CN107593448B

CN107593448B - Method for in-vitro rapid propagation by utilizing aseptic seedling stem segments of sedum aizoon

Info

Publication number: CN107593448B
Application number: CN201711026390.XA
Authority: CN
Inventors: 杨翠芹; 秦耀国; 付国召; 邓代辉
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2020-05-05
Anticipated expiration: 2037-10-27
Also published as: CN107593448A

Abstract

The invention discloses a method for in vitro rapid propagation by utilizing sedum aizoon aseptic seedling stem sections, which comprises the following steps: cutting the stem section of the aseptic rhodiola sachalinensis seedling into 1 node section with a length of 1cm, inoculating the section on a primary culture medium, and culturing the section in a culture room after inoculation to prepare callus or buds; respectively inoculating the callus generated by the stem segment in the primary culture on a callus differentiation culture medium to obtain cluster buds; then inoculating the bud or the cluster bud into a multiplication culture medium for culture; wherein the formula of the multiplication culture medium is the same as that of the primary culture medium; dividing buds obtained by cluster bud proliferation into single buds, inoculating the single buds on a rooting and strong seedling culture medium for culture to obtain tissue culture seedlings; transferring the tissue culture seedling to room temperature, hardening off the seedling for 3d, loosening the bottle mouth to continue hardening off the seedling for 4d, cleaning agar at the root of the tissue culture seedling, airing the surface moisture, and then transplanting the tissue culture seedling into a culture medium for culture. The invention solves the problems of low leaf budding rate and low rooting rate.

Description

Method for in-vitro rapid propagation by utilizing aseptic seedling stem segments of sedum aizoon

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for in vitro rapid propagation by utilizing sedum aizoon aseptic seedling stem sections.

Background

Sedum spectabile Boreau is an ornamental plant of Crassaceae, and is a variant of the long-acting eight-treasure (Hylotelephium spectabile). The pink variety is like a piece of pink cigarette when blooming, has excellent colony effect, is drought-resistant, low-temperature-resistant, shade-resistant and barren-resistant, has strong plants and extensive management, receives more and more attention in recent years, and has larger and larger popularization area in garden scenery. In addition, the sedum aizoon has a certain application prospect in repairing Hg and Co slightly polluted soil, and is hopeful to be used for greening Hg and Co polluted soil. Meanwhile, the ethanol extract of the leaves of the sedum aizoon has mild antibacterial activity, and the extract of the flowers of the sedum aizoon has certain antioxidant activity. The rhodiola crenulata is also a facultative Crassulaceae Acid Metabolism (CAM) plant and is a good material for physiological research. The two modes of tillering propagation and cutting propagation are commonly used in the current production, but the propagation coefficient is low, and the growing market demand is difficult to meet. By using tissue culture propagation, the propagation coefficient is high and is not limited by seasons. In addition, in the southwest region, the plant is subjected to a certain amount of variation due to flower forcing, so that the germplasm also needs to be propagated and preserved by means of tissue culture.

The reports about the tissue culture of the sedum aizoon are less, namely, young leaves of the sedum aizoon are used as explants for Zhang xiao yan and Cheng yun qing, and then cluster buds are obtained through callus induction and subsequent differentiation (Zhang xiao yan, Cheng yun qing. tissue culture and rapid propagation of the sedum aizoon. journal of the university of Jilin (Nature science edition). 2:60-62.), but the rooting effect is poor; we have also used leaves as explants to obtain regenerated plants (Cuiqin Yang, Yaoguo Qin, Xin Sun, et al.propagation of Sedum spectabile Boreau in Leaf Culture in vitro. not Bort Horti Agrobo,2012,40(1): 107-. The tissue culture propagation system of the sedum aizoon needs to be further improved to meet the requirement of production.

Disclosure of Invention

Aiming at the problems of low leaf budding rate and low rooting rate in the prior art, the invention provides a method for in vitro rapid propagation by utilizing the stem section of aseptic sedum aizoon seedlings. The method is a simple, convenient and efficient rapid propagation system to meet the increasing market demands.

In order to solve the technical problems, the invention discloses a method for in vitro rapid propagation by utilizing sedum aizoon aseptic seedling stem sections, which comprises the following steps:

step 1, obtaining stem segments and primary culture:

cutting the stem section of the aseptic rhodiola sachalinensis seedling into 1 node section with a length of 1cm, inoculating the section on a primary culture medium, and culturing the section in a culture room after inoculation to prepare callus or buds;

step 2, differentiation of callus:

respectively inoculating the callus generated by the stem segment in the primary culture on a callus differentiation culture medium to obtain cluster buds;

and 3, propagation culture:

then inoculating the bud obtained in the step 1 or the cluster bud obtained in the step 2 in a proliferation culture medium for culture; wherein the formula of the multiplication culture medium is the same as that of the primary culture medium;

step 4, rooting and seedling strengthening of buds:

dividing buds obtained by cluster bud proliferation into single buds, inoculating the single buds on a rooting and strong seedling culture medium for culture to obtain tissue culture seedlings;

step 5, hardening off and transplanting:

transferring the tissue culture seedling to room temperature, hardening off the seedling for 3d, loosening the bottle mouth to continue hardening off the seedling for 4d, cleaning agar at the root of the tissue culture seedling, airing the surface moisture, and then transplanting the tissue culture seedling into a culture medium for culture.

Furthermore, the primary culture medium is MS + BA 0-2mg/L + TDZ 0-1mg/L + NAA 0-2mg/L, and sucrose 30g/L and agar 5.8g/L are added, and the pH value is adjusted to 5.8.

Furthermore, the callus differentiation medium is MS + BA 0-4mg/L + TDZ 0-1.5mg/L + NAA0.2mg/L, and sucrose 20g/L and agar 5.8g/L are added, and the pH value is adjusted to 5.8.

Further, the rooting and seedling strengthening culture medium is MS + PP₃₃₃0-5.0mg/L + IBA0-0.1mg/L + SA 0-10mg/L, and adding sucrose 30g/L and agar 5.8g/L, and adjusting pH to 5.8.

Further, in the proliferation culture in step 3, the tissue with the shoot base when the shoot obtained in step 1 is cut off is inoculated into a proliferation medium.

Further, in the proliferation culture in the step 3, the clumpy buds obtained in the step 2 are not separated and inoculated into the proliferation culture medium together.

Further, the culture conditions in steps 1-4 are as follows: the light irradiation is 40-60 mu mol s^-2·h^-1The light period is 14h/d, and the temperature is 20-24 ℃.

Further, the culture medium in the step 5 is composed of turfy soil and nutrient soil according to the mass ratio of 1: 1.

Compared with the prior art, the invention can obtain the following technical effects:

1) the invention uses the salicylic acid as the seedling strengthening agent of the rhodiola sachalinensis tissue culture seedling for the first time, and has good effect. Although paclobutrazol is used for strengthening seedlings of many tissue culture seedlings of orchids and the like, paclobutrazol is not suitable for strengthening seedlings of tissue culture seedlings of sedum spectabilis.

2) The invention uses the tissue culture seedling to perform in vitro rapid propagation, and has higher propagation coefficient than the in vitro rapid propagation using the potted seedling as the explant.

3) The invention takes the tissue culture seedling as an experimental material, and the vitality of the tissue culture seedling is re-stimulated by adding the growth regulator, thereby preventing the aging phenomenon of the tissue culture seedling in the long-term subculture process.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is the development of the stem segment morphology of the present invention; wherein, A-E represent the treatments of the control, example 2 and example 7, example 8, example 10, respectively, in Table 2; F-I represents the treatment of TDZ/NAA in Table 3, example 16, example 17, example 14, example 20, respectively;

FIG. 2 is the differentiation of callus according to the present invention; wherein, A-F are pictures of BA 1, BA2 and BA 4 and TDZ 0.5, BA 1 and TDZ 1.5mg/L processed for 40d respectively;

FIG. 3 is a photograph showing the proliferation of multiple shoots according to the present invention;

FIG. 4 shows the rooting and strengthening of the shoots of the present invention, wherein A to E represent MS in Table 7, treatment with IBA alone, and addition of PP in an amount of 0.05, 0.5, 5mg/L, respectively₃₃₃The treatment of (1);

FIG. 5 is a PP according to the invention₃₃₃Comparing the strong seedling effect with SA; wherein A to E represent MS and 0.05mg/LPP in Table 8, respectively₃₃₃And SA0.1, 1, 10 mg/L.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

The invention discloses a method for in vitro rapid propagation by utilizing sedum aizoon aseptic seedling stem sections, which comprises the following steps:

step 1, obtaining stem segments and primary culture:

cutting the stem of aseptic seedling of Sedum Babayan into 1 node section with length of 1cm, inoculating on primary culture medium (MS + BA 0-2mg/L + TDZ 0-1 m)g/L + NAA 0-2mg/L, adding sucrose 30g/L and agar 5.8g/L, and adjusting pH to 5.8; inoculating 2-3 stem segments in each bottle, and irradiating under the light of 40-60 mu mol s^-2·h^-1Culturing in a culture room with a light period of 14h/d and a temperature of 22 +/-2 ℃ to prepare callus or buds;

step 2, differentiation of callus

Inoculating the callus produced by stem segment in primary culture on callus differentiation culture medium; the callus differentiation culture medium is MS + BA 0-4mg/L + TDZ 0-1.5mg/L + NAA0.2mg/L, and sucrose 20g/L and agar 5.8g/L are added, and the pH value is adjusted to 5.8; preparing cluster buds;

step 3, enrichment culture

wherein the tissue with the shoot base when the shoot obtained in step 1 is cut off, and the cluster shoot obtained in step 2 is inoculated into a proliferation medium without being separated.

Step 4, rooting and strengthening of the buds

Dividing buds obtained by cluster bud proliferation into single buds, inoculating the single buds on a rooting and strong seedling culture medium for culture to obtain tissue culture seedlings; the rooting and seedling strengthening culture medium comprises: MS + PP₃₃₃0-5.0mg/L + IBA (indolebutyric acid) 0-0.1mg/L + SA 0-10mg/L, and further adding 30g/L sucrose and 5.8g/L agar, and the pH value is 5.8.

Step 5, hardening off and transplanting:

and 4, transplanting the tissue culture seedling obtained after subculturing the seedling cultured on the MS culture medium without the growth regulator for one month by dividing the cluster buds into single buds in the step 4 to room temperature, hardening the seedling for 3d, loosening the bottle mouth for continuing hardening the seedling for 4d, cleaning agar at the root of the tissue culture seedling, airing the surface water, and transplanting the seedling to a culture container with a mass ratio of 1:1 and nutrient soil are mixed in a culture medium for culturing.

Example 1

Step 1, obtaining stem segments and primary culture:

cutting the stem of aseptic Sedum spectabile seedling into 1-node section with length of 1cm, inoculating to primary cultureOn a culture medium, the primary culture medium is MS + BA 0.5mg/L, and is additionally provided with sucrose 30g/L and agar 5.8g/L, and the pH value is adjusted to 5.8; inoculating 2-3 stem segments in each bottle, and irradiating under the light of 40-60 mu mol s^-2·h^-1Culturing in a culture room with a light period of 14h/d and a temperature of 22 +/-2 ℃; the observation is carried out once every 3 days, and the indexes of the recovery rate, the sprouting number and the like are recorded at 30 days.

Step 2, differentiation of callus

Respectively inoculating the callus generated by the stem segment in the primary culture on a callus differentiation culture medium; the callus differentiation culture medium is MS + BA2mg/L + NAA0.2mg/L, 20g/L of cane sugar and 5.8g/L of agar are additionally added, and the pH value is adjusted to 5.8; inoculating 3-6 pieces of callus to each bottle; MS without the regulator was used as a control. 15 bottles are inoculated in each treatment, and 3-6 blocks are inoculated in each bottle. The observation is carried out once every 3d, and indexes such as bud differentiation rate and differentiated bud number are recorded at 30d or 40d respectively.

Step 3, propagation of clumpy buds

Selecting a proliferation culture medium, and then inoculating the cluster buds into the proliferation culture medium for culture; fold of proliferation was recorded at 30 d.

Step 4, rooting and strengthening of the buds

Dividing buds obtained by cluster bud proliferation into single buds, inoculating the single buds on a rooting and strong seedling culture medium, and culturing; the rooting and seedling strengthening culture medium comprises MS, IBA0.1mg/L and PP₃₃₃0.05mg/L, adding 30g/L of cane sugar and 5.8g/L of agar, and adjusting the pH value to 5.8; and 15 flasks of 6 strains per flask were inoculated with each formulation using MS medium without growth regulator as a control. And after 30 days, counting strong seedlings and rooting conditions.

Step 5, hardening off and transplanting:

transferring the tissue culture seedling to room temperature, hardening off the seedling for 3d, loosening the bottle mouth to continue hardening off the seedling for 4d, cleaning agar at the root of the tissue culture seedling, airing the surface moisture, and transplanting the tissue culture seedling to a container with a mass ratio of 1:1 and nutrient soil are mixed in a culture medium for culturing.

The differences between examples 2 to 38 and the control group and example 1 are shown in Table 1, and the other conditions are the same as in example 1.

TABLE 1 culture Medium formulations for examples 2-38 and control

Examples 1-39 effects of different planting methods on the culture parameters of Sedum spectabile (regel):

first, the effect of examples 1-12 on the morphogenesis of stem segments is shown in Table 2.

TABLE 2 Effect of examples 1-12 on morphogenesis of Stem segments

The highest rate of stem outgrowth was found in example 10 (see table 2). For total germination, all combinations with NAA 1.0 and 2.0mg/L other than example 8 did not reach 100%; and the other combinations except the combination of the example 4 reach 100 percent.

The number of abnormal shoots transferred to MS medium without regulator was 5.55 per section.

Second, the effect of examples 13-24 on the morphogenesis of stem segments is shown in Table 3.

TABLE 3 Effect of examples 13-24 on the morphogenesis of stem segments

For the healing rate, the combinations of examples 16-18 were higher (see table 3), with 0 for both the treatment without NAA and the control, significantly lower than for the combined treatment.

Whereas example 21 reached 100% for total germination, significantly higher than the other combinations, but with a lower number of sprouts. The highest total number of sprouts, when the sprouting rate and the number of sprouts were considered together, was example 16 (the average number of sprouts was 2.15).

The development of stem morphology is shown in FIG. 1, where BA in combination with BA/NAA favours shoot development and TDZ in combination with TDZ/NAA favours callus development.

Third, example 25-30 Effect on Stem callus differentiation

TABLE 4 Effect of different combinations of regulators on Stem callus differentiation

For callus differentiation rate and number of shoots, the combination of BA was superior to that of TDZ, which was superior to the control without regulator (table 4). For callus differentiation rate, there was no significant difference between combinations of BAs; in the combination of TDZ, the medium-low concentration ratio is better than the high concentration combination effect. For the number of shoots, no difference was significant between the combinations of BA or TDZ.

The shoots obtained in the primary culture were excised together with the basal tissue and inoculated on a BA/NAA 1.0/1.0 combined medium without division, and after one month, the average multiplication factor was counted to 2.5.

TABLE 5 Calal differentiation pathway fold proliferation calculation

TABLE 6 calculation of multiplication factor of shoot multiplication pathway

According to the highest callus induction rate, callus differentiation rate, germination number and material number obtained in the above tests, it can be calculated that 1 aseptic seedling with 3 complete nodes can be proliferated 46.2 times in one cycle through callus differentiation (table 5). The total number of buds obtained by the cluster bud proliferation pathway was calculated from the highest bud induction rate, the number of buds emerged, and the multiple of cluster bud proliferation, and found to be 41.6 (Table 6). Therefore, the callus differentiation pathway has a slightly better proliferation effect than the cluster bud proliferation pathway, and the proliferation fold of the callus differentiation pathway is 4.6 higher than that of the cluster bud proliferation pathway. The data in the table are only based on short-term effect, while the effect of the callus differentiation pathway at the later stage is better than that at the earlier stage, and the differentiated bud number is greatly increased besides the callus differentiation rate is increased.

As can be seen from fig. 2, the BA combination treatment is superior to the TDZ combination treatment. As is clear from FIG. 3, the clumpy buds obtained in the stem morphogenesis experiments proliferated after subculture and also increased.

Fourth, Effect of examples 31 to 39 on rooting and strengthening seedlings

TABLE 7 Effect of examples 31-34 on rooting and strengthening seedlings

Note: the rooting rate of each treatment in the table reaches 100%.

As is clear from Table 7, PP was added at a high concentration₃₃₃The number of roots in the treatment is least, and the difference among other treatments is not obvious; control group without regulator, IBA alone and 0.05mg/L PP₃₃₃The treatment of (a) is significantly greater in root length than the other two treatments, the differences between the three treatments being insignificant; 5mg/L PP₃₃₃The number of axillary buds per section of the treatment was significantly greater than the other treatments, and 0.05mg/L PP₃₃₃Significantly more in tillering number than other treatments, indicating low concentrations of PP₃₃₃The tillering is promoted, and the axillary buds are promoted at high concentration; the difference among the treatments is not significant in the aspects of plant height, stem thickness and internode length. The above results show PP₃₃₃The growth of roots is inhibited, the influence on some characters of overground parts is not obvious, but the seedlings have the character of water loss and wilting in later period.

Table 8 effects of examples 35-38 on strong seedlings

As can be seen from Table 8, different treatments had a significant effect on the plant height, stem thickness and leaf area of the seedlings. The plant height of example 37 was significantly higher than the other treatments; the stem thickness values for examples 36-38 were greater than the control without the modifier and the treatment of example 35; the leaf area was the largest for example 36 and the smallest for example 35.

As can be seen from FIG. 4, both MS and IBA treatment had normal growth, good rooting effect and longer roots; is additionally provided with PP₃₃₃The leaves are dehydrated, the roots are short, and the roots are less when the concentration is higher; as can be seen from FIG. 5, the SA-treated strong seedling effect was superior to that of the MS control and PP₃₃₃And (6) processing. In addition, the statistical survival rate can reach more than 99 percent after the seedlings are trained and transplanted for 1 week.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for in vitro rapid propagation by utilizing the stem section of aseptic seedlings of sedum aizoon is characterized by comprising the following steps:

step 1, obtaining stem segments and primary culture:

cutting the stem section of the aseptic rhodiola sachalinensis seedling into 1 node section with a length of 1cm, inoculating the section on a primary culture medium, and culturing in a culture room after inoculation to prepare callus;

step 2, differentiation of callus:

and 3, propagation culture:

then inoculating the cluster buds obtained in the step 2 into a proliferation culture medium for culture; wherein the formula of the multiplication culture medium is the same as that of the primary culture medium;

step 4, rooting and seedling strengthening of buds:

step 5, hardening off and transplanting:

transferring the tissue culture seedling to room temperature, hardening off the seedling for 3d, loosening the bottle mouth to continue hardening off the seedling for 4d, cleaning agar at the root of the tissue culture seedling, airing the surface moisture, and then transplanting the tissue culture seedling into a culture medium for culture;

the primary culture medium is MS + BA2mg/L + NAA 0.5mg/L or MS + TDZ0.2mg/L + NAA 1mg/L, sucrose 30g/L and agar 5.8g/L are additionally added, and the pH value is adjusted to 5.8;

the callus differentiation culture medium is MS + BA 1-4mg/L + NAA0.2mg/L or MS + TDZ 0.5-1mg/L + NAA0.2mg/L, and 20g/L of cane sugar and 5.8g/L of agar are added, and the pH value is adjusted to 5.8;

the rooting and seedling strengthening culture medium is MS + PP₃₃₃0-0.5mg/L + IBA0.1mg/L, and further adding 30g/L of sucrose and 5.8g/L of agar, and the pH value is 5.8.

2. The method for rapid propagation in vitro by using the stem segments of aseptic seedlings of sedum aizoon as claimed in claim 1, wherein in the propagation culture in step 3, the multiple buds obtained in step 2 are inoculated into the propagation culture medium without being separated.

3. The method for in vitro rapid propagation by utilizing the aseptic seedling stem segments of sedum aizoon according to claim 1, wherein the culture conditions in the steps 1-4 are as follows: the light irradiation is 40-60 mu mol s^-2·h^-1The light period is 14h/d, and the temperature is 20-24 ℃.

4. The method for in vitro rapid propagation by utilizing the aseptic seedling stem segments of sedum aizoon according to claim 1, wherein the culture medium in the step 5 consists of turfy soil and nutrient soil according to a mass ratio of 1: 1.