bottleneck, sterilizing 25 minutes under pressure is 0.1MPa, the culturing room that blake bottle is placed in intensity of illumination 2500Lx, temperature is about 21 DEG C, humidity is 40% cultivates, illumination every day is cultivated for 24 hours, within 8 ~ 10 days, can induce the formation of root.Culturing room can be pulled out when a bottle seedling of taking root grows to 25 ~ 30 days and carry out natural daylight acclimatization and transplants.
(5) training tissue culture seedling is transplanted
A bottle seedling of taking root can pull out culturing room and move in greenhouse by solar heat when growing to 25 ~ 30 days, first hardening 5 days in greenhouse, the running water hardening 2 days of 0.5cm is added after opening sealing, when offspring leaf color to be generated is slightly deepened, take out from blake bottle, wash away the medium in shoot root portion, with 600 times of carbendazim libation at an ancient wedding ceremony roots, transplant extremely with in the vermiculite seedling-raising cup of bactericidal agent process, each seedling-raising cup is planted 3 strains, spread and sink in after cultivation permeable, be placed in the Small plastic shed built in greenhouse, temperature 20 DEG C, early stage shelters from heat or light spraying, humidity remains on 80%, take off film after 10 days gradually to ventilate, Small plastic shed canopy film is thrown off after 15 days, spray 1000 times of carbendazim solution spray nursery stocks during this period, when nursery stock obviously grows, nursery stock individual plant is moved into soil, husky, decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, outside immigration greenhouse in shade, nursery stock is made to adapt to open country growing environment gradually, when growing to about 10cm Deng seedling in nutritive cube, root system band soil mud enters land for growing field crops, cultivation management is with reference to field seedling raising method.
Embodiment 2
A kind of Mains Zumi tissue-culturing rapid propagation seedling-cultivating method, it comprises the following steps:
(1) raw-material process
Raw-material process: the annual treelet terminal bud of the vigorous excellent Mains Zumi of clip grown in field or lateral bud, by running water flow wash 10 minutes, then by bud on superclean bench with 75% alcohol surface sterilization 30 seconds, 0.1% mercuric chloride sterilizing 10 minutes, 15% hydrogen peroxide process 15 minutes, aseptic water washing 4 times, blots with aseptic filter paper, stand-by as explant.
(2) plantlet in vitro Initial culture
Explant after sterilization is seeded on inducing culture and carries out differentiation-inducing cultivation, and this inducing culture for adding following raw material and proportioning is made in 1 liter of MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
6-benzyl aminopurine 0.3mg
The Initial culture base configured is divided in blake bottle, often bottled 35 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.15MPa, being inserted by explant material has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 2500Lx, temperature is 25 DEG C, humidity is 50% cultivates, and irradiates 13 hours every day; Newly connecing material was transferred in new medium every 5 days, repeated 3 times, when medium no longer brown stain time, bud is proceeded in growth medium and grows.
(3) plantlet in vitro Multiplying culture
The stem section of growing thickly of Fiber differentiation be transferred in proliferated culture medium and carry out Fast-propagation, this proliferated culture medium for adding following raw material and proportioning is made in 1 liter of MS minimal medium.
The proliferated culture medium configured is divided in blake bottle, often bottled 35 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.15MPa, being inserted by induced bud has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 3000Lx, temperature is 25 DEG C, humidity is 50% cultivates, and irradiates 13 hours every day, illumination cultivation 25 angel seedling and bud proliferation; In order to expanding propagation quantity, be repeatedly transferred to by the bud seedling after propagation in the proliferated culture medium of new configuration, squamous subculture condition is identical with first generation condition of culture, and by production task, propagation bud seedling is carried out culture of rootage during 8 ~ 20 generation by breeding.
(4) plantlet in vitro culture of rootage
The stem apex of the 3cm of squamous subculture robust growth is put into root media and carries out culture of rootage, this root media for adding following raw material and proportioning is made in 1/2MS minimal medium.
White granulated sugar 10g
Agar powder 4.5g
Indolebutyric acid 0.2mg
Root media is divided in blake bottle, 35 milliliters every bottle, seals bottleneck, sterilizing 25 minutes under pressure is 0.15MPa, the culturing room that blake bottle is placed in intensity of illumination 3000Lx, temperature is 25 DEG C, humidity is 50% cultivates, and illumination every day is cultivated for 24 hours, within 8 ~ 0 days, can induce the formation of root.Culturing room can be pulled out when a bottle seedling of taking root grows to 25 ~ 30 days and carry out natural daylight acclimatization and transplants.
(5) training tissue culture seedling is transplanted
A bottle seedling of taking root can pull out culturing room and move in greenhouse by solar heat when growing to 25 ~ 30 days, first hardening 7 days in greenhouse, the running water hardening 3 days of 1.0cm is added after opening sealing, when offspring leaf color to be generated is slightly deepened, take out from blake bottle, wash away the medium in shoot root portion, with 800 times of carbendazim libation at an ancient wedding ceremony roots, transplant extremely with in the vermiculite seedling-raising cup of bactericidal agent process, each seedling-raising cup is planted 4 strains, spread and sink in after cultivation permeable, be placed in the Small plastic shed built in greenhouse, temperature 25 DEG C, early stage shelters from heat or light spraying, humidity remains on 100%, take off film after 10 days gradually to ventilate, Small plastic shed canopy film is thrown off after 15 days, spray 1000 times of carbendazim solution spray nursery stocks during this period, when nursery stock obviously grows, nursery stock individual plant is moved into soil, husky, decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, outside immigration greenhouse in shade, nursery stock is made to adapt to open country growing environment gradually, in nutritive cube, about 10cm is grown to Deng seedling, root system band soil mud enters land for growing field crops, cultivation management is with reference to field seedling raising method.
Embodiment 3
A kind of Mains Zumi tissue-culturing rapid propagation seedling-cultivating method, it comprises the following steps:
(1) raw-material process
Raw-material process: the annual treelet terminal bud of the vigorous excellent Mains Zumi of clip grown in field or lateral bud, by running water flow wash 15 minutes, then by bud on superclean bench with 75% alcohol surface sterilization 20 seconds, 0.1% mercuric chloride sterilizing 7 minutes, 15% hydrogen peroxide process 12 minutes, aseptic water washing 4 times, blots with aseptic filter paper, stand-by as explant.
(2) plantlet in vitro Initial culture
Explant after sterilization is seeded on inducing culture and carries out differentiation-inducing cultivation, and this inducing culture is identical with embodiment 1.
The Initial culture base configured is divided in blake bottle, often bottled 30 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.12MPa, being inserted by explant material has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 2200Lx, temperature is 23 DEG C, humidity is 45% cultivates, and irradiates 11 hours every day; Newly connecing material was transferred in new medium every 4 days, repeated 2 times, when medium no longer brown stain time, bud is proceeded in growth medium and grows.
(3) plantlet in vitro Multiplying culture
The stem section of growing thickly of Fiber differentiation be transferred in proliferated culture medium and carry out Fast-propagation, this proliferated culture medium is identical with embodiment 2.
The proliferated culture medium configured is divided in blake bottle, often bottled 30 milliliters, seal bottleneck, sterilizing 25 minutes under pressure is 0.12MPa, being inserted by induced bud has gone out in the medium of bacterium, the culturing room that blake bottle is placed in intensity of illumination 2800Lx, temperature is 23 DEG C, humidity is 45% cultivates, and irradiates 12 hours every day, illumination cultivation 23 angel seedling and bud proliferation; In order to expanding propagation quantity, be repeatedly transferred to by the bud seedling after propagation in the proliferated culture medium of new configuration, squamous subculture condition is identical with first generation condition of culture, and by production task, propagation bud seedling is carried out culture of rootage during 8 ~ 20 generation by breeding.
(4) plantlet in vitro culture of rootage
The stem apex of the 1.5cm of squamous subculture robust growth is put into root media and carries out culture of rootage, this inducing culture of this root media is identical with embodiment 2.
Root media is divided in blake bottle, 25 milliliters every bottle, seal bottleneck, sterilizing 25 minutes under pressure is 0.1MPa, the culturing room that blake bottle is placed in intensity of illumination 2500Lx, temperature is about 21 DEG C, humidity is 45% cultivates, illumination every day is cultivated for 24 hours, within 8 ~ 10 days, can induce the formation of root.Culturing room can be pulled out when a bottle seedling of taking root grows to 25 ~ 30 days and carry out natural daylight acclimatization and transplants.
(5) training tissue culture seedling is transplanted
A bottle seedling of taking root can pull out culturing room and move in greenhouse by solar heat when growing to 25 ~ 30 days, first hardening 5d in greenhouse, the running water hardening 2 days of 0.5cm is added after opening sealing, when offspring leaf color to be generated is slightly deepened, take out from blake bottle, wash away the medium in shoot root portion, with 600 times of carbendazim libation at an ancient wedding ceremony roots, transplant extremely with in the vermiculite seedling-raising cup of bactericidal agent process, each seedling-raising cup is planted 3 strains, spread and sink in after cultivation permeable, be placed in the Small plastic shed built in greenhouse, temperature 20 DEG C, early stage shelters from heat or light spraying, humidity remains on 80%, take off film after 10 days gradually to ventilate, Small plastic shed canopy film is thrown off after 15 days, spray 1000 times of carbendazim solution spray nursery stocks during this period, when nursery stock obviously grows, nursery stock individual plant is moved into water, husky, decomposed manure volume ratio is in the nutrient matrix of 1: 1: 0.5, outside immigration greenhouse in shade, nursery stock is made to adapt to open country growing environment gradually, in nutritive cube, about 10cm is grown to Deng seedling, root system band soil mud enters land for growing field crops, cultivation management is with reference to field seedling raising method.
Claims (1)
1. a Mains Zumi tissue-culturing rapid propagation seedling-cultivating method, it is characterized in that, it comprises the following steps: (1) raw-material process clip land for growing field crops annual Mains Zumi treelet terminal bud or lateral bud, stand-by as explant after using alcohol, mercuric chloride solution, hydrogen peroxide surface sterilization sterilizing respectively; (2) described explant is seeded on inducing culture and cultivates by Initial culture, this inducing culture for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.2mg in 1 liter of MS minimal medium, condition of culture is: temperature is 23 ± 2 DEG C, intensity of illumination 2000 ~ 2500Lx, light application time 10 ~ 13 hours/day, humidity is 40%-50%; (3) the bud seedling after grow thickly stem apex or the propagation of Initial culture is transferred in proliferated culture medium and cultivates by Multiplying culture, this proliferated culture medium for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g 6-benzyl aminopurine 0.8mg indolebutyric acid 0.2mg in 1 liter of MS minimal medium, condition of culture is: temperature is 23 ± 2 DEG C, intensity of illumination 2500 ~ 3000Lx, light application time 10 ~ 13 hours/day, humidity is 40% ~ 50%; (4) 1.5 ~ 3cm stem section of Multiplying culture is put into root media and is cultivated by culture of rootage, this root media for adding following raw material and proportioning is made: white granulated sugar 10g agar powder 4.5g indolebutyric acid 0.2mg in 1/2MS minimal medium, condition of culture is: temperature 23 ± 2 DEG C, humidity 40 ~ 50%, intensity of illumination 2500 ~ 3000Lx, light application time 24 hours/day; (5) acclimatization and transplants comprises following link: close bottle and take exercise, uncork is taken exercise, and is transplanted to vermiculite matrix, is transplanted in the Nutrition Soil be made up of soil, sand, decomposed manure.
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| CN105830927B (en) * | 2016-04-20 | 2018-03-30 | 甘肃德美地缘现代农业集团有限公司 | Salt-soda soil apple grafting cultivation method |
| CN106212274B (en) * | 2016-07-15 | 2018-07-06 | 武汉市农业科学技术研究院林业果树科学研究所 | The method for tissue culture of special beautiful Malus spectabilis |
| CN111194693B (en) * | 2020-01-19 | 2022-03-25 | 南京林业大学 | Tissue culture rapid propagation method for new variety of Chinese flowering crabapple |
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| CN1288656A (en) * | 2000-11-09 | 2001-03-28 | 中国科学院石家庄农业现代化研究所 | Fast seedling growing mehtod for Chinese flowering crabapple zhumei |
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| CN102106260A (en) * | 2010-11-29 | 2011-06-29 | 天津滨海国际花卉科技园区股份有限公司 | Inoculation method for tissue culture of Rieger Begonia |
| CN102217549A (en) * | 2011-06-03 | 2011-10-19 | 张曦予 | Culture method of rieger begonias test tube flowers |
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| WO2007121518A1 (en) * | 2006-04-21 | 2007-11-01 | The State Of Western Australia Through Its Department Of Agriculture | Improved plant culture methods using a modified auxin treatment step |
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| US6127182A (en) * | 1998-10-23 | 2000-10-03 | Agritope, Inc. | Rapid recovery of shoots through thin stem slices after preconditioning of micropropagated fruit tree shoots |
| CN1288656A (en) * | 2000-11-09 | 2001-03-28 | 中国科学院石家庄农业现代化研究所 | Fast seedling growing mehtod for Chinese flowering crabapple zhumei |
| CN101292630A (en) * | 2008-06-18 | 2008-10-29 | 深圳职业技术学院 | Rieger Begonia tissue culture quick replication method |
| CN102106260A (en) * | 2010-11-29 | 2011-06-29 | 天津滨海国际花卉科技园区股份有限公司 | Inoculation method for tissue culture of Rieger Begonia |
| CN102217549A (en) * | 2011-06-03 | 2011-10-19 | 张曦予 | Culture method of rieger begonias test tube flowers |
Non-Patent Citations (1)
| Title |
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| 珠美海棠的组织培养与快速繁殖技术;张希太;《农业科技与信息》;20110520(第10期);第2.2.1-2.2.3、3.1节 * |
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