CN103340153A - Tissue culture method taking masson pine in-vitro mature embryo as explant - Google Patents
Tissue culture method taking masson pine in-vitro mature embryo as explant Download PDFInfo
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- CN103340153A CN103340153A CN2013102757100A CN201310275710A CN103340153A CN 103340153 A CN103340153 A CN 103340153A CN 2013102757100 A CN2013102757100 A CN 2013102757100A CN 201310275710 A CN201310275710 A CN 201310275710A CN 103340153 A CN103340153 A CN 103340153A
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Abstract
The invention relates to a tissue culture method taking a masson pine in-vitro mature embryo as an explant. The tissue culture method comprises the following steps of: adventitious bud induction, namely, inoculating the masson pine in-vitro mature embryo on an inducing medium, culturing for about 20 days, and after inducing out bud primordia from a culture, carrying out the next step; differentiation and elongation of the buds: namely, (1) transferring the obtained culture to a differential medium, culturing for several days, after forming budlets through differentiation, inoculating the budlets on a bud elongation culture medium for carrying out bud elongation, culturing for 30-40 days, and when plantlets grow to heights required for a conventional rooting procedure, carrying out the next step; and rooting: namely, taking down the obtained plantlets obtained in the step (2) from the roots, transferring the plantlets to a conventional rooting medium, after culturing for 28-35 days, white short roots begin to occur at the roots of the plantlets, at the moment, transferring the rooting plantlets to a root elongation culture medium, culturing for about 30 days, then transplanting and finally growing into normal masson pine plants.
Description
Technical field
The present invention relates to a kind of is the method for tissue culture of explant with the masson pine isolated mature embryo, belongs to woody plant tissure culture technique field.
Background technology
Masson pine originates in China, is one of important green barren hill, paper making raw material woods and the resin tapping woods in each province, important species of natural land woods on the south the Qinling Mountains, is the widest seeds of China's distribution area.Its growth is rapid, the productivity height, and adaptability is strong.Characteristics such as mechanical property is good also extensively are used to engineerings such as building, mine timber, sleeper, bridge, also can be used as the raw material of fiber board, particle board, plywood industry, have important economic value.At present the masson pine breeding is mainly based on the sexual propagation of seed orchard and seed production stand.Yet sexual propagation exists the problem of differentiation, can not improve the genetic improvement gain to greatest extent, and vegetative propagation can overcome the above-mentioned deficiency of sexual propagation.But the present still difficulty of traditional vegetative propagation technique of masson pine (cuttage and grafting) satisfies the demand of afforestation, has restricted the process of masson pine stock breeding.
Adopt the tissue culture technique breeding plant can obtain very high reproductive efficiency, plant tissue culture technique is that to utilize totipotency of plant cell (be whole hereditary information that each cell has this plant, cells in-vitro has the potential ability that develops into whole plant under certain condition of culture), take the cell mass in the plant corpus, the small part of meristematic tissue or nutrition organs, by artificial preparation Different Nutrition composition and hormone regulating and controlling, make these histocytes form whole good genetic character in thousands of plantlets and the maintenance parent, this kind method can obtain a large amount of test-tube plantlets at short notice, can in the workshop, carry out, the enforcement batch production is produced, the seedling that obtains is left in the manually operated culturing room, can produce throughout the year.And in few area, carry out large-scale production, thereby land occupation not.If utilize tissue culture technique breeding masson pine, not only for expanding propagation masson pine breeding provides a new approach, also can be the breeding of masson pine MOLECULE DESIGN and create conditions, significant.
Yet the masson pine tissue is cultivated difficult, induce the bud of generation to be difficult for the differentiation elongation, and the seedling rooting rate is extremely low.Huang Jianqiu, Wei Zhiming etc. carry out somatic embryo to the masson pine mature embryo and induce and obtain regeneration plant, coinduction produces 27 cotyledonary embryos, finally only produce the complete plantlet of 2 strains, all the other all can only extend or stop growing slightly, and the frequency that somatic embryo converts plantlet to only is 7.4%; Zhang Yu is, and masson pine is organized cultivation, and certain bud induction rate is arranged, but rooting rate only is 70%, can't satisfy the requirement of production.For this reason, the present invention is intended to develop and is suitable for the masson pine mature embryo as the high efficiency breeding technology of explant.
Summary of the invention
The present invention wants the technical solution problem to be: overcome the problem that prior art exists, provide a kind of reproduction coefficient high, be the method for tissue culture of explant with the masson pine isolated mature embryo fast, may further comprise the steps:
1, adventitious bud inducing
The masson pine isolated mature embryo is inoculated on the inducing culture, cultivated about 20 days, treat that culture is induced the original hase that sprouts after, namely begin next stage.
2, the differentiation of bud and elongation
The culture of the 1st step gained is transferred on the differential medium, cultivated a couple of days, after waiting to differentiate budlet, budlet can be inoculated into the elongation of carrying out bud on the bud elongation medium, cultivated 30-40 days, and when treating that seedling length is taken root the program desired height to routine, entered next step.
3, take root
The neat root of seedling of the 2nd step gained is taken off, go in the conventional root media, cultivated 7 days under the dark condition, change illumination cultivation 21-28 days again over to, the seedling root begins to occur the short root of white, and at this moment, the seedling of will taking root moves on in the root elongation medium, cultivate afterwards about 30 days, can transplant and grow up to normal masson pine plant.
In the technique scheme, the composition of described inducing culture is the conventional minimal medium of DCR, 6-benzyl purine 0.5-2 mg/litre, methyl 0.05-0.15 mg/litre or indolebutyric acid 0.05-0.15 mg/litre, sucrose 30 grams per liters.
In the technique scheme, the conventional minimal medium of the DCR of described differential medium, composition are 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters and agar 5.6-6.0 grams per liter.
In the technique scheme, the composition of described bud elongation medium is the conventional minimal medium of DCR, methyl 0.01-0.1 mg/litre or indolebutyric acid 0.01-0.1 mg/litre or active carbon 0-2.0 grams per liter, sucrose 30 grams per liters.
In the technique scheme, the composition of described root media is the conventional minimal medium of 1/2DCR, methyl 0.5-2.0 mg/litre or indolebutyric acid 0.5-2.0 mg/litre, sucrose 20 grams per liters.
In the technique scheme, the composition of described elongation cultivation is the conventional minimal medium of DCR, active carbon 0.5 grams per liter.
Beneficial effect provided by the invention is to have adopted two kinds to be different from the medium that other coniferous species tissue is cultivated, be inducing culture and differential medium, hormone-content is higher in the inducing culture, easily induce culture to start and break up the original hase that sprouts, inductivity is up to 90.63%, after the bud original hase induces, should not be on the medium of high concentration hormonal readiness long-term cultivation, otherwise the easy callusization of the bud that induces in the medium that in time changes the hormone concentration reduction over to, is conducive to differentiation and the elongation of bud; Take root the stage, the present invention has also adopted and has been different from the method that other coniferous species is taken root, at first, be fit to the take root seedling of height of routine being elongated to, transfer to earlier in the DCR minimal medium that only adds active carbon and cultivated 7 days under the dark condition, changed illumination cultivation again over to 23 days, transfer to again and carry out culture of rootage in the root media, this measure can improve rooting rate greatly, and rooting rate reaches 92.5%; After treating that root grows, add the 0.5g/L active carbon, be conducive to the elongation growth of root, can make dark surrounds because of the interpolation of active carbon, make the growing environment of root more close to nature, so be conducive to the elongation growth of root.
In a word, the present invention has the following advantages: the bud original hase starts fast, bud coefficient of differentiation height, the seedling elongation is fast, growth is consistent and healthy and strong, rooting rate is high, root is long and strong, growth cycle weak point, do not have a lopsided seedling substantially.
Embodiment
Embodiment 1
Present embodiment be the method for tissue culture of explant with the masson pine isolated mature embryo, may further comprise the steps:
The first step, draw materials: get the masson pine mature seed, soaked overnight, and under flowing water, rinse well, peel off the outer shell of planting with tweezers.
In second step, materials disinfection: the seed after shelling with 75% ethanol disinfection 30s, with 0.1% mercuric chloride sterilization 10-15min, is used aseptic water washing 3-5 time more at last in superclean bench.
The 3rd step, inoculation and adventitious bud inducing: in superclean bench, method with conventional sterile working, use scalpel and tweezers that embryo is peeled off out, level is inoculated on the inducing culture, want careful careful when peeling off embryo, avoid embryo is damaged as far as possible, wherein the conventional minimal medium of consisting of of inducing culture: DCR, 6-benzyl purine 0.5 mg/litre, methyl 0.05 mg/litre, sucrose 30 grams per liters.
The 4th step, the differentiation of bud and elongation: the embryo that will exsomatize is inoculated on the inducing culture, cultivate and just can see a large amount of bud of generation on the cotyledon that expands after 15 days, then it is transferred on the differential medium, wherein the conventional minimal medium of consisting of of differential medium: DCR, 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters, agar 5.6 grams per liters.
In order to make the masson pine embryo culture induce grow thickly bud further growth and the elongation of generation, can in medium, add an amount of active carbon.Though active carbon can adsorb the disadvantageous secondary metabolite of bud elongation growth, excessive active carbon can produce inhibitory action to elongation and the growth of bud, and this may be because active carbon has also adsorbed mineral element when being adsorbed with harmful substances.Therefore, bud elongation medium composition is: the conventional minimal medium of DCR, active carbon 1.0 grams per liters, sucrose 30 grams per liters, this medium has promoted the bud elongation growth.
The 5th step, take root: when the bud length of growing thickly that differentiates is taken root desired height to routine, cut with scissors is single, be inoculated in the root media, wherein the component of root media is: the conventional minimal medium of 1/2DCR, methyl 0.5 mg/litre, sucrose 20 grams per liters, cultivated 7 days under dark condition earlier, change illumination cultivation again over to, the illumination condition of this medium and cultivation is conducive to root induction.
For making the root elongation growth better that induces, the seedling of taking root can be transferred in the root elongation medium, the composition of root elongation medium is: the conventional minimal medium of DCR, active carbon 0.5 grams per liter, the root elongation growth is obvious in this medium.
Cultivation temperature and illumination condition are: temperature is 25 ± 1 ℃; Illumination is 2000 luxs, 16 hours/day.
In the 6th step, transplant: the seedling of taking root growth is after about 35 days, and root system is relatively more flourishing, generally can grow 4-5 bar main root, has the generation of fibrous root on the main root, and can transplant this moment.
The method of transplanting is: open sealing film earlier, hardening is about 1 week in culturing room, take out seedling, clean the agar of seedling root, be transplanted in the matrix that vermiculite and rice chaff ash mix (matrix use before autoclaving), be put into culturing room after watering sufficient water, covered with plastic film was opened film after 24 hours, took a breath twice, prolong every day later on and open the time, till not covering fully.Select growth vigorous, stem is thicker, the little transplantation of seedlings that profile is good, and survival rate can improve greatly.
Embodiment 2
Be with the difference of above-described embodiment, the masson pine isolated mature embryo is inoculated on the inducing culture, cultivated about 20 days, after treating that culture is induced the original hase that sprouts, namely begin next stage, wherein the component of inducing culture is the conventional minimal medium of DCR, 6-benzyl purine 2.0 mg/litre, methyl 0.15 mg/litre, sucrose 30 grams per liters; Culture is transferred on the differential medium, cultivate a couple of days, after waiting to differentiate budlet, budlet can be inoculated into the elongation of carrying out bud on the bud elongation medium, cultivated 30-40 days, when treating that seedling length is taken root the program desired height to routine, enter next step, wherein the component of differential medium is: the conventional minimal medium of DCR, 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters, agar 6.0 grams per liters, the component of bud elongation medium is: the conventional minimal medium of DCR, methyl 0.1 mg/litre, sucrose 30 grams per liters, this medium has promoted the bud elongation growth; The neat root of seedling is taken off, go in the conventional root media, under dark condition, cultivated 7 days earlier, change illumination cultivation 21-28 days again over to, the seedling root begins to occur the short root of white, at this moment, the seedling of will taking root moves on in the root elongation medium, cultivate afterwards about 30 days, can transplant and grow up to normal masson pine plant, wherein the component of root media is: the conventional minimal medium of 1/2DCR, indolebutyric acid 0.5 mg/litre, sucrose 20 grams per liters.
Embodiment 3
Be that with the difference of above-described embodiment the component of inducing culture is the conventional minimal medium of DCR, 6-benzyl purine 1.0 mg/litre, methyl 0.10 mg/litre, sucrose 30 grams per liters; The component of differential medium is: the conventional minimal medium of DCR, 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters, agar 5.7 grams per liters; The component of bud elongation medium is: the conventional minimal medium of DCR, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters; The component of root media is: the conventional minimal medium of 1/2DCR, indolebutyric acid 2.0 mg/litre, sucrose 20 grams per liters.
Embodiment 4
Be that with the difference of above-described embodiment the component of inducing culture is the conventional minimal medium of DCR, 6-benzyl purine 1.5 mg/litre, indolebutyric acid 0.10 mg/litre, sucrose 30 grams per liters; The component of differential medium is: the conventional minimal medium of DCR, 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters, agar 5.9 grams per liters; The component of bud elongation medium is: the conventional minimal medium of DCR, methyl 0.05 mg/litre, sucrose 30 grams per liters; The component of root media is: the conventional minimal medium of 1/2DCR, methyl 2.0 mg/litre, sucrose 20 grams per liters.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. one kind is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: may further comprise the steps:
(1) adventitious bud inducing: the masson pine isolated mature embryo is inoculated on the inducing culture, cultivated about 20 days, treat that culture is induced the original hase that sprouts after, namely begin next stage;
(2) differentiation of bud and elongation: the culture of (1) step gained is transferred on the differential medium, cultivate a couple of days, after waiting to differentiate budlet, budlet can be inoculated into the elongation of carrying out bud on the bud elongation medium, cultivated 30-40 days, when treating that seedling length is taken root the program desired height to routine, enter next step;
(3) take root: the neat root of seedling of (2) step gained is taken off, go in the conventional root media, cultivated 28-35 days, the seedling root begins to occur the short root of white, and at this moment, the seedling of will taking root moves on in the root elongation medium, cultivate afterwards about 30 days, can transplant and grow up to normal masson pine plant.
2. according to claim 1 is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: the composition of described inducing culture is the conventional minimal medium of DCR, 6-benzyl purine 0.5-1.5 mg/litre, methyl 0.05-0.15 mg/litre or indolebutyric acid 0.05-0.15 mg/litre, sucrose 30 grams per liters.
3. according to claim 1 is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: the composition of described differential medium is the conventional minimal medium of DCR, 6-benzyl purine 0.5 mg/litre, indolebutyric acid 0.05 mg/litre, sucrose 30 grams per liters and agar 5.6-6.0 grams per liter.
4. according to claim 1 is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: the composition of described bud elongation medium is the conventional minimal medium of DCR, methyl 0.01-0.1 mg/litre or indolebutyric acid 0.01-0.1 mg/litre or active carbon 0-2.0 grams per liter, sucrose 30 grams per liters.
5. according to claim 1 is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: the composition of described root media is the conventional minimal medium of 1/2DCR, methyl 0.5-2.0 mg/litre or indolebutyric acid 0.5-2.0 mg/litre, sucrose 20 grams per liters; Under dark condition, cultivated 7 days earlier, change illumination cultivation again over to.
6. according to claim 1 is the method for tissue culture of explant with the masson pine isolated mature embryo, it is characterized in that: the composition of described elongation cultivation is the conventional minimal medium of DCR, active carbon 0.5 grams per liter.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104663445A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | In-vitro rapid propagation technology of high lipid-producing pinus elliottii |
| CN106135005A (en) * | 2016-08-26 | 2016-11-23 | 李军 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN111621520A (en) * | 2020-06-22 | 2020-09-04 | 南京林业大学 | Application of masson pine PmCOMT gene |
| CN111657150A (en) * | 2020-07-24 | 2020-09-15 | 广西壮族自治区林业科学研究院 | Method for improving rooting capacity of masson pine based on endogenous hormone |
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| CN101785430A (en) * | 2009-11-18 | 2010-07-28 | 南京林业大学 | Tissue culture technology using Pinus massoniana isolated mature embryo as explant |
| CN101849505A (en) * | 2010-02-10 | 2010-10-06 | 南京林业大学 | Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana |
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| CN101785430A (en) * | 2009-11-18 | 2010-07-28 | 南京林业大学 | Tissue culture technology using Pinus massoniana isolated mature embryo as explant |
| CN101849505A (en) * | 2010-02-10 | 2010-10-06 | 南京林业大学 | Method for inducing embryonal-suspensor mass (ESM) regeneration plant from immature seed of pinus massoniana |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104663445A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | In-vitro rapid propagation technology of high lipid-producing pinus elliottii |
| CN106135005A (en) * | 2016-08-26 | 2016-11-23 | 李军 | A kind of little Semen Pini Tissue Culture Regeneration System construction method |
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN111621520A (en) * | 2020-06-22 | 2020-09-04 | 南京林业大学 | Application of masson pine PmCOMT gene |
| CN111657150A (en) * | 2020-07-24 | 2020-09-15 | 广西壮族自治区林业科学研究院 | Method for improving rooting capacity of masson pine based on endogenous hormone |
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Application publication date: 20131009 |