CN104186317A - Tissue culture rapid propagation method for Lagerstroemia intermedia Koehne - Google Patents

Tissue culture rapid propagation method for Lagerstroemia intermedia Koehne Download PDF

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CN104186317A
CN104186317A CN201410394862.7A CN201410394862A CN104186317A CN 104186317 A CN104186317 A CN 104186317A CN 201410394862 A CN201410394862 A CN 201410394862A CN 104186317 A CN104186317 A CN 104186317A
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seed
yunnan
root
crape myrtle
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CN104186317B (en
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张启翔
王轲
潘会堂
蔡明�
程堂仁
王佳
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Beijing Forestry University
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Beijing Forestry University
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Abstract

The invention relates to a tissue culture rapid propagation method for Lagerstroemia intermedia Koehne. The method comprises the following steps: picking up lignified and uncracked fruits in the seed maturation season of Lagerstroemia intermedia Koehne, storing the fruits for 2 weeks under the condition of 4 DEG C, sterilizing the whole fruits, splitting the fruits, taking out the seeds, removing seed wings, inoculating a 1/2MS minimal medium containing 0.5g/L of activated carbon, performing dark culture for 1-2 weeks until the seeds germinate, and culturing in the sun; inoculating a multiple-shoot induction culture medium with germinated seedlings of which the height exceeds 2cm, and culturing for 5-6 weeks, thereby obtaining lots of multiple shoots; and inoculating a rooting medium with the multiple shoots, culturing for 6-8 weeks until all sterile seedlings root, hardening the rooting seedlings, and transplanting out of the bottle. Starting from propagation of Lagerstroemia intermedia Koehne, the problems that seed germination is difficult and the propagation coefficient is low under natural conditions are solved by utilizing a tissue culture method, and a foundation is laid for preservation of germplasm resources of Lagerstroemia intermedia Koehne and deeper research.

Description

The tissue culture and rapid propagation method of Yunnan crape myrtle
Technical field
The present invention relates to plant tissue culture technique, specifically, relate to the tissue culture and rapid propagation method of Yunnan crape myrtle.
Background technology
Yunnan crape myrtle (Lagerstroemia intermedia Koehne) is Lythraceae (Lythraceae), and the evergreen megaphanerophyte of Lagerstroemia, is state three level protecting plant.At present there are approximately 55 kinds of plant of Lagerstroemia in the whole world, is mainly distributed in east Asia, the southeast and Australia northern, and China originates in 18 kinds of plant of Lagerstroemias, after from Southeast Asia, introduce and cultivate 3 kinds, totally 21 kinds.Plant of Lagerstroemia and other flowers and trees Comparatively speaking, the bright-coloured beauty of pattern, the florescence is long, the life-span is long, also has in addition contamination resistance strong, adapts to the advantages such as urban environment, therefore very popular, in afforestation, is widely used.
In city, common plant of Lagerstroemia has crape myrtle, banaba, Fujian crape myrtle, Guizhou Province, river crape myrtle etc. at present, Yunnan crape myrtle is an important kind of plant of Lagerstroemia, be special product of China seeds, be mainly distributed in Simao, Yunnan and billows deep blue, mix in the shaw of being born in 1800 meters.Compare with other plant of Lagerstroemias, Yunnan crape myrtle not only has beautiful very large flower, and its florescence early, enters full-bloom stage May, and the crape myrtle of Beijing area has just just sprouted May, so it can be used as the important parent of plant of Lagerstroemia early blossoming breeding research; Another important feature of Yunnan crape myrtle is that it is evergreen megaphanerophyte, be only a kind of evergreen kind in plant of Lagerstroemia, so the introducing of Yunnan crape myrtle germ plasm resource has great significance to the improvement of plant of Lagerstroemia fancy points.
Yunnan crape myrtle plant is tall and big tall and straight, and blade ovalize or square circle shape are oval, and blade is equally large and beautiful with flower, has ornamental value, be rare early blossoming seeds and evergreen species, and plant is tall and big, and plant type is tall and straight.Though natural conditions are descending fruit power strong, naturally upgrade badly, sylvan life treelet is rare, and this may be that the hard and seed wing of seed skin due to Yunnan crape myrtle causes more greatly, is therefore difficult to a large amount of seedling of acquisition.Tissue culture technique is a kind of common plant fast propagation means, for breeding under field conditions (factors) means that difficult kind can train by group mostly, realizes numerously soon, thereby make germplasm preserve and produce to promote, becomes possibility.
Because Yunnan crape myrtle is China's endemic tree, only on Simao, Yunnan billows deep blue and other places, distribute, the current research about Yunnan crape myrtle is also in space state.
Summary of the invention
The tissue culture and rapid propagation method that the object of this invention is to provide Yunnan crape myrtle.
In order to realize the object of the invention, the tissue culture and rapid propagation method of Yunnan provided by the invention crape myrtle, in Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit, under 4 ℃ of conditions, store 2 weeks, then whole fruit is carried out to sterilizing, cut fruit open, take out seed, remove seed wing, be inoculated in containing in the 1/2MS minimal medium of 0.5g/L active carbon, secretly cultivate 1-2 week until then seed germination forwards under light and cultivate; The sprouting seedling that highly surpasses 2cm is inoculated on inducing clumping bud medium, cultivates 5-6 week, obtain a large amount of Multiple Buds, Multiple Buds is inoculated on root media, cultivates 6-8 week, until all aseptic seedling are all taken root, then the seedling of taking root is carried out to hardening, transplant bottle outlet.
Particularly, said method comprising the steps of:
(1) 11-12 month, Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit, in 4 ℃ of refrigerators, store 2 weeks, with the clean spirit of washing of 100 times of dilutions, soak 30min, with cleaning ball, grind off pericarp, then more than rinsing 2h under flowing water, in superclean bench, with 75% alcohol sterilizing 1min, then use 4% liquor natrii hypochloritis's sterilizing 30min, with aseptic water washing 5-6 time;
(2) cut the fruit of sterilizing open, take out seed, with aseptic scalpel, cut away seed wing, to expose seed, be advisable, be inoculated in the 1/2MS minimal medium containing 0.5g/L active carbon, secretly cultivate 1-2 about week, seed starts to sprout seedling, is then transferred under light and cultivates;
Minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(3) select highly to surpass the aseptic seeding seedling of 2cm, by sterile scissors, cut off root and unnecessary blade, be inoculated in inducing clumping bud medium, carry out inducing clumping bud;
Inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, pH5.2-5.4; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(4) treat that inducing clumping bud cultivates 5-6 after week, select highly over 3cm, and the Multiple Buds of robust growth is inoculated in root induction medium, carry out root induction;
Root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(5) when aseptic seedling root growth is intact, reach 3-4cm length, and while being no less than 5, open sealed membrane and train indoor hardening 1 week in group, then with sterile water, wash away the medium of group training shoot root portion, transplant to the sterilized peat composed of rotten mosses and the perlite volume ratio mixed-matrix that is 3:1, bagging maintenance humidity, and keep spray water every day 2-3 time, then reduce gradually humidity and increase intensity of illumination, after 2 weeks, after transplanted seedling survives, in greenhouse, carrying out cellar culture.
The present invention is also provided for the minimal medium of Yunnan crape myrtle group training, and described minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0.
The present invention is also provided for the inducing clumping bud medium of Yunnan crape myrtle group training, and described inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, pH5.2-5.4.
The present invention is also provided for the root induction medium of Yunnan crape myrtle group training, and described root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0.
The present invention starts with from the breeding of Yunnan crape myrtle, the method of utilizing tissue to cultivate solves its problem that seed germination is difficult under field conditions (factors), reproduction coefficient is lower, utilize the inventive method, every seedling from seed can a disposable acquisition 4-7 seedling, preserves and carry out more deep research to lay the foundation for follow-up Yunnan crape myrtle germ plasm resource.
(1) the present invention provides the group culturation rapid propagating technology of the distinctive plant of Lagerstroemia of a set of complete China Yunnan crape myrtle first.
(2) with the seed of Yunnan crape myrtle as explant, easily obtain, and convenient transportation, suitablely different, local it is studied.The method of utilizing tissue to cultivate, can obtain a large amount of Multiple Buds in a short time, has solved Yunnan crape myrtle and has bred under field conditions (factors) difficult problem.
(3) the invention provides a kind of simple, economic, efficient tissue culture and rapid propagation method, the germ plasm resource that is conducive to Yunnan crape myrtle is preserved and applies.Also can be generalized to Lagerstroemia other are difficult for kind and the kind of breeding simultaneously.
Accompanying drawing explanation
Fig. 1 is the ripe but fruit schematic diagram that do not ftracture of Yunnan crape myrtle in the embodiment of the present invention 1.
Fig. 2 is Yunnan crape myrtle aseptic seeding and seed germination schematic diagram in the embodiment of the present invention 1.
Fig. 3 is Yunnan crape myrtle inducing clumping bud schematic diagram in the embodiment of the present invention 1.
Fig. 4 is Yunnan crape myrtle group training seedling rooting schematic diagram in the embodiment of the present invention 1.
Fig. 5 is that in the embodiment of the present invention 1, Yunnan crape myrtle group training shoot root is schematic diagram.
Fig. 6 is Yunnan crape myrtle group training transplantation of seedlings schematic diagram in the embodiment of the present invention 1.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The tissue culture and rapid propagation method of embodiment 1 Yunnan crape myrtle
1,11-12 month, Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit (Fig. 1) is taken back laboratory, in the refrigerator of 4 ℃, low-temperature storage is 2 weeks.With the clean spirit of washing of 100 times of dilutions, soak 30min, with cleaning ball, grind off pericarp, more than then rinsing 2h under flowing water, in superclean bench, with 75% alcohol sterilizing 1min, with 4% liquor natrii hypochloritis's sterilizing 30min, use aseptic water washing 5-6 time.
(2) cut the fruit of sterilizing open, take out seed, with aseptic scalpel, cut away seed wing, to expose seed, be advisable.Be inoculated into containing in the 1/2MS minimal medium of 0.5g/L active carbon, secretly cultivate 1-2 about week, seed starts to sprout seedling, is then transferred to cultivation (Fig. 2) under light.
Minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, and Medium's PH Value is 5.8-6.0,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, can obtain the germination rate of 30-40%.
(3) select highly to surpass the aseptic seeding seedling of 2cm, by sterile scissors, cut off root and unnecessary blade, be inoculated in inducing clumping bud medium, carry out inducing clumping bud (Fig. 3).
Inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, Medium's PH Value is 5.2-5.4,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, each explant can obtain 3-5 regeneration bud.
(4) treat that inducing clumping bud cultivates 5-6 after week, select highly over 3cm, and the Multiple Buds of robust growth is inoculated in root induction medium, carry out root induction (Fig. 4).
Root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, Medium's PH Value is 5.8-6.0,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, rooting rate reaches 100%.
(5) when aseptic seedling root growth is intact, reach 3-4cm length, and while being no less than 5 (Fig. 5), open sealed membrane and train indoor hardening 1 week in group, then with sterile water, wash away the medium of group training shoot root portion, transplant to (Fig. 6) in the sterilized peat composed of rotten mosses and the perlite volume ratio mixed-matrix that is 3:1, bagging keeps humidity, and keep spray water every day 2-3 time, then reduce gradually humidity and increase intensity of illumination, after 2 weeks, after transplanted seedling survives, can in greenhouse, carry out cellar culture, transplanting survival rate reaches more than 70%.
The tissue culture and rapid propagation method of embodiment 2 Yunnan crape myrtles
(1) 11-12 month, Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit is taken back laboratory, in the refrigerator of 4 ℃, low-temperature storage is 2 weeks.With the clean spirit of washing of 100 times of dilutions, soak 30min, with cleaning ball, grind off pericarp, more than then rinsing 2h under flowing water, in superclean bench, with 75% alcohol sterilizing 1min, with 4% liquor natrii hypochloritis's sterilizing 30min, use aseptic water washing 5-6 time.
(2) cut the fruit of sterilizing open, take out seed, with aseptic scalpel, cut away seed wing, to expose seed, be advisable.Be inoculated into containing in the 1/2MS minimal medium of 0.5g/L active carbon, secretly cultivate 1-2 about week, seed starts to sprout seedling, is then transferred under light and cultivates.
Minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, and Medium's PH Value is 5.8-6.0,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, can obtain the germination rate of 30-40%.
(3) select highly to surpass the aseptic seeding seedling of 2cm, by sterile scissors, cut off root and unnecessary blade, be inoculated in inducing clumping bud medium, carry out inducing clumping bud.
Inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, Medium's PH Value is 5.2-5.4,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, each explant can obtain 4-7 regeneration bud.
(4) treat that inducing clumping bud cultivates 5-6 after week, select highly over 3cm, and the Multiple Buds of robust growth is inoculated in root induction medium, carry out root induction.
Root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, Medium's PH Value is 5.8-6.0,25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx, rooting rate reaches 100%.
(5) when aseptic seedling root growth is intact, reach 3-4cm length, and while being no less than 5, open sealed membrane and train indoor hardening 1 week in group, then with sterile water, wash away the medium of group training shoot root portion, transplant to the sterilized peat composed of rotten mosses and the perlite volume ratio mixed-matrix that is 3:1 bagging maintenance humidity, and keep spray water every day 2-3 time, then reduce gradually humidity and increase intensity of illumination, after 2 weeks, after transplanted seedling survives, can in greenhouse, carry out cellar culture, transplanting survival rate reaches more than 70%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (5)

1. the tissue culture and rapid propagation method of Yunnan crape myrtle, it is characterized in that, in Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit, under 4 ℃ of conditions, store 2 weeks, then whole fruit is carried out to sterilizing, cut fruit open, take out seed, remove seed wing, be inoculated in containing in the 1/2MS minimal medium of 0.5g/L active carbon, secretly cultivate 1-2 week until then seed germination forwards under light and cultivate; The sprouting seedling that highly surpasses 2cm is inoculated on inducing clumping bud medium, cultivates 5-6 week, obtain a large amount of Multiple Buds, Multiple Buds is inoculated on root media, cultivates 6-8 week, until all aseptic seedling are all taken root, then the seedling of taking root is carried out to hardening, transplant bottle outlet.
2. method according to claim 1, is characterized in that, comprises the following steps:
(1) 11-12 month, Yunnan crape myrtle seed maturity season, the lignification of gathering but uncracked fruit, in 4 ℃ of refrigerators, store 2 weeks, with the clean spirit of washing of 100 times of dilutions, soak 30min, with cleaning ball, grind off pericarp, then more than rinsing 2h under flowing water, in superclean bench, with 75% alcohol sterilizing 1min, then use 4% liquor natrii hypochloritis's sterilizing 30min, with aseptic water washing 5-6 time;
(2) cut the fruit of sterilizing open, take out seed, with aseptic scalpel, cut away seed wing, to expose seed, be advisable, be inoculated in the 1/2MS minimal medium containing 0.5g/L active carbon, secretly cultivate 1-2 week, seed starts to sprout seedling, is then transferred under light and cultivates;
Minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(3) select highly to surpass the aseptic seeding seedling of 2cm, by sterile scissors, cut off root and unnecessary blade, be inoculated in inducing clumping bud medium, carry out inducing clumping bud;
Inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, pH5.2-5.4; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(4) treat that inducing clumping bud cultivates 5-6 after week, select highly over 3cm, and the Multiple Buds of robust growth is inoculated in root induction medium, carry out root induction;
Root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0; 25 ± 2 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 2000-3000lx;
(5) when aseptic seedling root growth is intact, reach 3-4cm length, and while being no less than 5, open sealed membrane and train indoor hardening 1 week in group, then with sterile water, wash away the medium of group training shoot root portion, transplant to the sterilized peat composed of rotten mosses and the perlite volume ratio mixed-matrix that is 3:1, bagging maintenance humidity, and keep spray water every day 2-3 time, then reduce gradually humidity and increase intensity of illumination, after 2 weeks, after transplanted seedling survives, in greenhouse, carrying out cellar culture.
3. for the minimal medium of Yunnan crape myrtle group training, it is characterized in that, described minimal medium is: 1/2MS+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0.
4. for the inducing clumping bud medium of Yunnan crape myrtle group training, it is characterized in that, described inducing clumping bud medium is: WPM+0.5-1.0mg/L6-BA+0.025-0.1mg/L IBA+20g/L sucrose+6.5g/L agar, pH5.2-5.4.
5. for the root induction medium of Yunnan crape myrtle group training, it is characterized in that, described root induction medium is: 1/2MS+0.025-0.05mg/L6-BA+0.1-1.0mg/L IBA+0.5g/L active carbon+30g/L sucrose+6.5g/L agar, pH5.8-6.0.
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CN109169289A (en) * 2018-11-06 2019-01-11 湖南省林业科学院 A kind of crape myrtle tissue culture proliferation seedling rooting medicament and method
CN109392679A (en) * 2018-10-30 2019-03-01 浙江农林大学 A kind of aseptic seeding propagation method of Huang common vetch
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CN104604735A (en) * 2015-03-11 2015-05-13 华南农业大学 Tissue culture and rapid propagation method for American lagerstroemia indica pink velour
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CN109392679A (en) * 2018-10-30 2019-03-01 浙江农林大学 A kind of aseptic seeding propagation method of Huang common vetch
CN109169289A (en) * 2018-11-06 2019-01-11 湖南省林业科学院 A kind of crape myrtle tissue culture proliferation seedling rooting medicament and method
CN110313404A (en) * 2019-08-13 2019-10-11 湖南省林业科学院 A kind of construction method of crape myrtle Tissue Culture Regeneration System
CN113557954A (en) * 2021-07-06 2021-10-29 北京林业大学 Method for obtaining hybrid between lagerstroemia and lagerstroemia
CN113557954B (en) * 2021-07-06 2022-06-24 北京林业大学 Method for obtaining hybrid between lagerstroemia and lagerstroemia

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