CN102860260B - A kind of rapid propagation method of cedar tissue culture - Google Patents

Abstract

The invention discloses a cryptomeria fortunei tissue culture rapid-propagation method. The method comprises taking a cryptomeria fortunei explant stem segment, inoculating the cryptomeria fortunei explant stem segment on an inducing adventitious bud culture medium which comprises DCR+ 0.1-2.0mg/L of 6-BA+ 0.1-2.0mg/L of IBA+ 0.5-5.0g/L of AC+ 0.001-0.01mg/L of TDZ+ 30g/L of cane sugar, and performing inducing culture to generate adventitious buds; adopting two stages to take root for rooting culture, namely first placing a rooting culture medium 1 which comprises 1/2 of DCR+ 0.1-2.0mg/L of NAA+ 0.1-2.0mg/L of IBA+0.1-1.0mg/L of 6-BA+ 1.0-10.0g/L of AC+ 10.0-30.0g/L of cane sugar, first performing dark culture for 2-15d, performing culture for 5-30d under illumination, and transferring toa rooting culture medium 2 which comprises DCR+ 0.1-1.0mg/L of NAA+ 0.1-1.0mg/L of 6-BA+ 0.5-5.0g/L of AC+ 30.0g/L of cane sugar. By the method, the tender bud propagation coefficient is over 5.7, and the rooting rate is over 80%. The culture cycle is short, the propagation quantity is large, and the cryptomeria fortunei tissue culture rapid-propagation method is favorable for large-scale production.

Description

A kind of rapid propagation method of cedar tissue culture

technical field

The invention relates to the technical field of plant tissue culture propagation, in particular to a rapid propagation method of cedar tissue culture, in particular to a method of using cedar stems as explant materials, using a self-made medium to first induce cedar to produce adventitious buds, and then Rooting was induced to obtain regenerated plants.

Background technique

Cryptomeria fortunei ( Cryptomeria fortunei ) belongs to the genus Cryptomeria ( Cryptomeria D. Don ), also known as peacock fir, a tree with a height of 4m and a diameter at breast height of more than 2m; Whorls, flat or oblique; branchlets slender, often drooping, green, with longer leaves in the middle of branches, often gradually shortening towards both ends. The leaves are subulate and slightly curved inward, with stomatal lines on the four sides, 1-1.5 cm long. The leaves of fruiting branches are usually shorter, and the leaves of young trees and budding branches are up to 2.4 cm long.

Cedar is one of the important timber tree species in my country. In my country, cedar is mainly distributed in Fujian, Zhejiang, Jiangxi and other provinces. Driven by economic interests, cedar resources have been severely damaged in recent years, coupled with acid rain, air pollution and pest damage, there are only a handful of large diameter cedars. At present, cedar is mainly propagated by seeds, and cuttings can also be used, but usually the germination rate and qualified seedling rate are low. The research on its propagation is also mainly on the seedling breeding technology. Therefore, the use of biotechnology methods for artificial rapid propagation is of great significance to the resource protection of Cedar.

Zhang Cuiping (2008) preliminarily researched its tissue culture rapid propagation technology, but this study used Cedar seed embryos as explants, which was not suitable for the reproduction and culture of Cedar individuals with excellent genotypes, and only MS was used in the experiment. For the culture medium, the addition of exogenous hormones only adopted a single factor design, and no selective culture experiment was carried out for other basic medium and combination hormones. The applicant selected fine genotype cedar stems as explant materials, selected DCR as the most suitable basic medium through experiments, and carried out multi-factor design for the addition of exogenous hormones, and added activated carbon, etc. Rapid reproduction provides an effective way.

Contents of the invention

Purpose of the invention: Aiming at the deficiencies in the prior art, the purpose of the invention is to provide a method for rapidly propagating Cedar cedar by tissue culture.

Technical scheme: in order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:

A method for rapid propagation of cedar tissue culture, comprising the following steps:

(1) Take the stem section of Cedar explants and inoculate them on the medium for inducing adventitious buds, and induce culture to produce adventitious buds; among them, the medium for inducing adventitious buds is: DCR + 0.1~2.0mg/L 6-BA (6-benzyl Purine) + 0.1~2.0mg/L IBA (indole butyric acid) + 0.5~5.0g/L AC (activated carbon) + 0.001~0.01mg/L TDZ (thiadizuron) + 30.0g/L sucrose ;

(2) Rooting culture adopts two-stage rooting: first put the elongated and robust shoots into the rooting medium 1, after 2-15 days of dark culture, put them into the light for 5-30 days; then transfer In the rooting medium 2, continue to cultivate until rooting; Among them, the rooting medium 1 is: 1/2DCR+0.1~2.0mg/L NAA+0.1~2.0mg/L IBA+0.1~1.0mg/L 6-BA+1.0 ~10.0g/L AC+10.0~30.0g/L sucrose, rooting medium 2 is: DCR+0.1~1.0mg/L NAA+0.1~1.0mg/L 6-BA+0.5~5.0g/L AC+30.0 g/L sucrose.

In step (1), the medium for inducing adventitious buds is: DCR+1.0mg/L 6-BA+0.5mg/L IBA+0.01mg/L TDZ+3.0g/L AC+30g/L sucrose.

In step (2), the rooting medium 1 is: 1/2DCR+2.0mg/L NAA+1.0mg/L IBA+0.5mg/L 6-BA+0.5mg/L IBA+5.0g/L AC+25g/L sucrose.

In step (2), the rooting medium 2 is: DCR+0.5mg/L NAA+0.5mg/L 6-BA+3.0g/L AC+30.0g/L sucrose.

In step (2), in the 1/2DCR medium formula, the amount of macroelements is reduced by 1/2 compared with conventional DCR.

In step (2), the light time under the light is 14-16 hours, the light intensity is 1200-1300Lux, and the temperature is 25-28°C.

In this method, the stem segment of fine genotype Cedar cedar was selected as the explant material, and by using DCR as the most suitable basic medium, the addition of exogenous hormones was multifactorially designed, and activated carbon was added, which provided a rapid growth rate for the tissue culture of Cedar cedar. Reproduction provides an efficient route.

Beneficial effect: Compared with the prior art, the remarkable advantage of the present invention is: use the individual stem segment of Cedar cedar with excellent genotype as the explant, use DCR as the basic medium, and add different types and different concentrations of plant growth regulators and activated carbon to induce bud proliferation and rooting, through the method, the bud proliferation coefficient reaches more than 5.7, and the rooting rate is more than 80%. The cultivation period is short and the reproduction quantity is large, which is beneficial to large-scale production.

Description of drawings

The adventitious buds of Chinese cedar that are induced in Fig. 1;

Fig. 2 Complete cryptomeria plants formed after induction of rooting.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

Example 1

Select a robust cedar stem section, cut off the needles on the stem section, cut the stem section into about 4cm, wash it with running water for 1~2h, sterilize it in 75% ethanol for 30s, and then use 50% 84 disinfectant (Contains a small amount of Tween-20, add one drop per 50mL) Disinfect for 10 minutes, rinse with sterile water 8 times, then cut the stem segment into about 2cm, inoculate it on medium for inducing adventitious buds, and start to produce adventitious buds after 8 days of light-induced culture ; After the adventitious buds are produced, select the young shoots that are growing and strong and transfer them to the rooting medium 1, and then cultivate them in the dark for 2-15 days, then put them under the light and cultivate them for 2-30 days, and then transfer them to the rooting medium 2. 10d Started rooting. Among them, the medium for inducing adventitious buds is: DCR+0.1~2.0mg/L 6-BA+0.1~2.0mg/L IBA+0.001~0.01mg/L TDZ+0.5~5.0g/L AC; rooting medium 1 is : 1/2DCR+0.1~2.0mg/L NAA+0.1~2.0mg/L IBA+0.1~0.5mg/L 6-BA+1.0~10.0g/L AC+10.0~30.0g/L sucrose, rooting medium 2 is: DCR+0.1~1.0mg/L NAA+0.1~1.0mg/L 6-BA+0.5~5.0g/LAC+30.0g/L sucrose.

Example 2

Repeat the experiment according to the same steps as described in Example 1. At each induction culture stage, different doses of activated carbon (0.5-10.0 g/L) were added respectively, wherein 0.5-5.0 g/L of activated carbon was added to the medium for inducing adventitious buds, Adding 1.0-10.0 g/L of activated carbon to rooting medium 1 and adding 0.5-5.0 g/L to rooting medium 2 can obviously promote the growth and robustness of Cedar cedar, with green leaves, and effectively prevent the occurrence of yellowing and browning of seedlings. The induction rooting phase promotes rooting.

Example 3

Repeat the experiment according to the same steps described in Example 1. In the rooting induction stage, the macroelements in the 1/2DCR medium used are reduced by 1/2 compared with conventional DCR. The results are shown in Table 1, which can significantly promote the induction of rooting and increase the rooting rate to 85%.

Table 1 Effects of macroelements in medium on rooting of Chinese cedar

Example 4

The experiment was repeated in the same steps as described in Example 1. In the rooting induction stage, the sucrose concentration in the medium was reduced from 3% to 2% to reduce the osmotic pressure of the medium. The results are shown in Table 2, which effectively promotes plant rooting.

Table 2 Effect of sucrose concentration in medium on rooting of Chinese cedar

Claims (3)

1. a cryptomeria quick breeding method for tissue culture is characterized in that, may further comprise the steps:

(1) gets cryptomeria explant stem section, be inoculated in the evoking adventive bud medium, induce to cultivate to produce indefinite bud; Wherein, the evoking adventive bud medium is: DCR+1.0mg/L 6-BA+0.5mg/L IBA+0.01mg/L TDZ+3.0g/L AC+30.0g/L sucrose;

(2) culture of rootage adopts two stage root of hairs: the healthy and strong tender shoots that will extend and grow is first put into root media 1, after carrying out that 2 ~ 15d is dark and cultivating, puts under the illumination and cultivates 5 ~ 30d; Then transfer in root media 2, continue to be cultured to and take root; Wherein, root media 1 is: 1/2DCR+0.1 ~ 2.0mg/L NAA+0.1 ~ 2.0mg/L IBA+0.1 ~ 1.0mg/L 6-BA+1.0 ~ 10.0g/L AC+10.0 ~ 30.0g/L sucrose, and root media 2 is: DCR+0.1 ~ 1.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+0.5 ~ 5.0g/L AC+30.0g/L sucrose;

In the step (2), the light application time under the described illumination is 14 ~ 16h, and intensity of illumination is 1200 ~ 1300Lux, and temperature is 25 ~ 28 ℃; In described 1/2DCR culture medium prescription, macroelement reduces by 1/2 amount than conventional DCR.

2. cryptomeria quick breeding method for tissue culture according to claim 1, it is characterized in that: in the step (2), described root media 1 is: 1/2DCR+1.0mg/L 6-BA+0.1 ~ 2.0mg/L IBA+2.0mg/L NAA+5.0g/L AC+10.0 ~ 30.0g/L sucrose.

3. cryptomeria quick breeding method for tissue culture according to claim 1, it is characterized in that: in the step (2), described root media 2 is: DCR+0.1 ~ 1.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+3.0g/L AC+30.0g/L sucrose.

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