CN112868525A - Method for cultivating tetraploid incarvillea - Google Patents

Abstract

The invention discloses a method for cultivating tetraploid incarvillea, which comprises the following specific steps: step one, after seeds of artemisia incarvensis are sown and seedlings emerge in spring, when cotyledons of the artemisia incarvensis seedlings are flattened and the tips of true leaves are just exposed, soaking and coating the stem tips of the artemisia incarvensis in a colchicine solution; step two, retreating once every 12 hours, continuously processing for six times, and totally processing for 72 hours; and step three, performing cytological identification to obtain tetraploid incarvillea plants. The method for cultivating the tetraploid incarvillea provided by the invention can effectively obtain tetraploid incarvillea plants, wherein the polyploid plants have the nutrition mass, the method can expand incarvillea germplasm resources, increase the genetic diversity and cultivate new incarvillea varieties.

Description

Method for cultivating tetraploid incarvillea

Technical Field

The invention relates to the technical field of garden plant planting, in particular to a method for cultivating tetraploid incarvillea.

Background

The Incarvillea sinensis (Incarvillea sinensis) is an annual herbaceous plant in Incarvillea of Bignoniaceae, is an excellent wild flower variety in the north, is gorgeous in flower color and regular in plant, and has extremely high ornamental value in garden flowers. According to the research, the prior cultivated artemisia is diploid, and the chromosome number of somatic cells is 2 n-2 x-22. After the plants are multiplied, the enlargement of nutrition and reproductive organs is easy to occur, the content of secondary metabolites is improved, and the stress resistance and disease resistance are enhanced. Therefore, the homeotetraploid incarvillea is induced and cultured, and the ornamental quality, the stress resistance and the medicinal value of the incarvillea can be greatly improved.

Disclosure of Invention

The invention aims to provide a method for cultivating tetraploid incarvillea so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for cultivating tetraploid incarvillea comprises the following specific steps:

step one, after seeds of artemisia incarvensis are sown and seedlings emerge in spring, when cotyledons of the artemisia incarvensis seedlings are flattened and the tips of true leaves are just exposed, soaking and coating the stem tips of the artemisia incarvensis in a colchicine solution;

step two, retreating once every 12 hours, continuously processing for six times, and totally processing for 72 hours;

and step three, performing cytological identification to obtain tetraploid incarvillea plants.

As a further embodiment of the invention, in step two, the concentration of the applied colchicine solution is 0.3%.

As a further proposal of the invention, in the second step, the length of time for smearing the colchicine solution is 24 hours.

Compared with the prior art, the invention has the beneficial effects that: the method for cultivating the tetraploid incarvillea provided by the invention can effectively obtain tetraploid incarvillea plants, wherein the polyploid plants have the nutrition mass, and the method can expand incarvillea germplasm resources, increase the genetic diversity and cultivate new incarvillea varieties.

Drawings

FIG. 1 is a chromosome comparison of diploid and tetraploid of Artemisia scoparia cells.

FIG. 2 is a diagram of a comparison of plants diploid and tetraploid for caraway cells.

Detailed Description

The technical solution of the present patent will be described in further detail with reference to the following embodiments.

Examples

A method for cultivating tetraploid incarvillea comprises the following specific steps:

step one, after seeds of artemisia incarvensis are sown and seedlings emerge in spring, when cotyledons of the artemisia incarvensis seedlings are flattened and the tips of true leaves are just exposed, soaking and coating the stem tips of the artemisia incarvensis in a colchicine solution;

step two, retreating once every 12 hours, continuously processing for six times, and totally processing for 72 hours;

and step three, performing cytological identification to obtain tetraploid incarvillea plants.

Further, in the second step, the concentration of the applied colchicine solution is 0.3%.

Further, in the second step, the length of time for smearing the colchicine solution is 24 h.

Test examples

1. Materials and methods

1.1 test materials

The herba Incarvilleae Argutae is herbaceous flower of Incarvillea of Bignoniaceae.

1.2 Experimental methods

1.2.1 polyploid mutagenesis

When the leaves of the seedling of the artemisia horn are flattened and the true leaves are exposed to the tip, the stem tip of the artemisia horn is dip-coated with 0.3 percent colchicine solution, and after 12 hours, the processing is carried out once again for 6 times continuously for 72 hours.

1.2.2 identification of polyploids in plants

Picking up 2-4 mm plant stem tips by using tweezers at about 9:00 a.m., transferring the stem tips into a centrifugal tube of dichlorobenzene saturated aqueous solution for pretreatment for 3-5h, and then transferring into Carnot fixing solution (95% ethanol: glacial acetic acid: 3:1) for 24h of stem tip fixing treatment. During flaking, the stem tip of the incarvillea sinensis is placed into a centrifugal tube with 1mol/L HCl, the stem tip is dissociated in a water bath pot for 8-10min, the dripping Carbo Fuchsin staining solution is used for staining for about 20min, 1-2mm of the stem tip is taken for tabletting after staining, the quantity of incarvillea sinensis chromosomes is observed by using a Leica DM4000 microscope after the stem tip is placed for a period of time, and the picture is recorded.

1.2.3 morphometric assay

1.2.3.1 morphological observations: the height, pitch and the like of artemisia plants were measured with a graduated scale, the leaf length, leaf width and leaf area were measured, and 3 repetitions were set.

1.2.3.2 measurement of pore size and Density: taking artemisia leaf, tearing off the lower epidermis of the leaf by using tweezers, placing the lower epidermis on a glass slide, dyeing the lower epidermis of the plant by using 1% I-KI, covering a cover glass, observing by using an Olympus BH-2 optical microscope, observing the air hole state, measuring the length and width of the guard cell of the leaf, setting 5 times of repetition and calculating the average value; the pore density was measured using a mesh micrometer, photographed, and the average value was obtained.

2. Results and analysis

2.1 mutagenesis Effect of colchicine on Stem tips

By observing the mutagenesis effect of the artemisia argyi, the mutagenesis concentration and time are two main factors influencing the mutagenesis rate of the artemisia argyi seedlings, and when the mass concentration is 0.1%, the mutation rate of the artemisia argyi is the highest and is 46.1% when the artemisia argyi seedlings are treated by the agent for 72 hours; when the mass concentration is 0.2 percent and the treatment is carried out for 48 hours, the mutation rate is 62.9 percent; the mass concentration was 0.3%, and the highest mutation rate after 24 hours treatment was 68.1% (Table 2-1). By combining the analysis, the artemisia angustifolia has the optimal induction effect on the seedlings of the artemisia angustifolia when the soaking time is 24 hours and the ideal induction combination of the artemisia angustifolia among the three concentrations is 0.3 percent and 24 hours when the concentration is 0.3 percent.

TABLE 2-1 Induction Effect of Artemisia ordosica

2.2 identification of polyploids of Artemisia ordosica

2.2.1 identification of the number of chromosomes in Artemisia Selengensis plants

Tarragon plants were identified by the stem tip chromosome tabletting method, and of 60 identified plants, 23 were diploid (2 n-2 x-22, 29 were tetraploid (2 n-4 x-44), accounting for 38.3% and 48.3% of the total number of identified plants, respectively (shown in fig. 1).

2.2.2 comparison of diploid and tetraploid Properties

2.2.2.1 Effect on morphological indices

(1) The initial growth trends of the diploid plant and the tetraploid plant of the artemisia anomala are consistent. In the initial stage of colchicine treatment, the diploid plant and the tetraploid plant have the same height and are slightly higher than the tetraploid plant. With the increase of time, the plant height of the Incarvillea diploid is 5.41cm in 7-month and 10-day in 2019, the height of the tetraploid is 5.17cm, the plant heights of the Incarvillea diploid and the tetraploid are close to the same, and the difference is not obvious; the tetraploid plants grow slowly from No. 7/14 in 2019, and when the plants grow to No. 8/5, the height of the diploid plants of the artemisia argyi is 15.65cm, the height of the tetraploid plants is 10.05cm, the height of the diploid plants is 53.3% higher than that of the tetraploid plants, and the difference is extremely obvious (P is less than 0.01). After 8 months and 5 years, the diploid plant of the incarvillea maintains higher growth rate all the time, while the growth and development of the height of the tetraploid plant of the incarvillea is slower and nearly gentle, and the growth rate is reduced and is obviously lower than that of the diploid plant. The results show that the growth of the tarragon seedlings treated by colchicine is inhibited, so that the division speed of cells in the plant body is reduced, and the content of auxin is influenced.

(2) The leaves of the leaf-shaped artemisia angustifolia diploid plant are irregularly and finely split, and the number of the leaves is small; the leaf of the tetraploid incarvillea is obviously enlarged and rounded, and when the leaf is No. 8 and No. 5, the leaf length, the leaf width and the leaf area of the tetraploid plant are respectively 121.81 percent, 143.32 percent and 159.10 percent of those of the normal plant, and the difference is extremely obvious (P is less than 0.01); tetraploid plants became thick and dark in leaf, thick and short in stem, and increased in leaf number (shown in fig. 2).

(3) The size of guard cells is obviously changed, the guard cells of the leaves of the artemisia plants treated by colchicine are obviously larger, and the length and the width of the guard cells are respectively 140.37 percent and 155.69 percent of those of diploid plants. The number of stomata is greatly reduced, and the tetraploid is 33 percent of that of the diploid plant, and reaches a very significant level of difference (P < 0.01).

3. Results

(1) Soaking and coating 0.3% colchicine solution on the stem tip of Artemisia carota seedling for 24h to obtain tetraploid variant plant with variant rate of 68.1%.

(2) The carambola tetraploid variant plants show that the growth is accelerated, the leaves are thickened, the leaf shape index is changed, the leaf area is enlarged, the color of the leaves is deepened, the length and the width of guard cells in the leaves are increased, and the number of pores in unit area is reduced.

(3) The tetraploid artemisia annua karyotype is 2 n-4 x-44.

The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several variations and modifications without departing from the concept of the present invention, and these should be considered as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the utility of the patent.

Claims (3)

1. A method for cultivating tetraploid incarvillea, which is characterized by comprising the following specific steps:

step one, after seeds of artemisia incarvensis are sown and seedlings emerge in spring, when cotyledons of the artemisia incarvensis seedlings are flattened and the tips of true leaves are just exposed, soaking and coating the stem tips of the artemisia incarvensis in a colchicine solution;

step two, retreating once every 12 hours, continuously processing for six times, and totally processing for 72 hours;

and step three, performing cytological identification to obtain tetraploid incarvillea plants.

2. The method of claim 1, wherein the concentration of colchicine solution applied in step two is 0.3%.

3. The method of claim 1, wherein the colchicine solution is applied for a period of 24 hours in step two.

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