CN107593449B

CN107593449B - Method for inducing formation of flower buds in dendrocalamus parviflorus test tube

Info

Publication number: CN107593449B
Application number: CN201711032751.1A
Authority: CN
Inventors: 何俊; 李德铢; 程治英; 杨俊波; 李春芳; 李村富; 浦秀丽
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2017-10-30
Filing date: 2017-10-30
Publication date: 2020-04-03
Anticipated expiration: 2037-10-30
Also published as: CN107593449A

Abstract

Randomly selecting sterile cluster vegetative buds of the phyllostachys parviflora, inoculating the sterile cluster vegetative buds to a floral bud induction culture medium, subculturing once every 21 days, and performing 3 times of continuous floral bud induction culture and 1 time of recovery culture to obtain the floral buds on about 85 days. The method for inducing the test tube flower buds of the dendrocalamus parviflorus has the advantages of simple operation, short time consumption, high induction efficiency and good repeatability, provides research materials for deeply researching flowering prediction and flowering period regulation of woody bamboo plants, and further provides scientific guidance for reducing economic loss of bamboo forest management.

Description

Method for inducing formation of flower buds in dendrocalamus parviflorus test tube

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a method for inducing formation of flower buds in a dendrocalamus parviflorus test tube.

Background art:

the Dendrocalamus minor (Dendrocalamus barbatus Hsueh et D.Z.Li) belongs to Bambusa of Bambusoideae of Gramineae, is large sympodial bamboo, and is mainly distributed in tropical and southern subtropical areas of south Yunnan, Vietnam, Burma and Laos north. The phyllostachys parviflora is a good bamboo fiber raw material and becomes one of economic bamboo species with development prospect. However, the bamboo stalks die after the dendrocalamus parviflorus blooms, and even if individual bamboo clumps in a sheet bamboo forest bloom, the whole bamboo forest blooms and dies within a few years, which causes huge loss to operators. The large-area flowering of the bamboos has no obvious sign, and the natural updating needs 8-10 years after the blossoming and death, which causes troubles to practitioners in the bamboo forest industry.

The establishment of the bamboo test tube flowering experiment system provides a simple and controllable experiment platform for understanding the physiological metabolism and gene expression difference of bamboos before and after flowering, thereby providing possibility for effectively predicting flowering time and reducing economic loss of bamboo forest management.

So far, no report of successfully establishing a method for inducing the formation of flower buds in the dendrocalamus parviflorus test tube, which is simple and convenient to operate, short in time consumption, high in inducing efficiency and good in repeatability, is found in the prior art.

The invention content is as follows:

the invention aims to provide a method for inducing formation of flower buds in a phyllostachys parviflora test tube, which is simple to operate, short in time consumption, high in inducing efficiency and good in repeatability, and the flower buds can be obtained by inducing sterile cluster vegetative buds of the phyllostachys parviflora within 3 months.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

taking the clustered vegetative buds of the aseptic test tube of the dendrocalamus parviflorus as explants, inoculating 5 clustered buds (3-5 buds/cluster) in each bottle for in vitro culture at the culture temperature of 23 +/-2 ℃ and the illumination intensity of 40 mu mol/m²S, illumination time 12 h/d. The basic culture medium is MS, the PH value is 5.8, 3 percent of sucrose is added, 5.6g/L of agar is added for solidification, and the subculture period is 21 days. Continuously culturing in MS + TDZ 1.0mg/L culture medium for 3 times, transferring into MS + BA2.0+ NAA0.2 culture medium for 1 time, and culturing for 80-90 days until flower bud grows out.

Compared with the prior art, the invention has the following advantages:

the invention has the advantages that: randomly selecting sterile cluster vegetative buds of phyllostachys parviflora, inoculating the sterile cluster vegetative buds to a flower bud induction culture medium, subculturing once every 21 days, and performing 3 times of continuous flower bud induction culture and 1 time of recovery culture to obtain the existing flower buds about 85 days. The method for inducing the test tube flower buds of the phyllostachys parviflora established by the invention has the advantages of simple operation, short time consumption, high induction efficiency and good repeatability, provides research materials for deeply researching flowering prediction and flowering period regulation of woody bamboo plants, and further provides scientific guidance for reducing economic loss of bamboo forest management.

Drawings

FIG. 1 shows the growth of multiple shoots during the subculture in group d experimental treatment;

FIG. 2A shows flower buds, and B shows pistils and stamens obtained by dissecting the flower buds.

The specific implementation mode is as follows:

the following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the present invention.

Example 1:

1. establishment of sterile clones:

in 2009, the seeds of the phyllostachys pubescens (Dendrocalamus barbatus) were collected in Mengla county of Dai nationality of Xishuangbanna, and aseptically germinated the seeds to construct a cluster bud sterile clone of a single seed, and stored in vitro in the wild germplasm resource bank of southwest of China as test tube cluster buds. The method comprises the following specific operations: peeling off testa of collected Carcinia parvula seed, sterilizing with 75% ethanol for 1min, washing with distilled water for 2 times (5 min each time), and adding 0.1% mercuric chloride (HgCl)₂) The solution was sterilized for 10min, washed 5 times with sterile distilled water, and then sterilized seeds were inoculated on the medium (1)1/2MS for germination. The minimal medium is MS (Murashige)&Skoog, 1962) (1/2MS halved for MS medium macroelements, remaining components unchanged).

2. And (3) cluster vegetative bud proliferation:

and (3) transferring the plantlets obtained by aseptic germination of the seeds in the step (1) to a culture medium (2) MS + BA2.0 mg/L (the same in the unit below) + NAA0.2 for cluster bud proliferation, subculturing once every 28 days, and obtaining a large number of cluster nutritive buds after multiple subculturing to be used as a material for subsequent flower bud induction.

3. Flower bud induction and experimental treatment:

flower bud induction medium: (3) MS + TDZ 1.0, (4) MS + TDZ 2.0; recovering the culture medium: (2) MS + BA2.0+ NAA 0.2. Sucrose 3% is added into the culture medium, agar 5.6g/L is added for solidification, and the pH value of the culture medium is 5.8. The culture conditions were: the temperature is 23 +/-2 ℃, and the illumination intensity is 40 mu mol/m²S, illumination time 12 h/d. Five experiments were designed for flower bud induction: a. the culture medium (3) is independently and continuously subcultured; b. the culture medium (4) is independently and continuously subcultured; c. continuously culturing for 2 generations in (3) or (4), and then transferring to the culture in (2); d. continuously culturing for 3 generations in (3) or (4), and then transferring to the culture in (2); e. continuously culturing for 4 generations in (3) or (4), and then transferring to the culture in (2). In all five experiments, 21 days are taken as a subculture period, and the results of 6 subcultures are observed. At least 10 flasks were inoculated per experimental treatment, 5 clumps per flask (3-5 clumps/clump), 3 replicates per treatment. For each set of experiments, clumpy buds were dissected every 3 days under the olympus SZ61 scope, starting with 3 rd passage (day 43), until flower buds were found.

4. Flower bud induction results:

each experiment had the proliferation of multiple shoots, and the proliferation rate of the first subculture was approximately 1: 3-4, the secondary subculture proliferation rate is improved to a certain extent and can reach 1: 5-6. Wherein, when continuously cultured in low concentration TDZ (1.0mg/L), the cluster vegetative bud can continuously proliferate, but the cluster bud becomes short and whitens slowly. When cultured in high concentration TDZ (2.0mg/L) for more than 4 times, the cluster buds not only whiten but also begin to die. When the culture medium is transferred to the recovery medium (2) for continuous culture after 2-4 times of continuous culture in (3) or (4), the cluster vegetative buds continue to proliferate and recover from growth. Wherein, the medium is transferred into the step (2) after 2 times of continuous culture, the cluster buds are high in length and have small leaves, but no flower bud is seen in dissection; continuously culturing for 3 times, transferring into lotus plumule with high growth, and dissecting to find flower bud after the first culture (2) is finished (see figure 1); after 4 times of continuous culture, the albino cluster buds are transferred slowly to green and grow at high speed, but the proliferation rate is reduced, and the cluster buds are extremely weak and small and are extremely difficult to dissect. The results of five experiments are shown in table 1.

TABLE 1 growth of five experimental clumpy buds

5. And (3) dissecting flower buds:

the clumpy shoots induced by the group d experiments were used as an example of the results of dissecting clumpy vegetative shoots every 3 days under the olnbas SZ61 stereoscope, starting from 3 subcultures (day 43). By means of tracking dissection under a stereoscope, flower buds are not dissected from the 3 rd subculture to the 4 th subculture, and at the moment, the cluster buds are clustered into rosette buds. When the multiple nutritive buds are dissected continuously on the 85 th day, the flower buds appear, and then the culture is continued, and the proportion of the flower buds is gradually increased. And after 6 th subculture, the dissection result shows that nutritional buds are still mixed in the rosette-shaped clustered flower buds, the proportion of the flower buds is 70-90%, the proportion of the normal flower buds is about 60%, and the deformed flower buds are mainly shown in that the number of stamens and pistils is changed. In general, the number of stamens and pistils of the normal phyllostachys parviflora are respectively 6 and 1. The number of stamens of flower buds obtained in this group of experiments was 6 (60%), 7 (8.33%), 8 (16.7%), 9 (8.33%), 10 (8.33%), occasionally 2 (8.33%) pistils. The whole period from the nutrition clustering of the nutrition buds to the obtaining of the normal flower buds is about 85 days through multiple subculture.

To sum up the experimental results, the best method for inducing the formation of flower buds in the phyllostachys parviflora in the test tube is obtained as follows: taking the aseptic test tube cluster buds of phyllostachys pubescens as explants, inoculating 5 cluster buds (3-5 buds/cluster) per bottle for in vitro culture at 23 + -2 deg.C under illumination intensity of 40 μmol/m²S, illumination time 12 h/d. The basic culture medium is MS, the pH value is 5.8, 3 percent of sucrose is added, 5.6g/L of agar is added for solidification, and subculture is carried out once every 21 days. Continuously culturing in MS + TDZ 1.0mg/L culture medium for 3 times, transferring into MS + BA2.0+ NAA0.2 culture medium, and culturing for 1 time, wherein existing flower bud appears around 85 days. Based on the present invention, flower buds can be obtained within 3 months via sterile clumping vegetative buds of dendrocalamus parviflorus.

Claims

1. A method for inducing the formation of flower bud in the test tube of dendrocalamus parviflorus is characterized in that the method takes the multiple nutritive buds of the aseptic test tube of dendrocalamus parviflorus as explants, 5 multiple buds are inoculated to each bottle, each multiple bud is 3-5 buds/cluster, the isolated culture is carried out, the culture temperature is 23 +/-2 ℃, and the illumination intensity is 40 mu mol/m²S, the illumination time is 12h/d, the basic culture medium is MS, the pH value is 5.8, 3% of cane sugar is added, 5.6g/L of agar is added for solidification, a successive transfer cycle is 21 days, the culture is continuously carried out for 3 times in the culture medium of MS and TDZ 1.0mg/L, and then the culture medium is transferred into the culture medium of MS and BA2.0 mg/L and NAA0.2 mg/L for culture for 80-90 days until flower buds grow out.