BIOLOGY EXTENDED ESSAY

Investigating the effect of chitosan coating on the

1-aminocyclopropane-1-carboxylic Acid Oxidase (ACO-03)

ripening gene expression levels in the pulp and peel of harvested

Cavendish bananas (Musa acuminata)

Research Question: How does the use of chitosan coating affect

expression levels of the 1-aminocyclopropane-1-carboxylic Acid

Oxidase (ACO-03) ripening gene in the pulp and peel of harvested

Cavendish bananas (Musa acuminata)?

Group 4: Experimental Sciences – Higher Level Biology

Date of Submission: Thursday, 12 March 2020

Word Count: 4000 words

 1

Table of Contents
Table of Contents ........................................................................................................................... 1
1: Introduction ............................................................................................................................... 2
A. Research question .............................................................................................................. 2
2: Background Research ............................................................................................................... 4
A. Importance of Bananas ..................................................................................................... 4
B. Ethylene as a Fruit Ripening Hormone ........................................................................... 4
C. Chitosan Edible Coating ................................................................................................... 5
D. Hypothesis …..…………………………………………………………………………………………………..6
3: Methodology ............................................................................................................................... 7
A. Banana Samples ................................................................................................................. 7
B. Chitosan Coating and Banana Storage............................................................................ 7
C. ACO-03 Gene Expression Analysis .................................................................................. 8
1. RNA Isolation and Quality Inspection ...................................................................... 8
2. Synthesis of cDNA ....................................................................................................... 9
3. Polymerase Chain Reaction (PCR) .......................................................................... 10
4. Sequencing ................................................................................................................. 11
5. Analysis of Gene Expression using Quantitative Polymerase Chain Reaction
(qPCR) ........................................................................................................................ 12
4: Results and Discussion ............................................................................................................ 14
A. Processed Data ................................................................................................................. 14
B. Analysis of ACO-03 Gene Expression during by qPCR ………………….…....…………….……14
1. Pulp ............................................................................................................................. 15
2. Peel .............................................................................................................................. 16
3. Conclusion .................................................................................................................. 18
5: Evaluation................................................................................................................................. 19
A. Evaluation of Methodology ............................................................................................. 19
B. Evaluation of Data Analysis……...………………………………………..………...…………………………….20
6: Bibliography ............................................................................................................................. 21
7: Appendices ............................................................................................................................... 27
 2

1: Introduction

A. Research question

Since my early childhood, bananas

had always been me and my family’s

favorite fruit. As an Indonesian, I

recognize that there are many types

of locally produced bananas. One

variant that I have observed to be

(Figure 1) Cavendish Bananas Ripening Index
particularly successful is Cavendish (Edwards, 2002)

bananas [Musa acuminata (AAA group)] (Figure 1). Compared to many local variants, they

are less susceptible to disease and are more visually appetizing due to there usually being less

dark spots or bruising. Such traits are desirable, as people generally avoid purchasing bananas

with dark spots and prefer clean yellow ones, even if they are less sweet than their bruised

counterparts are, and their nutrients are not as abundant as other variants (Dwivany, 2011).

Since dark spots are the result of ripening, finding ways to delay ripening may improve the

quality and appeal of Cavendish bananas.

Through my research, I have found that several scientific papers investigating the use of

chitosan as an edible coating for fruits like bananas support the hypothesis that such treatment

can delay ripening in banana pulps and extended fruit shelf-life. However, it was more

challenging to find papers discussing whether the effect of chitosan edible coating differed

significantly between the pulp and peel of bananas. This curiosity led me to develop my

research question: How does the use of chitosan coating affect expression levels of the 1-

aminocyclopropane-1-carboxylic Acid Oxidase (ACO-03) ripening gene in the pulp and

peel of harvested Cavendish bananas (Musa acuminata)?

 3

To answer my question, I analyzed and compared the gene expression levels of 1-

aminocyclopropane-1-carboxylic Acid Oxidase (ACO-03), a gene that plays an important role

in ethylene biosynthesis and banana ripening, in the pulp and peel of Musa acuminata

(Cavendish bananas) through molecular analysis. The analysis was done using the quantitative

Polymerase Chain Reaction (qPCR) method to detect changes in ACO-03 gene expression

during Day-1 and Day-7 post-harvest ripening. The results of this study are also expected to be

the beginning of the application of simple post-harvest technology on Cavendish bananas,

which can be developed further in the future.

Through a family member that works in the Bandung Institute of Technology (ITB), I managed

to secure two months over the 2019 summer break to do my research in the labs of the School

of Life Sciences and Technology (SITH-ITB). One month to practice the procedure with

undergraduates doing the same methodology, and another for independent research of which

the process and results are discussed in this essay.

 4

2: Background Research

A. Importance of Bananas

Bananas are climacteric tropical fruits with nutritious benefits that allow them to be among the

four most important global food commodities with high exporting levels (Nelson et al., 2006;

Pro Ecuador, 2016). Developing countries in tropical regions are the biggest exporters of

bananas. However, Indonesia was only able to export 1,600 tons of bananas in 2015 (FAO,

2017), despite having a wide diversity of banana cultivars, which includes more than 325 types,

with several being potential cultivars for exports (Setyadjit et al., 2004).

B. Ethylene as a Fruit Ripening Hormone

Climacteric fruits continue to ripen post-harvest by experiencing high rates of respiration and

ethylene biosynthesis. Ethylene (C2H4) is a phytohormone produced in several plants that acts

as an important regulator in plant development and growth, such as fruit ripening, seed

germination, organ aging, and biotic/abiotic stress responses (Barry and Giovannoni, 2007;

Taiz and Zeiger, 2002; Xu and Zhang, 2015). Ethylene biosynthesis is primarily catalyzed by

ACC synthase (ACS) and ACC oxidase (ACO) enzymes. Two ACO enzymes (MaACO1-2)

exist in Cavendish bananas, coded for by nine similar ACO genes, one of them being the ACO-

03 gene (Dwivany, 2011). Cavendish bananas experience high levels of autocatalytic ethylene

biosynthesis in earlier stages of post-harvest ripening (Day 1-3) because of naturally high

expressions of the ACO genes (Patty, 2016; Liu et al., 1999). This leads to high productions of

ACO enzymes, which catalyzes the final stage of ethylene biosynthesis by converting ACC

precursors to ethylene (Figure 2) (Dwivany, 2011). In the later stage of post-harvest ripening

(Day 4-7), autocatalytic ethylene biosynthesis begins to drop (Patty, 2016; Liu et al., 1999).

When this happens, ACO enzymes need O2 as a secondary substrate to continue converting

ACC precursors into ethylene (Dwivany, 2011). The synthesized ethylene enters the signaling
 5

pathway and triggers fruit ripening

responses like fruit color and texture

changes, sugar metabolism, fruit

softening, aromatic compound

synthesis, and increased susceptibility

to pathogen attack, which causes fruit

quality to decrease (Barry and

Giovannoni, 2007). Therefore, post-

harvest engineering is required to

improve the quality of bananas.

C. Chitosan Edible Coating

Fruit post-harvest engineering is usually

done by altering the concentrations of (Figure 2) Yang Cycle of the ethylene

O2, CO2, or ethylene around the fruit
(Kader et al., 1989; Jiang et al., 1999;
Kudachikar et al., 2011). However,
biosynthetic pathway and the methionine cycle,
red circle is activation of ACO enzyme. Modified
from Yang & Hoffman (1984).

these methods are relatively expensive. Therefore, alternatives like edible coatings are needed.

Edible coating is known to be safe for consumption because it is non-toxic, biocompatible,

easily degradable, and affordable.

One example of potential edible

(Figure 3) Chitosan Chemical Structure (Sahib, 2014) coating is chitosan (Figure 3), a

natural linear polysaccharide obtained from chitin, a substance found in the exoskeleton of

crustaceans and fungal cell walls. It is the second most abundant polymer in nature after

cellulose (Jianglian and Shaoying, 2013; Luo and Wang, 2013) and is classified as Generally
 6

Recognized as Safe by the Food and Drug Administration (FDA). Chitosan can form a thin film

that prevents water loss and inhibit oxygen penetration, maintains product quality (aroma, taste,

and texture), and prevent microbial growth (Jianglian and Shaoying, 2013; Xing et al., 2016).

Chitosan coating has been applied to several banana cultivars, including in Musa sapientum

(Malmiri et al., 2011) and Musa acuminata (Pratiwi et al., 2015; Zaskia, 2016; Lustriane et al.,

2018), with results suggesting that chitosan delays fruit ripening by maintaining its quality. The

expression patterns of ACO genes have also been analyzed in chitosan-coated Cavendish

bananas, and results show that it delays changes in peel color, starch content, and total soluble

solids (TSS), suggesting that chitosan delays Cavendish banana ripening (Yamamoto, 2017).

D. Hypothesis

Based on the information above, the hypothesis for this investigation is as follows:

1. Chitosan coating on Cavendish bananas (Musa acuminata) can delay ripening and

maintain the quality of bananas in both its pulp and peel.

2. ACO-03 gene expression levels in the pulp and peel of Cavendish bananas are

influenced by chitosan coating.

 7

3: Methodology

A. Banana Samples

Individual mature green Cavendish bananas were selected for uniformity in size, color, and

absence of physical damage and fungal infection. Three replications (triplications) of each

sample, labeled according to Table A, were used. Two grams of each sample were submerged

into liquid nitrogen and refrigerated at -80°C. All chemicals used were analytical grade.

[Table A] Sample variations used in this investigation with labels.

Sample Day Post-

Treatment Part of Banana Sample Replicates
Label harvest
1UF Day 1 Control (Uncoated
7UF Day 7 with Chitosan, U)
Pulp (Flesh, F)
1CF Day 1 Treated (Coated Three replicates
7CF Day 7 with Chitosan, C)
(Example: 1UF-III is
1US Day 1 Control (Uncoated Day-1 Control Pulp
7US Day 7 with Chitosan, U) sample, 3rd replicate)
Peel (Skin, S)
1CS Day 1 Treated (Coated
7CS Day 7 with Chitosan, C)

B. Chitosan Coating and Banana Storage

The preparation of a 1.25% chitosan solution was done according to the Malmiri et al. (2011)

method with modifications and was stored at room temperature (27°C) until use. Bananas were

cleaned and weighed individually using an analytical digital scale, coated in the chitosan

solution for 2 minutes using the dipping method (Malmiri et al., 2011), dried at room

temperature (27°C) for 2 hours, and stored at 27°C with a relative humidity of approximately

82% for seven days.

 8

C. ACO-03 Gene Expression Analysis

The ACO-03 gene expression analysis began with isolating the total RNA from each sample

listed in Table A before its quality, purity, and concentration were examined for cDNA

synthesis. The ACO-03 gene was then amplified using the cDNA template through Polymerase

Chain Reaction (PCR) to confirm the success of ACO-03 gene amplification, using Actin as a

reference gene. Finally, the quantification of ACO-03 gene expression was done using qPCR.

1. RNA Isolation and Quality Inspection

Triplications of each sample variation (Table A) were used as RNA isolation

samples, which was done according to the Cordeiro et al. (2008) method with

modifications. After 2 grams of each sample was crushed with a mortar

containing liquid nitrogen to create fine powder, 800µL of RNA extraction

buffer (components listed in Table B) were heated at 60°C before 35µL β-

mercaptoethanol and samples were added. The mixture was then vortexed,

incubated (60°C, 15 minutes; homogenized every 5 minutes), and set until it

reaches room temperature. An 800µL chloroform:isoamilalkohol (24:1)

solution was added and vortexed. Samples were centrifuged (8,000rpm, 25

minutes, 4°C) and the supernatant was transferred to a new microtube, before

being extracted again with chloroform:isoamilalkohol (24:1) with the same

volume as the supernatant. The extracted supernatant was given 1/3 volume of

8M LiCl and was slowly turned over. Next, the sample was incubated (4°C, 18-

20 hours) before being centrifuged (12,000 rpm, 15 minutes, 4°C). The formed

pellet was dissolved in 500µL DEPC-treated cold water, 63.3µL Na-acetate 3M

(pH 5.2), and 1mL absolute ethanol, homogenized slowly and incubated (-20°C,

2 hours). Next, the samples were centrifuged (8,000rpm, 10 minutes, 4°C). The
 9

RNA pellets obtained were washed with 70% ethanol (1mL) and re-centrifuged

(8,000 rpm, 10 minutes, 4°C), dried at room temperature (+25 minutes) and

dissolved in 30µL DEPC-treated cold water before being refrigerated at -80°C.

[Table B] RNA extraction buffer component (Cordeiro et al., 2008 modified).

Final Concentration Volume required for

Solution Initial Concentration
in Solution reaction (µL)
NaCl 5M 2M 320
EDTA (pH 8) 0,5 M 25 mM 40
Tris-HCl (pH 8) 1M 100 mM 80
PVP 10% 2% 160
CTAB 10% 2% 160
Sterile Deion - - 40
Total 800

RNA sample quality was tested using gel electrophoresis on 1.5% agarose gel

(+75V for 30 minutes), with GelRed® (Biotium, Catalog No.41003-T) dye.

Pure RNA would produce two bands on the electrophoresis gel, representing

present 28S and 18S rRNA. The purity and quantity of RNA were tested using

a Biospectrometer from Eppendorf at 260nm, 280nm, and 230nm waves. The

RNA samples were then refrigerated at -80°C.

2. Synthesis of cDNA

The isolated RNA sample was first cleansed from genomic DNA contamination

with DNase using the DNaseI kit (Thermo Scientific, Catalog No.EN0521). The

procedure followed the manufacturer’s instructions. A total of 500ng RNA

samples were put into a sterile PCR tube, where 1µL 10x buffer, 1µL DNase

enzyme, and nuclease-free water were added until reaching a volume of 10µL.

The sample was then resuspended and incubated at 37°C for 30 minutes. To

stop DNase activity, 1µL 25mM EDTA was added and incubated at 65°C. The
 10

results of the DNase treatment were 11µL RNA samples. Synthesis of cDNA

was done after DNase treatment, using the BioRad’s iScript cDNA synthesis kit

(Catalog No.170-8890). The cDNA synthesis methodology followed the

manufacturer’s instructions with modification. A total of 11µL of RNA sample

solution from the DNase stage was added with 4µL of 5x iScript reaction mix,

1µL of reverse transcriptase enzyme, and nuclease-free water until reaching a

total volume of 20µL. The mixture was homogenized by resuspension and

incubated at 25°C (5 minutes), 42°C (30 minutes), and 85°C (5 minutes). The

cDNA obtained was refrigerated at -20°C for later use.

3. Polymerase Chain Reaction (PCR)

PCR was performed to amplify the ACO-03 gene from the Cavendish banana

cDNA. The primer (Table C) used was MaACO-03 (ACO-03 primer). The

Actin gene was used as a reference gene for normalization, using MaActin

primers. Primers have been optimized by Yamamoto (2017).

[Table C] Primers of ACO-03 genes for qPCR, according to Yamamoto (2017).

Gene ID* Coding Protein Primer F Primer R Source

Ma03_t02700.1 CGGTCATCGA TCGGAGCTGA Yamamoto
ACO-like
(XM_009392868.1) TTTCTCCAAG CCTTCTTCAC (2017)

*Banana Genome Hub (http://banana-genome-hub.southgreen.fr/home1)

The Cavendish banana cDNA sample was amplified using the GoTaq® Green

Master Mix reagent (Promega, Catalog No.M7122) with a thermolcycler. The

reaction material composition (Table D) were mixed into a 200µL microtube

until reaching a total volume of 15µL.

 11

[Table D] The composition and sequence of addition of the PCR reaction material.

Initial Final Concentration Volume

No. Reacting Materials
Concentration in Solution (µL)
1. GoTaq® Green Master Mix 2x 1 7,5
2. Forward Primer 5 µM 0,5 µM 1,5
3. Reverse Primer 5 µM 0,5 µM 1,5
4. Nuclease-free Water - - 1,5
5. cDNA Sample 2,5 ng/µL 0,5 ng/µL 3
Total 15

The PCR cycle (Figure 4) began with an initial denaturation at 94°C for 2

minutes, continued with 45 cycles of denaturation, annealing, and chain

extension. Denaturation was done

at 94°C for 30 seconds, annealing

at 60°C for 30 seconds, and

extension at 72°C for 30 seconds.

Next, the cycle enters the final (Figure 4) PCR cycle profile of the

extension at 72°C for 7 minutes. MaACO-03 and MaActin genes on the

pulp and peel of Cavendish bananas.
The results of the PCR process

were then confirmed by gel electrophoresis using 1% agarose gel in a 1% TAE

buffer, run at +75V for 50-60 minutes.

4. Sequencing

Confirmation of the reference gene (Actin) and target gene (ACO-03) was done

using DNA sequencing. The DNA bands obtained from gel electrophoresis

were then cut and purified using the Wizard® SV Gel kit and PCR Clean-Up

System (Promega, Catalog No. A9282). The DNA purification resulted in a total

volume of 30µL for each gene sample, with a concentration of 14.4 µg/mL for

the Actin gene sample and 11.4 µg/mL for the ACO-03 gene sample. The
 12

sequencing resulted in contig data that was processed using BioEdit Sequence

Alignment Editor, performed on the forward and reverse sequences of each

gene. The contig data processing results were then used for gene identification

using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The results showed

that the ACO-03 and Actin gene samples used are viable for qPCR processing.

5. Analysis of Gene Expression using Quantitative Polymerase Chain

Reaction (qPCR)

Quantitative Polymerase Chain Reaction (qPCR) or Real-Time PCR was used

to amplify and quantify the expression of the ACO-03 gene expression levels in

the pulp and peel of chitosan-coated and uncoated Cavendish banana samples.

After preparing the reaction mixture, all the samples were set in the qPCR

instrument set. The qPCR reaction was carried out according to manufacturer

protocols with modifications, and the composition was sequentially added

according to the order in Table E.

[Table E] The composition of the qPCR reaction mixture.

Initial Final Concentration Volume

No. Reacting Materials
Concentration in Solution (µL)
1. Nuclease-free Water - - -
2. Primer forward 5 µM 0,2 µM 0,4
3. Primer reverse 5 µM 0,2 µM 0,4

4. Thunderbird® SYBR® 2x 1x 2,2

qPCR Mix
5. Sampel cDNA 2,5 ng/µL 0,5 ng/µL 2
Total 10
 13

The qPCR results analysis included an examination of the amplification curves,

melting curves, and negative control. The data was then normalized and

quantified (Dorak, 2006). The amplification curve was used to set the threshold

value. Meanwhile, the melting curve was used to detect the presence of primary

dimers and PCR product specifications. Data from the qPCR quantification

results come in the form of Ct (threshold cycle) values, and changes in the

relative multiples of gene expression were analyzed using the relative

quantification method developed by Livak and Schmittgen (2001). The

calculation was done by entering the Ct values into the formula as follows:

ΔCt = Ct(GT) - Ct(REF)

ΔΔCt = ΔCt(Day-x) – ΔCt(Day-1 (control))

Value of relative multiples of gene expression = 2-ΔΔCt

GT is the target gene used (ACO-03). Meanwhile, REF is the reference gene

used (Actin), where expression values are considered the same in all tissues and

developmental conditions. The symbol x is the days passed of post-harvest

Cavendish banana ripening.

 14

4: Results and Discussion

A. Processed Data

Quantitative Real-time PCR was carried out to analyze levels of ACO-03 gene expressions

using cDNA synthesized from total RNA derived from both uncoated and chitosan-coated

Cavendish banana pulp and peel on Day-1 and Day-7. Each relative transcript abundance was

normalized by Actin using the 2-ΔΔCt method. The analysis was performed using sample

triplications to determine the consistency of results. Raw Ct Value results for ACO-03 gene

and Actin is in Appendix A. The calculation for values of relative gene expression using the

Livak and Schmittgen (2001) method is presented in Appendix B and were averaged between

triplications before being presented in a bar diagram (Figure 5) that displays changes in the

relative multiples of gene expression.

Relative Expression Level of ACO-03 gene in Chitosan-coated and Uncoated

Cavendish bananas (Musa acuminata) on Day-1 and Day-7 Post-harvest
3.50
Relative ACO-03 gene level

3.00 2.49
to Actin gene expression

1.97 1.71
2.50
2.00
1.08
1.50
1.00
0.50 0.18 0.34
0.07 0.07
0.00
Pulp (Day 1) Pulp (Day 7) Peel (Day 1) Peel (Day 7)
Banana Sample (Pulp or Peel) and Ripening Period (Day-1 or Day-7)

Control 1,25% Chitosan

[Figure 5] ACO-03 relative levels of gene expression in Cavendish bananas in Day 1 and Day 7 with
the treatment conditions of fruits coated with 1.25% chitosan (orange) and uncoated with chitosan or
B. Analysis of ACO-03 Gene Expression during by qPCR
control (blue). Error bars indicate the standard deviation (SD) of each sample variation (Appendix B.3).
 15

1. Pulp

Trends observed in Figure 5 show great similarity with trends discussed in Section

2B; In the earlier stages of post-harvest ripening (Day-1 to Day-3), the Cavendish

banana pulp experiences a surge in autocatalytic ethylene biosynthesis before

decreasing in the later stage of the ripening process (Patty, 2016; Liu et al., 1999).

This is supported by how in Figure 5, the uncoated Cavendish banana pulp showed

higher relative ACO-03 gene expression levels in Day-1 before decreasing by 6

folds in Day-7.

Chitosan-coated Cavendish banana pulp exhibits the same trend. However, chitosan

coating inhibits the pulp's interaction with the external atmosphere, which causes

the ethylene produced during the early stages of ripening to bounce back and be

trapped in the pulp. The abundance of ethylene within the pulp then further

promotes the expression of the ACO-03 gene in the early stages of catalytic ethylene

biosynthesis to produce more ACO enzymes, which further promotes ethylene

production. This is observable in Figure 5, where the ACO-03 gene expression

levels in chitosan-coated Cavendish banana pulps in Day-1 were 2.5 times higher

than that of its uncoated counterpart.

As discussed previously in Section 2B, the ACO-03 gene expression levels would

naturally decrease in the later stage of post-harvest ripening, resulting in the reduced

production of ACO enzymes, and eventually, the decrease in the autocatalytic

biosynthesis of ethylene. Usually, at this stage, oxygen can act as a secondary

substrate for the remaining ACO enzymes to keep producing ethylene. Although,

ethylene synthesis would not be as fast as the first days of post-harvest ripening.

However, since chitosan coating inhibits the pulp's interaction with the external
 16

atmosphere (Taiz and Zeiger, 2002; Ali et al., 2011; Xing et al., 2016) and that the

ACO enzymes need O2 as a substrate to convert ACC precursors into ethylene

(Kende, 1993), not enough oxygen is obtained by the ACO enzymes in the pulp of

chitosan-coated Cavendish bananas to produce more ethylene (Tezotto-Uliana et

al., 2014). This results in a much lower biosynthesis of ethylene, indicated by the

low level of ACO-03 gene expression in Figure 5, where ACO-03 gene expression

levels of Chitosan-coated pulp samples decreased by 2.57 folds in Day-7 compared

to its uncoated counterpart. This supports the hypothesis that chitosan coating

delays ripening in the pulp of Cavendish bananas, as the decrease in gene expression

indicates a delay in banana ripening (Dwivany, 2011).

Overall, it can be concluded that chitosan coating causes the relative ACO-03 gene

expression levels of Cavendish banana pulps to increase drastically at the beginning

of post-harvest ripening. At the same time, it decreases the gene expression levels

in the later stage of post-harvest ripening. This trend can be observed in Figure 5,

where the relative ACO-03 gene expression level of uncoated Cavendish banana

pulp only decreases by 6 folds between Day-1 and Day-7, compared to the

decreases of 36 folds in chitosan-coated samples.

2. Peel

Unlike Cavendish banana pulps, where the autocatalytic biosynthesis of ethylene

gradually decreases, the peel of Cavendish bananas experiences an increase in

autocatalytic ethylene biosynthesis over the post-harvest ripening period (Patty,

2016; Liu et al., 1999) typically. However, in chitosan-coated Cavendish banana

peels, the relative ACO-03 gene expression level at the beginning of post-harvest

ripening is 5 folds higher than that of its uncoated counterpart, as observed in Figure
 17

5. Such change in gene expression levels indicates the acceleration of banana peel

yellowing in the earlier stage of post-harvest ripening. This trend can be explained

by how chitosan coating inhibits the peel from interacting with its external

atmosphere, causing the ethylene produced through biosynthesis to be trapped.

However, Figure 5 also shows that chitosan coating does not significantly affect the

relative ACO-03 gene expression level in the later stage of ripening, as the gene

expression level of chitosan-coated Cavendish banana peel in Day-7 resulted in only

1.15 folds less than its uncoated counterpart. Additionally, the SD error bars for the

peel results overlap, indicating that the difference is not statistically significant.

Martino et al. (2007) also found this trend when 1-MCP was used to delay the

ripening of Musa sp., only to find that the peel of bananas used was not significantly

affected by the 1-MCP treatment.

In conclusion, the period in which the banana peel is yellow could be extended

using chitosan coating, because the phase in which it is green decreases, as it ripens

faster in the earlier stage of post-harvest ripening. This makes the bananas more

appealing even in the earlier stage of ripening. However, it does not significantly

affect the period in which it starts to develop dark spots, as Figure 5 shows that the

gene expression of chitosan-coated Cavendish banana peel in Day-7 resulted in only

1.15 folds less compared to its uncoated counterpart. These trends suggest that

chitosan coating does not significantly affect the Cavendish banana peel ripening,

but does suggest that the period in which the peel is yellow is extended.
 18

3. Conclusion

When the effect chitosan coating has on ACO-03 gene expression levels in the pulp

and peel of Cavendish bananas are compared, two conclusions can be deduced:

1. In Cavendish bananas, the pulp is more sensitive to changes caused by

chitosan coating compared to the peel, shown by how chitosan-coated

Cavendish banana pulp’s ACO-03 gene expression levels resulted in a

decreased by 2.57 folds in Day-7, compared to the peel where the

decrease was only by 1.15 folds. Martino et al. (2007) had also discussed

in their research that “pulp is more sensitive to ethylene compared to

peel, and the whole fruit.” This supports the hypothesis that chitosan

coating affects ACO-03 gene expression in the pulp, but not as

significant in the peel.

2. Despite results being more apparent in the pulp of Cavendish bananas,

ACO-03 gene expression levels in Day-7 for both pulp and peel has

decreased when compared to their uncoated counterpart, shown by how

Day-7 chitosan-coated Cavendish banana ACO-03 gene expression

levels resulted in a decrease of 2.57 folds in the pulp and 1.15 folds in

the peel. This supports the hypothesis that chitosan coating on

Cavendish bananas can delay ripening and maintain the quality of

bananas in both its pulp and peel.

 19

5: Evaluation

A. Evaluation of Methodology

The methodology used in this investigation was chosen because it allows minimal error to

occur, as each step requires the preceding step to be delivered accurately due to being change-

sensitive (Asif et al., 2000). The disadvantage of this methodology is how time-consuming it

is, as several repetition and modification of the methodology were required to carry it out

properly.

RNA isolation and amplification are required to evaluate gene expression. However, the

equipment for PCR and qPCR can only amplify DNA; thus, the conversion of target RNA into

cDNA is necessary. Amplifying cDNA by using PCR allows the optimal temperature for

annealing to be confirmed to avoid cDNA denaturation later on during qPCR (Asif et al., 2000).

This led to several repetitions of the PCR method to find the optimal temperature.

Another difficulty faced during RNA Isolation was the low purity of RNA (A260/A280 ratios

between 1,8-2,05 µg/mL), which meant that the isolation of RNA has to be repeated until

desired purity is obtained. If impure RNA were used in subsequent steps, the RNA/cDNA

sample would degrade quickly, and undesirable qPCR results (unusable melting peaks and

amplification results) would have been obtained (Asif et al., 2000).

The qPCR was repeated several times for each sample to obtain the desired melting peak, which

shows the specificity of the primer used; the expected result is for there to be one peak because

then that means it is amplifying only one gene. However, the presence of multiple peaks means

the amplification of multiple genes, which is not the desirable outcome. The multiplicity of

peaks can be caused by contamination in the procedure, or the primer is not specific enough.
 20

The solution to these challenges is to keep practicing and adjusting the methodology (including

the temperatures chosen and amount of materials used) to obtain the desired purity and for the

process to be more time-effective.

B. Evaluation of Data Analysis

The sample size was considerably small, with only three replications for each sample, and only

one representative gene of the ACO gene family was investigated. This does reduce not only

the confidence level of the study but also increases the margin of error. Smaller sample sizes

may be more affordable and feasible in the limited time available, but then research may have

to settle for less conclusive results. Additionally, the error bars for Day-7 were significantly

large, which may indicate that the data point was less reliable.

More repetition of each sample variation could be done to reduce the margin of error and

uncertainty, which would allow the SD to be closer to the true value (Cumming, 2007).

Performing a statistical test could also improve the analysis reliability, as SD error bars only

give clues for statistical significance.

Further research should include the study of fruit physiological quality parameters during

ripening to comprehensively investigate the effects of chitosan coating on Cavendish banana

ripening. Such physiological study should include visual observation of peel color change

during post-harvest ripening with a standard color chart (Li et al., 1997), pulp to peel weight

ratio, and starch-to-sugar conversion quantitative observation using total soluble sugar (TSS)

content analysis and iodine testing (Lustriane et al., 2018). Finally, analyzing patterns between

three or more ACO genes would have given more reliable results, as one is not enough to

represent an entire gene family.

 21

6: Bibliography

Asif, Mehar H., et al. “A Simple Procedure for the Isolation of High Quality RNA from

Ripening Banana Fruit.” Plant Molecular Biology Reporter, vol. 18, no. 2, 2000, pp.

109–115, doi: 10.1007/bf02824018. Accessed 6 Aug. 2019.

Barry, Cornelius S., and James J. Giovannoni. “Ethylene and Fruit Ripening.” Journal of Plant

Growth Regulation, vol. 26, no. 2, 2007, pp. 143–159, doi: 10.1007/s00344-007-9002-y.

Accessed 6 Aug. 2019.

Cordeiro, M.C.R., Silva, M.S., de Oliveira-Filho, E.C., de Miranda, Z.J., de Góis Aquino, F.,

de Rocha Fragoso, R., Almeida, J., and de Andrade, L.R.M. “Optimization of a method

of total RNA extraction from Brazilian native plants rich in polyphenols and

polysaccharides”, IX Simposio Nacional Cerrado, 12–17 October 2008, ParlaMundi,

Brazil, pp. 3–4. Accessed 6 Aug. 2019.

Cumming, Geoff et al. “Error bars in experimental biology.” The Journal of cell biology vol.

177, 1 (2007): 7-11, pp. 2–3, doi:10.1083/jcb.200611141. Accessed 4 Aug. 2019.

Dorak, Tevfik M. (Ed.). “Relative Quantification & Normalization.” Real-Time PCR, 1st,

Taylor & Francis, 2009, pp. 82–94. Accessed 12 Aug. 2019.

Dwivany, Fenny Martha. Molecular Genetics Study of Fruit Ripening. Penerbit ITB, 2011, pp.

24–34. Accessed 6 Aug. 2019.

Edwards, Don. “Banana Ripening Chart.” Fruit English - UC Postharvest Technology Center,

UC Davis, 2002,
 22

postharvest.ucdavis.edu/Commodity_Resources/Fact_Sheets/Datastores/Fruit_English/

?uid=9&ds=798. Accessed 6 Aug. 2019.

Food and Agriculture Organization (FAO). “Banana Facts and Figures.” Banana Facts, 2016,

www.fao.org/economic/est/est-

%20commodities/bananas/bananafacts/en/#.XmRWeKgzZKN. Accessed 6 Aug. 2019.

Food and Agriculture Organization (FAO). Banana Statistical Compendium 2015-2016. Food

and Agriculture Organization of the United Nations, 2017, pp. 5, www.fao.org/3/a-

i7409e.pdf. Accessed 6 Aug. 2019.

Food and Drug Administration (FDA). Freedom of Information Summary: Original Request

for Addition to the Index of Legally Marketed Unapproved New Animal Drugs for Minor

Species. Food and Drug Administration, pp. 4–5

https://www.fda.gov/media/83661/download. Accessed 6 Aug. 2019.

Jiang, Yueming, Joyce, D.C., and Macnish, A.J. “Extension of the Shelf Life of Banana Fruit

by 1-Methylcyclopropene in Combination with Polyethylene Bags.” Postharvest Biology

and Technology, vol. 16, no. 2, 1999a, pp. 187–193, doi: 10.1016/s0925-5214(99)00009-

5. Accessed 6 Aug. 2019.

Jiang, Yueming, Joyce, D.C., and Macnish, A.J. “Responses of banana fruit to treatment with

1-methylcyclopropene”. Plant Growth Regulation 28, pp. 77–82 (1999b).

https://doi.org/10.1023/A:1006222631666. Accessed 6 Aug. 2019.

Jianglian, Duan and Z. Shaoying. “Application of Chitosan Based Coating in Fruit and

Vegetable Preservation: A Review.” Journal of Food Processing & Technology, vol. 04,

no. 05, 15 Apr. 2013, pp. 1, doi:10.4172/2157-7110.1000227. Accessed 6 Aug. 2019.

 23

Kader, Adel A., Zagory, D., Kerbel, E.L. “Modified Atmosphere Packaging of Fruits and

Vegetables.” Critical Reviews in Food Science and Nutrition, vol. 28, no. 1, 1989, pp. 1–

30, doi: 10.1080/10408398909527490. Accessed 6 Aug. 2019.

Kende H. 1993. Ethylene Biosynthesis, Annual Review Plant Physiology & Plant. Molecular

Biology 44:283-307, pp. 299–300. Accessed 6 Aug. 2019.

Kudachikar, V. B., Kulkarni, S.G., and Prakash, M.N. “Effect of Modified Atmosphere

Packaging on Quality and Shelf Life of ‘Robusta’ Banana (Musa Sp.) Stored at Low

Temperature.” Journal of Food Science and Technology, vol. 48, no. 3, 2011, pp. 319–

324, doi: 10.1007/s13197-011-0238-y. Accessed 6 Aug. 2019.

Liu, Xuejun, et al. “Characterization of Ethylene Biosynthesis Associated with Ripening in

Banana Fruit.” Plant Physiology, vol. 121, no. 4, 1999, pp. 1257–1265,

doi:10.1104/pp.121.4.1257. Accessed 6 Aug. 2019.

Livak, Kenneth J., and Thomas D. Schmittgen. “Analysis of Relative Gene Expression Data

Using Real-Time Quantitative PCR and the 2−ΔΔCT Method.” Methods, vol. 25, no. 4,

2001, pp. 402–408, doi:10.1006/meth.2001.1262. Accessed 6 Aug. 2019.

Luo, Yangchao and Q. Wang. (2013). “Recent Advances of Chitosan and Its Derivatives for

Novel Applications in Food Science”. Journal of Food Processing & Beverages. 1. 13,

pp. 1. Accessed 6 Aug. 2019.

Lustriane, C., Dwivany, F.M., Suendo, V., and Reza, M. "Effect of chitosan and chitosan-

nanoparticles on post-harvest quality of banana fruits." Journal of Plant

Biotechnology 45.1 (2018), pp. 39–44, Science Central. Accessed 6 Aug. 2019.
 24

Malmiri, Hoda Jafarizadeh, et al. “Effects Of Edible Surface Coatings (Sodium Carboxymethyl

Cellulose, Sodium Caseinate And Glycerol) On Storage Quality Of Berangan Banana

(Musa Sapientum Cv. Berangan) Using Response Surface Methodology.” Journal of

Food Processing and Preservation, vol. 36, no. 3, 2011, pp. 989–997,

doi:10.1111/j.1745-4549.2011.00583.x. Accessed 6 Aug. 2019.

Martino, Giovanni de, et al. “Preliminary Investigation into the Uneven Ripening of Banana

(Musa sp.) Peel.” New Zealand Journal of Crop and Horticultural Science, vol. 35, no.

2, 2007, pp. 193–199, doi:10.1080/01140670709510185. Accessed 6 Aug. 2019.

Nelson, S.C., Ploetz, R.C., and Kepler, A.K. (2006): “Musa species (bananas and plantains)”,

ver. 2.2, 1-32, from Elevitch, C.R., ed., Species profiles for Pacific Island Agroforestry,

Permanent Agriculture Resources (PAR), Hōlualoa-Hawaii, pp. 2–4. Accessed 6 Aug.

2019.

Orchard, J. E. “Post-Harvest Qualities at Harvest.” Routine Post-Harvest Screening of

Banana/Plantain Hybrids Criteria and Methods, by B. K. Dadzie, IPGRI, 1997, pp. 7–

13. Accessed 7 Aug. 2019

Patty, K.L. “Isolasi, karakterisasi, dan analisis ekspresi gen MaACS1 dan MaACO1 selama

proses pematangan buah pada pisang tongkat langit (Musa troglodytarum) menggunakan

RT-PCR”. 2016, pp. 55–61. Tesis Program Magister Bioteknologi, Institut Teknologi

Bandung. Accessed 6 Aug. 2019.

Pillay, M. and Tripathi, L. “Fruits and Nuts.” Genome Mapping and Molecular Breeding in

Plants, by Chittaranjan Kole, Volume 4, Springer-Verlag, 2007, pp. 281–301. Accessed

6 Aug. 2019.
 25

Pratiwi, A.S., Dwivany, F.M., Larasati, D., Islamia, H.C., and Martien, R. “Effect of Chitosan

Coating and Bamboo FSC (Fruit Storage Chamber) to Expand Banana Shelf Life.” AIP

Conference Proceedings, vol. 1677, no. 1, 30 Sept. 2015, pp. 12–15,

doi:10.1063/1.4930763. Accessed 6 Aug. 2019.

Pro Ecuador. “Sector Analysis: Banano.” Avenida Francisco De Orellana, 2016, pp. 3–6,

www.bananalink.org.uk/wp-content/uploads/2019/04/PROEC_AS2016_BANANOI-

1.pdf. Accessed 6 Aug. 2019.

Sahib, Warraich. “Chitosan Chemical Structural Formula.” Wikimedia Commons, 25 May

2014, commons.wikimedia.org/wiki/File:Chitosan_chemical_structural_formula.svg.

Accessed 6 Aug. 2019.

Setyadjit, A., Dimyati, Lokollo, E.M., Kuntarsih, S., Basuki, R.S., Hidayat, A., Hofman, P.J.,

Ledger, S.N., and Woods, E.J. “Analysis of the Constraints to Banana Industry

Development in Indonesia Using the Supply Chain Concept.” (2004). ACIAR

Proceedings No. 119e, Bali, Indonesia, Johnson, G.I. and Hofman, P.J., ed., Australian

Centre for International Agricultural Research, pp. 59–68. Accessed 6 Aug. 2019.

Taiz, Lincoln, and Eduardo Zeiger. Plant Physiology. 3rd ed., W.H. Freeman, 2002, pp. 145–

152. Accessed 6 Aug. 2019.

Tezotto-Uliana, Jaqueline Visioni, et al. “Chitosan Applications Pre- or Postharvest Prolong

Raspberry Shelf-Life Quality.” Postharvest Biology and Technology, vol. 91, 31 Dec.

2014, pp. 72–77., doi:10.1016/j.postharvbio.2013.12.023. Accessed 6 Aug. 2019.

Xing, Y., Xu, Q., Li, X., Chen, C., Ma, L., Li, S., Che, Z., and Lin, H. “Chitosan-Based Coating

with Antimicrobial Agents: Preparation, Property, Mechanism, and Application

 26

Effectiveness on Fruits and Vegetables.” International Journal of Polymer Science, vol.

2016, 2016, pp. 1–24., doi:10.1155/2016/4851730. Accessed 6 Aug. 2019.

Xu, Juan, and Shuqun Zhang. “Ethylene Biosynthesis and Regulation in Plants.” Ethylene in

Plants, 2015, pp. 1–25., doi: 10.1007/978-94-017-9484-8_1. Accessed 6 Aug. 2019.

Yamamoto, K. (2017b): A multi-omics approach to investigate the effect of chitosan coating

on post-harvest quality of banana (Musa acuminata), pp. 20–24, Tesis Program

Magister, Institut Teknologi Bandung. Accessed 6 Aug. 2019.

Yamamoto, K., Amalia, A., Putri, S.P., Fukusaki, E., Dwivany, F.M. “Expression Analysis of

1-Aminocyclopropane-1-Carboxylic Acid Oxidase Genes in Chitosan-Coated Banana.”

Expression Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Genes in

Chitosan-Coated Banana, vol. 25, no. 1, 1 Jan. 2018a, pp. 12–20,

doi:10.4308/hjb.25.1.18. Accessed 6 Aug. 2019.

Yang, S F, and N E Hoffman. “Ethylene Biosynthesis and Its Regulation in Higher Plants.”

Annual Review of Plant Physiology, vol. 35, no. 1, 1984, pp. 155–189,

doi:10.1146/annurev.pp.35.060184.001103. Accessed 6 Aug. 2019.

Zaskia, H. “Studi ekspresi gen MaACS1 dan MaACO1 pada pisang Cavendish yang diberi

perlakuan edible coating kitosan dan suhu.” 2016, pp. 20–23. Tesis Program Magister,

Institut Teknologi Bandung. Accessed 6 Aug. 2019.

 27

7: Appendices

[Appendix A] Raw Ct Value Results for ACO-03 gene and Actin as Reference Gene

[Appendix A.1] Raw Ct Value Results for Actin (Reference Gene)

Raw Ct Value Results for Actin

Sample qPCR Sample Replication Standard
Average
I II III Deviation
1UF Day-1 Uncoated 16.15 17.06 17.17 16.61 0.64
Pulp 7UF Day-7 Uncoated 16.4 16.51 16.26 16.46 0.08
(Flesh, F) 1CF Day-1 Coated 16.51 16.26 17.19 16.39 0.18
7CF Day-7 Coated 15.383 15.739 15.495 15.54 0.18
1US Day-1 Uncoated 14.35 14.56 14.8 14.57 0.23
Peel 7US Day-7 Uncoated 16 16.04 15.75 15.93 0.16
(Skin, S) 1CS Day-1 Coated 15.46 16.22 16.33 16.00 0.47
7CS Day-7 Coated 16.44 15.91 16.23 16.19 0.27

[Appendix A.2] Raw Ct Value Results for ACO-03 (Target Gene)

Raw Ct Value Results for ACO-03

Sample qPCR Sample Replication Standard
Average
I II III Deviation
1UF Day-1 Uncoated 22.09 21.8 21.82 21.95 0.21
Pulp 7UF Day-7 Uncoated 25.73 23.04 24.4 24.39 1.90
(Flesh, F) 1CF Day-1 Coated 20.19 20.36 20.84 20.46 0.34
7CF Day-7 Coated 24.51 24.55 24.46 24.51 0.05
1US Day-1 Uncoated 23.42 23.38 23.87 23.56 0.27
Peel 7US Day-7 Uncoated 19.82 20.08 20.4 20.10 0.29
(Skin, S) 1CS Day-1 Coated 22.1 22.57 23.38 22.68 0.65
7CS Day-7 Coated 20.19 20.39 21.31 20.63 0.60
 28

[Appendix B] Calculating Relative Multiples of Gene Expression (with Normalization)

[Appendix B.1] Calculating Average ΔCt for Cavendish Banana Samples (with Normalization)

ΔCt
Sample Average ΔCt
I II III
1UF Day-1 Uncoated 5.94 4.74 4.65
Pulp 7UF Day-7 Uncoated 9.33 6.53 8.14
(Flesh, F) 1CF Day-1 Coated 3.68 4.10 3.65
7CF Day-7 Coated 9.13 8.81 8.97
5.11
1US Day-1 Uncoated 9.07 8.82 9.07
Peel 7US Day-7 Uncoated 3.82 4.04 4.65
(Skin, S) 1CS Day-1 Coated 6.64 6.35 7.05
7CS Day-7 Coated 3.75 4.48 5.08

[Appendix B.2] Calculating Average 2-(ΔΔCt) and Standard Deviation 2-(ΔΔCt)

ΔΔCt 2-(ΔΔCt) Average

Standard
Sample Deviation
I II III I II III 2-(ΔΔCt)
2-(ΔΔCt)
Day-1
1UF 0.83 -0.37 -0.46 0.56 1.29 1.38 1.08 0.45
Uncoated
Day-7
7UF 4.22 1.42 3.03 0.05 0.37 0.12 0.18 0.17
Pulp Uncoated
(Flesh, F) Day-1
1CF -1.43 -1.01 -1.46 2.69 2.01 2.75 2.49 0.41
Coated
Day-7
7CF 4.02 3.70 3.86 0.06 0.08 0.07 0.07 0.01
Coated
Day-1
1US 3.96 3.71 3.96 0.06 0.08 0.06 0.07 0.01
Uncoated
Day-7
7US -1.29 -1.07 -0.46 2.45 2.10 1.38 1.97 0.55
Peel Uncoated
(Skin, S) Day-1
1CS 1.53 1.24 1.94 0.35 0.42 0.26 0.34 0.08
Coated
Day-7
7CS -1.36 -0.63 -0.03 2.57 1.55 1.02 1.71 0.79
Coated

[Appendix B.3] Results for Relative Multiples of Gene Expression and Standard Deviation

Relative Multiples of Gene Expression Standard Deviation

Sample
Uncoated Coated Uncoated Coated
Pulp Day-1 1.08 2.49 0.45 0.41
(Flesh, F) Day-7 0.18 0.07 0.17 0.01
Peel Day-1 0.07 0.34 0.01 0.08
(Skin, S) Day-7 1.97 1.71 0.55 0.79