CN101743909B

CN101743909B - Tissue culture and propagation method of ''Haierlian'' of Illiciaceae plant

Info

Publication number: CN101743909B
Application number: CN2009102326669A
Authority: CN
Inventors: 谈建中; 颜世和; 方向明; 周伯岳; 刘文倩; 苏媚; 阚雪芹; 王海玲
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2009-12-04
Filing date: 2009-12-04
Publication date: 2012-06-20
Anticipated expiration: 2029-12-04
Also published as: CN101743909A

Abstract

The invention discloses a tissue culture and propagation method of illicium plant illicium benryi dies. In the method, an explant which is in tissue culture and propagation with a sprout on the top of a current-year branch of the illicium benryi dies is firstly subjected to surface treatment in an ethanol solution and then is subjected to sterilization treatment in an HgC12 solution; then a bud slice is cut down from the branch to be inoculated to an initial medium and cultured under the conditions of illumination for 12-15h everyday with the illumination intensity of 1500-2000LX and the temperature of 23 DEG C plus/minus 1 DEG C; and new leaves are transferred onto a subculture medium to be continuously cultured after germinating, and a tissue culture seedling is obtained. The invention solves the problems of less resources, acarpia, incapability of harvest seeds, difficult vegetative propagation and critical shortage of seedling of the traditional illicium benryi dies, can accelerate propagation, provide a new way to satisfy the market demands of new variety seedling, increase a new type as a rare ornamental plant and have a favorable application potential in landscaping.

Description

The tissue culture propagation of Winteraceae plant " my child lotus "

Technical field

The present invention relates to the tissue culture propagation of a kind of Winteraceae plant " my child lotus ", be specifically related to the selection of explant kind, initial culture method, successive transfer culture method and medium component thereof in " my child lotus " tissue culture procedures.

Background technology

Winteraceae (Illiciaceac) is a single genus section plant, about 50 kinds of the whole world, and there are 28 kinds and 2 mutation in China, mainly is distributed in China west and south to the eastern region; They are evergreen dungarunga or shrub, and fragrance is arranged, and like dark and damp environment, grow in woodland stream limit more; Flower both sexes, armpit are given birth to or give birth on nearly top, redness, and tepal is 7～21 pieces, the florescence is generally in 4～June, the ornamental value that tool is certain.The Winteraceae plant mainly leans on seminal propagation, and its fruit is poisonous mostly, all has certain toxicity like the fruits of Winteraceae plants such as thick grass, red fennel and anisetree bark, causes the application of this type of plant in afforestation to receive the restriction of Environmental security factor.

Rare ornamental plants " my child lotus " is Winteraceae Illicium (Illicium Linn.) plant, evergreen dungarunga, the about 3～8m of the height of tree, tree crown obovate; The bark taupe, single leaf alternate or closely verticillate, lanceolar, blade keratin degree is high, and doubling is easily broken, branches and leaves tool peat-reek; Flower shape lotus shape, pattern is ruddy, and the florescence, when blooming, pattern was ruddy in 4～May, was the reversal of the natural order of things lotus shape, and as small lantern, several are hung on the end of the branch, and it very is beautiful being embedded between the greenery, has very high ornamental plantation and is worth.Especially " my child lotus " do not observe its normal phenomenon as a result so far in Suzhou District, thereby can avoid people and animals to eat fruit by mistake and cause the defective of poisoning, and the greening and the garden that can be widely used in street, park beautify.

According to the "Taihu remarks," a book written: "The baby lotus, wood like laurel, flowers such as large pieces, shape and color with lotus flowers are not fragrant, Norway smell of its leaves, Xinfen like fennel.Wuzhong is not to there being this kind, and Dongshan Weng Hanjin is the west of a river, Yunnan county magistrate between Shunzi, takes and return, and the back owns for Xi Shi, and treasure is curiosa.The figured cloth knot, root does not have rudiment, its kind of tendency to develop non-availability ... ".Though " my child lotus " imported the history in existing more than 300 year in Jiangsu and Zhejiang Provinces area into, its resource is most deficient, and artificial plantation few at present, at Suzhou District poke strain bearing tree only, can be rated as rare tree species.This is because " my child lotus " is barren, can't gather in the crops seed; Grafting lacks stock, and the cottage propagation survival rate is very low, and the vegetative propagation difficulty causes a seedling difficulty to be asked, and these have all limited " my child lotus " application in afforestation.

In recent years; The application of plant tissue culture technique on ornamental plants more and more widely; Especially the application on the low plant of natural breeding rate enjoys people to pay attention to; A series of successfully report is also being arranged aspect the tissue-culturing rapid propagation of woody plant; Problems such as but in the Winteraceae plant, do not succeed so far, forefathers did not carry out system research to the key technique of " my child lotus " tissue culture yet, and were seriously polluted such as explant in its group training process, that the blastogenesis length that exsomatizes stops, easy brownization is withered are not effectively solved.

Summary of the invention

The purpose of this invention is to provide explant kind, Biocidal treatment method, initial culture method, successive transfer culture method and medium component thereof that a cover is applicable to Winteraceae plant " my child lotus " tissue culture; Solve that " my child lotus " explant pollutes easily, difficult problems such as " mashed core " and brownization, for the quick breeding of rare plant " my child lotus " provides effective technical support.

For realizing the foregoing invention purpose, technical scheme of the present invention is: a kind of Winteraceae plant---the tissue culture propagation of " my child lotus ", cultivate in vitro tissue, and comprise the steps:

1) bud of sprouting with " my child lotus " current-year branch top is a starting material, after cleaning solution immersion, flowing water flushing operation are cleaned, dries indoor, as the explant of tissue culture propagating;

2) above-mentioned explant is carried out the surface treatment in 5～6 seconds in concentration is the ethanolic solution of volume ratio 70～75% after, be the HgCl of w/v 0.1% in concentration ₂Carry out 5～6 minutes sterilization processing in the solution, fully clean 5～6 times with sterile water again, accomplish the sterilization of explant is handled;

3) branch behind the above-mentioned sterilization cuts the bud sheet; Be inoculated on the initial culture base; Illumination every day 12～15 hours; Intensity of illumination is that 1500～2000LX, temperature are to cultivate under 23 ℃ ± 1 ℃ the condition, treats to transfer to after young leaves is sprouted and continues on the subculture medium to cultivate, and obtains normotrophic tissue cultivating seedling.

Described initial culture base is White medium+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1, add sucrose and agar powder, volume ratio by weight, sucrose concentration is 3% of an initial culture base, agar powder concentration is 0.6% of initial culture base; Regulating pH is 5.6～5.8.

Described subculture medium is White medium+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.1mg.L ^-1, add sucrose and agar powder, volume ratio by weight, sucrose concentration is 3% of an initial culture base, agar powder concentration is 0.6% of initial culture base; Regulating pH is 5.6～5.8.

The present invention compared with prior art has advantage:

1, the vegetable material of the present invention's use is " a my child lotus "; It shows as the not result of only blooming in certain areas; These are different with the most poisonous situation of other Winteraceae fruits; " my child lotus " tissue cultivating seedling that adopts technical scheme of the present invention to provide can be used as a kind of ornamental plants new varieties, and the application in afforestation has better safety.

2, the technical method that proposes of the present invention has the characteristics of simple and easy to do, operability and favorable reproducibility.

Embodiment

Below in conjunction with embodiment the present invention is further described:

Embodiment: adopting the bud of not sprouting on " my child lotus " current-year branch top respectively is starting material with the bud of sprouting, and compares test.After cleaning solution immersion, flowing water flushing, using concentration as after carrying out the surface treatment in 5 seconds in the ethanolic solution of volume ratio 75% respectively above-mentioned two kinds of materials, is the HgCl of w/v 0.1% in concentration ₂Carry out 6 minutes sterilization processing in the solution, use sterile water wash at last 5～6 times, put and blot excessive moisture on the filter paper, cut explant at the sprout downside and be inoculated in medium.

Cultivation results is, the culture effect of " my child lotus " bud of not sprouting is relatively poor, is prone to produce endogenous and pollutes, and brownization rate is higher to be 22.2～46.9%, and survival rate is lower, is merely 2.8～6.2%.Secondly, be the occasion of explant at the employing bud of sprouting, use 0.1%HgCl ₂When solution sterilization was handled 3 minutes, pollution rate was up to 100%; When sterilization time was 9 minutes, though do not see pollution, sprout stem apex lethality reached 33.3%, and brownization rate reaches 50.0%; And sterilize 6 minutes the time, pollution rate is merely 8.3%, and survival rate can be up to 91.7%.Show that thus " my child lotus " bud of sprouting more is applicable to and the explant material of tissue-culturing rapid propagation helps setting up the aseptic culture system.

With MS (Murashige & Skoog, 1962) is minimal medium, and anti-brownization agent PVP, 6-BA and the NAA of additional variable concentrations compare test as the initial culture base.Wherein medium I is MS+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1, medium ii is 1/2MS+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1, medium ii I is B5 medium+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1, medium IV is White medium+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1Each medium all adds sucrose and agar powder, volume ratio by weight, and sucrose concentration is 3% of an initial culture base, agar powder concentration is 0.6% of initial culture base; Regulate pH 5.6～5.8.

Cultivation results is, the upgrowth situation of explant in medium I, II is relatively poor, and brownization rate is more than 90%; In medium ii I, IV, the explant that upgrowth situation is good accounts for more than 80%, and brownization rate is lower.In medium I, II, III; The explant lower cut-out all produces callus; Have only the explant lower cut-out among the medium IV not produce callus; And the explant ratio that upgrowth situation is good has reached 90.9%, shows that medium IV ratio is more suitable for the initial culture of " my child lotus " stripped bud, has also obtained similar cultivation results in successive transfer culture.

In subculture medium, add 500mg.L respectively ^-1And 1000mg.L ^-1Anti-brownization agent PVP is not to add PVP as contrast, brownization of the explant situation after the comparison different disposal.The result shows that when not adding PVP, whole brownization of explant lower cut-out spread to whole explant then; When adding 1000mg.L ^-1During PVP, though explant lower cut-out and itself brownization rate have reduction, its retarded growth; When adding 500mg.L ^-1When resisting brownization agent PVP, explant lower cut-out and itself brownization rate are all very low, and it is good to grow.Therefore, add 500mg.L ^-1Anti-brownization agent PVP explant is had preferably anti-brownization effect, and several nothings influences of growing that the bud that exsomatizes is cultivated.

Claims

1. the tissue culture propagation of a Winteraceae plant " my child lotus " is characterized in that, carries out in vitro tissue and cultivates, and comprises the steps:

3) branch behind the above-mentioned sterilization cuts the bud sheet; Be inoculated on the initial culture base; Illumination every day 12～15 hours; Intensity of illumination is that 1500～2000LX, temperature are to cultivate under 23 ℃ ± 1 ℃ the condition, treats to transfer to after young leaves is sprouted and continues on the subculture medium to cultivate, and obtains normotrophic tissue cultivating seedling;

Described initial culture base is White medium+500mg.L ^-1Anti-brownization agent PVP+6-BA 2.0mg.L ^-1+ NAA 0.5mg.L ^-1, add sucrose and agar powder, volume ratio by weight, sucrose concentration is 3% of an initial culture base, agar powder concentration is 0.6% of initial culture base; Regulating pH is 5.6～5.8;