This document is a thesis presented to the faculty of the Department of Biology at the Polytechnic University of the Philippines by Tiu, Lualhati S.D. and Javier, Amalia Carla Severina S. in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology. The thesis examines the in vitro cultivation of the Philippine native variety of Allium sativum Linn. (Ilocos White). Various concentrations of Murashige and Skoog media were tested, with and without the addition of plant growth regulators kinetin and NAA. The explants underwent direct organogenesis without a callus phase. Shoot and root formation and growth were evaluated under different treatment conditions.

1. In Vitro Cultivation of the Philippine Native Variety
of Allium sativum Linn. (Ilocos White)
A Thesis
Presented to
the Faculty of the Department of Biology
College of Science
Polytechnic University of the Philippines
Sta. Mesa, Manila
In Partial Fulfillment
of the Course Requirements for the Degree
Bachelor of Science in Biology
by
TIU, LUALHATI S.D.
JAVIER, AMALIA CARLA SEVERINA S.
2016

2. ACCEPTANCE AND APPROVAL SHEET
The undergraduate thesis entitled “In Vitro Cultivation of the Philippine Native
Variety of Allium sativum Linn. (Ilocos White),” prepared by AMALIA CARLA
SEVERINA S. JAVIER and LUALHATI S.D. TIU, presented to the faculty of
DEPARTMENT OF BIOLOGY, COLLEGE OF SCIENCE on 21st
of March 2016 in
partial fulfillment of the course requirements for the degree BACHELOR OF
SCIENCE IN BIOLOGY is hereby accepted.
MA. ELEANOR C. SALVADOR ARMIN S. CORONADO, MSc.
Adviser Chairman of the Panel
NICHELL M. VILLARTA ARCIBEL B. BAUTISTA
Member Member
Noted:
ARMIN S. CORONADO, MSc. DR. THERESITA V. ATIENZA
Chairman, Department of Biology Dean, College of Science

3. ACKNOWLEDGEMENT
The researchers would like to thank the following:
 to Mr. Ace Pascual of Bureau of Plant Industries for accommodating us and
authenticating our plant samples;
 to Professor Ma. Eleanor Calapatia-Salvador, our research adviser, for her
undying support, motivations and for simply believing us throughout the
course of this study;
 to our chairperson Professor Armin S. Coronado MSc. for allowing us to use
the isolation room of Science and Technology Laboratory in conducting our
research;
 to Professor Armin S. Coronado MSc., Chairman of the Department of
Biology and chairman of our panel; Prof. Arcibel B. Bautista, Research
Coordinator and Prof. Nichell M. Villarta, panel members for their
constructive criticisms and insights making this study better;
 to Pascua family for opening their door to accommodate us while writing
this paper;
 to Mr. Jhunel Vinarao our former professor who served as our research
consultant, for his magnificent ideas, kindness, words of encouragement
and bringing the best out of us;
 to our colleagues and friends, the BS Biology family, especially the original
BS Biology Section 2, SciTech Babies and Amalia & Friends (Lily, Jomabel,
Rose Anthonnette, Ruth, Jayson, and Charlene) who always bring smile to
us and always cheering us up when we’re down.
 to our family, our parents and extended family for their words of comfort,
inspiration and supporting us unconditionally from the beginning until to the
completion of our study; and finally,
 to Almighty God for giving us wisdom, knowledge and strength to keep
going and not to give up and finish this research study.
The Researchers

4. Table of Contents
Page No.
Acceptance and Approval Sheet ………………………………………. ii
Acknowledgement ……………………………………………………….. iii
Abstract …………………………………………………………………… v
List of Figures ……………………………………………………………. vi
List of Tables …………………………………………………………….. vii
List of Appendices ………………………………………………………. viii
Introduction …………………………………………………………….. 1
Background Information ……………………………………….. 2
Objectives of the Study ………………………………………… 3
Significance of the Study ………………………………………. 4
Scope and Limitations of the Study …………………………... 5
Review of Related Literature ………………………………………… 7
Allium sativum Linn. ……………………………………………. 7
Plant Description ………………………………………….. 7
Plant Nutritional Value ……………………………………. 8
Plant Uses …………………………………………………. 8
Planting Season and Planting Problems ……………….. 10
In Vitro Tissue Culture …………………………………………. 11
Murashige and Skoog Media …………………………………. 13
Macromolecule ……………………………………………. 13
Micromolecule …………………………………………….. 14
Iron EDTA …………………………………………………. 14
Vitamins ………………………………………………….... 14
Plant Growth Hormone ………………………………………… 14
NAA ………………………………………………………… 16
Kinetin ……………………………………………………… 16
Methodology …………………………………………………………… 18
1.0 Collection and Preparation of Samples …………………. 18
2.0 Preparation of Stock Solution ……………………………. 18
3.0 Preparation of the Culture Medium ……………………… 19
4.0 Planting of Explant to Medium …………………………… 20
4.1 Surface Sterilization …………………………………. 20
4.2 Placing of Explant to Medium ………………………. 20
5.0 Treatment Evaluation ……………………………………… 21
5.1 Effects of Concentration of Culture Medium ………. 21
5.2 Effects of Plant Growth Regulators ………………… 22
6.0 Maintenance of the Set-ups ………………………………. 23
7.0 Statistical Analyses ………………………………………… 23
Results and Discussion ……………………………………………… 24
Shoot …………………………………………………………….. 24
Roots …………………………………………………………….. 32
Summary, Conclusions and Recommendations ………………… 38
Literature Cited ………………………………………………………… 41
Appendices ……………………………………………………………… 48

5. In Vitro Cultivation and Optimization of the Philippine Native Variety
of Allium sativum Linn. (Ilocos White)
ABSTRACT
Allium sativum L. or more commonly known as garlic from family of
Amaryllidaceae is considered as one of the ten most important medicinal plants of
the Philippines by the Department of Health (DOH). In the Philippines only 8% of
the garlic in the market was being propagated locally and the rest was being
imported. Plant cell and tissue culture was widely known to be used in increasing
crop yield of important crops and enhance plant health quality.
The native variety of A. sativum L, Ilocos White. was subjected to different
cultural conditions in vitro namely effects of MS medium concentration and effects
of plant growth regulators. No callus phase was observed and the explants have
undergone direct organogenesis. All concentrations of the said factors were able
to induce shoot and root growth. Only one shoot per explant was observed.
Highest mean (32.19±23.30) shoot length was observed in full strength of
the medium for the effects of MS concentration. For the effects of PGR
concentration in shoot length, highest mean was observed in PGR12
(58.24±35.34) and lowest in PGR13 (13.03±14.44). In terms of root length highest
mean was recorded in 100% MS with 12.40±21.3 for the effects of MS
concentration and 8.00±4.94 in PGR13 for the effects of PGR concentration. In
terms of percentage response for root, medium with PGR stimulated better
response than PGR-free medium.

6. List of Figures
Figure No. Title Page No.
1 Direct Organogenesis of Explants in Growing Medium .. 25
2 Shoot Formation in MS Media without PGR ……………. 27
3 Shoot Length in MS Media ……………………………….. 28
4 Growth of One Shoot per Explant ……………………….. 28
5 Root Formation in MS Media …………………………….. 33
6 Root Length in MS Media ………………………………… 33

7. List of Tables
Table No. Title Page No.
1 Treatments for the varied concentrations and
combinations of plant growth regulators ……………….. 22
2 Effects of 100% MS media with different PGR
Concentrations on explants of Allium sativum L.
Ilocos White variety for Shoot …………………………… 30
3 Effects of 100% MS media with different PGR
Concentrations on explants of Allium sativum L.
Ilocos White variety for Root ……………………………. 34

8. List of Appendices
Appendix Title Page No.
A Macronutrient and Micronutrient composition in
Allium sativum Linn. ………………………………………
49
B Components of Murashige and Skoog Media ………… 51
C Raw Data …………………………………………………. 53
D Statistical Analysis ………………………………………. 62
E Images ……………………………………………………. 67

9. CHAPTER 1
INTRODUCTION
Background Information
Allium sativum L. or commonly known as garlic from the family
Amaryllidaceae is considered as one of the ten most important medicinal plants of
the Philippines by the Department of Health (DOH). Some of its medicinal
properties are anti-hypertensive, wound healing, anti-diabetic, anti-cancer, anti-
atherosclerosis and hypolipidemic, anti-microbial, anti-fungal, immunomodulatory,
antioxidant, anti-inflammatory, anthelmintic, anti-coagulant and fibrinolytic and
hepatoprotective (Londhe et al., 2011). Allium sativum L. is also widely used as
spice throughout the world and in agriculture it serves as bactericide and fungicide
compounds (Tokit et al., 2003).
The Philippines produces only 8% of the garlic supply in the market while
the rest is being imported from nearby countries like Taiwan and China due to low
crop yield. One of the main reasons for the inadequate supply of garlic in the
country is due to “single planting a year”. Also, garlic is exclusively being
propagated vegetatively (Novak, 1990), which often affect the health status of the
crop whereby almost all garlic cloves are being contaminated by one or more
pathogens, mainly viruses (Mehta et al., 2013) which cause around 3% to 45%
reduction in yield (Bhojwani, 1980). Boosting local production is not easy when
imports have driven garlic farmers out of the business for many years. Hence, it is
important to come up with a proper protocol for plant cell and tissue cultures in

10. in vitro cultivation of A. sativum L. for its fast crop production that is also free from
any plant diseases and contamination.
Plant cell and tissue culture plays an important role in the plant
regeneration, micropropagation and manipulation of plants for improved crop
varieties. Cells and tissues of many plant species are difficult to culture and also
to establish optimal growing conditions in vitro (Lee et al., 2009). A number of
chemical and physical factors like media components, light effect and plant growth
regulators have been studied extensively to contribute to the efficiency of in vitro
cultivation. Manipulation of physical aspects and nutritional elements in a culture
is perhaps the most fundamental approach for optimization of culture productivity
(Mulabagal and Tsay, 2003). Besides the optimization of physical factors, other
important aspects affecting plant regeneration includes explant type, the
physiological condition of the explant, genotype and the growth regulator
combination used in the culture medium (Scotton et al., 2013).
Different techniques in plant cell and tissue culture are presented by many
researchers, one of these is meristem culture technique. It has been widely used
for the production of virus-free plants because these meristem cells are free or
almost free of virus (Salomon, 2002). Explants that can be used in cell and tissue
culture in plants particularly in garlic are frequent such as stem disc meristem,
shoot apex, root tips and leaf explants (Peat and Jones, 2012; Roksana et al.,
2002; Haque et al., 1998; Feroel et al., 2002). Researchers that have conducted
experiments proved that the use of plant growth regulators is essential for shoot-
root multiplication and proliferation of A. sativum L. in cell and tissue culture

11. (Roksana et al., 2002). ). 6-furfurylaminopurine (Miller et al., 1955a, 1955b, 1956),
a plant hormone which is included in a class now referred to as cytokinins (Skoog
et al., 1965) was proposed to have the trivial name kinetin and it was described by
its ability to promote cell division (Amasino, 2005). Another sort of plant growth
regulator, a-naphthaleneacetic acid (NAA) which is a type of auxin that induce
callus formation and proliferation as well as somatic embryogenesis is commonly
used in plant cell and tissue culture (De Klerk et al.,1998).
Although many researches, have been made to subject different varieties
of A. sativum L. for in vitro culture, the Philippines is still in need of the proper
protocol of propagating different native varieties. There must be an easy way on
how this species can be mass propagated in good health condition, free from any
plant diseases in a short period of time in order to provide the country’s needs.
Objectives of the Study
The main goal of this study was to determine the best cultural conditions for
the in vitro cultivation of the Philippine native variety of Allium sativum L. Ilocos
white.
The specific objectives of this study were:
1. to determine the effects of varying concentrations of MS media without any
plant growth regulation and 100% MS media added with different
concentrations of plant growth regulators in terms of inducing formation of
new shoots and roots;

12. 2. to determine the effects of varying concentrations of MS media without
anyplant growth regulators in terms of shoot and root length;
3. to determine the effects of 100% MS media added with different
concentrations of plant growth regulators in terms of shoot and root length;
4. to evaluate which among the treatments gave best result in terms of both
shoot and root length.
Significance of the Study
With its prominent importance in the field of culinary, the countless
capabilities of garlic in the world of medicine is also conspicuous, making it one of
the most known and imperative crops in the world. The Philippines, unlike its
neighboring countries such as Taiwan and China has a low production of the said
crop. Garlic in the market are mostly imported from the countries mentioned earlier
and only 8% of these are native to the Philippines. In the country, garlic is being
cultivated in the field which makes it prone to the negative effects of environmental
conditions while other countries produce their crops in laboratories where
circumstances can be controlled, resulting to larger and good crop yield. The
‘single-planting a year’ and lack of technological innovation that focuses mainly on
garlic contributes to the insufficient production of the said crop in the country.
The Philippines is in need of ways to boost the production of garlic, in order
to diminish the amount of the crop being imported. Researches regarding mass
propagation of garlic by in vitro process are not new to other countries but in the

13. native varieties of A. sativum L. in the Philippines, this method is not yet very well
tested. Although researches are being steered in the country, no standard protocol
has been recognized yet that will solve the problem at hand.
The study was conducted to serve as a baseline research regarding in vitro
cultivation of the Ilocos variety of Allium sativum L. that can be used to increase
the amount of garlic being produced as well as to enhance its health quality in a
more convenient and faster way. Importing from nearby countries can be
prevented and there will be enough or even ample supply of garlic that can be
exported, which may help the economy of the Philippines. The beneficiaries of the
study are the Department of Agriculture (DA), Agribusiness and Marketing.
Scope and Limitations of the Study
This research dealt mainly on the determination of the optimum cultural
conditions for the in vitro cultivation of the Philippine native variety of A. sativum L.
(Ilocos white). It was based on the effect of concentration of medium and the effect
of plant growth regulators. The garlic samples were purchased from Divisoria and
was identified by the researchers. Authentication was through Mr. Ace Pascual, an
agriculturist from Bureau of Plant Industry. The research was exclusively
conducted in vitro and acclimatization on soil was not included.
For the effect of concentration of medium, the researchers discussed only
the effect of concentration of MS (Murashige and Skoog, 1962). The researchers

14. presented the results of MS salt medium in varying concentrations namely 100%,
75%, 50% and 25%.
For the effect of plant growth regulators (PGR), kinetin and a-
naphthaleneacetic acid (NAA) were added in to the culture medium singly or
combined, in different concentrations to induce shoot and root differentiation and
elongation.
All of the explants were placed in glass jars with plastic lid that was bought
from Divisoria and was sterilized using autoclave at approximately 1210
C, 15 psi
for 20 minutes. All of these set-ups were placed in a growing chamber in the
isolation room of the Polytechnic University of the Philippines Sci-tech which were
provided with white fluorescent light for about 16 hours a day. There was five trials
in each cultural condition with three replicates. Data was gathered at the 21st
day
after placing the explant in the media. Length of the shoot, root length as well as
the number of roots per explant were recorded. For the statistical analysis, SPSS
One-Way ANOVA with Post-Hoc and Tukey Test were used to determine if there
was a significant difference in the effect of the different cultural conditions. The
level of significance was set at p <0.05.

15. CHAPTER 2
REVIEW OF RELATED LITERATURE
Allium sativum Linn.
Allium sativum Linn. is commonly known as garlic. Other common names
include ajo in Spanish, ail in French, arishtha and lashuna in Sanskrit, lasan in
Hindu and Gujarat and vellaipundu in Tamil, Suan in Chinese, Toi in Vietnamese,
alho in Portugese.
Plant description
The taxonomy position of Allium and the related genera had been a
controversy for a long time (Fritsch and Friesen, 2002).
The genus Allium is widely distributed over the warm-temperate and
temperate zones of northern hemisphere and it occurs in boreal zone (Stavelikova,
2008). Garlic, is an herbaceous, annual (surviving only one growing season),
bulbous plant in the family Amaryllidaceae grown for its pungent, edible bulb of the
same name. The garlic plant can either have a short, woody central stem
(hardneck) or a softer pseudostem made up of overlapping leaf sheaths
(softneck). True stem is much reduced or very short and flattened which gives way
to a pseudostem. Bulbs are broadly ovoid, 2 to 4 centimeters in diameter and is
made up of 1-15 cloves, consisting of several, densely crowded, angular and
truncated tubers. The garlic plant can possess 6-12 linear, flat and blade-like
leaves that range up to 50 cm in length. Umbels are globose, many flowered.

16. Sepals are oblong, greenish white, slightly tinged with purple. Stamens are not
exerted from the perianth. Garlic is believed to originate from Asia.
Plant nutritional value
Garlic’s main core nutrients include high levels of vitamins C and B6
(Hedges and Lister, 2007), flavonol in garlic is mycetin. The bulk of garlic’s dry
weight is composed of fructooligosaccharides, followed by sulfur compounds,
protein, fiber and the free amino acids (Rahman and Lowe, 2006). Aside of sulfur
compounds, garlic also contains high levels of saponins, some phenolics and
moderate levels of provitamin A (Rahman and Lowe, 2006). Organosulfur
compounds is present in garlic gamma glutamylcysteines and cysteine sulfoxides.
Allicin, an intermediate breakdown of Allylcysteine sulfopoxide, or alliin, is thought
to be responsible for the odour of fresh garlic (Rahman, 2003 and Higdon, 2005).
The major flavonoids in garlic are the flavonols, myricetin and apigenin and, low
levels of quercetin (Lanzotti, 2006). It also contains phenolic compounds which
becomes the interest of many researches because of its antioxidant activity
(Hedges and Lister, 2007), A number of sapogenins (the aglycone base) and
saponins have also been identified in garlic, (Matsuura 2001; Lanzotti 2006).
Plant uses
The garlic is the second most important Allium species. It is grown
worldwide as an important spice and medicinal plant. The bulb, composed of few
to too many cloves, is the main economic organ. The fresh leaves, pseudostems

17. and bulbils (topsets) are also consumed (Fritsch and Friesen, 2002). The first
mention of garlic is 6,000 years old; Sumerians, Egyptians or Jews used it. The
parts of garlic plants were found also in Southern Moravia. They come from 2000
B.C. (Lužný and Vaško, 1982). Since ancient times, garlic and related species
have been widely used in many parts of the world as vegetables, as well as in
traditional folk medicine.
Garlic’s medical applications are recorded in ancient Egyptian, Greek,
Roman, Indian and Chinese writings, for a host of complaints from bee stings to
dog bites and headaches to hair loss (Hedges and Lister, 2007). The major health
issues that garlic is thought to protect against include cardiovascular disease,
cancer and other age-related problems such as loss of brain function (Hedges and
Lister, 2007). Furthermore, garlic has strong antimicrobial activity against a wide
range of organisms (Hedges and Lister, 2007), assortment of therapeutic effects
have also been reported, including hypolipidaemic, anti-atherosclerotic,
hypoglycaemic, anticancer, anticoagulant, as an antidote for heavy metal
poisoning, antihypertensive, liver protective, antimicrobial immunomodulatory
(Banerjee et al. 2003), antibacterial effect of garlic juices as described by Louis
Pasteur, antioxidant activity, prevention of diabetes, it can also prevent cataracts
and macular degeneration and arthritis, improve blood circulation and decrease
skin wrinkling (Hedges and Lister, 2007). In addition, recent studies about the anti-
microbial properties of garlic extract was investigated and being incorporated into
functional foods to replace synthetic preservatives (Hedges and Lister, 2007).

18. Planting season and planting problems
Garlic is grown in many the countries today: China, India and Republic of
Korea are its principal producers (Stavelikova, 2008). Russian Federation, Ukraine
and Spain are then the biggest producers of garlic in Europe (FAO, 2008). The
time of planting differs from region-to-region. Garlic is one of the most important
commodities in the Philippines not only today, but also during the pre-Hispanic era.
With its agro-climatic suitability, the Ilocos region is the main producer of garlic
bulbs in the Philippines with 65% of the country's average total production per year.
Ilocos White is the most common variety of garlic planted for commercial
production in the country, preferred over other varieties by local consumers
because of its pungent aroma and tangy taste, the Ilocos white is considered a
major cash crop by its growers. But even though the Ilocos white has good qualities
as a crop, its production is still relatively low and this compels the government to
import garlic from other countries to meet the local demand. The garlic planting
season in Ilocos Norte is from May to October.
Garlic can be a very easy-to-grow herb in the garden, however, it is also
prone to several diseases. These include, but are not limited to: Basal Rot
(Fusarium culmorum), White Rot (Sclerotium cepivorum), Downy Mildew
(Peronospora destructor), Botrytis Rot (Botrytis porri) and Penicillium Decay
(Penicillium hirsutum). Most of the major garlic diseases are soil-born, so proper
site assessment and yearly rotations are crucial in maintaining a healthy garden of
garlic (Plant Disease Diagnostic Clinic). In addition to these diseases, garlic is also
subject to damage by several genera of nematodes.

19. In vitro Tissue Culture
Garlic is vegetatively propagated by planting the clove into the soil and
because of this, virus or diseases from the mother plant can be transferred to the
emerging plantlet. Thus, yield is reduced. Another problem in planting crops in soil
is that they are susceptible to environmental factors that can affect good growth.
One of the tools that is exercised up to this day in producing virus-free plants and
increasing yield is tissue culture technology. Experimental systems based on plant
cell and tissue culture are characterized by the use of isolated parts of plants,
called explants, obtained from an intact plant body and kept on, or in a suitable
nutrient medium. Compared to the use of intact plants, the main advantage of
these systems is a rather easy control of chemical and physical environmental
factors to be kept constant and the growth and development of various plant parts
can be studied without the influence of remote material in the intact plant body
(Neumann et al., 2009).
There are different techniques that are used in tissue culture technology,
some are plant propagation, meristem culture and somatic embryogenesis. In this
approach, mostly isolated primary or secondary shoot meristems are induced to
shoot under aseptic conditions. Generally, this occurs without an interfering callus
phase, and after rooting, the plantlets can be isolated and transplanted into soil.
Thereby, highly valuable single plants can be propagated. The main application,
is in horticulture for mass propagation of clones for the commercial market, another
being the production of virus-free plants. Thus, this technique has received a broad

20. interest in horticulture, and also in silviculture as a major means of propagation
(Neumann et al., 2009).
Researches regarding cell and tissue culture in Allium sativum L. started in
1970, among the many studies conducted is about micropropagation of garlic. This
technique was proved to be advantageous over clove reproduction, as it only
requires cells or small tissue fragments to generate high number of plants
(Robledo-Paz and Tovar-Soto, 2012). Organ or meristem culture technique is one
of the morphogenetic ways to carry out micropropagation. This technique has been
widely used for the production of virus-free clones and will be practiced in this
study.
Virus elimination through meristem culture is based on the fact that these
meristematic cells are free or almost free of virus and therefore the plants
regenerated from them will also be virus-free (Salomon, 2002). The two distinctive
meristems (a group of stem cells) are the shoot apical meristem (SAM) and the
root apical meristem (RAM). The SAM develops shoot organs and tissues in
peripheral regions, whereas the RAM differentiates several types of root tissues in
proximal and distal directions to form the root proper and root cap, respectively
(Machida et al., 2013). Explants for the meristem culture in A. sativum L. can be
obtained from the different plant organs such as in the shoot, root as well as in the
inflorescences, cloves, bulbils and stem disc (Mariconi et al., 1990; Scotton et al.,
2013; Verbek et al., 1995 and Peat and Jones, 2012). The stem disc which is
situated in the basal part of the clove will be the explant for this study. The two
surface of the stem disc which is the basal part and the apical part which contain

21. meristematic tissues will likely develop into root tissue or into shoot tissue,
respectively. The same type of explant from the cultivar Fukuchi-howaito was used
by Ayabe and Sumi (1998) for micropropagation of garlic.
Murashige and Skoog Media
Optimal growth and morphogenesis of tissues may vary for different plants
according to their nutritional requirements (Saad and Elshahed, 2012). However,
tissues from different parts of the plants may also have different requirements for
satisfactory growth (Murashige and Skoog, 1962). Murashige and Skoog (MS)
medium is one of the basic medium that is frequently used. A plant tissue culture
media should generally contain some or all of the following components:
macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements,
source(s) of carbon, undefined organic supplements, growth regulators and
solidifying agents (Saad and Elshahed, 2012).
Macromolecule
Stock solutions of the macro nutrients are usually prepared of 10 times
concentration of the final strength and stored at +4 0
C temperature.
Micromolecule
Micronutrients stock solutions are generally made up of 100 times the
concentration of their final strength and stored at +4 0
C temperature.
Iron EDTA

22. Stock solution of iron are generally prepared at 10 times concentration of
the final medium and stored at +4 0
C temperature.
Vitamins
Vitamins are prepared 100 times the concentration of the final strength and
stored at -20 0
C temperature.
Plant Growth Hormones
Plant growth regulators are important in plant tissue culture since they play
vital roles in stem elongation, tropism, and apical dominance (Saab and Elshahed,
2012). They are generally classified into the following groups; auxins, cytokinins,
gibberellins and abscisic acid. Moreover, proportion of auxins to cytokinins
determines the type and extent of organogenesis in plant cell cultures (Skoog,
1957).
A. Auxins
The common auxins used in plant tissue culture media include: indole-3-
acetic acid (IAA), indole-3- butric acide (IBA), 2,4-dichlorophenoxy-acetic acid
(2,4-D) and naphthalene- acetic acid (NAA) (Saab and Elshahed, 2012). IAA is the
only natural auxin occurring in plant tissues There are other synthetic auxins used
in culture media such as 4-chlorophenoxy acetic acid or p-chlorophenoxy acetic
acid (4-CPA, pCPA), 2,4,5-trichloro-phenoxy acetic acid (2,4,5 T), 3,6- dichloro-2-
methoxy- benzoic acid (dicamba) and 4- amino-3,5,6-trichloro-picolinic acid
(picloram) (Torres, 1989).

23. Auxins differ in their physiological activity and in the extent to which they
translocate through tissue and are metabolized. In tissue cultures, auxins are
usually used to stimulate callus production and cell growth, to initiate shoots and
rooting, to induce somatic embryogenesis, to stimulate growth from shoot apices
and shoot stem culture (Saab and Elshahed, 2012).
B. Cytokinins
Cytokinins commonly used in culture media include BAP (6-
benzyloaminopurine), 2iP (6-dimethylaminopurine), kinetin (N-2-furanylmethyl-1H-
purine-6-amine), Zeatin (6-4-hydroxy-3-methyl-trans-2-butenylaminopurine) and
TDZ (thiazuron-N-phenyl-N-1,2,3 thiadiazol-5ylurea) (Saab and Elshahed, 2012).
In culture media, cytokinins proved to stimulate cell division, induce shoot
formation and axillary shoot proliferation and to retard root formation (Saab and
Elshahed, 2012).
C. Gibberellins
Gibberellins comprise more than twenty compounds, of which GA3 is the
most frequently used gibberellin. These compounds enhance growth of callus
(Vasil, 1998) and help elongation of dwarf plantlets (Torres, 1989). It enhances
shoot proliferation and inhibits later stages of embryo development (Anagnostakis,
1974). Other growth regulators are sometimes added to plant tissue culture media
as abscisic acid, a compound that is usually supplemented to inhibit or stimulate
callus growth, depending upon the species (Saab and Elshahed, 2012).

24. NAA
1-naphthaleneacetic acid (NAA) is the most effective auxin (Paek and
Yeung, 1991), increases the growth rate of the fruit (Ortola et al. 1990).
Kinetin
6-furfurylaminopurine (Miller et al., 1955a, 1955b, 1956), is a plant hormone
in a class now referred to as cytokinins (Skoog et al., 1965). They proposed the
trivial name kinetin for this substance, and described its ability to promote cell
division in test tissues from tobacco (Nicotiana tabacum) (Amasino, 2005). 6-
Benzyladenine, now a commonly used cytokinin, was the first of several highly
active cytokinins to be discovered by Skoog and Strong and their co-workers quite
soon after the discovery of kinetin. The effect of kinetin on developmental
processes that were also affected by red light, such as leaf expansion (Miller,
1956) and seed germination (Miller, 1958), was discovered. Other groups quickly
discovered other developmental processes that could be influenced by cytokinin,
such as leaf senescence (Richmond and Lang, 1957). Kinetin was the first highly
active member of the cytokinin class of plant hormones to be discovered (Amasino,
2005).

25. CHAPTER 3
METHODOLOGY
1.0 Collection and Preparation of Samples
The Philippine native variety of A. sativum L. namely the Ilocos White was
purchased from the local market at Divisoria. Cloves from the bulbs of A. sativum
L. Ilocos white variety were separated and dry scales were removed. The peeled
cloves were placed in a glass jar and were stored in a refrigerator at 4°C
approximately a day before it was planted in the media to enhance growth and
especially shoot development (Ayabe and Sumi 1998, 2001).
2.0 Preparation of Stock Solution
Stock solutions of Murashige and Skoog Premedium was first prepared.
Macronutrient stock solution was made by weighing specified amount of
ammonium nitrate (NH4NO3), calcium chloride (CaCl2. 2H2O), magnesium
sulphate (MgSO4. 7H2O), potassium phosphate (KH2PO4), and potassium nitrate
(KNO3) and was added to 1 liter of distilled water. For the micronutrients stock
solution, the chemicals boric acid (H3BO3), cobalt chloride (CoCl2. 6H2O), cupric
sulphate (CuSO4. 5H2O), manganese sulphate (MnSO4. 4H2O), potassium iodide
(KI), sodium molybdate (Na2MoO4. 2H2O) and zinc sulphate (ZnSO4. 7H2O) were
weighed and was dissolved in a 1 liter of distilled water. The same was done with
the Iron EDTA stock solution which is composed of sodium EDTA (Na2 EDTA.
2H2O) and ferrous sulphate (FeSO4. 7H2O). Vitamins stock solution was also

26. prepared consisting of myo-inositol, thiamine HCL, nicotinic acid, and pyridoxine
HCl. All of these stock solutions were placed in a refrigerator until it was used.
3.0 Preparation of Culture Medium
For the full strength concentration of MS media, 100ml of the macronutrients
stock solution, 10ml of micronutrients, 10ml of Iron EDTA and another 10 ml of
vitamins were mixed together in a graduated cylinder and distilled water was added
until it reached the marker of 1000ml. The resulting solution was added with 30g
of sugar and 0.02 mg of glycine. The mixture was heated then 9g of agar bar was
dissolved. About 30ml of the media was dispensed in glass bottles, capped tightly
and these were sterilized in the autoclave at 121o
C, 15 psi for 30 minutes.
The other different concentrations of MS media were prepared by
diminishing the macronutrients into 75ml, 50ml, and 25ml for the 75%, 50% and
25% concentrations, respectively, different amounts of kinetin and NAA namely
0.5mg/L, 1.0mg/L and 1.5mg/L were added singly or combined together with the
stock solutions of MS media before heating. The said plant growth regulators were
first dissolved in a small amount of NaOH before it was added to the solution and
the steps mentioned earlier were repeated.

27. 4.0 Planting of Explant to Culture Media
4.1 Surface Sterilization of Explant
Scalpel, forceps, beakers and petri dishes were autoclaved at 1210
C, 15psi at
about 30 minutes duration and were again moved over a flame to further sterilize
before using. Peeled cloves were subjected to 1:10 dishwashing liquid for 30
minutes, then transferred to a beaker with 70% ethyl alcohol for 5 minutes and was
soaked into 1:10 sodium hypochlorite solution for 30 minutes. The sterilized peeled
cloves were washed with double distilled water thrice. This process was done
inside a glass chamber which was fumed and spewed with anti-bacterial and anti-
fungal spray (Lysol disinfectant spray and Lysol disinfectant all purpose cleaner 4
in 1 spray).
4.2 Placing of Explant to Culture Medium
Sterilized scalpel was used to remove the hard and brown colored portion
at the basal part of the clove and from there 2mm of flesh clove was cut again
which was known as the stem disc (Peat & Jones, 2012) that served as the explant
for this study. The forceps was flared first over blue flame, was cooled for several
seconds and was used to put the explants in the glass jar which has the MS media
and was covered with a plastic lid. In a single glass jar, eight (8) explants were
placed.

28. 5.0 Treatment Evaluation
5.1 Effect of Concentration of Culture Media
MS (Murashige and Skoog, 1962) medium was used as the culture medium
in the study and it was prepared in full strength, 75%, 50% and 25%. The varying
concentrations differed only in the amount of macronutrients used in making the
media.
5.2 Effect of Plant Growth Regulators
Plant growth regulators were added to the medium to observe its effects on
the cultures. 6-furfurylaminopurine more commonly known as kinetin, a type of
cytokinin which induce mostly shoot and root differentiation and elongation, and a-
naphthaleneacetic acid (NAA) which is a type of auxin which induce callus
formation and proliferation as well as somatic embryogenesis (De Klerk et al,.
1998) were both added singly with 0.5 mg/L, 1.0mg/L, and 1.5mg/L concentrations
in the culture medium. Three concentrations of kinetin (0.5mg/L, 1.0mg/L, and
1.5mg/L) were combined with the same concentrations of NAA and the resulting
solution were added to the culture medium.

29. Table 1. Treatments of the varied concentrations and combinations of plant growth
regulators.
Treatment Kinetin Concentration
(mg/L)
NAA Concentration
(mg/L)
CONTROL 0 0
PGR1 0 0.5
PGR2 0 1.0
PGR3 0 1.5
PGR4 0.5 0
PGR5 1.0 0
PGR6 1.5 0
PGR7 0.5 0.5
PGR8 0.5 1.0
PGR9 0.5 1.5
PGR10 1.0 0.5
PGR11 1.0 1.0
PGR12 1.0 1.5
PGR13 1.5 0.5
PGR14 1.5 1.0
PGR15 1.5 1.5

30. 6.0 Maintenance of the Cultures
All the glass jars containing the media with the inoculated explants were
placed in the growing chamber inside the isolation room of the Science and
Technology laboratory in Polytechnic University of the Philippines, Sta. Mesa
Campus. The growing chambers were provided with white fluorescent tubes with
a 40v and were left open at approximately 16 hours per day. Daily monitoring was
done to record the growth of shoots and roots in each explant. Observation was
done virtually and presence of shoots and roots as well as the number of roots
from the explants in a single glass jar were noted.
7.0 Statistical Analyses
All the data collected specifically the shoot and root length and the number
of roots per explant in the varying cultural conditions were analyzed using SPSS
One-Way ANOVA with Post-Hoc and Tukey Test. Level of significance was set at
p<0.05 and mean and standard error mean.

31. CHAPTER 4
RESULTS AND DISCUSSION
Shoot
Daily monitoring was done and data were gathered and analyzed after 21
days of culture for both shoot and root growth in the different cultural conditions.
In all treatments, only one shoot per explant was recorded. Data for shoot length
for the effects of MS concentration and effects of PGR concentration were
summarized in Figure 3 and Table 2, correspondingly. Highest mean (32.19±2)
shoot length was observed in full strength of the medium, followed by 14.08±27.31
of 75% MS for the effects of MS concentration. For the effects of 100% MS with
varying PGR concentration, highest mean 58.24±3 was observed in 1.5mg/L Kn +
1.0mg/L NAA (PGR14) followed by 1.5mg/L Kn + 0.5mg/L NAA (PGR13) with
41.58±2. Lowest means were observed in 1.5mg/L NAA combined with varying
concentrations of Kn (PGR9, PGR12 and PGR15). It was observed that as the
concentration of NAA alone (PGR1, PGR2 and PGR3) increases, shoot length
significantly deacreses and no significant difference in the lengths of the shoot in
concentrations of Kn alone (PGR4, PGR5 and PGR6) was recorded.
All the explant did not undergo callusing and direct organogenesis occurred
as showed in Figure 2 which is considered an edge over other results in this field
of study. Luciani et al., (2006) grown tissue cultured A. sativum explants in
darkness for callus induction as well as Scotton et al., (2013). However,

32. Figure 2. Direct organogenesis of explants in growing medium.
according to Alizadeh et al., (2013) callusing is not desirable when the aim is to
propagate true to type material because it might lead to somaclonal variations. In
garlic, In vitro organogenesis occurs indirectly when explants are cultivated in the
dark, which provides conditions for callus formation and regeneration of
adventitious meristems from callus cells (Scotton et al., 2013).
All concentrations of MS media without any PGR were able to induce shoot
growth. It is evident based on Figure 3, that the 100% MS media without PGR has
the fastest response and Figure 4 showed that as the concentration of MS media
without PGR increases so does the shoot length. Although this showed an
increasing trend, only the 100% MS media concentration without PGR has a
significant difference to all other concentrations tested. It was stated by Saad &

33. Elshahed (2012) that full strength of salts in media proved good for several
species, but in some species the reduction of salts level to ½ or ¼ of the full
concentration gave better results in in vitro growth. In contrast to this finding,
Roksana et al., (2002) reported in the study they conducted which is in vitro bulblet
formation of A. sativum L. using shoot apex that no shoot proliferation was
observed in medium without any plant growth regulators. Furthermore, the full or
half strength of MS media without any PGR failed to induce regenerated shoots
according to Mehta et al., (2013) which conducted a study in in vitro propagation
of A. sativum L. through callus induction.
Plant hormones regulate many aspects of plant growth and development.
Both auxin and cytokinin have been known for a long time to act either
synergistically or antagonistically to control several significant developmental
processes, such as the formation and maintenance of meristem that are essential
to establish the whole plant body (Su et al., 2011).
It was shown in Figure 5 that only one shoot per explant was observed
during the entire period of the study which might be due to the cytokinin-auxin
interaction. Shimizu-Sato et al., (2009) stated that cytokinin-auxin interaction is
involved in the predominant shoot apex growth, which inhibits the outgrowth of
axillary buds. Consequently, cytokinin level might not be enough to induce axillary
bud growth as Su et al., (2011) reported that reduced levels of cytokinin increase
apical dominance and inhibit axillary bud formation. Results reported by Tanaka et
al., (2005) indicated that in apical dominance one role of auxin is to repress local

34. biosynthesis of cytokinin in the nodal stem which are thought to be derived from
the roots.
The SAM (shoot apical meristem) generates almost all of the aerial parts of
the plant and there is active cell division and cell differentiation in this that occurs
region. As mentioned earlier highest the shoot length that can be seen in Table 2
was means for observed in the combined concentrations of the two plant growth
regulators used in the study where Kn (cytokinin) has greater proportion
Figure 3. Shoot response of varying concentrations of MS media without PGR on explant
of Allium sativum L. Ilocos White.
0%
20%
40%
60%
80%
100%
120%
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21
growthpercentage
Shoot Formation In MS Media
25% MS 50% MS 75% MS 100% MS

35. Figure 4. Effects of varying concentrations of MS media on explant of Allium sativum L.
Ilocos White in terms of shoot length.
Figure 5. Explants from 25%, 50%, 75% and 100% MS media without PGR (2 explants
each from left-right respectively.
0
5
10
15
20
25
30
35
25% MS 50% MS 75% MS 100% MS
Lengthinmm
Treatments

36. over NAA (auxin) which are 1.5mg/L Kn + 1.0mg/L NAA (PGR14) and 1.5mg/L Kn
+ 0.5mg/L NAA (PGR13). It has been known for a long time that cytokinin plays a
major role in regulating meristem function. Studies on the classical chemical
regulation of growth and organ formation in in vitro plant tissues have indicated
that excess of cytokinin over auxin promotes shoot formation (Skoog & Miller,
1957). This suggests a positive action of cytokinin on SAM activity. Recent studies
have shown that cytokinin deficiency reduced shoot meristem size and activity
(Werner et al., 2003; Higuchi et al., 2004; Werner and Schmulling, 2009).
Lowest means of shoot length were recorded in the combined
concentrations of PGR where amount of NAA is higher than that of Kn which are
1.5mg/L NAA combined with varying amounts of Kn (PGR9, PGR12 and PGR15)
and 0.5mg/L Kn + 1.0mg/L NAA (PGR8) set-ups. It was stated by Nordstrom et al.,
(2004) that auxin has recently been shown to rapidly down-regulate cytokinin
biosynthesis in the shoot. In addition, auxin accumulation, facilitated by the efflux
or influx carriers to various organ initiation sites, suppresses the expression of STM
(SHOOTSTEMLESS) (Furutani et al., 2004; Heisler et al., 2005). According to
Clark et al., (1996); Endrizzi et al., (1996); and Long et al., (1996), STM is required
for the maintenance of stem cells in the meristem, and its expression in the whole
shoot meristem prevents stem cells from switching to organ-specific cells. Because
STM has been proved to be a positive factor of cytokinin biosynthesis (Jasinski et
al., 2005; Yanai et al., 2005), a model is proposed that

37. Table 2. Effects of MS media with different PGR concentrations on explant of Allium
sativum L. Ilocos variety for Shoot
Treatment
No.
Kn
(mg/L)
NAA
(mg/L)
Shoot Formation
Growth Rate (%)
Shoot Length (mm)
(mean±SE)
Control - - 100% 32±2.13def
PGR1 - 0.5 90.01% 36±1.35ef
PGR2 - 1.0 76.16% 29±1.28cde
PGR3 - 1.5 58.01% 17±2.03ab
PGR4 0.5 - 100% 33±2.15def
PGR5 1.0 - 100% 31±2.18cdef
PGR6 1.5 - 100% 38±2.76ef
PGR7 0.5 0.5 85.69% 25±2.04bcd
PGR8 0.5 1.0 51.93% 16±1.91ab
PGR9 0.5 1.5 49.33% 13±1.32a
PGR10 1.0 0.5 95.83% 28±2.16cde
PGR11 1.0 1.0 72.14% 34±2.59def
PGR12 1.0 1.5 65.53% 21±2.02abc
PGR13 1.5 0.5 100% 42±2.33f
PGR14 1.5 1.0 100% 58±33.23g
PGR15 1.5 1.5 88.71% 13±1.71a
auxin antagonizes cytokinin for organ initiation in the peripheral zone of the
meristem (Su et al., 2011). The significant decrease in shoot length as the
concentration of NAA alone (PGR1, PGR2 and PGR3) increases which is shown
in Table 2 can be associated with the aforementioned statement that auxin
accumulation suppresses expression of STM.
In the study conducted by Gull et al., (2014) they found out that Kn was an
ideal phytohormone for the shoot formation of garlic as it was noted that by

38. increasing the concentration of Kn to 1.5mg/L in MS media significant increase in
shoot length was observed but further increase in the concentration showed a
decline in the shoot length. Furthermore, studies conducted by Mehta et al.,
(2013a) and Mehta et al., (2013b) reported that 1.0mg/L Kn stimulated the best
response in shoot proliferation and elongation of A. sativum by callus induction
and using node explants. This report was not in line with the researchers’ finding
that there was no significant difference observed in the shoot length of the explants
treated in the three (3) varying concentrations of Kn namely 0.5mg/L, 1.0mg/L and
1.5mg/L (PGR4, PGR5 and PGR6).
For shoot elongation, it was found out that adding PGR in the medium is
best to apply. Highest mean of shoot in 100% MS media without any PGR
increased almost two-fold when supplied with PGR. Furthermore, it was also found
out that adding combined PGR to the media werein cytokinin is higher in
concentration that auxin in in vitro culture of Allium sativum resulted to longer
shoots per explant instead of using only one type of PGR. In the study conducted
by Roksana et al., (2002) combination of cytokinins and auxins is better for early
establishment of shoot apex than using cytokinin alone, also Kondo et al., (2000)
proposed that medium supplemented with cytokinin and auxin promoted the best
rates of regeneration for all cultivars of Allium sativum from Brazil that were tested
in their study. In addition, Scotton et al., (2013) reported that regeneration
frequency for media containing auxin alone varied between 6.6%-8.9%, whereas
the combination of auxin plus cytokinin resulted in 30%-48% regeneration.

39. Roots
Highest mean (12.40±2) root length was observed in full strength of the
medium, followed by 9.08±1 of 75% MS for the effects of MS concentration. For
the effects of 100% MS with varying PGR concentration, highest mean (32.19±2)
was observed in Control followed by 8.00±0.5 in PGR9 (0.5mg/L Kn + 1.5mg/L
NAA). Lowest means were observed in PGR8 (0.5mg/L Kn + 1.0mg/L NAA). It was
observed that as the concentration of NAA alone (PGR1, PGR2 and PGR3)
increases root length increases. However, there is no significant difference
between the three concentrations. While in the concentration of Kn alone (PGR4,
PGR5 and PGR6) it was observed that as the concentration increases the root
length decreases. PGR4 (0.5mg/L Kn) showed significant difference between
PGR5 and PGR6 (1.0mg/L Kn and 1.5mg/L Kn respectively).
It is evident that as the concentration of MS increases without PGR, root
percentage response and root length also increases as shown in Figure 5 and
Figure 6, respectively. However, based on statistical analysis, full strength and
75% MS are not significantly different with each other but both concentrations are
significantly different to 25% and 50% MS concentration. In some plant species,
MS media without PGR will not promote shoot and root formation. However, the
results obtained from the study shows that the root induction was observed in
almost all the compositions even in the hormone free MS media. Same with the
results of Gull and colleagues (2014), who studied about the micropropagation of
A. sativum.

40. Figure 5. Root response of varying concentrations of MS media without PGR on explant
of Allium sativum L. Ilocos White.
Figure 6. Mean values of root length for the effects of MS concentration.
0%
20%
40%
60%
80%
100%
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21
Root Formation In MS Media
25% MS 50% MS 75% MS 100% MS
0
2
4
6
8
10
12
14
25% MS 50% MS 75% MS 100% MS
Lengthinmm
Treatments

41. Data gathered for roots was summarized in Table 3. Results for varying
concentrations of NAA alone, revealed increasing concentration exhibited
increasing root length, contrary to the results of Kn which showed that as the
concentration increases, root length decreases. It was shown in Table 7 that the
highest mean of root length treated with NAA alone was observed in 1.5mg/L NAA
(PGR3) but it doesn’t show significant difference compared with those treated with
0.5mg/L NAA and 1.0mg/L NAA (PGR1 and PGR2). NAA belongs to the group of
auxins which are usually use to stimulate growth from shoot apices and shoot stem
culture (Saab & Elshahed, 2012).
Table 3. Effect of 100% MS media with different PGR oncentrations on explant of Allium
sativum L. Ilocos White for roots
Mean±SE, n=120
Means sharing the same superscript are not significantly different from each other (Tukey’s, p
<0.05)
Treatment No. Kn (mg/L) NAA (mg/L) Growth Rate (%) Root Length (mm) (mean±SE)
Control - - 23.08 32.19±1.98c
PGR1 - 0.5 42.4 1.97±0.18ab
PGR2 - 1 58.21 4.21±0.22ab
PGR3 - 1.5 50.16 4.70±0.28b
PGR4 0.5 - 52.48 7.86±0.98c
PGR5 1 - 28.71 3.74±0.41ab
PGR6 1.5 - 23.44 2.95±0.40ab
PGR7 0.5 0.5 20.55 2.65±0.27ab
PGR8 0.5 1 11.9 1.46±0.18a
PGR9 0.5 1.5 68.43 8.00±0.45c
PGR10 1 0.5 37.28 2.77±0.22ab
PGR11 1 1 22.51 2.90±0.26ab
PGR12 1 1.5 38.16 2.41±0.24ab
PGR13 1.5 0.5 25.29 1.72±0.21a
PGR14 1.5 1 35.7 2.57±0.24ab
PGR15 1.5 1.5 57.57 3.08±0.20ab

42. In Kn alone, highest mean of 7.86±1 for root length was observed in 0.5mg/L
Kn (PGR4) which is significantly different from all other mean root length in
concentrations of NAA and Kn alone (PGR1, PGR2, PGR3, PGR5 and PGR6). Kn
is a type of cytokinin that is proven to stimulate cell division, induce shoot formation
and axillary shoot proliferation and promote root formation. Cytokinin was proved
more effective as compared to media fortified with different auxins in garlic (Gull
et. al., 2014).
When NAA is combined with Kn, it is evident that there is a significant
difference in the highest mean for root length (8.00±0.5) observed in 0.5mg/L Kn
+ 1.5mg/L NAA (PGR9) with the other combined treatments. The balance of the
amounts of auxin and cytokinin concentration is necessary in determining the
optimal amount of PGR which varies in every plant (Su et. al., 2001). Interaction
of auxin-cytokinin in in vitro regulates meristem development (Su et. al., 2001) and
in garlic NAA-cytokinin combination is required (Patena, et. al., 1991). Rapid
elongation and differentiation of cells leaving the root meristem has been
demonstrated to be controlled by auxin (Rahman, et. al. 2007) thus higher amount
of NAA than Kn is desired. The decision of whether a cell culture stays in the
proliferating status of new organs, such as shoots or roots, depends on the
concentration ratio between these 2 plant hormones (Skoog and Miller, 1957).
Root induction was observed in almost all the concentrations even in the
hormone free 25% MS media same with the results obtained in the study of Gull
et. al. (2014), on the micropropagation of Allium sativum which is in contrast to the
results of other studies that, full or half strength of MS media without any PGR

43. failed to induce rooting (Mehta, et. al., 2013). Furthermore, some reports stated
that the rooting of garlic does not need phytohormones and that it can be easily
achieved on hormone free MS media (Lu and Dong, 1982; Metwally and Zanata,
1996).
As shown in Table 3, highest response for root formation in NAA alone was
observed in PGR2 (1.0mg/L NAA) same with the results of Gull et. al. (2014), who
found that the best concentration of NAA for rooting response is 1.0mg/L NAA
using shoot meristem as the explant. Concentration of NAA below and above
1.0mg/L decreases rooting reponse (Gull et. al. 2014). However, studies showed
that as the concentration of Kn increases rooting reponse using shoot meristem as
an explants also increase (Gull et. al. 2014). In contrast to the results obtained
showed in Table 7, that as the concentration increases rooting reponse decrease
using stem disk. For the combined NAA and Kn, PGR9 (0.5mg/L Kn and 1.5mg/L
NAA) showed the highest rooting reponse of all the treatments (MS without PGR,
100% MS with added varying concentratons of NAA and 100% MS with added
varying concentratons of Kn alone and 100% MS Media combined with different
concentrations of NAA and Kn). Cytokinin functions as a positive regulator of auxin
(generally for root formation) biosynthesis and auxin, however, represses cytokinin
biosynthesis (Su, et. al., 2011).
For root elongation, it was found out that 100% MS adding PGR in the
medium is best to apply. Highest mean of root was in control containing 100% MS
media without PGR followed by PGR9 (0.5mg/L Kn + 1.5mg/L NAA) however
response increased almost twice when 100% MS supplied with combined auxin-

44. cytokinin. PGR13 (0.5mg/L Kn + 1.5mg/L NAA) had the highest rooting response
to all treatments.
The results of this study showed considerable variation over other findings
in the in vitro propagation of Allium sativum reported by Roksana et al., (2002);
Mehta et al., (2013a) and Mehta et al., (2013b). In in vitro propagation of A. sativum
L., the most important factors affecting plant regeneration are the explant type, the
physiological condition of the explant, the genotype and the growth regulator
combination used in the culture medium (Myers & Simon 1998, 1999; Barandiarian
et al., 1997, 1999a, b, c; Robledo Paz et al., 2000; Luciani et al., 2001; Sata et al.,
2000; Fereol et al., 2002). The said factors might have cause the variations in the
results obtained.
In the study conducted by Luciani et al., (2006), to determine effects of
explants in in vitro culture of A. sativum, it was found out that root tips and immature
umbels were less responsive and showed delayed callus induction and plant
regeneration, compared with basal plates and meristems which performed better
as initial explants. Moreover, Scotton et al., (2013) reported that analyses of
variance revealed statistical differences for the eight (8) cultivars of A. sativum from
various Brazilian market groups, they tested for the in vitro cultivation of
marketable garlic cultivars, thus supporting the earlier claim that genotype causes
variation in the results. In addition to that, Barandiaran et al., (1999a) stated that
genotype influences in vitro responsiveness, suggesting that protocols should be
optimized for each cultivar.

45. CHAPTER 5
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Allium sativum L. or commonly known as garlic from the family
Amaryllidaceae is considered as one of the ten most important medicinal plants in
the Philippines by the Department of Health (DOH). The Philippines produces only
8% of the garlic supply in the market while the rest is being imported from nearby
countries. Low crop yield of garlic is due to the ‘single planting a year’ and almost
all garlic cloves are being contaminated with pathogens, mainly viruses (Mehta et
al., 2013) which caused around 3% to 45% reduction yield (Bhojwani, 1980)
because it is propagated vegetatively. Plant and tissue culture can be used in order
to boost yield of garlic and also to produce virus-free plants (Salomon, 2002). The
main goal of this study was to define the optimum cultural conditions for the in vitro
culture and micropropagation of the Philippine native variety of Allium sativum L.
Ilocos white. Specifically it aimed: (1) to determine the effects of MS media
concentration and varied concentration of plant growth regulators in terms of shoot
length, root length, and the number of root growth per explant; to evaluate which
among the different cultural conditions gave the best result in terms of the shoot
and root length and growth per explant and; to serve as a baseline study in the
field of in vitro culture of the Philippine native variety of Allium sativum L. (Ilocos
White).
The plant specimen was purchased from Divisoria and authenticated at
Bureau of Plant Industries by Mr. Ace Pascual, an agriculturist. Peeled cloves of
garlic were placed in a glass jar and was stored in a refrigerator at 4 o
C a day

46. before planting. Stock solutions of Murashige and Skoog medium were prepared
and were placed in a refrigerator until used. For the effects of MS media without
PGR, four (4) concentrations were prepared namely 100%, 75%, 50% and 25%
and 15 treatments for the effects of PGR concentration which is composed of Kn
(cytokinin) and NAA (auxin). Peeled cloves were sterilized in dish washing liquid,
ethyl alcohol and sodium hypochlorite, three times in double distilled water
consecutively and stem disc was excised. Explants were then placed in glass jars
containing different concentrations of MS medium with or without PGR. All glass
jars were situated in a growing chamber with 16h photoperiod per day supplied
with 40v fluorescent tubes.
All concentration of MS media without PGR and 100% MS media added
with varying concentrations of PGR induced shoot and root growth. In all
treatments, only one shoot per explant was recorded and multiple root formation
was observed. For the effects of MS media concentration, highest response for
shoot was recorded in the full strength while explants in 75% MS medium exhibited
best response. Highest response for shoot and root for the effects of PGR
concentration were recorded in the combination of 1.0mg/L NAA + 1.5mg/L Kn and
1.5mg/L NAA + 0.5mg/L Kn, respectively.
Based on the results, the following were concluded:
1.) all concentration of MS media free from phytohormone and 100% MS added
with varying concentrations of plant growth regulators were able to induce
shoot and root formation;

47. 2.) for shoots, 100% MS media with 1.5mg/L Kn + 1.0mg/L NAA (PGR14) is
best for promoting fastest response and longest roots;
3.) for roots, 100% MS media with 0.5mg/L Kn + 1.5mg/L NAA (PGR9) is best
for promoting fastest response and longest shoots;
4.) for both shoot and root induction and elongation 100% MS without any plant
growth regulator showed best result.
Upon the completion of this study, the following are recommended:
1.) use of other varieties of Allium sativum, explant type, plant growth
regulators and different combinations as well as culture medium can be
practiced to determine variations in the results obtained;
2.) plant growth regulators for shoot initiation must be introduced first followed
by plant growth regulators for the induction of roots;
3.) longer experimental period and sub-culturing of explant to fresh medium
after several weeks can be performed for better response;
4.) age and size of the explants must also be considered in in vitro culture;
5.) transferring of in vitro cultivated plantlets from explants of Allium sativum
to soil can be performed to determine survival rate; and,
6.) other factors such as effects of light, temperature, and carbohydrate source
can be added to the tested aspects to determine better response.

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55. APPENDICES

56. APPENDIX A
Micro and Macronutrient composition in Allium sativum Linn.
Table 1. Micro and Macronutrient compositions in Allium sativum L. Athar et al.
(2004).
Composition Garlic cloves, raw, peeled
Water 64.3 g
Energy 97 kcal
Protein 7.9 g
Total fat 0.6 g
Carbohydrate, available 15 g
Dietary fiber (Englyst 1988) 8 g
Ash 1.5 g
Sodium 4 mg
Phophorus 170 mg
Potassium 620 mg
Calcium 19 mg
Iron 1.9 mg
Beta-carotene equivalents T μg
Total vitamin A equivalents T μg
Thiamin 0.13 mg
Riboflavin 0.04 mg
Niacin 0.4 mg
Vitamin C 17 mg
Cholesterol 0 g
Total saturated fatty acids 0.122 g
Total monounsaturated fatty acids 0.015 g
Total poly unsaturated fatty acids 0.342 g
Dry matter 35.7 g
Total nitrogen 1.27 g
Glucose 0.4 g
Fructose 0.6 g
Sucrose 0.57 g
Lactose 0 g
Maltose 0 g
Total available sugars 1.6 g
Starch 13.4 g
Alcohol 0 g
Total niacin equivalents 1.5 mg

57. Soluble non-starch polysaccharides 5.5 g
Insoluble non-starch polysaccharide 2.5 g
Energy 402 kJ
Magnesium 25 mg
Manganese 500 μg
Copper 0.06 mg
Zinc 1 mg
Selenium 2 μg
Retinol 0 μg
Potential niacin from tryptophan 1.1 mg
Vitamin B6 0.38 mg
Folate, total 5 μg
Vitamin B12 0 μg
Vitamin D 0 μg
Vitamin E 0.01 mg

58. APPENDIX B
Components of Murashige and Skoog Media:
Table 1B. Macronutrient Stock Solution (10X)
Ingredient Amount Per litre in MS
(g/L)
Amount of per litre of
stock (g/L)
NH4NO3 1.65 16.50
KNO3 1.9 19.00
CaCl2•2H2O 0.44 4.40
MgSO4•7H2O 0.37 3.70
KH2PO4 0.17 1.70
Table 2B. Micronutrient Stock Solution (100X)
Ingredient Amount Per litre in MS
(g/L)
Amount of per litre of
stock (g/L)
H3BO3 0.0062 0.62
MnSO4•4H2O 0.0223 2.230
ZnSO4•H2O 0.0086 0.860
KI 0.000083 0.0083
NaMO4•2 H2O 0.00025 0.025
CuSO4•5H2O 0.000025 0.0025
CoCl2•6H2O 0.000025 0.0025
Table 3B. Iron-EDTA Stock Solution (10X)
Ingredient Amount Per litre in MS
(g/L)
Amount of per litre of
stock (g/L)
FeSO4•7H2O 0.0278 0.278
Na2EDTA•2H2O 0.0373 0.373
Table 4B. Vitamin Stock Solution (100X)
Ingredient Amount Per litre in MS
(g/L)
Amount of per litre of
stock (g/L)
Nicotinic acid 0.0005 0.05
Pyridoxine•HCl 0.0005 0.05
Thiamine•HCl 0.0001 0.01
Myo-inositol 0.01 1

59. Table 5B. Other Components
Ingredient Amount Per litre in MS
(g/L)
Amount of per litre of
stock (g/L)
Glycine (Amino Acid) 0.002 0.02
Sucrose ( Carbon
Source)
30
Agar (Solidifying Agent) 9

60. APPENDIX C
Raw Data
Shoot and Root Monitoring Every 3 days in Varying Concentrations of MS without PGR.

62. Shoot and Root Monitoring Every 3 days in 100% MS Media Added with Varying
Concentrations of Plant Growth Regulators.

69. APPENDIX D
Statistical Analyses
Mean Shoot Length per Explant, Standard Deviation and Standard Error in MS
Concentration without PGR
Mean Std. Deviation Std. Error
25% MS 7.7250 12.50409 1.14146
50% MS 12.7000 19.26577 1.75872
75% MS 14.0750 27.31490 2.49350
100% MS 32.1917 23.29543 2.12657
Total 16.6729 23.17905 1.05797
One Way Analysis of Variance using Shoot Length for MS Concentrations without PGR
Shoot_length
Sum of Squares df Mean Square F Sig.
Between Groups 41211.606 3 13737.202 30.253 .000
Within Groups 216140.042 476 454.076
Total 257351.648 479
Tukey’s (Homogeneous Subsets) for Shoot Length for MS Concentrations without PGR
Shoot_length
MS_Concentration N Subset for alpha = 0.05
1 2
Tukey HSDa
25% 120 7.7250
50% 120 12.7000
75% 120 14.0750
100% 120 32.1917
Sig. .098 1.000
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 120.000.

70. Mean Shoot Length, Standard Deviation and Standard Error for 100% MS with varying
concentrations of PGR
One Way Analysis of Variance using Shoot Length for PGR Concentrations
Sum of Squares df Mean Square F Sig.
Between Groups 251818.866 15 16787.924 30.507 .000
Within Groups 1047782.758 1904 550.306
Total 1299601.624 1919
Tukey’s (Homogeneous Subsets) for Shoot Length for MS Concentrations without PGR
Mean Std. Deviation Std. Error
Control 32.1917 23.29543 2.12657
0.5mg/L NAA 36.3250 14.80130 1.35117
1.0mg/L NAA 28.8333 14.05412 1.28296
1.5mg/L NAA 17.0500 22.29438 2.03519
0.5mg/L Kn 33.2500 23.57190 2.15181
1.0mg/L Kn 31.3250 24.99652 2.28186
1.5mg/L Kn 38.2250 30.24868 2.76131
0.5mg/L NAA + 0.5mg/L Kn 25.7083 22.43099 2.04766
0.5mg/L NAA + 1.0mg/L Kn 28.1417 23.63387 2.15747
0.5mg/L NAA + 1.5mg/L Kn 41.5750 25.47751 2.32577
1.0mg/L NAA + 0.5mg/L Kn 16.2667 20.89097 1.90708
1.0mg/L NAA + 1.0mg/L Kn 34.0500 28.42116 2.59448
1.0mg/L NAA + 1.5mg/L 58.2417 35.34321 3.22638
1.5mg/L NAA + 0.5mg/L Kn 13.0250 14.44169 1.31834
1.5mg/L NAA + 1.0mg/L Kn 21.0667 22.10697 2.01808
1.5mg/L NAA + 1.5 Kn 13.0667 18.75393 1.71199
SHOOT_LENGTH
PGR_CONC N Subset for alpha = 0.05
1 2 3 4 5 6 7
Tukey
HSDa
1.5mg/L NAA +
0.5mg/L Kn
120 13.0250
1.5mg/L NAA + 1.5
Kn
120 13.0667

71. Mean Root Length per Explant and Standard Deviation in MS Concentration without
PGR
Mean Std. Deviation Std. Error
25% MS .5978 .63663 .05812
50% MS .5885 .83663 .07637
75% MS 9.0842 10.92798 .99758
100% MS 12.4049 21.30551 1.94492
Total 5.6689 13.03499 .59496
One Way Analysis of Variance using Root Length for MS Concentrations without PGR
1.0mg/L NAA +
0.5mg/L Kn
120 16.2667 16.2667
1.5mg/L NAA 120 17.0500 17.0500
1.5mg/L NAA +
1.0mg/L Kn
120 21.0667 21.0667 21.0667
0.5mg/L NAA +
0.5mg/L Kn
120 25.7083 25.7083 25.7083
0.5mg/L NAA +
1.0mg/L Kn
120 28.1417 28.1417 28.1417
1.0mg/L NAA 120 28.8333 28.8333 28.8333
1.0mg/L Kn 120 31.3250 31.3250 31.3250 31.3250
Control 120 32.1917 32.1917 32.1917
0.5mg/L Kn 120 33.2500 33.2500 33.2500
1.0mg/L NAA +
1.0mg/L Kn
120 34.0500 34.0500 34.0500
0.5mg/L NAA 120 36.3250 36.3250
1.5mg/L Kn 120 38.2250 38.2250
0.5mg/L NAA +
1.5mg/L Kn
120 41.5750
1.0mg/L NAA +
1.5mg/L
120 58.2417
Sig. .361 .125 .057 .297 .068 .058 1.000
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 120.000.

72. Sum of Squares df Mean Square F Sig.
Between Groups 13027.698 3 4342.566 30.238 .000
Within Groups 68359.636 476 143.613
Total 81387.334 479
Tukey’s (Homogeneous Subsets) for Root Length for MS Concentrations without PGR
MS_ROOT_LENGHT
MS_CONCENTRATION N Subset for alpha = 0.05
1 2
Tukey HSDa
50% MS 120 .5885
25% MS 120 .5978
75% MS 120 9.0842
100% MS 120 12.4049
Sig. 1.000 .140
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 120.000.
Mean Root Length, Standard Deviation and Standard Error for 100% MS with varying
concentrations of PGR’
Treatment Mean Std. Deviation Std. Error
Control 10.7914 21.70949
0.5mg/L NAA 1.9702 1.91806 1.98180
1.0mg/L NAA 4.2087 2.37984 .17509
1.5mg/L NAA 4.6964 3.03729 .21725
0.5mg/L Kn 7.8592 10.72320 .27727
1.0mg/L Kn 3.7433 4.53348 .97889
1.5mg/L Kn 2.9529 4.37884 .41385
0.5mg/L NAA + 0.5mg/L Kn 2.6517 2.99234 .39973
0.5mg/L NAA + 1.0mg/L Kn 2.7738 2.44958 .27316
0.5mg/L NAA + 1.5mg/L Kn 1.7193 2.34214 .22361
1.0mg/L NAA + 0.5mg/L Kn 1.4646 1.98031 .21381
1.0mg/L NAA + 1.0mg/L Kn 2.8950 2.86881 .18078
1.0mg/L NAA + 1.5mg/L Kn 2.5740 2.62269 .26188
1.5mg/L NAA + 0.5mg/L Kn 7.9976 4.93527 .23942
1.5mg/L NAA + 1.0mg/L Kn 2.4149 2.67439 .45053
1.5mg/L NAA + 1.5mg/L Kn 3.0835 2.23973 .24414

73. One Way Analysis of Variance using Root Length for 100% MS with varying
concentrations of PGR
Sum of Squares df Mean Square F Sig.
Between Groups 12523.772 15 834.918 18.539 .000
Within Groups 85748.941 1904 45.036
Total 98272.713 1919
Tukey’s (Homogeneous Subsets) for 100% MS with varying concentrations of PGR
ROOT_LENGTH
PGR_CONCENTRATION N Subset for alpha = 0.05
1 2 3
Tukey HSDa
1.0mg/L NAA + 0.5mg/L Kn 120 1.4646
0.5mg/L NAA + 1.5mg/L Kn 120 1.7193
0.5mg/L NAA 120 1.9702 1.9702
1.5mg/L NAA + 1.0mg/L Kn 120 2.4149 2.4149
1.0mg/L NAA + 1.5mg/L Kn 120 2.5740 2.5740
0.5mg/L NAA + 0.5mg/L Kn 120 2.6517 2.6517
0.5mg/L NAA + 1.0mg/L Kn 120 2.7738 2.7738
1.0mg/L NAA + 1.0mg/L Kn 120 2.8950 2.8950
1.5mg/L Kn 120 2.9529 2.9529
1.5mg/L NAA + 1.5mg/L Kn 120 3.0835 3.0835
1.0mg/L Kn 120 3.7433 3.7433
1.0mg/L NAA 120 4.2087 4.2087
1.5mg/L NAA 120 4.6964
0.5mg/L Kn 120 7.8592
1.5mg/L NAA + 0.5mg/L Kn 120 7.9976
Control 120 10.7914
Sig. .109 .116 .058
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 120.000.

74. APPENDIX E
Images
Sample Materials
MS Medium Stock Solution

75. Plant Growth Regulators
Planting of Explants to MS media

76. Maintenance of Culture

77. Plantlets from in vitro culture of Allium sativum