IN VITRO REGENERATION AND ANTIMICROBIAL ANALYSIS OF

DIOSCOREA ALATA L.

MUHAMMAD FIRDAUS BIN HANAPI

SET130009

INSTITUTE OF BIOLOGICAL SCIENCE

FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2015/2016

i
IN VITRO REGENERATION AND ANTIMICROBIAL ANALYSIS OF
DIOSCOREA ALATA L.

MUHAMMAD FIRDAUS BIN HANAPI

SET130009

THIS THESIS WAS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE BACHELOR OF SCIENCE
DEGREE

INSTITUTE OF BIOLOGICAL SCIENCE

FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2015/2016

ii
 UNIVERSITY OF MALAYA
ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: MUHAMMAD FIRDAUS BIN HANAPI

(I.C/Passport No) : 94085-03-6001
Registration/Matric No: SET130009
Name of Degree: BACHELOR OF SCIENCE (BSc.)
Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):
IN VITRO REGENERATION AND ANTIMICROBIAL ANALYSIS OF
DIOSCOREA ALAT L.
Field of Study: BIOTECHNOLOGY
I do solemnly and sincerely declare that:
(1) I am the sole author/writer of this Work;
(2) This Work is original;
(3) Any use of any work in which copyright exists was done by way of fair dealing
and for permitted purposes and any excerpt or extract from, or reference to or
reproduction of any copyright work has been disclosed expressly and sufficiently
and the title of the Work and its authorship have been acknowledged in this
Work;
(4) I do not have any actual knowledge nor do I ought reasonably to know that the
making of this work constitutes an infringement of any copyright work;
(5) I hereby assign all and every rights in the copyright to this Work to the
University of Malaya (“UM”), who henceforth shall be owner of the copyright in
this Work and that any reproduction or use in any form or by any means
whatsoever is prohibited without the written consent of UM having been first had
and obtained;
(6) I am fully aware that if in the course of making this Work I have infringed any
copyright whether intentionally or otherwise, I may be subject to legal action or
any other action as may be determined by UM.

Candidate’s Signature Date:

Subscribed and solemnly declared before,

Witness’s Signature Date:

Name:
Designation:
iii
 ABSTRACT

Dioscorea species known as water yam is widely grown worldwide and considered as

high value crop. Several common uses Dioscorea species are as staple food,

pharmaceutical usage as synthesis of sex hormone, steroid production and nutritional usage

as source of carbohydrate, dietary fiber, reducing obesity and diabetes. In this experiment,

mass propagation of Dioscorea alata is focused. As Dioscorea alata is commonly stay

dormant for a period of time, the present investigation is to grow the plantlet of Dioscorea

alata in vitro using Murashige and Skoog (MS) medium supplemented with multiple

phytohormones. The explant part used in this experiment is petiole, leaf and stem segment.

Five hormones were utilized which consist of Kinetin (KN), 2,4-Dichlorophenoxyacetic

acid (2,4-D), alpha-Naphthalene acetic acid (NAA), Indole-3-acetic acid (IAA) and 6-

Benzylaminopurin (BAP). Best result of shoot proliferation was observed in MS medium

containing 2.0 BAP with highest rate of shoot multiplication at percentage of 70 ± 0.10 a.

The best root regeneration was observed on MS medium supplemented with 1.0 mg/L IAA

with percentage of 15±0.08a. Antimicrobial analysis was conducted on stem, tuber and leaf

of Dioscorea alata of different bacteria and fungi. Inhibition zone can be seen on Bacillus

Aureus, Escheria Coli, Bacillus Subtilis, Fusarium sp. and Penicilium sp.

Keyword: Dioscorea alata, in vitro culture, Inhibition zone

i
 ABSTRAK

Spesis Dioscorea atau juga dikenali sebagai ‘ubi badak’ ditanam meluas di seluruh

dunia dan dianggap sebagai tanaman yang mempunya nilai yang tinggi. Antara contoh

kegunaan spesis Dioscorea ialah sebagai makanan ruji, bidang farmaseutikal seperti

penghasilan hormon seks, penghasilan steroid dan kegunaan nutrisi sebagai sumber

karbohidrat, serat diet, mengurangkan kadar obesiti dan diabetes. Di dalam eksperimen ini,

pembiakan Dioscorea alata secara in vitro telah difokuskan. Dioscorea alata biasanya

kekal dalam tidak aktif buat tempoh masa tertentu. Kajian ini ingin mengfokuskan untuk

menumbuhkan bahagian anak pokok Diosocrea alata secara in vitro dengan menggunakan

medium Murashige and Skoog (MS) dibekalkan dengan beberapa jenis hormon. Eksplan

yang digunakan dalam eksperimen ini adalah petiole, daun dan batang. 5 jenis hormone

telah digunakan iaitu Kinetin (KN), 2,4-Dichlorophenoxyacetic acid (2,4-D), alpha-

Naphthaleneacetic acid (NAA), Indole-3-acetic acid (IAA) dan 6-Benzylaminopurine

(BAP). Hasil pertumbuhan pucuk yang terbaik telah dapat diperhatikan pada media MS

bersama 2.0 BAP mg/L dengan kadar pertumbuhan sebanyak 70 ± 0.10a peratus. Hasil

pertumbuhan akar yang terbaik telah dapat diperhatikan pada media MS bersama 1.0 IAA

dengan kadar pertumbuhan sebanyak 15±0.08a peratus. Analisis antimikrob telah

dijalankan pada batang, daun dan ubi Dioscorea alata menggunakan bakteria dan kulat.

Zon penyekatan dapat dilihat pada Bacillus Aureus, Escheria Coli, Bacillus Subtilis,

Fusarium sp. and Penicilum sp.

Kata kunci: Dioscorea alata , kultur in vitro, zon penyekatan.

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 ACKNOWLEDGEMENTS

I would like to show my appreciation to my beloved supervisor, Prof Dr Rosna binti

Mat Taha for her support, advise and encouragement in my final year project for this two

semesters.

I would also like to thank the postgraduate students under Prof Dr Rosna , Mr . Asif

Khan, Miss Sakinah, Miss Aisyah and Miss Lydia for their guidance in the tissue culture

process and tips and suggestion in carrying out my final year project. To the lab assistant,

Pn Nurul Ain for proving me the integral item for culturing. Not to forget, my lab partner,

Farhan Faiz who has given me a tremendous effort in order to help me finishing this

studies.

To my friends and family, thank you from the bottom of my heart for encouraging me

and for a constant companionship in my final year project.

Thank you very much.

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 TABLE OF CONTENT

Title Pages

ABSTRACT......................................................................................................................i

ABSTRAK........................................................................................................................ii

ACKNOWLEDGEMENTS...........................................................................................iii

LIST OF SYMBOLS AND ABBREVIATION..........................................................viii

LIST OF FIGURES.........................................................................................................x

LIST OF TABLES........................................................................................................xii

1.0 INTRODUCTION.....................................................................................................1

1.1 General Introduction.............................................................................................2

1.2 Overview Of Dioscorea Alata.............................................................................3

1.3 Taxonomic Classification Of Dioscorea Alata..................................................7

1.4 Growing Season..................................................................................................7

1.5 Plant Tissue Culture...........................................................................................8

1.5.1 History And Development Of Plant Tissue Culture.................................9

1.5.2 Laboratory Facilities And Equipment....................................................13

1.5.3 Sterilization Method...............................................................................15

1.5.4 Aseptic Technique In Sterilization Of Materials....................................17

1.5.5 Media Preparation..................................................................................18

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 1.5.6 Ms Media Preparation............................................................................23

1.6 Objectives……................................................................................................24

2.0 LITERATURE REVIEW......................................................................................25

2.2 Disadvantages Of Plant Tissue Culture..............................................................27

2.3 Plant Regeneration Pathways............................................................................28

2.4 Economic And Social Importance Of DIOSCOREA ALATA.........................31

2.4.1 Health Benefits........................................................................................34

2.5 Dioscorea alata Harvesting...............................................................................35

2.6 Limitation In Dioscorea alata Production........................................................39

2.6.1 Disease In Dioscorea alata.....................................................................39

2.6.2 Post Harvest Lost....................................................................................44

3.0 MATERIALS AND METHOD..............................................................................46

3.1 List Of Material And Apparatus.........................................................................47

3.2 Laboratory Facilities...........................................................................................51

3.2.1 Media Preparation Area............................................................................52

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 3.2.2 Transference Area...................................................................................53

3.3 Laminar Flow Chamber Regulation.................................................................54

3.4 Stock Solution Preparation...............................................................................55

3.5 Transferrring Media Into Sterile Tube.............................................................56

3.6 Sterilization Method.........................................................................................57

3.6.1 Apparatus And Media Sterilization.........................................................57

3.6.2 Subculturing Of Explant..........................................................................57

3.6.3 Explant Sterilization.................................................................................58

3.6.4 Preparation For Cutting The Explant.......................................................59

3.6.5 Washing The Explant................................................................................59

3.7 Media Preparation.............................................................................................60

3.8 Antimicrobial Test..............................................................................................61

3.9 Statistical Analysis………………………………………………………………58

4.0 RESULTS.................................................................................................................64

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 4.1 Percentages Of Shoot Formation......................................................................65

4.2 Percentages Of Root Formation.....................................................................66

4.3 Antimicrobial Test.........................................................................................81

4.3.1 Bacteria.................................................................................................81

4.3.2 Fungi….................................................................................................81

5.0 DISCUSSION...........................................................................................................88

6.0 CONCLUSION........................................................................................................98

7.0 REFERENCE........................................................................................................102

8.0 APPENDICES........................................................................................................111

vii
LIST OF SYMBOLS AND ABBREVIATION

MS Murashige and Skoog (1962)

PGR Plant Growth Regulators

KN Kinetin

2,4-D Dichlorophenoxyacetic acid

NAA Naphtalaneacetic acid

IAA Indole-3-acetic acid

BAP 6-Benzlaminopurine

2-iP 6-(gamma,gamma-dimethylallylamino) purine

GA3 Gibberelin

IBA Indole-3-butyric acid

EDTA Ethylenediaminetetra-acetic acid

NaOH Sodium Hydroxide

HCL Hydrocloric acid

HgCL2 Mercury chloride

H2O Water

NaCIO Sodium hypocloride

G Gram

viii
M Meter

Mg Milligram

L Liter

mL Milliliter

% Percentage

°C Degree Celcius

v/v Volume per volume

w/v Weight per volume

Tween20 Polyoxyethylene sorbitan monolaurate

ix
LIST OF FIGURES

Figure 1.1: Intact plant of Dioscorea alata grown at University of Malaya green house.

Figure 1.2: Tuber Dioscorea alata collected from Tumpat, Kelantan.

Figure 1.3: Amount of element in MS medium (Lindsey, 2013).

Figure 2.1: Phases in indirect organogenesis phase.

Figure 2.2: Direct organogenesis pathway.

Figure 2.3.1 : Anthracnose on Dioscorea alata

Figure 2.3.2 : Dry rot disease on Dioscorea alata

Figure 2.3.3 : Mealybug on Dioscorea alata

Figure 2.3.4 : Root knot nematode

Figure 2.3.4 : White Scale Insects on Dioscorea alata tuber .

Figure 4.1 : Graph showing percentage of shoot formation in different medium

Figure 4.2 : Graph showing percentage of root formation in different medium

FIGURE 4.7.1 : Inhibition zones for methanol extract of Dioscorea alata tuber against

bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp.

Figure 4.7.2 : Inhibition zones for methanol extract of Dioscorea alata tuber against

bacteria (a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

Figure 4.7.3 : Inhibition zones for methanol extract of Dioscorea alata stem against

bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp.

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Figure 4.7.4 :Inhibition zones for methanol extract of Dioscorea alata stem against bacteria

(a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

FIGURE 4.7.5 : Inhibition zones for methanol extract of Dioscorea alata leaf against

bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp

FIGURE 4.7.6 : Inhibition zones for methanol extract of Dioscorea alata leaf against

bacteria (a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

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 LIST OF TABLES

Table 3.1 : List of material and apparatus

Table 4.1: Percentage of shoot formations from the explant when supplemented with

different types and concentrations of hormone in MS medium .

Table 4.2 : Percentage of root formations from the explant when supplemented with

different types and concentrations of hormone in MS medium .

Table 4.2.1 : Observation of the effect of plant growth regulators on leaves of Dioscorea

alata

Table 4.2.2 : Observation of the effect of plant growth regulators on petioles of Dioscorea

alata

Table 4.3 : Antibacteria test on different Dioscorea alata parts .

Table 4.4 : Antifungal test on different Dioscorea alata parts .

TABLE 4.5 : Diagrams of shoot formation from stem segments of Dioscorea alata

TABLE 4.6 : Diagrams of root formation from stem segments of Dioscorea alata

xii
 1.0
INTRODUCTION

1
1.1 GENERAL INTRODUCTION

Plants has lived and evolved in the earth since pre-historic period. As same as other

organisms, it also having evolution that changes many aspects of the plant itself so it can

survive the changes especially in environmental surrounding condition.

Right now, there are more than 280,000 species of plants are living in the world.

Even though some of the species such as seaweed that return to its original habitat during

the stages of evolution, most of the other species lived in various environment such as

desert, grassland and jungle. Currently, all of these plants are considered as land plant even

though some of them still referred as the land plant, even if some of them are aquatic plant.

This is to make the taxonomy and classification of other group such as Protista is easier to

differentiate.

There are four group of plants consist of Angiosperm, Gymnosperm,Pteridophyte and

Bryophytes . Most of Bryophytes are consist of moss, for Pteridophytes, it consists of fern.

Pine and Conifer are on Gymnosperm group and most of Angiosperm are come from

flowering plant. Flowering plant are considered as one of the most diverse group in the land

plants. The studies of these four group give us wider perspective so that more

understanding can be obtain about the general history and knowledge, because different

group come from different intervals time.

2
1.2 OVERVIEW OF DIOSCOREA ALATA

Dioscorea alata are yearky tuber-bearing and climbing plants that are classified

under the family of Dioscoreaceae. Besides Dioscorea alata, it also known as

D.atropurpurea Roxb., Greater yam, white yam and D.sativa Del. It often known as Water

Yam and in Malaysia, it is called as Ubi Badak. Some cultivars of Dioscorea are native

from Africa before scattering out to other region of the world while some are said to be

originated from Asia and have spread to Africa (Hahn et al., 1987). Dioscorea alata is

very famous because of its consumption as the main source of food and become the staples

food in lot of countries where it contributes to 200 dietary calories the consumer with

various source of energy and nutrients.

Morphology of Dioscorea alata is very prominent and easy to be recognized. From

the vine that grown from the tube, the vine itself can either be uniform or spiky. The length

of the vine itself can grow up to 10m (30ft) or more, but this depend on the varieties of the

yam. It is freely branching above. For the leaves of the plant, the colour can be either green

or purple or even intermediate between this two colour. For Dioscorea alata L, the colour

of the leaves are green while for Dioscorea alata L. var. purpurea, the leaf is purple in

colour. So the leaf colour are basically will depend on the variation of the plants itself. The

shape of the leaf can either be oval or heart shape, with long petioles, the opposite side are

often consistent with one leaf, the blade length up to 20cm (8 inches) or longer, also come

with basal lobes that usually angular. A few of them contain pikes located at the bases of

the leaf. The plant itself can produce a singular tuber or even more than one tuber that

elongated from stolon from a central corm based on the species itself.

3
Figure 1.1: Intact plant of Dioscorea alata grown at University of Malaya green house.

Figure 1.2: Tuber Dioscorea alata collected from Tumpat, Kelantan.

4
 Nowadays, Dioscorea alata itself are grown extensively in many different

regions such as Asia, Africa and Europe. Biological diversity of yam is very wide,

currently more than 600 species are known in the world (Burkill, 1960; Coursey, 1967). In

Africa, there are six species of yam are being grown in Africa, mainly based in West and

Central region including Dioscorea alata. Tetteh and Saakwa (1994) stated that some wild

types of yam are still present and consume as food source after it experiencing processing

during the season of hunger. The main species of yam is used for medicinal purposes as

Dioscorea alata itself are known as traditional herbal medicine (Farombi et al., 1997;

Albrecht and McCarthy, 2006).

In any plant, the environmental condition always plays important role in supporting

the growth and development of the plant, and Dioscorea alata is no exception. Dioscorea

alata is grown in the region of tropical climates and it cannot stand cold and frosty

condition (Coursey, 1967). It has been reported that if the environment temperature is -20

°C or lower, the growth of the plant is inhibited. The ideal temperature for the growth of

Dioscorea alata should be between 25°C and 30°C which is consider as the optimum

temperature. Besides that, the light intensity is the other factor that will affect the growth

and formation of tuber. To induce tuber formation, the short days which is between 10-11

hour is consider as ideal while for vine growth, the days should be longer than 12 hours. To

promote the growth of yield, Dioscorea alata vines are stacked so that the light is being

block by the leaves from the vine (Coursey, 1982; Okezie, 1987). The ideal annual rainfall

for Dioscorea alata cultivation is around 1000 mm in the period of five to six months. The

soil characteristic that are deep, fertile, easy to crumble and well drained is also considered

as ideal for Dioscorea alata cultivation (IITA, 2009). The best yield is reported in areas

where the rainy season is long and continuous. Usually, Dioscorea alata are vegetatively
5
progated from the whole tuber, or big size tuber pieces known as setts, and also minisetts

(Otoo et al., 1985). Most of the Dioscorea alata growth are initiated with a sprout from the

tuber that already past it dormant phase (Passam, 1977; Onwueme, 1984). The pattern of

growth showed by the Dioscorea alata are sigmoidal growth pattern which are no longer

strange to annual plant (Craufurd et al. ,2001; Sobulo ,1972). The pattern that a show is

showing a slow growth period continued by rapid exponential growth and finally the

decline phase marks the senesces of canopy. Maturity is very important trait in plant

production as it is one of the quality indices for the production to the consumer. Usually,

the dryness of vines is the one the main point to measure its maturity, but overall, it cannot

be well defined (Okoli, 1980). Besides that, the post-harvest factor such as storage period

and food quality trait are often affected by the physiological condition of Dioscorea alata

tuber (Osagie and Opute ,1981).

Tuber is the most important part in Dioscorea alata as it is the part where many

people consume the tuber as the main source of food. The tuber part of Dioscorea alata is

different in size and species. This will depend on factors such as cultivars and growing

conditions. Usually, the tuber come in shape as cylindrical, sometimes almost resemble

foot of human itself, with present of some root ‘hairs’. Some of the consumer might suffer

some allergic reaction when Dioscorea alata are eaten without being cook or even touch

directly with the skin. This is due to present of tannin cells and also cell containing bundle

of crystal (raphids). The outer layer of tuber is brown in colour and act as protection from

many pathogens attack, water loss and lesion. Inside the inner part of the tuber, it is

consisting of parenchyma tissue. Carbohydrate in form or starch is formed inside the tissue

which is mainly why it is eaten by the consumer as staple food. Asiedu (1986) reported that

6
the different similarity in growing environment, storage method, stage of maturation and

type of species is the main factor that affecting the factor of tuber variation.

1.3 TAXONOMIC CLASSIFICATION OF DIOSCOREA ALATA

Scientific classification Dioscorea alata.

Kingdom : Plantae

Phylum : Angiosperms

Class : Monocots

Order: Dioscoreales

Family: Dioscoreaceae

Genus: Dioscorea

Species: D. alata

1.4 GROWING SEASON

The growing season of Dioscorea alata depend on its geographical location. Mostly of

the Dioscorea alata usually growing in East Coast part of Malaysia especially in Kelantan

and Terengganu. The timing of growing is usually during Monsoon season between

November and December. All around the world, D.alata is commonly grow at best in with

7
partial sun, semi shade and fertile , crumbly soil but at the same time , they grow dormant

after rainy season for a period of two to three month .

Most of the yams are annual plant, mean that they are only grow ones in year, harvest

only one season. Some of the perennial tubers are showing size that keep on growing but

with the vines dying during the last period of growing season but under optimum condition,

it will regrow.

However, Discorea plant are getting destroyed from natured due to over-usage by

mankind. Dioscorea alata are normally grow for 8-10 months, after that it will goes

dormant for 3-4 months (Martin and Rhodes 1977). It also grown commercially in Japan,

and said to survive the cold winter season if planted deep enough.

1.5 PLANT TISSUE CULTURE

Tissue culture is defined as the conservation or growth of tissue in vitro that permits

the differentiation and maintenance of the structure and function (Tones, 1989). The

purpose of the tissue culture to use to cultivate certain parts of the plant (cells, organs,

tissue) under controlled environment that is clean and free from pathogen inside the proper

synthetic medium in vitro. Nowadays, the latest interest in the usage of plant tissue culture

techniques is to be applied in various range of research problems and the main aims is to be

applied in the agricultural and horticultural field for the rapid plant production, pathogen-

free plants production, germplasm storage and also for various usage in industrial scale

such as synthesis of pharmaceutically-useful metabolites by commercial scale. It also used

in basic science knowledge in order the understand and find the proper solution in plant

physiology and others field. There a lot of scientific method that being applied and

8
developed to use in cloning of the cell, generation of the cell, screening of desirable

characteristic, and also increasing the range of genetic variability.

1.5.1 HISTORY AND DEVELOPMENT OF PLANT TISSUE CULTURE

Many of the knowledge in tissue culture is applied in science and technology

to support the growth of plant cells, tissue or organ under controlled environment in the

proper media composition that contain nutrients, hormones and vitamins. One of the

earliest work in the plant tissue culture is done by Duhumel Du Monceau, which starting

his investigation in wound healing plant, and also observing callus formation (Gautheret,

1985; Smith, 2012). After a lot of studies for a period time, the concept of Totipotency.

Totipotency is defined as ability of an organism to divide and differentiate to produce one

complete organism. In 18 January 1838, Scheiden and Schwann proposed this cell theory in

which it discussing about the characteristic of cell totipotency. They insisted that that each

cell is a free living organism and cells that differentiate had become multicellular organism

that still had the same information which contain in original single cell, which is fertilized

egg cell. Since that, it become a very integral point in history of plant biotechnology and

plant tissue culture in which it given a great attention at late 1970s. Sachs proposed that

plant can synthesis compounds that can expand into organ and it will spread in polarized

way in 1882.

From the theory that has been proposed by Scheiden and Schwann, which is the

concept of in vitro cell culture is developed by Haberland. In 1902, Haberland try to do

tissue culture for the first time ever using monocot tissue, but the attempt was failed.

Haberland attempt to culture the leaves of lamium purpureum, which is chloroplast-

9
containing differentiated cells, cells from petioles of Eichhornia crassipes, glandular hairs

of Pulmonaria and Urtica, and stamen hair of Tradencantia , in Knop’s (1865) salt solution

in hanging drop cultured . However, there is no cell division occur but only cell increasing

in size. The culture is then lost to infection (Vasil ,2008). The cause of the failure is said to

be on the medium, which is lack of nutrition is given inside the Knop’s salt solution. Since

then, Haberland is known as Father of Tissue Culture, following his contribution in this

field. First ever embryo culture had been tried at 1904 using selected Crucifer species. In

1909, Fusion of protoplast is done even the product failed to expand. Attempt had been

done by Custer and it partner, Molliard had obtain cultivation of fragment from plant

embryo, and success is done in 1921.

Many new information and knowledge is obtained from the Haberland

experiment, one of them are the rate of success is can be dependent on the media, which is

the nutritional content and hormones applied in the tissue culture process. For the past

years, there been many reports and studies on the alteration for more than 2 dozens of basic

medium composition compositions (Street and Shillito, 1977: Pierik, 1997; Torres, 1989;

Gamborg and Phillips, 2013). The growth and development of the plant is stimulated when

cultured in vitro in the suitable medium. The medium itself are composed of important

element from Micronutrients and Macronutrients such as amino acid, glucose, vitamin and

so on (George, Hall and De Klerk, 2007).

Asymbiotic seed germination from seed of orchid had been done by

Knudson in 1922. At the same year, Robbin had done in vitro culture at root tip. In 1924,

formation of callus induction from explant of carrots root is obtain by adding lactic acid

into the media. This is proven by Blumenthal and Meyer. In 1925 saw that the two

significant success in cell tissue culture segment, which is involve culture of embryo by the
10
intention of interspecific hybrid in Limus spp by Laibach, while the second success is

involve study of Knudson that successfully making symbiotic germination into the seed of

orchid.

Laibach and Hered has done intensive experiment to overcome the problem in their

previous study which involve Linum spp, in where they manage to produce embryo culture

to avoid the instability problem in the culture of that species. In 1934 saw that attempt to

culture the cambium tissue from multiple tree and plant shrubs in vitro, however no success

is obtained. Effort is done in multiple way and by many scientist, one of them is Guatheret.

However, one positive sign has been obtained by White when his tomato culture had

obtained a success because it can grow for a good period of time.

One of the most important discovery on the same year is the identification of first

plant hormone, which is, IAA that contribute to growth of cell. The same success is

attained Kogl and friend. In 1936, LaRue had done embryonic culture using variety of

Gymnosperm. The effort of Gautheret (1936) to culture the cadmium cell of carrot and

tobacco finally worth of victory when the cells growing continuously. After that, Guatheret

cultured the cambium tissue of Ulmus for experiment of adventitious shoot.

In 1941, Overbeek and friend has using coconut water for culture in intention for

growth and development of young Datura embryo. Gutheret had done an observation into

secondary metabolites in Culture of callus in 1942. Skoog (1944) successfully culture the

tobacco cell which is used earlier for the purpose of study of formation of adventitious root

in vitro. Loo (1945) obtained cultivation from cutting of cell tip of Asparagus plant. Ball

(1946) had manage to obtained regeneration of plant tissue Lupinus and Tropacolum from

shoot tip obtaining one complete plant.

11
 Formation of shoot and root in tobacco is obtained from culture of Skoog and Tsui

in 1948. Miller (1955) and friend had managed to find the structure and technique of

synthesis of Kinetin as a hormone.

Murashige and Skoog (1962) has successfully managed to formulated the MS media

which at the same time becoming one of the most important basic media in Plant Tissue

culture. At the same year, first ever haploid plant is obtained from androgenesis of Datura.

Among the first plant that had been culture after the creation of media MS is Populous

tremuloides through regeneration of shoot and root from callus of the plant. Since then, the

MS media become the most commonly used media. At first, MS media is developed for

tobacco callus ideal growth and then their development is followed by a huge number of

different responses which depend for the amount of various essential minerals.

Figure 1.3:
Amount of element in MS medium (Lindsey, 2013).

12
 In 1973, Pierik had proven that Cytokinin hormone had proven to stop the dormancy of

Gerbera plant

Murashige (1974) had manage to induce branching at the shoot tip of Gerbera using

cytokine hormone.

After 1974, Recombinant DNA technology is started to be introduced into tissue culture.

As an example, Ti Plasmid had become ‘Tumor Inducing Principle in Crown Gall ‘ .

Before this, tissue culture only for the regeneration purpose.

1.5.2 LABORATORY FACILITIES AND EQUIPMENT

It is very important to divide the laboratory facilities and equipment to a

specific area (Lindsey, 2013). The specific area such as media preparation, autoclave area

and culture room is very important to ensure aseptic condition.

In plant tissue culture operation, the required tools must reach the certain criteria that

had been standardize. Overall design must be taken care the aspect of maintaining the

aseptic condition in the surrounding environment.

Present of dust and spores at the surface of working area and in air can cause

contamination, which can lead to failure in culture. Loss can occur at small percentage until

reaching 50%, and this will lead to decrease in efficiency in culture.

Because of that, facility for tissue culture must have a good design that emphasize on

aseptic condition such as removal of dust and dirt in effective way. Air that been brought

from outside must pass through specialized air filter with high efficiency, such as HEPA

13
filter. Installation of tool that have ability to produce positive air pressure is a great

advantage, but not necessary to be compulsory.

In each tissue culture lab must contain at least one autoclave machine. It functions in

sterilization of tools and nutrient media which stabilized to heat. Usually, the media is

autoclave at 121°C and pressure of 103 kPa (15 psi 2) for 12 to 20 minutes. This depend on

volume of media inside the container.

Laminar air-flow cabinets will supply a continuous flow of air across around the

environment (Singh and Kumar, 2009). Laminar Flow is a chamber that have a motor that

moved the air through a very fine filter. This will ensure that the air move inside it is clean

and free from contamination. HEPA (High Efficiency Particulate Air) filter is function to

ensure the clean air is flow continuously at fix speed rate which is 27m/min.

The laminar flow chamber should be open 15 minutes before any work is done inside it.

This is to ensure any foreign particle is removed out and leaved clean air inside it.

One of the thing that should be consider while using laminar flow chamber is not to

put many unnecessary tools that are not required in culture process such as bottle or excess

sterile tube inside it so that the air flow is not disturbed and at the same time reducing the

risk of contamination. The culture process should be done far into the cabinet. This is to

ensure that work is done inside a clean air flow condition. Besides that, the movement must

be against air flow and not following air flow.

pH meter is also need to ensure to be at 4.0 or 7.0. This can be maintaining be dipping

the pH rod meter into the buffer solution. This is used to measure pH in the Media solution.

pH value that intended depend on species that want to be culture.

14
 In the process of making media, mixture of nutrient and others needed to be mix evenly

by using magnetic stirred.

To store tools and materials of making the media such as pipette, magnetic stirrer and

micropipette tip, the incubator tools is used to ensure all of these tools is in sterile

condition. Temperature in incubator is set to 40° C.

1.5.3 STERILIZATION METHOD

There are a few techniques that available to be applied in plant tissue culture. For

example, there are dry sterilization, steam distillation by using autoclave, Sterilization by

filtration, radiation and Chemical Sterilization.

Dry sterilization Is used incubator functioning as to sterilize glass apparatus in media

preparation. As an example of apparatus is pipette, micropipette tips and magnetic stirrer.

In Tissue culture lab, the temperature of incubator is set to 40 ° C.

Besides that, in laminar flow chamber there is a tools called microcarrier beads that used

to sterilized microtome blade and forceps during culturing process. In Principle, this tools

are dry sterilization tool because it involves heating in microcarrier beads at a very high

temperature.

In principle, autoclave function as steam distillation. It is used to sterilized the media and

culturing apparatus such forceps, filter paper and so on. The usage of autoclave in

sterilization process is better because it can be done in easy and quick way. Besides that,

sterilization level of media apparatus is more guaranteed because all microorganism is

destroyed at a very high pressure. However, there are a few things that needed to be taken

care during sterilization of media when using autoclave. This is because there is a
15
possibility that the composition of media itself that can change after being autoclave. pH of

the medium can change after it been autoclaved. Sucrose in a medium can change into

caramel at a very high temperature. If it became brown in colour and became toxic to the

culture. Organic salt can precipitate if it is exposed for too long at high pressure.

Solidifying agent such as agar also having polymerization, besides than other tools that

sensitive to heat that become damaged or chemically change. At the same time, autoclave

is often used to produce autoclave distilled water.

In process of sterilization of material that sensitive toward the heat, Sterilization through

filtration can be used to ensure that the chemical composition of the material remain

unchanged. Example of material that sensitive to heat is Giberellic Acid (GA 3), Zeatin,

antibiotic enzyme, vitamin and Colchicine. All the microorganism particle and virus will

separate when the it size is bigger than diameter of filter pores that used. Usually, the

diameter of the filter is 0.22μm which is enough for tissue culture. Example of filter that

usually used is Millipore.

Two others sterilization technique is rarely used in culture project because of it has high

risk, dangerous and need a suitable environment for the process that involve radiation and

the usage of chemical substances.

16
1.5.4 ASEPTIC TECHNIQUE IN STERILIZATION OF MATERIALS

In sterilization process of glass equipment which is usually used in making media,

usually we use dry sterilization. Glass material should be wrapped in aluminum foil before

be kept in incubator. The temperature is must be kept at 40 ° C.

In steam sterilization which is using autoclave, there are many materials that can be

sterilized by using this method. As an example, culture equipment, media and autoclave

water is prepared by using autoclave.

In culturing process, example of material use is microtome, forceps, conical flask and

others is used and also if sterilization and cleaning need to be done, one container is needed

for the waste material from the cleaning process. Before the material is autoclave, it should

be wrapped with aluminum foil. This is to ensure that the sterile condition of the tools can

be kept for couple of days before the culture process is done. For material such as conical

flask and baby jar, the tip of the material must be wrapped up for the same purpose.

For sterilization of medium, if the medium is poured inside a gem jar, right after it being

poured into each of jam jar, the jam jar must be cover then labelled to avoid any confusion

during making of media. After it being sealed, media is then put into autoclave for

sterilization process. After the media is sterilize, then it will be let to be cooled in the

laminar flow which is already being open for 15 minutes. After that, each gem jar is sealed

with parafilm to avoid any small insect such as ant into a media.

If the media is made in other container such as conical flask and then poured into sterile

tube, the tip of the conical flask must be sealed with aluminum foil. This will ensure that

the media will remain sterile when carried from autoclave to laminar flow chamber. This is

17
because the exposure to open air in the lab can contain many contaminations which can

inhibit the growth of the explant.

The sterile distilled water is poured inside Schott bottle that has volume of 1 L, then it

will be put into autoclave according to same pressure and temperature such as sterilization

process of media and apparatus. Before sterilization is done, the cover of sterile distilled

water must not be sealed too tight.

However, at the end of sterilization, the pressure inside autoclave must be ensure that it

not decreasing too fast becoming atmospheric pressure. If this happen then the plug can

come off and boiling liquid will come out of the bottle.

1.5.5 MEDIA PREPARATION

One of the factor that will determine the achievement of plant tissue culture is based on

the usage of suitable medium for the culture process itself. So, to get the expected result,

the media formulation has an integral role in determining the final result. In order to obtain

the best result, the medium should contain all the component such as micronutrient,

macronutrient, vitamin, amino acid, carbon source, solidifying agent, pH buffer and plant

growth regulator (PGRs)

Macronutrients is one the mixture that store the main ions that needed for the Plant

growth and development. It acts as soil toward the tissue. Inside micronutrient, there are six

main components which is source of Nitrogen, Phosphate, Potassium, Calcium, Magnesium

and Sulphur. Source of Nitrogen can present in the form of Nitrate or Ammonium.

However, the source of Nitrogen mostly used ammonium as because it causing Nitrate tend

to cause media to become more alkali.

18
 Carbohydrate which is a macronutrient is one the most important element, is usually

added in form of sucrose in order to substitute the carbon. Usually, plant fixed carbon from

the normal environment in the photosynthesis process as an energy source. To fix this,

Sucrose is then use. Besides that, the cellular tissue of explant is not completely well

developing due to the fact of less number of chlorophyll that cause limited gas exchange

and Carbon Dioxide in sterile tube. To increase the growth rate, the media is also added

with organic substances.

Essential elements that present in micronutrients are such as Iron, Manganese, Zinc,

Boron, Copper and Cobalt. All these elements are important component in cell and act as

source of metabolite and physiology of the tissue. Before that, Iron source usually

supplemented in the form of active compound, as an example in Iron Sulphate. However,

this kind of mixture has an affinity to form precipitation inside the media, and it cannot be

use totally by the plant tissue. So, another initiative is which is proposed by Murashige and

Skoog (1962) is by making the Iron in the form of Fe-EDTA. EDTA is known as chelating

agent; which can make iron absorb more inside the plant.

Vitamin is also need in tissue culture, but only in small quantity. This is because most of

the plant can synthesize vitamin by itself. Its function as to increase the development of

tissue culture. Example of vitamin that usually used in medium of plant tissue culture is

Thiamine, Nicotinic acid and Pyridoxine. Due to addition of vitamin in culture did not give

any negative effect, so the usage of additional vitamin is often used. Example of vitamin

that can used is Myo-inositol, Pantothenic acid, Folic acid, Riboflavin and Ascorbic acid.

Usually, Myo-inositol is added to the medium separately and not prepared in stock vitamin.

The organic substances are very integral in supporting the growth and development of

media. Example of organic substances are trace amounts, vitamins and also plant growth
19
regulators. Vitamins is very important for supporting metabolic processes as cofactor so

that the enzyme can function in optimal condition.

There is a lot source that can be used to produce amino acid. Amino acid and urea is

known as good source of Nitrogen in plant tissue culture. Both can function as stimulus

toward plant growth and development, morphogenesis and somatic embryogenesis.

Usually, source of amino acid is Alanine, Aspartic acid, Proline and Glutamic acid.

Activated charcoal is another substance that are often supplemented to function as both

promotion and inhibition. For examples, in orchid, the addition of activated charcoal will

induce growth while in soybean, it will cause inhibition. The benefit of activated charcoal

for most species is it will absorb the brown black pigment and function to oxidized

phenolic compound, which reduce the rate of toxicity in the culture. The mechanism of

activated charcoal it that it contains a very thin network of pores with big inner surface area

making the substances to be absorbed easily (Thomas, 2008).

The source of carbon that usually used in plant tissue culture is sucrose with optimum

concentration at 2-4%. Sucrose is simple sugar and chosen because it can be easily

synthesizing by plant for metabolism process. The alternative sources of carbon are

Fructose, Glucose and Maltose also can be used.

pH range that can be used in growth and development of the culture is between 4.0 to

7.0. The instability in pH can cause some of nutrient source cannot be absorb by tissue.

Besides that, the source can become precipitate beside making the agar cannot solidify.

Agar can be used as solidifying agent in a solid medium. This is because it properties

which is inert toward any chemical reaction in media composition. It also has high

consistency because it properties cannot be digested by the plant enzyme. Agar has boiling
20
point at 100 ° C and freezing point at 45 ° C. The optimum concentration pH the medium is

between 0.7% to 1.0 % (w/v). Example of other solidifying agent that can replace agar is

Gelatin, Alginate and Gelrite.

Auxin and Cytokinin is known as plant growth regulator that are the most widely use.

Both are playing important role that the absence of this two hormone can cause failure for

culture to develop. However, the presence of endogenous hormone in tissue culture should

also be consider. Endogenous hormone is hormone that are already presence in the plant.

Because of the difficulty to detect presence of endogenous hormone, plant growth regulator

is still used in the media to avoid the problem lacking of hormone which can lead to stunted

growth. Beside from Auxin and Cytokinin, two plant growth regulator that can be used is

Gibberrelin and Abscisic acid. Skoog and Miller is one of the first that state that the ratio of

Auxin and Cytokinin that can really influence the type and development of organogenesis

in tissue culture. Both are very important in morphogenesis, even though universally the

ratio of the hormone of shoot and root development are relatively the same.

Example of Auxin that used in media of pant tissue culture is Indole-3-acetic acid (IAA),

Indole-3-butyric acid (IBA), 2,4-Dichlorophenoxyacetic acid (2,4–D) and 1-

Naphthaleneacetic acid (NAA). The only Auxin that presence naturally inside tissue of the

plant is IAA. The only Auxin that presence naturally inside plant tissue is IAA. Synthetic

Auxin hormone show function of Auxin at different level. Previous study shows that 2,4 –

D show 8-12-time activity of IAA while NAA has 2-time activity of IAA. However,

usually 2,4-D is usually being used to stimulate cell proliferation, but the usage at high

concentration will cause morphogenic activity stunted. In general, auxin function to

stimulate callus formation and cell growth, and formation of shoot and root at the same

time. Besides that, it all can promote somatic embryogenesis.

21
 Cytokinin is also usually used in culture of media. For example, 6-Benzylaminopurine

(BAP) or 6- benzyl adenine, isopentenyl adenine (2ip) , N6-furfuryladenine (Kinetin ) , (E)-

2-methyl-4-(7H-purin-6-ylamino)but-2-en-1-ol (Zeatin) . Zeatin and 2iP is known as

natural Cytokinin , while BAP and Kinetin is synthethic version of Cytokinin . The

function of Cytokinin inside medium is to stimulate cell division, induction of shoot

formation and shoot proliferation beside inhibition of root formation. However, the

mechanism of Cytokinin is still not well understood in detail, but the proposed theory is

that it can activate the synthesis of RNA and stimulate the protein activity and enzyme in

certain tissue.

Gibberelin (GA3) and Abscisic acid (ABA) is two of plant growth hormone that Plant

Growth Regulator that are rarely used in media of tissue culture. The tissue culture is

usually can develop with or without GA 3 or ABA, however, some of species needed either

of this two to increase the growth and development. Generally, GA 3 function to increase the

development of low density cell culture, formation of callus and increase the length of

stunted or small saplings . Abscisic acid is put in media either to inhibit or stimulate the

growth of the callus depend on species , stimulate the proliferation shoot or buds , and

inhibit the development of embryo at the final stage .

Mixture that usually used in media such as Thidiazuron (TDZ) . It is known as hormone

that act as Cytokinin . Usually it is applied toward the woody plant species for stimulation

of shoot proliferation and accelerate the shoot formation.

22
1.5.6 MS MEDIA PREPARATION

The step of MS media preparation is according to following step. 500ml of distilled water

is measured using measuring cylinder of 500 ml then poured into conical flask of 1000ml.

Stock solution of macro nutrition with 10x concentration is measured to 100ml using

measuring cylinder of 100ml. Then, stock solution of micronutrient, vitamin, Iron with

100x concentration is measured with 10ml volume using pipette. All the stock solution is

which had been measure then poured into the same conical flask which contain distilled

water solution.

The same conical flask then is shaken for a while to ensure all the solution is mixed

evenly. Then, it will put on the magnetic stirred which is then turned on. This to ensure all

the solution become homogenous.

Then, sucrose is weighted at 30g, myo-inositol 0.1 g and agar for 8g respectively by

using electronic balance. Each of the measurement need to ensure to be accurate so that the

media formulation is correct which is crucial toward the goal of plant tissue culture.

Sucrose is poured carefully inside the conical flask that contain solution and it will be let

to dissolve first. This is because the sucrose crystal has a very large size that will take some

time to dissolve. Then, myo-inositol is then poured inside the mixture to let it to be

dissolve.

pH meter is used to measure the pH of the solution which is set to 5.8 that usually

optimum for most of the tissue culture. For Dioscorea alata, the value of pH is set up to

5.8. To change the value of the PH we use Hydrochloric Acid (HCL) of 1.0 M to lower the

pH and Sodium Hydroxide (NaOH) to increase the value of pH.

23
 After the value of pH is set to 5.8, agar is then poured inside the conical flask and let it

to dissolve again by using magnetic stirrer for a while. This is to ensure all the component

mixed evenly in the media.

In the preparation of MS media with half of the volume, all the value of the component is

set to half. For example, the concentration of hormone is set to half, weight of component

like sucrose or agar is set to half.

1.6 OBJECTIVES

1. To study the effects of Plant Growth Regulators on the explants of Dioscorea alata.

2. To determine the best explant for in vitro culture.

3. To carry out antimicrobial analysis on parts of the Dioscorea alata.

4. To compare and contrast differences of in vitro and in vivo culture of Dioscorea

alata

24
2.0 LITERATURE
REVIEW

25
2.1 BENEFIT OF PLANT TISSUE CULTURE

There are several advantages of plant tissue culture that had been identified by various

scientist throughout the timeline of Plant tissue culture. This are some of advantages of

plant tissue culture (Razdan,2003).

1. Produced huge amount of clones from one single seed or explants can be produced

which can be applied to make a plant with better quality of flowering, smell, fruits

and more important trait for the consumer.

2. Tissue culture offering a feasible selection of an intended trait directly from the

culture.

3. Tissue culture able to decrease the total quantity of space needed for field

experiment since for tubes containing clones viable for trials can be produced.

4. Tissue culture is time effectiveness where it only required short period of time and

the need of waiting for the complete life cycle of seed development is no required

5. Rapid Propagation is available for species that required longer time of generation,

seed production is at very low levels and the seeds are not ready to germinate.

6. Tissue culture able to overwhelm seasonal restriction for plant growth.

7. Allow pollen preservation and collection of cell from the plant that been propagated

8. Tissue culture allows for international exchange of sterilized plant materials thus

eliminating the needs for quarantine.

9. Tissue culture able to remove the pathogen that causing plant diseases.

10. Large number of viable plants can be stored inside the cold storage which required

only small space.

26
 11. Able to produce plants anytime although the climates unsuitable and inappropriate

for the plant to grow. If the seed is unavailable, this technique can be very useful.

12. Tissue culture is an important technique and very useful in investigating the

genetically modified organism which need a lot of studies.

13. Tissue culture is a controlled physiochemical environment such as temperature and

osmotic pressure and defined physiological condition constitution of medium.

14. Tissue culture is cost effectiveness (economical) since smaller quantities of reagent

are needed than in vivo.

15. The equipment needed for tissue culture works are inexpensive when compared to

the animal ell culture works.

16. Tissue Culture has almost no legal moral and ethical questions compare to the

animal experiments.

2.2 DISADVANTAGES OF PLANT TISSUE CULTURE

Even though plant tissue culture has multitude of advantages, however, it still had its

own disadvantages. Here are some of them (Razdan,2003)

1. The cost of equipment will increase as the scale of production increase.

2. Each of the procedure required different technique and the step will depend on the

type of species so for each species, it required trial-and-error kind of experiment if

there are no previous studies is done before. This also required a lot of money.

3. Individual must be dedicated throughout the experiment because each of the step in

culture required a good technique and meticulous observation.

4. The identity of the organism might have an error after culture.

27
 5. There will be spread of disease and infection if the precaution is not being carefully

measuring at the time of experiment.

6. Tissue culture also able to decrease genetic variability of the plant.

7. Expertise is needed to interpret and regulated the behavior of cells in culture.

8. Tissue culture also constitute to unstable aneuploidy chromosome condition.

9. Tissue culture may cause an introduction of unknown pathogens or appearance of

an unobserved mutant to the plant.

10. Probability of an error occurs in maintaining the plant identity.

Therefore, the scientist or researcher must conduct one proper plan to achieve a

successful tissue culture technique. This is very important to avoid any lost that should

avoidable.

2.3 PLANT REGENERATION PATHWAYS

The needs to studies and production of commercial application is one of the

main goals in plant tissue cultured since it is very useful method for this type of situation

(Thorpe, 2006). In plant tissue culture scope, it is very important to study the plant

regeneration pathway since the explant that been cultured will regenerate in different

pathway which will produce different organs or cells. The concept of totipotency is the

main concept used in induction of plant parts since from the tissue culture technique,

organs such as shoots, roots or even callus will regenerate. In Plant regeneration of

pathway, the role of Plant Growth Regulators such as Auxin and Cytokinin amount is one

of the main factor that will influence the pathway. For example, when the amount of Auxin
28
and Cytokinin is almost equal, callus will be formed. If the amount of Cytokinin is more

than Auxin, shoot proliferation will occur while in contradictory to this in which the

amount of Auxin is greater than Cytokinin, root regeneration will be induced.

Organogenesis and Somatic embryogenesis is the two type of pathway where plant will

regenerate. In Organogenesis, it is different when compared to embryogenesis in which the

plant organ such as shoot will start, extend and increase in amount which followed by

formation of root, when the root formation treatment (Aitken-Cristie et al., 2013).

Generally, organogenesis is easily defined as direct formation of plant organ from explant.

Trigiano and Gray (2011) stated that a few plant cells will preserve their capability to

differentiate again from their recent form, state of function and they will initiate a different

developmental path toward other structure end-points. There are two type of organogenesis,

which is direct and indirect developmental stages. The phases in which organogenesis

process involved are including dedifferentiation, induction and differentiation (Chritianson

and Warnick, 1985; Trigiano and Gray, 2011).

Explant Organ

1 2 3

(Dedifferentiation) (Induction) (Differentiation)

Figure 2.1: Phases in indirect organogenesis phase.

Indirect organogenesis is involving formation of callus. Callus is a mass of

undifferentiated cell tissue, in a more flexible morphologically and it is the starting form of

new organogenesis. There are two different type of callus; embryogenic and non-

29
embryogenic callus. Somatic embryogenesis is form from embryogenic callus and it can

produce any organs in the culture. Non-embryogenic callus is type of callus that cannot

differentiate and form organs from the explant. When the explant is wounded, it can give

rise to a callus as a response. Morphologically, callus cell can be differs in size, shape and

also expression of genetic. Callus has a huge central vacuole and the position of nucleus is

to the side. This form is totally different when compare with undifferentiated, meristematic

cell that are contain a smaller size, lack of clear vacuole , cytoplasmic and contain a very

big central nucleus (Smith, 2012). Direct organogenesis is process in which the explant

form organs such as shoot or roots when a suitable amount of Plant Growth Regulators is

supply without the formation of callus at any stage of the differentiation process. The

sequences of Direct organogenesis are explant, meristemoid and organ primodium .

Primary explant Meristemoid Organ Primodium

Figure 2.2: Direct organogenesis pathway.

Besides fertilized egg, embryogenesis regeneration pathway are

applicable to many others different types of cell. The process can be done either naturally

or artificially. Physiological and developmental factor are the one that responsible to

regulate the ability of species and genotype to induce Somatic embryogenesis. The

discovery of somatic embryogenesis is tough to be documented even the first reported

30
discovery is in the umbellifer Oenanthe aquatic where it is observe to induce somatic

embryogenesis (Vasil and Vasil, 1972; Gautheret, 1983, Haperin, 1995; Jain et al., 2013) .

Most of the documented studies are only in in vitro process. The pioneer is the somatic

embryogenesis studies is Harry Waris study (Krikorian and Simola, 1990; Jain et al, 2013).

Somatic embryogenisis is the process where the competent cell will initiate a response to

different condition along with embryogenic growth. Fehér, (2006) stated that factor such as

changes in amount of Auxin exogenous or endogoneous and stress of the plant will

influence the process of Somatic Embryogenesis. In this process, the physical alteration can

be observed at multiple stages. The different characteristic shown in Somatic

Embryogenesis making it is one of the main studies and investigation in plant which

include categories of morphological, molecular, physiological and biochemical studies that

happened on the development of embryogenesis in plants (Quiroz-Figueroa et al., 2006).

2.4 ECONOMIC AND SOCIAL IMPORTANCE OF DIOSCOREA ALATA

Dioscorea alata is an annual plant that has a certain dormancy period. Different than

other tropical crops, the dormancy period do give advantage for a longer period of storage,

which lead to a guaranteeing source of food especially at the time of universal scarcity.

Usually, storage time of Dioscorea alata can be up to six month or longer but this will

depend on certain factor such as storage environment, handling and so on. Dioscorea alata

also consider cash crop which mean it can produce money when kept and when the prices

are higher, yam can be sold.

31
 There are a lot of famous usages of Dioscorea alata especially for their

nutritional values and also medicinal values. Opara (1999) reported that Dioscorea alata

fall next to Cassava as the most significant tropical root yield. Actually, some of nutritional

content of Dioscorea alata are better when compare next to cassava such as it has content

of vitamin C (40-120 mg/g) and for content of crude protein content (40-140 g/kg dry

matter) . Many research has been done to investigate the Nutritional benefit of Dioscorea

alata (Bradbury and Holloway, 1988; Opara, 1999; Alves, 2002; Afoakwa and Sefa-

Dedeh, 2001) .Opara (1999) reported The health benefit of Dioscorea alata can be

directly in south pacific , where it served in various amount at 20% , 8.1% and 4.6% of

total dietary calorie intake in Kingdom of Tonga , Solomon island and Papua New

Guinea.

Dioscorea alata are often associated with high nutritional value besides being a

staple food for a certain region such as Africa and so on. This is important because for

country who are always associated with hunger and starvation crisis, Dioscorea alata can

be the main food source to fill the daily intake need to continue the living process. In

Filipina, Dioscorea alata are made as Ube Cake, in Trinidad and Tobago, they used it as

mashed meal while many traditional food in Africa such as fufu are using Dioscorea alata

as their ingredient. As a staple food, Dioscorea alata must have a good quality such as

taste and texture of the food itself. Dioscorea alata has a good carbohydrate and fiber

value which is important to give the energy and digestion to human. Dioscorea alata is

considered as rich source of carbohydrate and has content of important micronutrients such

as vitamins and mineral. For mineral status, usually the ash content of Dioscorea alata is

measured (Osagie, 1992). Dioscorea alata tubers have great amount of moisture, dry

matter and starch. For natural food, they provide good source of some minerals. In studies
32
conducted by Osagie and Eka, (1998), they found out that Dioscorea alata contain a

decent amount of Potassium. Potassium is a minerals source that help to stabilize the blood

pressure. Consumption of Dioscorea alata is suggested for consumer who suffered high

blood pressure but it is not recommended for one that has renal failure disease.

It has been reported that Dioscorea alata flour contain a greater

amount of fiber when compared with potato flour (Woolfe, 1987), wheat flour, maize and

rice. Instead of simple carbohydrate, Dioscorea alata provide complex carbohydrates and

fiber which digested at a very slow rate which also making the rate of sugar released and

absorbed into the bloodstream lower. Besides that, Dioscorea alata can provide

micronutrients such manganese, a type of trace mineral which can aid the carbohydrate

metabolism and also cofactor for many enzymes that serve in energy production and also

defense of antioxidant. Barquar and Oke (1977) reported that it also has traces of vitamin

B-complex. Vitamin C and Vitamin A is very useful micronutrients that aids in healthy

skin and promotes good vision. Beta-carotene which is a precursor of vitamin A is also

abundant in Dioscorea alata cultivar (Osagie, 1992). In study conducted by Bradbury and

Singh (1986) , amount of total Ascorbic acid of Dioscorea alata tuber is 50% more when

compared with cassava . Besides that, Dioscorea alata also contain limiting amino acids

such as Isoleucine and Sulphur-containing amino acids.

Other micronutrients inside Dioscorea alata tubers are Phosphorus which is more

than 200mg/100g, but most of them are in form of Phytic Acid. Dioscorea alata starches

are said to have three to four times of phosphorus that Cassava and Aroids (Moorthy,

1994). This is supported by Peroni et al (2006) that stated that there is abundant content of

Phosphorus in Dioscorea alata (0.022%) in Dioscorea alata when comparison is made

with other tropical root and tuber crops. In Dioscorea alata, the amount of Phosphorus is
33
said to be higher when compare with D.rotunda and D.Cayenesis .There are a lot of factor

that effect the nutrient and minerals content in the Dioscorea alata . Factors such as type

of soil where Dioscorea alata was harvested from, content of moisture and crop maturity

will play an integral role to the beneficial value of water Dioscorea alata. There also

calcium content in Dioscorea alata in which they morphologically occur as raphide

bundles-crystal that surrounding the whole cells as calcium oxalate. They also contain in

starch grains where they play a role as storage and serve as nuclei for starch deposition

(Okoli and Green, 1987).

2.4.1 Health Benefit

Besides being a staple food for many countries, Dioscorea alata also serves as source of

nutrition. Dioscorea species had contain of important micronutrient such vitamin C and B 6

(Bhandari, Kasai et al. 2003) . One of the major cause of death and disease in the whole

world is the lack of micronutrient or also known as micronutrients deficiencies (Clarke and

Zhang 2013) . Usually, the one who need a lot of nutrient for grow especially children, will

be the one that will have affected because lack of nutrient can cause their growth

development and system stunted. with diet that include Dioscorea alata , the will get their

source of nutrients along with others healthy food such as vegetables, fruit and meat

Dioscorea alata has contain Thiocynate that have the ability to treat sickle cell

anemia .(Agbai 1986) . Anti-sickling agent or also known as SCN is reported to be high in

Dioscorea alata flour which is 50 to 60 mg/100 g. It can be a beneficial amount of of

SCN to prevent the sickle cell anemia disease. In Africa, they usually consume 1 g of SCN,

more than twice amount of it to prevent the case of sickle cell anemia disease.

34
 Dioscorea alata contain secondary metabolites that are widely used in the field

medicine and pharmaceutical. It contain Diosgenin which able to be extracted from the

Dioscorea alata plant and utilized for the production of drugs especially hormonal drugs

(Albrecht and McCarthy, 2006) such as prognenolone , progesterone , cortinsone and other

steroid product . Diosgenin is generally used as synthesis of sex hormones and

corticostedroid . This compound are used to prevent anti-inflammatory , androgenic and

contraceptive drugs (Sautour, Mitaine-Offer et al. 2007) .Besides Diosgenin , Dioscorea

alata also contain Dioscorin , which is the major storage of protein in the tuber and has

effective effect against angiotensin . Angiotensin can the enzyme that responsible for

causing hypertension. Water Soluble Polysaccharides in Dioscorea alata can lower the

blood pressure or known as Hypoglycemic effect. It also being reported that certain species

have alkaloid and steroid derivatives, which can produce the tuber bitter and has possibility

to be poisonous if it is used without an appropriate processing (Purseglove, 1976). Farombi,

(1998) and Farombi et al ( 2000) has reported that brown Dioscorea alata flour contain

antioxidant activity which can be used to stabilize the bulk oils , emulsion and biological

membrane against lipid peroxidation .

2.5 DIOSCOREA ALATA HARVESTING

Dioscorea alata is usually harvested mainly in region where it become staple

food such as Africa which is known as main producer. However, there are not too many

modern technologies are applied in this area, which mainly depend on man work. Thus,

harvesting of Dioscorea alata can be a very labor-intensive operation because it need a lot

of people with a good technical skill so that the efficiency of harvesting is good. For area

35
such as rainforest, there are many dangerous threat such as insect and root of trees which

has high potential to give a problem and can give physical damage. There are a lot of

deformation to the harvest as the result form the difficulties that being impose. The quality

of tubers is getting lower. For manual harvesting, the tuber is dig from the soil by using

either spades, diggers or sticks. All of these material need to be replace time by time and

also need to be in large quantity. Manual plucking from the vine is applied if the intended

product are aerial tubers or bulbils. There is an effort to introduce mechanical Dioscorea

alata harvesting as being reported by Nystrom et al, (1987) which is the main aim is for

pharmaceutical usage, but this technology is not applicable in industrial scale and only for

the research and demonstration purposes only. Besides, there are suggestion that for the use

of potato spinner to harvest small tubers (Onwueme, 1997). The newest crop production

technique and species give a substantial obstacle to be effective in mechanization of

Dioscorea alata production, especially to the farmers that run a small-scale in rural

production. A lot of things required to be changed such as the latest traditional cultivation

practices, architecture and physical properties of tuber. Post-harvest technique to measure

quality is also required such maturity indices is important. For example, time of harvest is

one of the most important trait to give the tuber a good maturity. This also depends on

period of cultivar from planting or time to break dormancy which is about 6-7 months or

even 6-10 months. Martin et al. (1984) and Onwueme, (1977) stated that for double-

harvesting, there are recommended eight to ten months’ period and four to five months

from planting to maturity to be applied. However, Bencini (1991) also reported to harvest

the Dioscorea alata first at five to six month after planting and then three to four month

later.

36
 There is different amount of time of harvest that are usually used; single

harvesting and double harvesting which is done to get at first and second year of harvest.

For single harvesting, the number of time of the Dioscorea alata is harvested only once

and this happened at the end of the season where all the crops have reach a satisfying

levelled of maturity. Single harvest is usually done at the end of the season when all the

vines have died, before the yam is harvest. The crop is only harvested once, not twice. The

process of harvesting is usually started with plowing around the tuber to ensure that the soil

is separated and loosen from it, lifting the tuber slowly and then finally, the vine is cut

while the corm is attached to tuber. The first harvest is usually involving the removal of

soil from the tuber meticulously and the lower portion is cut, in order to leave the upper

part of tuber to continue to grow. The soil is then put back into the plant and the tuber is

left to grow until the end of the other season for the second harvest. By doing this,

Dioscorea alata cultivars can produce a few small-size tubers in the second growth

following the initial harvest. Diosocorea alata, Dioscorea rotundata and Diosocrea

Cayenensis is said to be most suitable for harvesting. The yield for single and double

harvesting is said to be quite similar but there are been report by Onwueme and Charles,

(1994) that the single-harvested tuber will give a better quality of eating when compare to

double-harvested tuber. In Diosocorea alata , two type of practices are involved in term of

timing of harvest . For double harvesting, the Dioscorea alata plant is usually harvested

twice. The first harvest is applied around five months after emergence in which during this

time, the farmer will carefully dig the soil to expose the tuber without damaging the roots

of the plant. Then, the tuber is detected from the whole plant at the point just below the

corms. The tuber is then removed and soil will cover back the roots. The plant will carry on

growing and then a new tuber is produced. The second harvest is done when the plant is

37
died at the end of the season. There is changed in term of texture and morphology when the

tuber is recovered in the second harvest in which a firmer texture and irregular shape is

obtained when comparing with the tuber from the first harvest.

Besides single harvesting, double harvesting is also having many distinct benefit

of producing a new crop available for consumption in the earlier season, and at the same

time, supplementing good planting material at the second harvest. Like single harvesting, it

also has several disadvantages. First, the labor-demand which already a big problem is

needed in double of work since the harvesting is done at the same time. Then, the first

batch of double harvest is needed to be handle gently because if the handling is poor, many

damage such as root damage will occur. This also why the mechanization of Dioscorea

alata production is not applicable to a wide scale. Third, the tuber that been produces from

double harvesting are poorer for usage than the tuber from single harvesting. The quality

from the first harvest is not good enough since the tuber are often watery and no completely

mature. The second harvest tuber are woody in the head region. The double harvesting

technique is suitable only for traditional agriculture and in future, the single harvesting is

more appropriate. For a better future, mechanical harvesting should be studies and practice

more. There is work and progress in region of West Indies and West Africa in

mechanization of Dioscorea alata production. The selection of quality indices is also being

standardize.

38
2.6 LIMITATION IN DIOSCOREA ALATA PRODUCTION

2.6.1 DISEASE IN DIOSCOREA ALATA

Fungus is one the main microorganism that effect Dioscorea alata (I.O. Ezeibekwe and

A.E. Ibe , 2010) . Different Genera of fungi are being involved with the storage

deterioration in Dioscorea alata tuber. (Noon, 1978; Okigbo and Ikediugwu, 2000). It had

been reported that are over 50% of Dioscorea alata tuber is lost in storage due to infection

during production and harvesting process (Onayemi ,1983). As a staple food in many

country, a disease in Dioscorea alata could cause many of bad effect such as the reduce in

quantity of yam and also the appearance which can affect the consumer. Amusa (2003)

reported that Dioscorea alata is vulnerable to contamination from many seedling stage

until harvesting stage which shown . Information of how the mechanism of fungi infection

will be very useful since the knowledge can be applied to find the cure and preventative

measure in the future in order to produce a high quality Dioscorea alata.

Usually, Dioscorea alata production always been affected by environmental problem

such as pest, disease and so on. One of the disease that infected the leaves of the Dioscorea

alata itself is Anthracnose (Scorch). It is the disease that cause many observable symptoms

such as blotches, defoliation, leaf spots and many others on the plant itself (Koetter R. and

Grabowski M.). It does not cause permanent damage to established trees but can cause

many other harmful effect in future. The cause of this infection are usually from Fungi, and

if the condition is getting worse, the use of fungicide can be applied

39
FIGURE 2.3.1 : Anthracnose on Dioscorea alata

Besides Antrachnose , Dry rot disease that had been caused by yam nematode is also

usually affect the Dioscorea alata itself . Instead of the vine, it effects the tuber itself.

Inside the tuber, it will show sign of rotting. Without this infection, the colour of tuber is

dark brown, but with the infection, the dry rot is showing the colour of cream and light

yellow lesion. This colour can be seen on the outer skin just outside the skin. If prowling,

the disease can leach into the deeper region and will affect the content of tuber. It can cause

the entry of fungus which can be seen if with the growth of fungus inside the tuber itself.

40
Figure 2.3.2 : Dry rot disease on Dioscorea alata

Mealybugs

Mealybugs is an insect that can be found on the Dioscorea alata when it is infected. It

scientific name is Rastrococcus spp. And known as foot tree mealybugs. It can be found

in moist, warm climates where this is the best climate to grow Dioscorea alata. Mealybugs

is known as pest by feeding on plant juices as their main source of food. It can be

considering as transporter of other several plant diseases. Mealybugs is very small and

white in colour, has oval to round disk-like insect that can be found in many plant parts. It

can attract ants to the plant itself. Growth of fungal that cause to formation of sooty mold is

also the main symptom that been induced by mealybugs because it will released the sugary

honeydew. Ant is considering as their host because of it sugary honeydew secretion.

41
 Figure 2.3.3 : Mealybug on Dioscorea alata

Root Knot Nematode

Root knot nematode is type of disease that infected plant that are accompanied with

very low growth rate. The amount of portion in tuber that are edible which is the one of the

main part is reduce. Tuber will become abnormal with the sign of deformation and

abnormal rootlet. It can cause a lot of loss of yield up to more than 50% and it is reported

in Nigeria, it will reduce it the value and quality of tubers itself .

Galled tubers with crazy roots

42
 Figure 2.3.4 : Root knot nematode

White Scale Insects

Aspidiella hartii or yam scale is known as the white scale insects is one of the insect that

will affect the Dioscorea alata storage production. Besides Dioscorea alata, it also infected

ginger (Zingiber) and turmeric (Ronald F.L. M, Jayma L. M, K, 1992). The mechanism of

this insect is by taking its feed from the Dioscorea alata. If the small amount of Aspidiella

hartii is present, the damage that is set is not critical. But if the population is become bigger

until it reaches a certain level, a clear damage can be seen. Example of sign that the yam

had been infected are yellowing, loss in a quality of tuber, defoliation. The store Dioscorea

alata also can become encrusted with turmeric root scale which will make it become dry

and fibrous. A clear white scales can be seen covered with small white scales on the leaves

and tuber from the planting field up until production level. It also said to delay or stopped

germination. tube shrivel also can occur due to infestation.

43
 Yam tuber in

insect (2)FfFigure 2.3.4 : White Scale Insects on Dioscorea alata tuber .

Yam mosaic disease

Instead of insect, there is also virus that can affect Dioscorea alata production.

The virus is known as Yam mosaic potyvirus. The virus rarely occurs alone and is often

accompanied with other disease, such as, yam mild mosaic virus, and cucumber mosaic

virus. Dioscorea alata are one of the plant that prone to virus infection. This will lead to

loss of desired quality of Dioscorea alata tuber such as quantity, size and also their starch

content. The germplasm movement also will be blocked due to the virus infection.

Example of sign of infection of this virus is narrow yellow leaves, yellow and green pattern

44
on the leaves, distorted margin disease and backward curling (Grahame Jackson, 2014) .

The yellow and green pattern on the leaves are called mosaic, with the pattern are position

on the vein and showing a visible vein banding. the growth of the plant can be stunted

besides the reduction of starch content. Some plants may recover from the virus infection

soon after first symptom but virus may survive in plant and reduce the vigor.

2.6.2 POST HARVEST LOST

Besides problem in Dioscorea alata , there are also limitation that lead to loss of

crop which is during storage. Some of factors that contribute to this is rotting, tuber

respiration and also tuber sprouting. The respiration of the crop during the storage and

sprouting will consume the stored material of the tubers. The moisture is also decrease

from the tuber. There are report stating a significant amount of Dioscorea alata where

often lost between harvests and market. By the project conducted in 1995 to study the

relationship between handling and marketing system in Ghana to study important thing

such as nature, causes and implication of the losses to give a complete study. This also to

find a technologies and knowledge on how to reduce the losses. This project is continuing

on 2000 to find a way to grade Dioscorea alata by quality, and to eliminate the diseased

found in tuber. There is a system known as “electronic nose” system used to identify

diseased tubers in batches which are used for exporting material from Ghana. The studies

also found that the cold storage for Dioscorea alata tubers can be advantageous in

reducing storage losses due to rotting, sprouting and respiration. The problem with this

technique is the cost where the amount of refrigeration is quite exuberant and the stored

45
temperature are below 10°C which has a tendency to become brown and not suitable for

market quality of Dioscorea alata (Onwueme, 1997).

46
3.0 MATERIALS AND METHOD

47
MATERIALS AND METHOD

3.1 List of material and apparatus

In this experiment, several materials and apparatus were utilized for the tissue culture

works. Generally, most of the apparatus and machine were already available at the Tissue

Culture laboratory. However, there are a few apparatuses which not available in the

laboratory and needed to be purchased. The table below (Table 1) shows the materials and

apparatus utilized in this experiment with their general functions.

Material / Apparatus Function Status

Yam / Discorea alata -Main materials for the in vitro Purchased in Tumpat ,

tissue culture and provide multiple Kelantan

part of explant for culture

Clorox (Sodium -Different concentration of Clorox Available at Lab

Hypochlorite ) (70%, 50% ,30& and 10%) used as

the sterilization solution in

sterilization process

-Used as detergent during washing

apparatus after being used

95% and 70% Ethanol -Disinfect the apparatus and -Available at the

materials before entering the Tissue Culture

Lamina Flow chamber Laboratory

-As sterilization solution in the

sterilization process

48
Tepol (soap) -washing all the apparatus after available at the tissue

being used and used in sterilization culture laboratory

Distilled water -to rinse all the apparatus after available at the tissue

washing and remove the foam culture laboratory

produced during the sterilization

process

-Added to the medium , stock

solutions and sterilization solutions

to reach the desired volume

Sterile Distilled water -cooling down the forceps and available at the tissue

blades during the tissue culture culture laboratory

works

-Rinsed and remove foam produce

during sterilization process

Twen-20 Aided more contact between available at the tissue

explant and 705 alcohol during culture laboratory

sterilization

Hormones ( 2,4-D , NAA , Main hormone used in this available at the tissue

IAA , Kinetin , BAP ) experiment culture laboratory

NaOH and HCL -adjusting the pH of media to 5.7- available at the tissue

5.8 culture laboratory

-NaOH used to dilute the

hormones in hormones stock

solution preparation

49
Sucrose Functioning as carbon source in available at the tissue

the media culture laboratory

Forceps Transferring the explants into the available at the tissue

sterile tube culture laboratory

Blade Cutting the explants into smaller available at the tissue

piece culture laboratory

Tissue To wipe the surfaces of Laminar available at the tissue

flow chamber when disinfecting culture laboratory

process with 70% alcohol

Sterile Tissue Absorbing the excess water from available at the tissue

the explants before transferring culture laboratory

into the medium

Sterile Tube Act as place for media will be used available at the tissue

in in vitro culture and tissue culture laboratory

culture works

Jam Jar -As alternative to store media if available at the tissue

culture laboratory
sterile tube is unavailable

-Forceps and blade will be air dry

inside the jam jar after dipped into

the sterile distilled water during

tissue culture work

Conical flask -1L conical flask used in 1L media available at the tissue
culture laboratory
preparation

50
 -250ml conical flask used to stored

hormones stock solution

Aluminum and masking tape -wrap the apparatus needed to be available at the tissue
culture laboratory
inserted into the Autoclave

machine

-Masking tape will possess a black

line after undergo autoclaving

process

Petri dish Serve as place for antimicrobial available at the tissue

culture laboratory
activity

Laminar Flow Chamber Provide sterile environment for available at the tissue
culture laboratory
tissue culture works

Autoclave machine Sterilizing all the apparatus media

available at the tissue
culture laboratory
Stirrer Stirring the media and stock available at the tissue
culture laboratory
solutions to form homozygous

solution

Weight balance Weighing materials, apparatus and available at the tissue

culture laboratory
callus

Refrigerator Act as a place to store stock available at the tissue

culture laboratory
solutions

Glass rod Stirring the hormone during stock available at the tissue
culture laboratory
solution preparation to get

colorless solution

Table 3.1 : List of material and apparatus

51
3.2 Laboratory Facilities

Some special general consideration and facilities are need to achieve successful Plant

Tissue culture technique. In Tissue culture procedures, the experiment need to be done in

special area such as location of tissue culture laboratory is not adjacent to laboratories that

handle microorganism or facilities which are used to stores the yam or other plant

materials. This is done as we did not want any contamination from air vent and high foot

traffic which help spread contaminants.

The tissue culture area should be clean most of the time to avoid any kind of

contamination from various source that can affect the result of experiment. Contaminant

such as bacteria, virus, fungi and other kind of microorganism can affect cultures and

reproducible result of the plant. Thus, the experimental area for tissue culture in smooth

traffic flow generally can be arranged into three work area which is comprises of;

1. Media Preparation area

2. Transference area

3. Environmentally controlled culture

3.2.1 Media preparation area

Media Preparation area known as general laboratory for media preparation and autoclaving

of media. This section also includes many activities that relate to the handling of tissue

culture materials. Regarding of that, this area generally knows as central section of the

52
laboratory where most of the activities area performed. This area also provided with spaces

for common work which does not necessarily need a very aseptic condition. In media

preparation area, there are essential equipment items of:

1. Laminar air flow hood

2. Autoclave machine

3. Bench

4. Gas outlet

5. Magnetic Stirrer with hot plate

6. Analytical and top-loading balances

7. pH meter

8. Refrigerator

9. Freezer

10. Microwave oven for heating agar media

11. Water bath with temperature control

12. Shaker, gyratory with platform and clips for different size flasks

13. Water purification and storage system

14. Dish-washing area

15. Desktop centrifuge

Therefore, based on equipment that is equipped for media preparation work, culture

media may be conveniently prepared on a laboratory chemical bench with balances, pH

meter and a sink in close proximity. The refrigerator or freezers is used to keep stock

solutions used in the media and also to keep the chemical reagents which can be oxidized

easily and they are most conveniently arranged either in alphabetically or in smaller

53
baskets. The glassware of pipette, conical flask, biker, measuring cylinder and jam jars are

arranged properly inside the plastic basket and cabinet.

A low bench and file cabinet are used for culture evaluation and record – keeping. A

desktop computer is essential for writing up reports to keep a detailed of all the chemical

including the name of manufacturer and the grade.

3.2.2 Transference Area

Tissue culture technique can be successfully done in an aseptic transfer area for aseptic

manipulation of plant material. This aseptic transfer area carried out in cleanliness of

laboratory and dry atmosphere with protection from air –borne microorganisms. Therefore,

essential equipment items are needed in aseptic transfer area include of

1. Laminar air flow transfer hood

2. Gas outlet

3. Vacuum lines

4. Disposable and sterile tissue

5. Spatulas, forceps, scalpels and disposable blades

Generally, the laminar airflow hood is mostly used for aseptic manipulations work. This

equipped the place where the explants are trimmed, divided and transferred into medium of

cultures. The apparatus such as hot bead sterilizer inside the laminar flow cabinet is

necessarily used to sterilize the forcep and scalpel at all times they were used.

54
3.3 Laminar Flow Chamber Regulation

Laminar flow is a chamber which provide a sterile and clean environment for tissue

culture work. There aer several regulations must be followed to ensure no bacteria or fungi

available and cause contamination which are

1. The laminar flow must be switched on and UV light applied at least 15 minutes

before using

2. The laminar flow surfaces must be disinfected by using 70% alcohol before carried

out any works

3. All apparatus or equipment must be disinfected by using 70% alcohol before

entering the Laminar flow

4. After culturing, Laminar flow must be empty and free from any apparatus or

equipment

5. After using Laminar flow, once again the surfaces must be disinfected by using

70% alcohol

3.4 Stock Solution Preparation

Stock hormone solution is required since there are four different concentrations of each

hormone utilize in this experiment. The concentration of this stock hormone solution is for

depend on the desired concentration that required during media preparation. The steps in

preparing stock hormone solution are:

55
 1. Approximately 0.05g or 50mg of hormone was weighed.

2. The hormone transferred into small test tube.

3. Two to three drops of NaOH were added.

4. The hormone was diluted until a clear solution form by using glass rod.

5. The clear solution was transferred into conical flask.

6. The test tube was rinsed using distilled water.

7. The distilled water was used for rinsing the test tube transferred into conical flask.

8. Distilled water was added until the solution reach 50ml.

9. The conical flask was covered with aluminum foil.

10. The stock hormone solution was stored in the -20° C refrigerator.

3.5 Transferring media into sterile tube

After autoclaving the media must be transferred into sterile tube by the following steps

1. Two bag of sterile tube was disinfected with 70% alcohol and transferred into

Lamina flow

56
 2. It is not advisable to put more than two bags of sterile tube inside the Laminar flow

because sufficient space needed for transferring the media from conical flask to the

sterile tube

3. The conical flask which contain media was disinfected with 70% alcohol and

transferred into Lamina rflow

4. The media was transferred into the sterile tube by pouring slowly until the mark

first mark on the sterile tube reach

5. The sterile tube cap must be losing hard enough to ensure no air which contain

bacteria and fungi can pass through

6. All the sterile tubes was brought out from the lamina flow and organize on the table

7. Each sterile was labelled by writing down name, date and respective hormone

concentration

8. If no contamination was observed after two to three days, the media will be used for

tissue culture works

3.6 STERILIZATION METHOD

3.6.1Apparatus and Media Sterilization

Tissue culture works required highly sterile environment and also sterile apparatus.

Therefore, all the apparatus and media must be sterile according to the steps below
57
 1. All the apparatus such as forceps, jam jar, tissue, test tube and conical flask

wrapped with aluminum foil and masking tape

2. Each apparatus and media was labelled by writing down and date

3. All the wrapped apparatus and media was inserted into Autoclave machine

4. The apparatus and media was organized properly inside the Autoclave machine to

avoid any damage during the sterilization process

5. The sterilization process will take about 2 hours and 30 minutes at 121 °C

3.6.2 Subculturing of Explant

After one week of in vitro germination, the best result explant will be subculture into the

new media. Below are the following steps for sub culturing the explant

1. Sterile tubes from each concentration of hormone and culturing apparatus was

disinfected with 70% alcohol and transferred into Lamina flow

2. All the sterile tube and culturing apparatus must be organized properly to avoid any

accident during the culturing process

3. The sterile tubes containing explants was disinfected with 70% alcohol and

transferred into Lamina flow

4. The forceps and blade were sterilized with hot beads sterilizer and dipped into

sterile distilled water to cool it down

5. The explants were transferred out from sterile tube to the petri dish by using forceps

6. Approximately 1cm of stem was cut out by using blade and transferred into

respective concentration of hormones

7. It is highly recommended to put the stem at the center of media

58
 8. Step 4 repeated for every cutting and transferring process

9. All the sterile tube was cultured inside the culture room for one month at

appropriate condition

3.6.3 Explant sterilization

Materials

1. Explant of Discorea alata (Nodes, internodes, Leaves).

2.Gem Jar.

3.Aluminium Foil.

4. Distilled water.

5.Sterile Distilled water.

6.Twen – Twenty.

7 .70% Clorox.

8 .50% Clorox.

9. 30% Clorox.

10 .70% ethanol.

3.6.4 PREPARATION FOR CUTTING THE EXPLANT

59
 1. Part of the explant was cut according to the intended part (Nodes, internodes,

leaves).

2. The explant was washed gently running tap water.

3. The explant was washed with Dettol 3 times.

4. The explant inside the gem jar was then covered with aluminum foil, 3 small holes

is made.

5. The explant was washed with running tap water for 15 minutes.

3.6.5 WASHING THE EXPLANT

1. 70% Clorox was put inside the gem jar along with 2 drops of twen–twenty and

shaken for around two minutes

2. Autoclave distilled water was put inside the gem jar and shaken for one minutes

3. Step (1) to (3) was repeated by using 30% and 50 % Clorox

4. 70% alcohol was put inside the same gem jar and shaken for 1 minutes

5. Autoclave distilled water was used to wash the explant inside the gem jar 3 times,

one minutes each.

3.7 MEDIA PREPARATION

MATERIAL

1. 4.4g of MS powder.

2. 1 Litre conical flask.

60
3. Distilled water.

4. 30 g of sucrose.

5. 8g of agar (gelling agent).

6. Measuring cylinder.

7. Volumetric flask.

8. Small glass container and labels.

9. Hydrochloric acid and Sodium Hydroxide.

The medium that will be used for culturing the Discorea alata explant is the formula

created by Murashige and Skoog (1962) . The formula can be easily prepared inside the

plant tissue culture lab and also available in form of powders (commercially prepared

media.

1 .1 Litre of conical flask was filled with 600 ml of distilled water.

2 .MS media was dissolve in the distilled water.

3. The magnetic stirrer was turned on for mixing process.

4 .30g of sucrose was measure into the solution.

5 .8g of agar is added into a solution as gelling agent.

5. The MS media solution was transfer into a conical flask and filled with distilled

water until it reach 1 Litre . At this point, the magnetic stirrer was turned off.

6. The pH of the solution was adjusted to 5.8. Hydrochloric acid and sodium hydroxide

was added to the solution to obtain the desired pH.

61
 7. The MS media was autoclave to obtain a sterile media and to dissolve all the mixture

in the solution.

8. After autoclave for about 2-3 hours, the media was let to be cool for a while.

9. When the media is still in liquid form, a small amount of media was pour into sterile

tube under aseptic condition.

10. The lid is of each sterile tube was closed and labelled. The media is ready.

3.8 ANTIMICROBIAL TEST

Extraction

1. 30g of each samples (tuber, stem,and leaf) was grinded into the powder form

2. 20ml of 95% Methanol is extracted

3. The Methanolic extract was left incubated at room temperature for 24 hours

4. The sample inside centrifuge tubes were placed in the centrifuge for 5 minutes. This

was done to separate pellet and supernatant.

5. The supernatant was removed and the samples were poured into sterile universal

container

Microbial test

1. Bacto agar plate and potato dextrose agar plate was prepared fot the antimicrobial

test.

2. 15g of Bacto-agar powder was added into 1 L of distilled water to prepare Bacto

agar plate.

62
 3. 39g of potato dextore agar powder was added into 1L of distilled water for potato

dextrose agar.

4. For each of agar, the pH is adjusted to 5.6 ±0.1 and 6.8 ±0.1 using NaoH to increase

the pH value and HCL to lower the pH value.

5. The solutions were then autoclaved.

6. After the solution is done autoclaved, the agar was poured into sterile petri dish and

sealed with parafilm.

BACTERIA AND FUNGI TEST

1. The test fungi and bacteria were separated from the agar plate by using a cork borer

in the laminar flow chamber which is then position on potato dextrose agar plate for

fungi and bacto agar plate for bacteria.

2. Sterilized filter paper disc was soaked in the methanolic extracts (20mg.ml) and

placed in each plates.

3. Carbendezine is used for positive control of fungi while Chloramphenicol is used

for positive control of bacteria. Methanol is used as negative control.

4. The plates with the test fungi were incubated at room temperature (25 ±2.0 ±) while

the test bacteria plates were incubated at (37 ±2.0 ±) for two days.

5. Inhibition zones diameter were measured using a ruler.

STATISTICAL ANALYSIS

The explants inside the sterile tubes were monitored for 7 weeks. The results were analysed

by using IBM SPSS Statistic version 23 software, One-way ANOVA test with Duncan’s

Test at 95% significance.

63
4.0 RESULTS

64
65
4.1 PERCENTAGES OF SHOOT FORMATION

Plant Growth Regulator Concentration (mg/L) Number of Shoot growth

Control 0.0 10 ± 0.07cd

2,4 - D 0.5 0 ±0.00d

1.0 15 ± 0.08cd

1.5 10 ± 0.07cd

2.0 15 ±0.08cd
KN 0.5 0 ±0.00d

1.0 10 ± 0.07cd

1.5 0 ± 0.00d

2.0 0 ± 0.00d
IAA 0.5 0 ± 0.00d

1.0 15 ± 0.08cd

1.5 0 ± 0.00d

2.0 15 ± 0.08cd
BAP 0.5 25 ± 0.10c

1.0 50 ± 0.11b

1.5 25 ± 0.09c

2.0 70 ± 0.10a
NAA 0.5 0 ± 0.00d

1.0 0 ± 0.00d

1.5 0 ± 0.00d

2.0 0 ± 0.00d
Mean ± Standard error, n=20. Mean with different letters in the same column different significantly at
percentage=0.05. The numbers in bold show the best result of this explant.

Table 4.1: Percentage of shoot formations from the explant (stem) when supplemented
with different types and concentrations of hormone in MS medium.

66
 80
70

Percentages of shoot formation

60
50
40
30
20
10
0
Con- MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+
trol 0.5 1.0 1.5 2 0.5 1 KN 1.5 2 KN 0.5 1 1.5 2 0.5 1 1.5 2 0.5 1 1.5 2
2,4D 2,4D 2,4D 2,4D KN KN IAA IAA IAA IAA BAP BAP BAP BAP NAA NAA NAA NAA
Media

Figure 4.1 : Graph showing percentage of shoot formation in different medium

67
4.2 PERCENTAGES OF ROOT FORMATION

Plant Growth Regulator Concentration (mg/L) Number of root growth

Control 0 0±0.00b
2,4 - D 0.5 0±0.00b

1.0 10±0.07ab

1.5 10±0.07ab

2.0 10±0.07ab
KN 0.5 0±0.00b

1.0 0±0.00b

1.5 0±0.00b

2.0 0±0.00b
IAA 0.5 0±0.00b

1.0 15±0.08a

1.5 0±0.00b

2.0 10±0.07ab
BAP 0.5 5±0.05ab

1.0 10±0.07ab

1.5 0±0.00b

2.0 0±0.00b
NAA 0.5 0±0.00b

1.0 0±0.00b

1.5 0±0.00b

2.0 0±0.00b
Mean ± Standard error, n=20. Mean with different letters in the same column different significantly at
percentage=0.05. The numbers in bold show the best result of this explant

Table 4.2 : Percentage of root formations from the explant (stem) when supplemented with
different types and concentrations of hormone in MS medium .

68
 16

14

Percentages of root formation

12

10

0
Con- MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+
trol 0.5 1.0 1.5 2 0.5 1 KN 1.5 2 KN 0.5 1 1.5 2 0.5 1 1.5 2 0.5 1 1.5 2
2,4D 2,4D 2,4D 2,4D KN KN IAA IAA IAA IAA BAP BAP BAP BAP NAA NAA NAA NAA
Media

Figure 4.2 : Graph showing percentage of root formation in different medium

69
Plant Growth Regulator Concentration (mg/L) Observation

Control 0.0 Remain Unresponsive

2,4 - D 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
KN 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
IAA 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
BAP 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
NAA 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive

Table 4.2.1 – Observation of the effect of plant growth regulators on leaves of Dioscorea
alata

70
Plant Growth Regulator Concentration (mg/L) Observation

Control 0.0 Remain Unresponsive

2,4 - D 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
KN 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
IAA 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
BAP 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
NAA 0.5 Remain Unresponsive
1.0 Remain Unresponsive
1.5 Remain Unresponsive
2.0 Remain Unresponsive
Table 4.2.2 – Observation of the effect of plant growth regulators on petioles of Dioscorea
alata

71
 SHOOT FORMATION

Hormone Observation
s

MSO

1.0 2,4-D

1.5 2,4-D

72
2.0 2,4-D

1.0 KN

73
1.0 IAA

2.0 IAA

74
0.5 BAP

75
1.0 BAP

1.5 BAP

76
2.0 BAP

TABLE 4.5 : Diagrams of shoot formation from stem segments of Dioscorea alata

77
 ROOT FORMATION

Hormones Obsevation

1.0 2,4-D

1.5 2,4-D

78
2.0 2,4-D

1.0 IAA

79
2.0 IAA

0.5 BAP

80
1.0 BAP

TABLE 4.6 : Diagrams of root formation from stem segments of Dioscorea alata

81
4.3 ANTIMICROBIAL TEST

4.3.1 Bacteria

Penicilum sp Aspergillus sp. Fusarium sp.

Carbendezine (+) 0.70 ± 0.00b 4.05±0.05a 3.75 ± 0.25a

Methanol (-) 0.00 ± 0.00c 0.00±0.00b 0.00 ±0.00c

Tuber 0.65 ± 0.05b 0.00±0.00b 0.00 ±0.00c

Stem 0.75 ± 0.05ab 0.00±0.00b 1.40 ±0.10b

Leaf 0.85 ± 0.05a 0.00±0.00b 1.85 ±0.15b

Table 4.3: Antibacteria test on different Dioscorea alata parts .

4.3.2 Fungi

Escherichia coli Bacillus subtilis Bacillus aureus

Chloramphenicol (+) 3.65 ± 0.35a 0.85 ± 0.15b 0.80 ± 0.00ab

Methanol (-) 0.00 ± 0.00d 0.00 ±0.00c 0.00 ±0.00c

Tuber 1.50 ± 0.20b 0.95 ±0.05b 1.05 ±0.15a

Stem 1.95± 0.05b 0.85 ±0.15b 1.00 ±0.00ab

Leaf 0.70± 0.10c 1.90 ±0.10a 0.75 ±0.02b

Table 4.4: Antifungal test on different Dioscorea alata parts .

82
 ANTIMICROBIAL TEST

FIGURE 4.7.1 : Inhibition zones for methanol extract of Dioscorea alata tuber against
bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp.

83
Figure 4.7.2 : Inhibition zones for methanol extract of Dioscorea alata tuber against
bacteria (a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

84
Figure 4.7.3 : Inhibition zones for methanol extract of Dioscorea alata stem against
bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp.

85
Figure 4.7.4 :Inhibition zones for methanol extract of Dioscorea alata stem against
bacteria (a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

86
FIGURE 4.7.5 : Inhibition zones for methanol extract of Dioscorea alata leaf against
bacteria (a) Penicilum sp (b)Aspergillus sp. (c)Fusarium sp.

87
FIGURE 4.7.6 : Inhibition zones for methanol extract of Dioscorea alata leaf against
bacteria (a)Escherichia coli (b) Bacillus subtilis (c) Bacillus aureus

88
5.0 DISCUSSION

89
 There are many studies and investigations that had being conducted to study the tissue

culture of monocotyledon species. But there are not too many in vitro culture of Dioscorea

alata L. species that can be compare with the present studies. From the findings, most of

the journals that are published for the studies of Dioscorea species are from country where

Dioscorea alata is considered as important source of food such as countries in Africa. The

need to grow the Dioscorea species in vitro is very important as the usage of the Dioscorea

alata is not limited and at the same time, the quality yam can be consumed by the people,

worldwide.

Present investigation is about the tissue culture studies of Dioscorea alata species.

Since there are a lot of limitation towards the consumption of this species, a dynamic effort

is put in this studies is to grown the Dioscorea alata in vitro so that we can improve its

quality and growth. This is also due to the fact that Dioscorea alata has many beneficial

effects beside acting as important source of food, it has many medicinal and industrial uses.

One of the limitation of Dioscorea alata is the disease that infected the plant itself.

There are two type of disease that can infect the yam itself; field disease and storage

disease. Example of field disease are Anthracnose, Mosaic virus, and other diseases. while

in the storage, there are dry, soft and wet rot disease. To investigate the effect of the

disease, the yam are grown in two different kind of environment, one is in the laboratory

under optimum condition while the other is grown outside of the laboratory, in the natural

environment where it will depend on the sunlight and exposed to many kind of risk such as

pest, weed and so on .

90
 The explant that been used are cut from the vine that are grown massively. There are

many kind of disease can be seen infecting the leaves and the vine itself. The plant need to

be kept in a safe area since the tuber is one of the food that been feeding the animal and

insect surrounding such as rat, mealybugs and so on. Many diseases are seems to be

infecting the vines of the yam itself such as Anthracnose, Mealybugs and so on. In Nigeria,

Anthracnose is considering as the biggest disease that been vastly infected the yam. In the

other hand, Yam mosaic virus disease is the one that cause the loss of yam in term of

quantity and quality (Mignouna, Mank et al. 2002).

Result shown that the disease that usually infect the leaves of the yam is Anthracnose.

Anthracnose can be indicate by the sign of small size dark brown spot and the leaf, or it can

also be surrounded by black lesion that accompanied by a chlorotic halo on the leaf, and

stem, necrosis of the leaf, stem dieback, scorched appearance and withered leaves (Amusa,

1991, 1997 ; IITA, 1993). The leaf shown obvious dark brown spot and almost completely

destroyed the leaf. The leaf that shown the sign of this symptoms are not recommended to

be cultured in vivo. Anthracnose is one of the most popular disease that always infecting

the yam production (Nwakiti and Arene, 1978; Simon, 1983). Fungus infection is reported

as the main cause of Anthracnose.

From this study, the effect of plant growth regulators is being

investigated. The main type of hormone that being used are auxin and Cytokinin . Auxin is

very important component in plant tissue culture because with the present of Auxin, it will

cause the adventitious roots formation, callus formation by cell division, cell elongation

and tissues swelling, inhibit the formation of adventitious and axillary shoot formation,

apical dominance and also somatic embryogenesis (Davies, 2013) . Cytokinin on the other

hand, is also integral hormone that has important function as plant growth regulators such
91
as formation of shoot and promotes cell division. When the concentration of Cytokinin is

increase, then the formation of shoot also increases while in Auxin, the higher the

concentration of Auxin, the then adventitious root will formed. When both of the

concentration of Auxin and Cytokinin are equal, then callus can be formed.

The basic medium that used in the present study was MS (Murashige and Skoog,

1962 ) which were supplement with various types and concentration of single plant growth

regulators As one of the main objective of this study , there are five different type of Plant

growth regulators is used and one control . As been stated, two type of hormone is used

which is auxin and Cytokinin . For auxin, the hormone used is IAA, NAA and 2,4-D

whereas for Cytokinin , Kinetin and BAP is used . All of this hormone are supplemented

into media containing MS medium in sterile tube. The concentration used is standardize up

to four different concentrations which is 0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2.0 mg/L for

each media. MS basal media, which is media without any Plant Growth Regulator was used

as control throughout the experiment

For this experiment, the observation is made after the explant is culture for 7 weeks.

The plant is placed in optimum environment that will support the growth which is in the

culture room. Aspect such as temperature, lighting and air is very important because the

growth of the plant will be supported besides the aseptic condition of the environment

itself. For each treatment, 20 replicate are made for each explants. This is to standardize

the observation to be made.

For shoot proliferation, from six different type of media, only one media did not

show any response toward the shoot formation which is NAA. MS basal is the media

without any exogenous hormone shown a shoot proliferation in two weeks. The percentage

92
of shoot grown is 10 percent. This mean that the endogenous hormone inside the plant itself

can support the formation of root and it contain a good Cytokinin value. It required up to 2

weeks for the explant to grow up the shoots. Das, Choudhury et al. (2013) also observed

formation of shoot of explant when MS basal is used with the mean value is 1.5. From this

experiment also stated the number of shoot formation is very slow compared to the media

that has been supplemented with exogenous hormone.

For shoot proliferation, the use of MS medium supplemented with 2.0 BAP is

showing the highest percentage of shoot growth by 70 ± 0.10a. This is the highest growth

for shoot formation when compared with other hormones. With only concentration of 0.5

mg/L, BAP has already managed to form the shoot even with 25 ± 0.10c. This is because

BAP is a synthetic Cytokinin hormone which support the formation of adventitious shoot

(Krikorian, 1995) . This can be said that for shoot formation, BAP is the best hormone to

use. Belarmino and Gonzales (2008) managed to obtain adventitious shoot when the

explant is transferred to MS media supplemented with 1.0 mg/L BAP. It indicates that with

1.0 mg/L concentration, the shoot is already start to shown a response for growth, but with

the result that obtained shown that with 0.5 mg/L concentration of BAP, the explant is

already start to show a response. This is probably due to the fact that in experiment done by

Belarmino and Gonzales, they did not use MS medium, instead they use D- 571 medium.

The use of 1.0 mg/L of BAP shows higher percentage of shoot recovery (Borges, Ceiro et

al. 2004) supporting the fact for shoot proliferation, the best media is BAP.

According to the result that obtained from experiment, BAP, which is a Cytokinin

hormone, did showed an increase in number of shoot form when the concentration of the

hormone is increase from 0.5 mg/L up to 2.0 mg/L. This shown a pattern where the

concentration of BAP is directly proportional to the percentage of growth of shoot.

93
However, this relationship is only applied to BAP, and not applied to other four hormones.

For example, in Kinetin, only MS medium supplemented with 1.0 mg/L concentration is

the only concentration that support that growth of shoot, while other concentration did not

manage to show the same effect towards the explant.

Percentage of root formation however, it not as impressive as percentage of shoot

formation. The best percentage of root formation 15±0.08a when the explant is grown on

MS medium supplemented with 1.0 IAA. For root formation, the only PGRs that support

the formation of root is 2,4-D, IAA and BAP. Most of the root formation are when the use

of 2,4-D where only 0.5 mg/L concentration of IAA do not induce the regeneration process.

In IAA, 1.0 mg/L and 2.0 mg/L concentration of IAA manage to break the dormancy of the

explant. This result is expected since both IAA and 2,4-D are synthethic Auxin, where

auxin is known to support the root formation. Das, Choudhury et al. (2013) reported that

for root formation, the best was observed in MS medium supplemented with 2.5 mg/L IAA.

To study the best explants for in vitro culture of Dioscorea alata are being

investigated which consist of leaf, stem and petiole. The reason why these three explants

were chosen was because they establish for most parts of the plant which is the vine, that

are grow for vigorously. This making the explant to be easily obtained for the experiment.

Ability of these three explants to grow a plantlet when supplemented with proper media

and plant growth hormone in vivo was investigated. The regeneration that being targeted

are both direct and indirect regeneration, where direct regeneration, the explant with

directly grown into root or shoot before becoming the complete plant. The indirect

regeneration pathway is where the plant will form callus first before forming an organ and

then also transforming into complete plant. The mean number of plantlet form from explant

is determined for all three explants. The production of vine will take up to 1 to 2 month.
94
The plant is being watered at most twice a week. The vine is showing a twinning pattern,

growing from tuber. Not all the tuber can grow a long vine since some of them remain

dormant. It is placed in the area where the sunlight and air are sufficient enough to support

its growth.

From the result obtained, only stem which consist of nodes and internodes

are showing a positive response towards the plant growth and development. From this

investigation, it is considered as the best explants for both shoot and root formation. This is

due to the fact that when using the same medium, the same result is not as progressive as

the stem part when leaves and petioles is used. From the previous experiment, Belarmino

and Gonzales (2008) has produced multiple adventitious shoots that occurred after the same

in vitro nodal explant when transferred to MS media supplemented with 1.0 mg/L BAP.

This is indicating that the nodal segment is proven to be a good explant for mass

propagation of Dioscorea alata. Besides that, Maximum shoot proliferation is obtained

with nodal segment from previous investigation of Das, Choudhury et al. (2013). In this

experiment, all result is showing unresponsive response when the leaves and petioles are

used. This is probably due to the fact that the sterilization method is not suitable because of

it is intact plant, which is very hard to remove all the contaminations and debris. The tissue

of this explant is easily get burned during sterilization process making it is not suitable for

in vitro tissue culture.

Since Dioscorea alata are very famous for their medicinal properties, an

antimicrobial test is carried out to investigate the antimicrobial properties of the part of

Dioscorea alata. Three main part of the Dioscorea alata is investigating which consist of

tuber, where it is edible part and mainly consume, Leaf and also stem. Different type of

fungus and bacteria is used to investigate the properties of Dioscorea alata toward the
95
inhibition of microbes. The inhibition zone in the petri dish will indicate the effectiveness

of Dioscorea alata against the microbes which is measured its length by ruler.

Carbendezene is used as positive control against fungus, and Chloramphenicol is used

against bacteria. Positive control is the control that already known to have the antimicrobial

properties toward each respective microbe. Methanol is used in both antifungal and

antibacterial test which act as negative control which known to have known effect in

antimicrobial analysis.

To check the antifungal properties, there type of fungi is used which is Penicilium sp,

Aspergillus sp. and Fusarium sp . Fungi has a potential to be a detrimental microorganism

either to human or the plant itself. Mycotoxin that synthesize by fungi have many

hazardous effect on animal and human, while for Dioscorea alata or other plant it can

cause disease that resulted into lossed of crop quality and quantity. Aspergillus species can

lead to disease in respiratory system of human being (Hedayati, Pasqualotto, Warn,

Bowyer, & Denning, 2007) . Zmeili and Soubani (2007) stated that it can cause invasive

pulmonary and chronic necrotizing aspergillosis. Fusarium species also will lead to

infection in human health. It has been reported in the case of immunocompetent patients,

infection by Fusarium known as Fusariosis is the main factor. Woodman's disease is caused

by hypersensitivity pneumonitis when the exposure of Penicillium sp., is type of disease

associated with this type of fungi. All this fungus can be a very dangerous threats toward

the health of human itself.

The result obtained in antifungal test is showing that all the

Carbendezine , which is the positive control are showing inhibition zone to all three part of

fungus and none of inhibition zone are showed in negative control . This is the expected

result. All three parts of the Dioscorea alata are showing an inhibition zone towards
96
Penicilium sp., but the leaf are showing a higher inhibition zone when compare with

Carbendezine. This tell us that the leaf is more effective to resist the infection of Penicilium

sp. when compare with Carbendezine and the other two parts of the Dioscorea alata. For

Fusarium sp, all parts of the explants are showing the inhibition zone towards these

microbes. However, the same result cannot be obtained with Aspergillus sp, because there

are no inhibition zones that can be seen on part of the Dioscorea alata, only on

Carbendezine. This indicate that in Dioscorea alata , this three part of the plant cannot be

used if want to make a resistance against this microbes.

Bacteria can be good and can be bad for the consumer depends on the type

of bacteria. For example, gut microbiota, a strict anaerobe located in human intestine, will

aid in digestion and also help immune system. Escherichia coli or E.coli is one the famous

bacteria that are commonly infected human . It’s a rod shape gram negative bacteria that

respire in facultative anaerobic mode and can be found in the warm-blooded organism

lower intestine. Even though some of E.coli stains are harmless but some of the strain are

known to cause fatal in babies and also in young calves because of different type gastro-

enteritis.(Roberts, COWIE, Abelson, Bolton, & Britten, 1955) . They are also known for

the cause food contamination (Nazem, Abdelshahid, & Aleem, 2016). Different compare to

Escherichia coli, Bacillus species is from gram-positive that has rod shape morphology.

There are two type of Bacillus, one that are not harmful and one pathogenic species. One of

the most important that Bacillus usage, is that it can produce enzymes that useful for

production. There are also Bacillus infection detected in cancer patients, causing anthrax

and also food poisoning.

97
 The same procedure is used with the antibacterial test. Only the difference is

Chloramphenicol is used instead of Carbendezine because Chloramphenicol is the chemical

that are known to have antibacterial properties. Different type of bacteria is used;

Escherichia coli, Bacillus subtilis and Bacillus aureus. All parts of Dioscorea alata are

showing the Antimicrobial properties as inhibition zone can be detected in the petri dish.

For Bacillus subtilis , leaf part are showing inhibition zone that are higher than positive

control ,which is up to 1.90 ±0.10a , compare to chloramphenicol which only showing 0.85

± 0.15b of inhibition zone . This indicate that the leaf part is more effective against these

bacteria. Overall, it can be said that all this three part can be use as antibacterial agent.

There are not many previous studies are reported on the antimicrobial activity of

Dioscorea alata. Wiastuti et al. (2016) reported that the association of Dioscorea alata

starch film and ginger essential oil has allowed the films to have antibacterial and radical

scavenging activities. Aderiye et al. (1996) had used the peel of Dioscorea alata which also

produced inhibition zone in the antifungal test. Since there are different part of the plant is

used, It is not a the most suitable comparison even though Dioscorea alata proven both

antifungal and antibacterial properties.

98
6.0 CONCLUSION

99
 Since Dioscorea alata has a lot of importance values for the consumer from

nutritional, pharmaceutical and daily uses due to their beneficial content, it is very

important to improve its quality and quantity so that the amount of consumer demands can

be fulfill. For example, in Africa, Dioscorea alata is always needed in a huge amount since

it has been a staple food for that region at the same time the quality of the plant is required

to be in the optimum condition to meet the consumer demands. Even though it has a lot of

limitation such as diseases, pathogens attack and dormancy period, the use of in vitro

culture can improve this problem.

Plant tissue culture is a technique used to grow the explants under a control aseptic

condition, lead to formation of clones of a plant, depends on characteristic of the plant that

able to differentiate and regenerate one new complete plant known as concept of

Totipotency . In Dioscorea alata , there are many works is done to culture the plant in vivo

by using different explants , different medium and different environments.

For Dioscorea alata , the regeneration pathway that are shown are direct organogenesis

where the explants are capable of dividing and differentiate to produce a whole plant

without formation of callus. There is no Somatic Embryogenesis is observed in this study.

This is probably due to the fact that the amount of hormone supplied to the explants in the

medium in which if the amount of Auxin is higher than Aytokinin, it will produce root and

in the other hand , if the amount of Cytokinin is more than Auxin , it will induce shoot

formation .

Shoot formation can be observed by using hormones 2,4-D, IAA, BAP, Kinetin and

MS Basal on the MS medium. Only NAA cannot induce shoot formation. The best shoot

100
formation is observed on MS medium supplemented with 2.0 mg/L BAP with 70 ± 0.10a

percentage of shoot formation. The observation is made for 7 weeks. BAP is the showing a

consistent pattern in inducing shoot formation when the higher concentration of BAP, the

higher the percentage of root formation.

For root formation, the best medium to be used is 1.0 IAA supplemented with MS

medium where 15±0.08a percentage of growth is observed. The mediums that are

supporting the root formation on Dioscorea alata is BAP, IAA and 2,4-D.

The best explant is stem which consist of nodes and internodes. This is due to the

fact that the other two explants which is petioles and leaves, are remain unresponsive

eventhough the same medium is used. This is probably due to the fact of that the

sterilization technique of the intact plant is very hard on leaves and petioles because the

tissue can get easily burned.

There are still a lot of works and prospects regarding in vitro culture of Dioscorea

alata . For the future studies, the explants should be able to easily regenerate from the

explant up to one complete plant so that it can be acclimatize and finally able to produce in

abundant amounts. Antimicrobial studies of others species of microbes should also be done

to determine the antimicrobial properties of Dioscorea alata to more types of species of

microbes especially the one that impose a constant threat to human health. Cytological

studies is another studies that can be imply for Dioscorea alata . The studies on

chromosomal number and genotype of Dioscorea alata is important. Root tip meristem is

good for Cytological studies since it contains a lot of cell that actively dividing. Genetic

modification technique is one of the best prospects in Biotechnology can also be studies

with cultivars of Dioscorea alata in order to produce more variation.

101
 From the shoots and roots that grown from the explants of Dioscorea alata, pigment

analyzing and extraction of chemical compounds can be done to compare the structure of

the organ that grown in vivo and in vitro. This can be done by using GCMS (Gas

chromatography Mass Spectroscopy) and also HPLC (High performance Liquid

Chromatography). All of this analytical techniques are using state-of-the-art technologies

that can detect various compounds by separation of the components individually.

102
7.0 REFERENCE

103
REFERENCE

1. Aderiye, B. I., Ogundana, S. K., Adesanya, S. A., & Roberts, M. F. (1996). Antifungal

properties of yam (Dioscorea alata) peel extract. Folia microbiologica, 41(5), 407-

412.Afoakwa, E. O., & Sefa-Dedeh, S. (2001). Chemical composition and quality

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8.0 APPENDICES

116
Formulations of MS (Murashige and Skoog) Media (1962)
Component mg/L
NH4NO3 1650.00
H3BO3 6.2
CaCl2 332.2
CoCl2 0.025
CuSO4.6H2O 0.025
Na2EDTA 37.26
FeSO4.7H2O 27.80
MgSO4 180.70
MnSo4.H2O 16.90
Na2MoO4 · 2H2O 0.25
KI 0.83
KNO3 1,900.00
KH2PO4 170.00
ZnSO4.H2O 8.60

Vitamins:
Myo-Inositol 100.00
Nicotene Acid 0.50
Pyridoxine HCL 0.50
Thiamine HCL 0.10

Other Components
Glycine (Free Base) 2.00
MES 500.00
Sucrose
30,000.00

117