Philippine Science High School

Bicol Region Campus

Evaluation for the Presence of Kinetin in Rice

(Oryza sativa)

Rafael I. Gaite
Romeo Robert C. Indico IV
Joshua Ivan A. Roldan

March 2019 i
Evaluation for the Presence of Kinetin in Rice (Oryza sativa)

by

Rafael I. Gaite
Romeo Robert C. Indico IV

Joshua Ivan A. Roldan

Submitted to the Faculty of the

Philippine Science High School – Bicol Region Campus
in Partial Fulfillment of the Requirements in
Science and Technology Research 3

March 2019
ii
 ABSTRACT

Kinetin is a hormone found in plant and animal tissues. Studies

have extracted kinetin from plant tissues with focused on the fruit of the
plant only. While rice (Oryza sativa) is a staple crop and is used as main a
source of food, the stems and roots of the rice plant however have little
use because only the fruit is being consumed. This study aimed to
address this by determining the presence of kinetin in rice, and
consequently its optimum yield from the different parts of the plant –
fruit, roots, leaves, and stem to avoid waste. Samples were extracted from
the different plant parts using ethanol extraction method. To investigate
the effect of a different solvent on the yield, methanol was also used in the
extraction and compared with ethanol sample yield. The percentage yield
of the samples for the two solvents was taken with the bran containing
the highest while the leaves had the lowest for both solvents. Independent
t-Test result showed that there is no significant difference between the
ethanol and methanol yields. To evaluate the concentration of kinetin in
the sample, Ninhydrin reagent and spectrophotometry were used. All
samples tested were negative for Ninhydrin reagent. The samples were
tested in 268 mµ ultraviolet light which has the maximum absorbance
point for kinetin. The roots in ethanol solvent tested were positive with
maximum absorption. The results had shown that the roots, leaves and
stem of rice tested were negative for kinetin .

Key words: optimum, plant parts, presence, kinetin

iii
 APPROVAL SHEET-

This research work entitled, “Evaluation for the Presence of Kinetin in Rice (Oryza

sativa)” by Rafael I. Gaite, Romeo Robert C. Indico IV, and Joshua Ivan A. Roldan,

presented to the Faculty of the Philippine Science High School – Bicol Region Campus in

partial fulfillment of the requirements in Science and Technology Research 3, is hereby

accepted.

Alpha Rowena O. Pimentel

Research Adviser

iv
 TABLE OF CONTENTS

Page

Approval Sheet......................................................................................................................... iv

Table of Contents ...................................................................................................................... v

List of Tables ........................................................................................................................... vi

List of Figures ......................................................................................................................... vii

Introduction ............................................................................................................................... 1

Background of the Study ...................................................................................................... 1

Statement of the Problem ...................................................................................................... 1

Significance of the Study ...................................................................................................... 2

Scope and Limitations........................................................................................................... 2

Materials and Methods .............................................................................................................. 6

Results and Discussion ........................................................................................................... 10

Summary and Conclusion ....................................................................................................... 12

Bibliography ........................................................................................................................... 14

Appendices ............................................................................................................................. 16

v
 List of Tables

Table Title Page

1 Yield of Solution after Filtering Process 21

2 Average Yield of Solution after Distillation Process 21

3 Absorbance Rates of Samples in UV light spectrophotometer 22

vi
 List of Figures

Figure Title Page

1 Causal Model 6

2 Process Flowchart 7

3 Procurement of Rice Plant 8

4 Separated Rice Stem and Leaves 8

5 Separated Rice Fruit 8

6 Separated Rice Roots 9

7 Soaking of Rice Fruit in Alcohol 9

8 Distillation of the solution 10

9 Application of Ninhydrin reagent 11

10 Use of spectrophotometer 11

11 Average Yield of Distilled Samples 13

vii
 INTRODUCTION

Background of the Study

Kinetin is a plant hormone that plays a vital role in inhibiting growth to plants. It

inhibits chlorophyll production of plants and decreases the rate of cell division, effectively

reducing their growth rate (Amasino, 1955). Fruits can be treated with this compound to

further extend their shelf life. Application of kinetin on Kinnow mandarins shows that it can

reduce their rate of rotting, thus extending their shelf life as shown in the research

conducted by Kaur et al in 2014.

Kinetin is a cytokinin that can be found in all plant and animal tissues. A study

conducted by Miller (1960) suggested that the presence of a kinetin-like compound could be

found in corn. This kinetin could be extracted and used in extending the shelf life of fruits.

Rice (Oryza sativa) is the most important food crop in the world, directly feeding

more people than any other crop. According to CGIAR Research Program on Climate

Change, Agriculture and Food Security (CCAFS), nearly half of the world’s population has

relied on rice every day in 2012. However, its importance is focused mainly on its fruit,

which is utilized as staple food in almost all countries. The stems and roots are usually

neglected and did not establish any use at all. With rice being a plant with high potential

content for kinetin from its fruit, stem, and roots, prompts the researchers to make use of

these by products by extracting a potential amount and quality from it.

Statement of the Problem

The study has sought to answer the following questions:

1
General Problem:

Which part of the rice plant (Oryza sativa)tested : roots, stem, or fruit, would be positive for

the presence of kinetin?

Specific Problems:

1. Which part of the rice plant (Oryza sativa) would produce the highest percent

yield of kinetin?

2. Which part of the rice plant (Oryza sativa) would produce the best quality of

kinetin?

3. Would the solvent used in the extraction affect the kinetin yield of the rice plant?

4. Which part of the rice plant (Oryza sativa) would produce the optimum kinetin

production level?

Significance of the Study

This study aimed to contribute to the overall study of kinetin and can be used as a

basis for other studies in a similar or related field. In addition, this study would also benefit

the rice industry, as it would put more importance on the whole rice plant if it is a possible

source of kinetin. By doing this, it can reduce the amount of waste caused by the disposal of

the unusable rice plant parts.

Scope and Limitations

This study has focused on the rice plant (Oryza sativa) as a natural source of kinetin.

The kinetin was extracted using the ethanol extraction method. This study has assessed the

2
efficiency of the rice plant as a source based on the percent yield and the quality of the

kinetin. The quality of extracted kinetin was evaluated using the Ninhydrin Test and

Spectrophotometer. Ethanol being the most commonly used as solvent , this study however

has utilized methanol as an alternative solvent in the extraction of kinetin.

3
 REVIEW OF RELATED LITERATURE

Kinetin

Kinetin is a chemical compound with the formula C10H9N5O. It is widely used in

cosmetics and is employed in the manufacturing of medicine. Kinetin is a hormone found in

almost all plant and animal tissues. A study by Miller et al in 1955, as cited in an article by

Amasino in 2005, isolated the first kinetin sample from herring sperm. Another study by

Miller in 1956 extracted kinetin from coconut and brewer’s yeast. Kinetin is a type of

cytokinin, which are compounds that promote cytokinesis, or the physical process of cell

division. A study by Lou et al in 2012 indicated that kinetin could prolong the harvest date of

loquat fruits by preventing cell deterioration. According to another study by Kaur et al in

2014, kinetin slows down the spoilage of fruits through the reduction of several chemicals,

such as polygalactouranose and cellulose enzymes, that induce deterioration in kinnow fruits.

Most studies have focused on the optimal source of extraction of kinetin using several plants.

A study by Miller in 1956 investigated the optimum yield of kinetin from three (3) sources –

coconut, brewer’s yeast, and DNA. Another study by Miller in 1960 extracted a kinetin-like

compound from corn. However, there have been no studies that have focused specifically on

the parts of the plant. This prompts the researchers to investigate on the optimum yield of

kinetin from different parts of a plant.

Rice

Rice (Oryza sativa) is the second most produced crop globally. It is a staple in all

common households in almost every country. It is a grainy crop which is similar to corn (Zea

mays), which was used in Miller’s study. It has three parts – the roots, the stems, and the
4
bran. These factors make the rice plant an ideal sample for investigation on the optimum

yield for kinetin.

Ethanol Extraction

Ethanol extraction is a commonly used method in the extraction of pure substances

from various materials. It involves the soaking of the material in ethanol and then heating the

solution to evaporate the ethanol, a process known as distillation. However, ethanol is used

because it has a low evaporation temperature. Other alcohols may also be used which also

have low melting points, such as methanol, which may affect the yield of the extract. This

prompts the researchers to investigate the effect of the solvent used in extraction on the yield

of the extract.

5
 MATERIALS AND METHODS

RESEARCH DESIGN

Figure 1. Causal Model

The source of kinetin and the solvent used were used as the independent

variables since they would determine the kinetin yield and quality of the samples, which

were the dependent variables in the setup. The extraneous variables were the rice growing

methods and conditions as these were included in the setup. However, all the plants were

taken from the same harvest batch.

6
PROCESS FLOWCHART

Plant Samples
Preparation

Distillation Process

Ninhydrin Test

Spectrophotometry

Data Analysis

Figure 2. Process Flowchart

Preparation of Plant Samples

The rice plant was procured in a farm located in Tagongtong, Goa, Camarines Sur.

The researchers had specifically searched for rice plants that were 100 days old. Rice plants

7
enter its ripening age during this period and it is the time when kinetin levels were at its peak

(Miller, 1956).

After procurement, the rice plant was separated into its four parts namely: rice fruit

(pre-mill rice grain), leaves, stem, and roots. The rice plants were washed to remove physical

contaminants. After washing, the rice plant parts were air dried until completely void of

water.

The researchers had weighed 10g of bran, leaves and roots which were soaked in

100mL ethanol and methanol,then the solutions were left to stand for 24 hour.

After letting the methanol and ethanol solutions stand for 24 hour, the samples were

filtered using No.1 Whatman filter paper and a funnel. The volume of the filtrate was

measured using a graduated cylinder afterwards.

Distillation of Filtrates

The filtrates were distilled using a simple setup which consisted of a hot plate,

condenser, connecting glass tubes, and two flasks. The distilling temperatures were 125°C

and 155°C for the methanol and ethanol solutions respectively. The concentrated extract after

distillation was measured using a graduated cylinder to determine the volume. This process

was repeated for the other plant parts and their respective replicates

Ninhydrin Test

Six test tubes were prepared for the Ninhydrin test. Pipettors were used to measure

and transfer the liquids. Each test tube contained 1mL of each solution diluted with 3mL

8
distilled water; then 1mL of the Ninhydrin reagent was added. The test tubes were soaked in

a water bath at 90°C for 15min. The presence of Ruhemann’s purple color served as an

indicator in determining the presence of kinetin.

Spectrophotometry

2 mL of the filtrates were transferred to cuvettes via a pipettor. Using a UV-visible

spectrophotometer, the cuvettes containing the solutions were analyzed with each filtrate

having 3 replicates, for a total of 24 samples analyzed. UV light at 268 nm wavelength was

passed through the cuvettes and the absorbance of each sample was measured.

DATA ANALYSIS

For the third objective, the Independent t-Test was used, as there was only one

independent variable, the type of solvent, and one dependent variable, percent yield, which

was a ratio variable. The Independent t-Test evaluates if there was a significant difference

between two types of samples using the formula below.

𝑥̅𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙 − 𝑥̅𝑒𝑡ℎ𝑎𝑛𝑜𝑙
𝑡=
1 1
𝑆𝑝 √ +
𝑛𝑚𝑒ℎ𝑎𝑛𝑜𝑙 𝑛𝑒𝑡ℎ𝑎𝑛𝑜𝑙

9
 RESULTS AND DISCUSSION

The percent yield after the distillation process was taken to show which combination

of plant part and solvent produced the highest amount of the remaining unknown compound

after removal of the alcohol solvent. The results showed that the samples which contained the

bran and roots produced the highest yields of the remaining unknown compound for both

methanol and ethanol solvents. Moreover, it produced a significantly higher yield as

compared to the other methanol filtrates. The leaves filtrates contained the next highest

percentage yield with the ethanol solution having the higher percentage over its methanol

counterpart. The root filtrates had the least percent yield with a lesser ethanol filtrates than

their methanol counterpart. This suggested that the solvent used in extraction affects the

percent yield. However, results from the Independent t-test showed that there was no

significant difference in the yield between solutions with different solvents.

30

25
Volume of Filtrates

20

15

10

0
Roots Leaves Stem Fruit
Rice Plant Part

Ethanol Methanol

10
 Figure 11. Average Yield of Samples after Distillation.

The Ninhydrin Reagent tested was negative in all the samples, as Ruhemann’s purple,

which indicated the presence of amines in the filtrate. This indicated that the filtrates did not

contain kinetin (Miller, 1960).

Based on the absorbance values, only the bran and root filtrates in ethanol have a

maximum absorbance at 268 nm. This was similar to a previously mentioned study by Miller

(1960) wherein a kinetin-like compound was extracted from corn. The aforementioned

study’s extract also tested negative in Ninhydrin Reagent but had a similar absorbance with

kinetin in ultraviolet light. The extract from Miller (1960), albeit not being kinetin, also

exhibited cytokinin properties. However, this was not tested in the samples.

11
 SUMMARY AND CONCLUSION

Based on the findings, the bran and roots of the rice plant (Oryza sativa) yielded the

highest percentage yield of the unknown compound after extraction as compared to the fruit

and the roots. In addition, the results from the Independent t-test show that there was no

significant difference in the percent yield between the solvents used. However, after testing

for the presence of kinetin using Ninhydrin test, no color change was detected, indicating that

there were no amines in the samples present in kinetin. Spectrophotometry indicated that

none of the samples both in ethanol and methanol had the same absorbance as kinetin, both

having a maximum at 268 nm.

Based on the findings, the researchers conclude that none of the samples contained

kinetin. Therefore, rice (Oryza sativa) is not a viable source for kinetin based from the

results for both the Ninhydrin and spectrophotometry test results.

12
 RECOMMENDATIONS

It is recommended that the researchers should improve the method of separating the

plant parts. Separating the rice bran from the husk using a rice miller was suggested by one

of the researchers. Another option is purchasing the husks and the bran separately but it is

feared that it may affect the data as the parts come from different sources.

For further study, the researchers recommend that the cytokinin properties of rice

roots be tested, as they have similar properties to a kinetin-like compound extracted by Miller

in 1960, which promoted cell division and prolonged the shelf life of plants. It is also

recommended that other methods of extraction for kinetin from plants be studied, which will

contribute further to the study of cytokinesis and kinetin.

13
 BIBLIOGRAPHY

Nagar, P. K. (1993). Effect of Some Ripening Retardents on Fruit Softening Enzymes of

Kinnow Mandarin Fruits. Indian f. Plant Physiol Vol XXXVII. No.2 pp. 122-124.

Amasino, Richard (2008). Inhibition of Leaf Senescence by Autoregulated Production of

Cytokinin. SC'IENJOE, VOL. 27.

Letham, D. S. (1963) Zeatin, a Factor Inducing Cell Division Isolated from Zea mays. Fruit
Research Division, Department of Scientific and Industrial Research.

Miller, C. O., Skoog, F., Von Saltza, M. H., & Strong, F. M. (1955). Kinetin, a cell division
factor from deoxyribonucleic acid1. Journal of the American Chemical Society,
77(5), 1392-1392.

Miller, C. O. (1961). A kinetin-like compound in maize. Proceedings of the National

Academy of Sciences, 47(2), 170-174.

Kaul, K. & Sabharwal, P. S. (1971). Effects of sucrose and kinetin on growth and chlorophyll
synthesis in tobacco tissue cultures. Plant physiology, 47(5), 691-695.

Dadzie, B.K. & Orchard, J.E. (1997). Routine Post Harvest Screening of Banana/Plantain
Hybrids: Criteria and Methods. INIBAP Technical Guidelines 2. International Plant
Genetic Resources Institute, Rome, Italy; International Network for the Improvement
of Banana and Plantain, Montpellier, France; ACP-EU Technical Centre for
Agricultural and Rural Cooperation, Wageningen, The Netherlands.

Backer, H. D. (1996). Effect of heat on the sterilization of artificially contaminated water.

Journal of travel medicine, 3(1), 1-4.

14
15
 APPENDIX A

METHODOLOGY DOCUMENTATION

Figure 1. Procurement of Rice Plant

Figure 2. Separated Stem and Leaves

16
Figure 3. Separated Rice Fruit

Figure 4. Separated Rice Roots

17

Figure 8. Use of Spectrophotometer

Figure 5. Soaking of Rice Fruit in Alcohol

Figure 6. Distillation of the solution

18

Figure 7. Application of Ninhydrin

 APPENDIX B

TABLES

Table 1: Yield of Solution after Filtering Process

Plant Part Replicate Methanol Solution Ethanol Solution
Extracted (mL) Extracted (mL)

1 89 74

Fruit 2 78 79

3 88 79

1 88 80
`Leaves
2 82 81

3 83 80

1 88 83

Stem 2 91 81

3 82 79

1 79 87

Roots 2 85 76

3 88 80

Table 2: Average Yield of Filtrates after Distillation Process

Plant Part Average Methanol Average Ethanol
Solution Extracted Solution Extracted
(mL) (mL)

19
 Fruit 25 20

Leaves 17 12

Stem 15 11

Roots 20 15

Table 3: Average Absorbance Rates of Samples in UV light spectrophotometer

Plant Part Solvent Wavelength (nm) Absorbance

Fruit Methanol 268 2.299

Ethanol 268 2.404

Leaves Methanol 268 3.000

Ethanol 268 3.000

Stem Methanol 268 2.983

Ethanol 268 2.922

Roots Methanol 268 2.411

20
Ethanol 268 2.698

21
 APPENDIX C

COMPUTATIONS FOR RESULTS

t-test:

Independent Variable = Solvent used (Methanol, Ethanol)

As all samples were taken from the same batch of the rice plant, equal variances were
assumed.

𝑥̅𝑚 − 𝑥̅𝑒
𝑡=
1 1
𝑆𝑝 √ +
𝑛𝑚 𝑛𝑒
With

2
(𝑛𝑚 − 1)𝑆𝑚 + (𝑛𝑒 − 1)𝑆𝑒2
𝑆𝑝 = √
𝑛𝑚 + 𝑛𝑒 − 2

Where
Confidence level = 95%
α = 0.05

x̅m = mean of first sample (Methanol)

x̅e = mean of second sample (Ethanol)

nm = sample size of first sample

ne = sample size of second sample

Sm = standard deviation of first sample

Se = standard deviation of second sample

22
 Sp = pooled standard deviation

df = degree of freedom

Calculation of Sm and Se:

𝑁
1
𝜎 = √ ∑(𝑥𝑖 − 𝜇)2
𝑁
𝑖=1

Sm = 16.3
Se = 9.6
Subsitution to formula for Sp:

(12 − 1)264.1 + (12 − 1)92.2

𝑆𝑝 = √
12 + 12 − 2

2905.1 + 1014.2
𝑆𝑝 = √
22

𝑆𝑝 = 13.3

Substitution to formula for t:

1021 − 959
𝑡=
1 1
13.3√ +
12 12
62
𝑡=
5.4
𝑡 = 11.5
Calculation for df:

𝑑𝑓 = 𝑛1 + 𝑛2 − 2
23
 𝑑𝑓 = 12 + 12 − 2
𝑑𝑓 = 22

Critical t value for α = 0.05 and df = 22: 1.717

11.5 > 1.717
Calculated t < Critical t value
Accept H0

Average Percent Yield of Solution after Distillation Process (in %):

Rice Fruit

Methanol Solution
25 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 29.4%
85 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Ethanol Solution
20 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 25.9%
77.3 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Rice Plant Leaves

Methanol Solution
17 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 20.2%
84.3 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Ethanol Solution
12 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 14.9%
80.3 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

24
Rice Stems

Methanol Solution
15 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 17.2%
87 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Ethanol Solution
11 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 13.6%
81 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Rice Plant Roots

Methanol Solution
20 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 23.8%
84 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Ethanol Solution
15 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
× 100 = 18.5%
81 𝑚𝐿 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

25