CN102523785A - Application of safranine T in seed dye mark tracing aspect - Google Patents

Abstract

The invention discloses the application of saffron T in the aspect of seed dyeing mark tracing. The invention adopts saffron T to dye and mark the seeds, is harmless to the seeds, does not affect the activity of the seeds, and has a long-lasting dyeing effect. It can be applied to the study of plant seed dispersal and seed banks in various ecosystems such as farmland ecosystems, river and lake ecosystems, grassland ecosystems, and forest ecosystems.

Description

Application of Saffron T in Seed Dyeing Marking and Tracing

technical field

The present invention relates to a new application of saffron T, in particular to the application of saffron T in seed dyeing and marking. the

Background technique

Seed dispersal, dispersal, etc., linking the end of the reproductive cycle of mother plants with the establishment of their offspring populations is now widely recognized to have profound effects on vegetation structure. Seed dispersal has many meanings to plant populations, communities and ecosystem biology. It not only affects the dynamics and continuous growth of populations, genetic compartmentalization, affects the interaction between organisms, but also changes the richness and distribution of species, thereby affecting the community. structure has an impact. Moreover, seed dispersal is also of great significance to weed flora in agricultural ecosystems. To study the influence of various media on the propagation and diffusion of plant seeds, the method of plant survey seed bank quantity and spatial distribution dynamics or the method of small ball simulation is generally used. These methods can qualitatively reveal the effects of various dispersal routes on plant seed dispersal and the spatial dynamics of plant seed banks, but cannot determine the dispersal proportion of weed plant seeds, and it is difficult to link plant seed dispersal with communities in various ecosystems stand up. The first reason is that the seed dispersal cycle is complex: there are many intermediate steps and processes between seed production and the formation of a new generation of adult plants; another reason is that seed dispersal is difficult to track, and it is difficult for researchers to track the seeds produced by the mother plant spread to new places, making their fate difficult to determine. In recent years, the application of some new technologies and methods to mark and trace the dispersing seeds has shown good prospects in the correspondence between seeds and seedlings and mother plants. These labeling and tracing techniques include: radioisotope labeling, fluorescent dye labeling, stable isotope analysis, and molecular genetic markers. Using these methods and successfully marking often requires larger seeds with rich endosperm, and the detection of these marked seeds requires special instruments and relatively complicated operations, which are inconvenient to use, time-consuming and labor-intensive. Among them, fluorescent dye labeling and radioactive Isotopes have safety and environmental pollution problems. In addition, these methods are relatively expensive, so their application range is limited. the

Contents of the invention

The purpose of the present invention is to provide a method for using saffron T to mark and trace seeds in order to solve the defects in the prior art. the

In order to achieve the above object, the present invention provides an application of saffron T in the aspect of seed dyeing mark tracing. When Safranin T is used for dyeing immature and exfoliated seeds, the following steps are included:

(1) Preparation of saffron T staining solution: take saffron T or saffron T and ethanol, add water, and mix evenly to fully dissolve saffron T to prepare saffron T staining solution; The weight percentage concentration of T is 0.5～5%, and the volume percentage concentration of ethanol is 0～70%;

(2) Seed dyeing and marking: Before the plant seeds mature and fall off, take the saffron T staining solution prepared in step (1), spray or repeatedly brush the saffron T staining solution on the plant seeds in situ until The seeds are dyed red.

When Saffron T is used for dyeing mature seeds, it includes the following steps:

(1) Preparation of saffron T staining solution: take saffron T or saffron T and ethanol, add water, and mix evenly to fully dissolve saffron T to prepare saffron T staining solution; The weight percentage concentration of T is 0.5～5%, and the volume percentage concentration of ethanol is 0～70%;

(2) Seed dyeing and marking: collect mature plant seeds, let them dry naturally indoors, soak them in the saffron T staining solution prepared in step (1) for 0.5-5 hours, then remove and drain the staining solution, and dry them indoors The plant seeds are dyed red.

Among them, the seeds used for dyeing come from farmland weeds, forest trees, marsh or aquatic plants, and specifically come from the following plants: Poaceae such as Japanese kangaroo, kanko, oyster grass, weedy rice, wild oats, Paspalum, barnyardgrass, barnyardgrass, crabgrass, big foxtail, foxtail, pennisetum, bermudagrass, old awn wheat, ryegrass, zoysia, clubhead grass, long awned clubhead grass, Gunn's hard Grass, Brome, Bluegrass, Goosegrass, Miscanthus, Grass, Reed, Weed Grass, Bentgrass, Weedy Rice, Caryophyllaceae such as Beef Chickweed, Curly Fructus, Polygonaceae such as Row's Foot, Dentated Sorrel , Polygonum oleifera, Ranunculaceae, such as Shilong Rui, Fennel Garlic, Chenopodiaceae, such as Chenopodium, Scrophulariaceae, such as Tongquancao, Artemisia spp., Persian Grass, Erect Grass, Grass, Beishui Kuiping, willow leaves Vegetables such as Polygonum lilac, Asteraceae such as rice chamomile, chicory, wild mugwort, mud mustard, cocklebur, solidago canadensis, parsley, Sumen white wine grass, dipper, annual capsicum, amaranth Family such as Amaranthus retroflexus and Achyranthes bidentata, Malvaceae such as Abutilon, Labiatae such as Sarcophagus, Rubiaceae such as Swamp, Brassicaceae such as blueberry and shepherd's purse, Cyperaceae such as sedge and cow felt . Trees, Aceraceae, such as triangular maple, pentagonal maple, and chicken claw; Juglandaceae, such as Cyclocarya, Maple Poplar; Rosaceae, such as Potentilla; Chaotian Potentilla, Euphorbiaceae, such as Dijin, Dadijin, and Bandi Brocade, the family of Trichosanthaceae is such as festival vegetables, and the family of Umbelliferae is such as Cnidium, etc. the

Compared with the prior art, the present invention has the following advantages: saffron T is also called safranin O, which is a natural dye extracted from saffron stigma. Saffron T is used for seed dyeing and marking, which is harmless to seeds, does not affect the activity of seeds, and has a long-lasting dyeing effect. Using the invention to dye and mark seeds has simple operation, low cost and no pollution. It can be applied to the study of plant seed dispersal and seed banks in various ecosystems such as farmland ecosystems, river lake ecosystems, and forest ecosystems. the

Detailed ways

Below in conjunction with example the present invention is described in further detail. the

Example 1

Before the safflower seeds mature and fall off (at the end of May), use the saffron T staining solution to in-situ stain and mark the seeds of the saffron plants in the wheat field, and use a soft brush dipped in the saffron T staining solution to repeatedly coat the saffron seeds Brush until the original pale yellow sage seeds are evenly dyed to red.

Example 2

1. During the seed maturity stage of summer weeds, collect mature seeds of weeds Beckmannia syzigachne and Alopecurus japonicus in wheat fields, and soak them in saffron T staining solution for 2 hours after they are naturally air-dried indoors Take it out and let it air dry indoors. The original light yellow sagegrass and Japanese kangaroo were actually dyed red.

2. The weight of weed seeds before and after being marked, the comparison of germination rate and the comparison of floating ability

Determination of seed weight: Weigh the thousand-grain weight of grass seeds before and after being marked, and repeat 5 times.

Determination of seed germination rate: Spread filter paper with a diameter of 10 cm in a petri dish of the same diameter, soak the filter paper with water, then place 100 unmarked and dyed-marked weed seeds evenly on the filter paper, cover, 5 times Repeat, place in a plant incubator at 20±1°C, light:dark=12:12. Keep the filter paper moist throughout the experiment. The number of germinated weed seeds was counted every 2 days until no more weed seeds germinated. the

The weight of the dyed Smilax and A. japonica seeds was slightly increased compared with the undyed, but the difference was not significant (Table 1), indicating that the dyeing treatment only slightly increased the weight of these two weed seeds, and did not change their floating properties. The germination rate of the dyed S. japonica and A. japonica seeds was slightly lower than that of the undyed, and the difference was not significant (Table 1), indicating that the dyeing had no significant effect on the germination rate of these two weed seeds, that is, the dyeing did not damage them Vitality, staining solution safety. the

Table 1 Comparison of the weight and germination rate of the seeds of A. japonica and A. japonica before and after dyeing

Note: The same letters after the mean mean that the difference is not significant at the 0.05 level.

Example 3

Prepare different concentrations of saffron T staining solutions: take 0.5g, 1g, 2g, 3g, 4g, 5g of saffron T and add them to 100ml ethanol solution with a concentration of 50% by volume, and mix well to fully dissolve saffron T , prepared as Saffron T staining solution. Soak the saffron seeds in different saffron T staining solutions, remove them after 1 hour, and let them dry naturally in the room. The original light-colored saffron seeds are dyed red, and compare the germination rate of the saffron seeds before and after dyeing Variety.

Prepare 1% Safranin T staining solutions with different ethanol concentrations: Take 1g of Safranin T, add respectively 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70% 100ml of ethanol solution, mixed evenly, to fully dissolve saffron T, and prepare safflower T staining solution. Soak the saffron seeds in different saffron T staining solutions, remove them after 1 hour, and let them dry naturally in the room. The original light-colored saffron seeds are dyed red, and compare the germination rate of the saffron seeds before and after dyeing Variety. the

When the germination rate is measured, disperse the Smilax seeds in a plate with wet filter paper, 50 seeds per plate, repeat 5 times, place the plate at 20±1°C, light:dark=12:12 plant culture in the box. Keep the filter paper moist throughout the experiment. The number of germinated Smilax seeds was counted every 2 days until the seeds no longer germinated. the

Table 2 Comparison of germination rate of Saffron seeds stained with different concentrations of Safranin T

Note: The same letter after the mean value (±SE) indicates that the difference is not significant at the 0.05 level.

Table 3 Comparison of germination rate of Saffron T-stained Saffron seeds prepared with different percentage concentrations of ethanol

Note: The same letter after the mean value (±SE) indicates that the difference is not significant at the 0.05 level.

There is no significant difference in the germination rate of saffron T after staining with different concentrations of saffron T staining solution, indicating that 0.5%-5% saffron T can be used for dyeing saffron, and the germination of saffron will not be affected after dyeing ( Table 2). the

There is no significant difference in the germination rate of Saffron after staining with 1% saffron T staining solution prepared with different concentrations of ethanol, indicating that water or ethanol with a volume percentage concentration not higher than 70% can be used for the preparation of the staining solution ( table 3). the

Example 4

Collect mature weedy rice seeds in the farmland in autumn, after drying, soak the seeds in saffron T dyeing solution for 1 hour, remove them, and let them dry naturally in the room. The originally light-colored weedy rice seeds are dyed red. After the seeds were dried, the germination rate of weedy rice seeds before and after dyeing was compared. In addition, the dyed weedy rice seeds were respectively buried in 5 cm, 10 cm, and 15 cm soil of the paddy field, and the color change of the dyed weedy rice seeds was observed after 1 year.

When the germination rate is measured, the weedy rice seeds are dispersed on a plate equipped with wet filter paper, 50 seeds per plate, repeated 5 times, and the plate is placed at 20±1°C, light:dark=12:12 plants in the incubator. Keep the filter paper moist throughout the experiment. The number of germinated weed seeds was counted every 2 days until the weedy rice seeds no longer germinated. the

In the end, the germination rate of weedy rice before dyeing was 76.2±9.5%, and the germination rate after staining with saffron T staining solution was 74.8±10.4%. The dyeing solution is safe for weedy rice seeds and does not affect their germination and emergence. The experiment found that the weedy rice seeds stained with saffron T can be clearly distinguished after being buried in 5 cm, 10 cm, and 15 cm soil of paddy fields for 12 months, indicating that the dyeing can be maintained for a long time. the

Example 5

Collect the linden samara that fell into the pre-set net bag after ripening and falling off in autumn, soak the samara in saffron T dyeing solution for 3 hours, remove it, and let it dry naturally in the room. Both the elm seeds and fruit wings are dyed is red, and compares the germination rate changes of elm seeds before and after dyeing.

When determining the germination rate, disperse the seeds before and after dyeing of elm trees in a plate with wet filter paper, 25 seeds per plate, repeat 4 times, place the plate at 25±1°C, light:dark=12:12 plants in the incubator. Keep the filter paper moist throughout the experiment. Take the radicle breaking through the seed coat by 1mm as the standard for seed germination, observe and record every day during the germination process, pay attention to keep the filter paper moist, until no new seeds germinate for 2 consecutive days, it is regarded as the end of germination, count the number of germinated seeds before and after dyeing, and calculate the germination rate, germination index. Calculated as follows:

Germination rate (G ) = total number of seeds germinated / number of tested seeds × 100 %

Germination index (G _i ) =∑(G _t / D _t )

Among them: G _t — germination rate at time t; D _t — corresponding germination days

The germination peak of elm seeds before and after dyeing was 2-4 days, and the whole germination process lasted about 15 days. The germination rate before dyeing was 89±9.5%, and the germination index was 6.2±1.4%; The rate was 84±3.3%, and the germination index was 5.7±0.6%. There was no significant difference in the germination rate and germination index of elm seeds before and after dyeing, indicating that the saffron T staining solution was safe for elm seeds and did not affect their germination and emergence.

Example 6

Collect the mature seeds of Artemisia marsh in autumn, dry them naturally, soak the seeds in saffron T staining solution for 1 hour, remove them, put them in the room and dry them naturally, and then the seeds will be dyed red. Comparison of the germination rate of the seeds of Artemisia marmoscens before and after dyeing.

For the determination of germination rate, disperse the seeds of Artemisia marshmallows in a plate with wet filter paper, 50 seeds per plate, repeat 5 times, place the plate at 20±1°C, light:dark=12:12 plants in the incubator. Keep the filter paper moist throughout the experiment. The number of germinated weed seeds was counted every 2 days, and the number of germinated seeds before and after dyeing was counted respectively until the seeds of S. marshmallow no longer germinated, and the germination rate was calculated. the

The germination rate of Artemisia marsh seeds before dyeing was 69±7.8%, and the germination rate after staining with saffron T staining solution was 65±8.2%. The T staining solution was safe to the seeds of Artemisia marsh, and did not affect the germination and emergence of seedlings. the

Example 6

Collect the ripe fruit (fruit and seeds) at the turn of spring and summer or in autumn, after natural air-drying, soak the fruit in saffron T dyeing solution for 1 hour, then remove it, put it in the room and dry it naturally, and the seeds will be dyed red .

The dyed and undyed seeds were respectively buried in the soil at a depth of 10 cm in the paddy field, and the water depth was kept at 5 cm in the paddy field. After 30 days, 90 days, and 150 days of burial, more than 20 seeds of each kind of dyed and non-dyed seeds were dug out for observation Changes in seed color were recorded. After the seeds are dug out and washed, they are dried in a drying oven at 30 ± 2 ^○ C for 10-15 days. If the difference between dyed and non-dyed seeds can be distinguished by naked eyes, take 5 seeds of each kind and take pictures of these seeds under the same conditions. , and then use Adobe Photoshop 6.0 software to analyze and judge the color of the seeds.

As can be seen from Table 4, the dyed weed seeds in all tables were clearly discernible when buried for 30 days, only a very small number of weed seeds faded when buried for 90 days, and more than half of the dyed and non-dyed seeds were still clearly visible after buried for 150 days. difference. the

Table 4. Variation of color of dyed plant seeds with burial time

Note: The weed seeds were buried in the paddy field with a burial depth of 10 cm, and a water depth of 5 cm was maintained in the paddy field. Different lowercase letters after the mean (±1SD) indicate significant differences at the 0.05 level.

Claims (5)

1. Application of saffron T in seed dyeing and marking. 2. the application of saffron T according to claim 1 in the aspect of seed dyeing mark trace, is characterized in that: comprise the following steps: (1) Preparation of saffron T staining solution: take saffron T or saffron T and ethanol, add water, and mix evenly to fully dissolve saffron T to prepare saffron T staining solution; The weight percentage concentration of T is 0.5～5%, and the volume percentage concentration of ethanol is 0～70%; (2) Seed dyeing and marking: Before the plant seeds mature and fall off, take the saffron T staining solution prepared in step (1), spray or repeatedly brush the saffron T staining solution on the plant seeds in situ until The seeds are dyed red. 3. the application of saffron T according to claim 1 in the aspect of seed dyeing mark trace, is characterized in that: comprise the following steps: (1) Preparation of saffron T staining solution: take saffron T or saffron T and ethanol, add water, and mix evenly to fully dissolve saffron T to prepare saffron T staining solution; The weight percentage concentration of T is 0.5～5%, and the volume percentage concentration of ethanol is 0～70%; (2) Seed dyeing and marking: collect mature plant seeds, let them dry naturally indoors, soak them in the saffron T staining solution prepared in step (1) for 0.5-5 hours, then remove and drain the staining solution, and dry them indoors The plant seeds are dyed red. 4. The application of saffron T according to any one of claims 1 to 3 in the aspect of seed dyeing, marking and tracing, characterized in that: the seeds are derived from farmland weeds, forest trees, marsh or aquatic plants. 5. the application of saffron T according to claim 4 in the aspect of seed dyeing, marking and tracing, is characterized in that: the seeds are derived from Gramineae, Caryophyllaceae, Ranunculaceae, Chenopodiaceae, Scrophulariaceae, Willow Leaf Vegetables Asteraceae, Compositae, Amaranthaceae, Malvaceae, Lamiaceae, Rubiaceae, Cruciferae, Cyperaceae, Pittogoniaceae, Magnoliaceae, Buxus, Ulmaceae, Tiliaceae, Aceraceae, Walnut Family, Rosaceae, Euphorbiaceae, Chitridaceae or Umbelliferae.