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CONFERENCE PROCEEDING
The Efficacy of Black Glutinous Rice (Mhor37) Extract for Use in Plant Chromosome Staining
Farhana Buwaeyusoh1*, Sitthisak Jantarat2
1Islamic Sciences Demonstration School, Prince of Songkla University, 94000, Muang, Rusamilae, Pattani, Thailand
2Department of Science, Faculty of Science and Technology, Prince of Songkla University, 94000, Muang, Rusamilae, Pattani, Thailand
*Corresponding author: [email protected]
ABSTRACT
Black glutinous rice (Mhor37) is one of the plants that contain anthocyanin. Which is a naturally occurring pigment and will be able to change color according to the pH level. The efficacy of black glutinous rice (Mhor37) extract was studied by using a solvent of acetic acid and ethanol at concentrations of 30%, 45%, and 60% with a ratio of 1:1 (g/ml) for 12 hr and 24 hr. After that, take the extracts extracted with acetic acid at a concentration of 30% for 12 hr adjusted the pH level to 1.5, 3.0, 4.5, 6.0, and 7.5. The experimental results show that the extract from black glutinous rice (Mhor37) extracted with 30% acetic acid at pH 1.5 was efficient in chromosome staining at onion roots and it can clearly observe each phase of cell division. This project is expected to reduce the use of chemicals in chromosome staining, it may result in less chemical contamination in the environment and it also reduces the budget for the purchase of expensive chemical dyes.
Keywords: Black glutinous rice, Anthocyanin, Solvent extraction, Chromosome staining, Chromosomal dyes
INTRODUCTION
The study of cell division and the behavior of chromosomes in plant cells is critical in genetics biology. For example, study to observe abnormalities both inside and outside the cell, study the evolution of plants, study to improve plant species or increase economic value, etc. In the study of the cell division of plants, chromosome staining was performed. Most of them use imported chemical paints that are expensive and it can also cause chemical contamination to the environment. Therefore, the extraction of natural dyes from various plants was initiated to reduce the budget and reduce the use of chemicals in chromosome staining. A pigment that can be stained with chromosomes is anthocyanin. It is in the group of phenolic compounds. polyphenol group as shown in figure 1. It has good water solubility but is insoluble in solvents that do not contain hydroxyl groups. Easily decomposed by heat, therefore unstable and will change color according to pH. If the acidity is high, it will be red and when it becomes more alkaline, it becomes blue as shown in figure 2. This pigment can be found in many common plants such as black glutinous rice seeds, red thorns, and dragon fruit rinds. Roselle flower, butterfly pea flower (Wanpen Kaewpuk, 2015, p.
1435). This project uses black glutinous rice (Mhor37), which is native rice from Phatthalung Province. It was extracted as a natural dye and studied its efficacy in chromosome staining. This is to reduce chemical contamination in the environment and reduce the budget on chromosome dyes.
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METHODOLOGY
Dye extraction from black glutinous rice (Mhor37)
Take the black glutinous rice (Mhor37) seeds to extracted with acetic acid and ethanol solvents at concentrations of 30%, 45%, and 60% by soaking black glutinous rice (Mhor37) seeds at a ratio of 1:1 (g/ml) at room temperature for 12 hr and 24 hr. Then filtered with a thin white cloth and filtered again with filter paper.
The chromosome staining of the onion root tip with black glutinous rice (mhor37) extracts by squash technique
Use the extract to chromosome staining at the onion root tips by using the squash technique, starting by cutting off only the cloudy white root tip of the onion root tip soaked in 70% ethanol solution. Then placed it on a clean slide for 1–2 roots, rinse it with distilled water and then blot-distilled water with tissue paper. Then drop 1 N hydrochloric acid on the onion root and leave it for 5 minutes. After the time is up, rinse it with distilled water again, then blot-distilled water with tissue, and then drop the extract from the black glutinous rice (Mhor37) extracted with acetic acid 30% at the onion root tip and leave it for 10 minutes and use the tip of the teasing needle to rub the cells to spread and closed with a glass slide. Then, repeat all 12 types of extracts and look at them under a light microscope. After that, observe the chromosome at the onion root tip, take pictures and record the results.
Adjusting the pH level of black glutinous rice (mhor37) extracts
Adjust the pH level of black glutinous rice (Mhor37) extract extracted with 30% acetic acid for 12 hr to 1.5, 3.0, 4.5, 6.0, and 7.5, then use them to dye chromosome at the onion root tip. After that, observe the chromosome staining characteristics to compare the chromosome staining efficiency of black glutinous rice (Mhor37) extract and aceto-orcein chemical dye and record the results of the experiment.
RESULTS AND DISCUSSION
The efficacy of black glutinous rice (Mhor37) extract extracted with different solvents and concentrations on plant chromosome staining
From extraction of black glutinous rice (Mhor37) extracted with acetic acid and ethanol with concentrations of 30%, 45%, and 60% for 12 hr and 24 hr were stained on the onion root tip. The result shows that the extracted with an acetic acid was stain on chromosomes better than those extracted with ethanol. Additionally, from a total of 12 extraction patterns, the extraction of black glutinous rice with 30% acetic acid for 12 hr was considered the best extraction method for chromosome staining.
The efficacy of black glutinous rice (Mhor37) extract extracted with acetic acid 30% at different pH levels in chromosome staining
From extraction of black glutinous rice extracted with acetic acid 30% for 12 hr was adjusted to the pH level of 1.5, 3.0, 4.5, 6.0, and 7.5. The results show that the extract in the acidic condition was effective in chromosome staining better than extracts in the neutral and the base condition. Additionally, the result shows that the black glutinous
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rice (Mhor37) extract adjusted pH to 1.5 showed the best chromosome staining from all pH level because the anthocyanin in black glutinous rice was more stable under more acidic conditions.
Comparison of chromosome staining efficiency between aceto-orcein dye and extracts from black glutinous rice (Mhor37) at pH 1.5
Chromosomes were stained with aceto-orcein and black glutinous rice (Mhor37) extract extracted with acetic acid 30% for 12 hr with pH adjusted to 1.5. The results showed that both types of dyes can clearly stain chromosomes. It can also clearly show the changes in each cell division stage and there is a small amount of dye in the cytoplasm. Therefore, the chromosome staining efficiency of black glutinous rice extract extracted with acetic acid 30% for 12 hr was comparable with the chromosome staining efficiency of aceto-orcein. This result is consistent with the research by Chamnien & Pohma (2021) studied the efficacy of dye from red dragon fruit in chromosome staining of onion roots. concluded that Pigment extraction from red dragon fruit by acetic acid 60% solvent at pH 1.5, but extracted at a ratio of 10:1 g: ml.
for 24 hr, the chromosomes can be stained clearly and have similar efficacy to aceto- orcein dyes and it is also consistent with the research of Tongsrisuk, et al., (2017) studied the efficacy of plant chromosome dyes with pigments extracted from purple sweet corn. From the experiment, it was found that dye extraction from purple sweet corn by using a solvent of acetic acid 45% with a pH 2, the chromosome staining from the mitosis of the onion root tip was the best method of extraction.
CONCLUSION
The natural dye extraction from black glutinous rice (Mhor37) with a 30%
concentration of acetic acid solvent at a ratio of 1:1 (g/ml) for 12 hr at pH 1.5 was effective in dyeing and it is most clearly observed chromosomes close to synthetic aceto-orcein making it possible to save costs. Reduce the use of chemicals and reduce the harm to users in plant chromosome staining.
ACKNOWLEDGEMENT
This project was supported by Science Classroom in University Affiliated School (SCiUS). The funding ofSCiUS is provided by Ministry of Higher Education Science.
Research and Innovation.
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