sign in sign up
<<
Home , Acetic acid, Naphthalene

PH0400003

The Effects of Varying of Growth Regulators Benzyladenine and Acetic and Gamma Irradiation on the Gross Plantlet Morphology of Garcinia mangostana L.

Ma. Fatima Nona M. Bonsol

Jade Marie Edenvirg F. Lasiste

Ma. Franchesca S. Quinio

A Research Paper Submitted to the Faculty of the Philippine Science High School Diliman Campus in Partial Fulfillment Of the Requirements in Research 2

February 2004 APPROVAL SHEET

This research work entitled "The Effects of Varying Concentrations of Growth

Regulators Benzyladenine and Naphthalene and Gamma Irradiation on the

Gross Plantlet Morphology of Garcinia mangostana L.n by Ma. Fatima Nona M. Bonsol,

Jade Marie Edenvirg F. Lasiste, and Ma. Franchesca S. Quinio, which was presented to the faculty of the Philippine Science High School Diliman Campus in partial fulfillment of the requirements in Science and Technology Research 2, is hereby accepted.

Gerel Roa Research Adviser

11 ACKNOWLEDGEMENT

The group would like to express their utmost gratitude:

To the Almighty God; if not for the mental strength He supplied the group with they would have given up on this research long ago.

To the parents, for their overwhelming support throughout the conduction of the project; for providing free transportation and lending a financial hand.

To the Philippine Nuclear Research Institute's (PNRI) Nuclear Training Group (NTG), who helped in arranging all the papers needed to be able to work in the laboratory.

To Mr. Apolinar B. Asencion, who tried to find a research that can be worked on.

To Mrs. Avelina G. Lapade, who shared her written materials to increase the group's knowledge on the subject, and for allowing us to work on part of their research; and to Mrs. Marie Veluz and Kuya Manny, who were always around to give advice on the different laboratory techniques, and for guiding the members every step of the way.

To Mr. Gerel Roa, who tried to make the members see their mistakes as best as he could; for the encouragement, support, and guidance.

To Mr. Jun Samson, for allowing the group to maximize the use of the computer, and for lending them the glassware and chemicals needed.

To. Mrs. Alma Calud and Mr. Ellery Jose, for being ready to give advice at all times.

To Mr. Bert Barcelo, Mr. Rafael Santiago, Ms. Joanne Pe Benito, and Ms. Malou Capundag, for giving the group permission to juggle their class schedules in order to work at the PNRI whenever needed.

To Mr. Alex Alix, Mr. Gerel Roa, Mr. Delfin Angeles, Mrs. Mercy Sanchez, Ms. Melissa Cardenas, Mrs. Alma Calud and Mrs. Sofia Docto, for buying the excess mangosteen from the group.

To classmates and friends, whose little deeds aided us in getting through the year, amid the hustle.

in ABSTRACT

Mangosteen (Garcinia mangostana L.) is regarded as one of the best- flavored fruits in the world. Though this fruit yields high income and has great potential as an export, productivity in the Philippines is still limited due to its slow and difficult conventional propagation. To increase the plants' productivity, growth regulators can be

added, the seeds can be exposed to irradiation to cause genetic mutations, and

micropropagation techniques can be used. This study seeks to determine the effects of these three variables on the general morphological characteristics of the plantlets grown both in vitro and in vivo.

The first set of mangosteen seeds was inoculated and grown in Murashige and

Skoog (MS) basal culture medium with different treatment combinations of growth

regulators, naphthalene acetic acid (NAA) and benzyladenine (BA). Treatment

concentrations ranged from 0 parts per million (ppm) to 6 ppm. The treatment that best

induced stem, leaf and root formation, and produced plantlets with long stems and roots,

was MS with 6 ppm NAA combined with 6 ppm BA. A second set of seeds was then

exposed to varying levels of gamma radiation and propagated in vivo and in vitro. For in

vivo, it was observed that the length of stem of the plantlet decreases with increasing

radiation dose; length of leaves of the mangosteen plantlets was best noted in seeds

exposed to 10 Gy gamma rays. In in vitro, the two treatment concentrations that best

helped seeds to develop callus, which were 0 ppm BA with 4 ppm NAA and 4 ppm BA

with 6 ppm NAA, were added to MS basal medium. Seeds exposed to 5 Gy and grown in

MS basal medium with 0 ppm BA and 4 ppm NAA were observed to have developed the

most callus.

IV TABLE OF CONTENTS

TITLE PAGE

Title i

Approval Sheet ii

Acknowledgement iii

Abstract iv

Table of Contents v

List of Tables vi

List of Figures vii

Introduction 1 Background of the Study 1 Statement of the Problem 1 Significance of the Study 2 Scope and Limitations 2

Review of Related Literature 4

Methodology 13

Results and Discussion 22

Summary and Conclusion 33

Recommendations 35

Bibliography 36

Appendices 37 Appendix A: Complete Data Tables 37 Appendix B: T-Test Calculations 47 LIST OF TABLES

TABLE TITLE PAGE

1 Chemicals Used in the Preparation of Stock Solutions 14

2 Other Chemicals Used in the Preparation of MS Basal Medium 15

3 Volume of BA and Distilled Added to Treatments 15

4 Volume of NAA and Distilled Water Added to Treatments 16

5 The Sixteen Treatments of Growth Regulators for Optimization 17

6 Treatment Combinations for Callus Formation 18

7 Averages Observed for the Optimization of Growth Regulators 22

8 Averages Observed for Callus Formation 23

9 Averages Observed for In Vivo Planting 23

10 Summary of Optimum Treatments 32

11 Complete Data for the Optimization of Growth Regulators 37

12 Complete Data for Callus Formation 40

13 Complete Data for Stem Length in In Vivo Planting 41

14 Complete Data for Leaf Length in In Vivo Planting 44

15 T-Test Calculations for In Vitro Root Length. 47

16 T-Test Calculations for In Vitro Stem Length. 47

17 T-Test Calculations for In Vitro Leaf Length. 48

18 T-Test Calculations for In Vivo Stem Length. 48

19 T-Test Calculations for In Vivo Leaf Length. 48 vi LIST OF FIGURES

FIGURE TITLE PAGE

1 In Vivo Planting (Block 1) 19

2 In Vivo Planting (Block 2) 20

3 In Vivo Planting (Block 3) 20

4 Average Area of Seed Covered with Callus of In Vitro Plants in 24 Different Combinations of Growth Regulators 5 Average Root Length of In Vitro Plants in Different 24 Combinations of Growth Regulators 6 Average Stem Length of In Vitro Plants in Different 26 Combinations of Growth Regulators 7 Average Leaf Length of In Vitro Plants in Different 26 Combinations of Growth Regulators 8 Average Root Number of In Vitro Plants in Different 27 Combinations of Growth Regulators 9 Average Number of Stems of In Vitro Plants in 27 Different Combinations of Growth Regulators 10 Average Leaf Number of In Vitro Plants in 28 Different Combinations of Growth Regulators 11 Percentage Formation of the Parts of In Vitro Plants in Different 29 Combinations of Growth Regulators 12 Size of Callus for Two Growth Regulator Treatments at 29 Different Doses of Irradiation 13 Average Leaf and Stem Length of Mangosteen Exposed to 30 Different Amounts of Irradiation

Vll INTRODUCTION

Background of the Study

One of the most praised of tropical fruits and certainly the most esteemed in the family GiUifferae, the mangosteen, Garcinia mangostana L, is almost universally known. It is usually eaten fresh or preserved into candies. It also has a variety of uses, since it can be used medicinally and at the same time as a raw material for building objects. Filipinos employ a decoction of the leaves and bark as a febrifuge to treat , dysentery and urinary disorders. In Thailand, it has been used to make handles for spears, pounders and is employed in construction and cabinetwork.

Though mangosteen yields high income, the Philippines still has limited productivity of its fruits due to the fact that this crop is seasonal and only bears fruits during its peak season. Furthermore, the Philippines is finding it hard to propagate mangosteen due to the fact that its conventional vegetative propagation is difficult while various methods of grafting have been unsuccessful. There is also a low of this fruits due to poor cultural management practices and lack of awareness of this crop.

Statement of the Problem

There are three ways of increasing the plants' productivity. One is through the use of growth regulators, the other involves exposing the seeds to irradiation to cause genetic mutations, and the last is by using micropropagation techniques. This study aims to determine the growth regulator concentrations that would best induce long and numerous roots, stems and leaves, and the most formation of callus, in seeds planted in vitro. It also seeks to establish the growth regulator treatment and irradiation dose combination that would produce seeds planted in vitro with the most callus formed, and to ascertain the

1 effects of irradiation on the stem and leaf lengths of the seeds grown in vivo. Suitability of tissue culture for mangosteen will also be examined.

It is hypothesized that there is a significant difference in the morphological characteristics observed among the different treatments of growth regulators. A

significant difference is also expected among the seeds exposed to varying levels of gamma irradiation, both in vivo and in vitro.

Significance of the Study

The morphological changes that are expected from the irradiation of the plants'

seeds, and from the treatment with growth regulators, include development of plantlets with long and multiple roots, stems, and leaves, and the formation of the most callus.

Long and numerous roots, stems and leaves are signs of greater productivity for the plants, as they function for higher absorption of nutrients, stronger vertical support, and

more through photosynthesis, respectively. In addition, longer leaves with the same

width also reduce the plants' water loss due to transpiration; plants like these minimize

water use in irrigation. Having the most callus formation will help in the faster

propagation of the plant. On the other hand, tissue culture, if proven successful in the propagation of mangosteen, can solve the problem of limited production for the plant.

Collectively, successful results may signify a higher chance of mangosteen production for

exports, and may thus help in the alleviation of the economy.

Scope and Limitations

Mangosteen seeds of the Davao variety, with the same size and plumpness, were

used throughout the whole experiment. Stock solutions of micronutrients, vitamins, and

the growth regulators benzyladenine (BA) and naphthalene acetic acid (NAA), both with a of 20 parts per million (ppm), were prepared prior to MS medium preparation. Only the macronutrients were reweighed every time a new set of medium was made, as storage of its stock solution caused precipitate to form. Absolute© distilled water and Biolife© agar was used. The 70% solution and 20% Chlorox© solution that was needed for surface sterilization also came from their respective stock solutions.

The cotton plugs were a combination of Care© and Purity© cottons.

The optimization part of the experiment involved sixteen treatment combinations of the growth regulators BA and NAA, and ten replicates for each treatment. The in vitro planting of irradiated mangosteen seeds involved two treatments for the established optimum growth regulators treatments for callus formation, and six treatments for the amount of radiation to which the seeds were exposed. There were twelve replicates for each combined treatment of irradiation and concentration of growth regulators. After inoculation, the test tubes will be placed in a room with cool , high humidity, and sufficient fluorescent light.

For the in vivo planting of the irradiated mangosteen seeds, there were six doses of gamma radiation treatments, the same dose treatments used in in vitro culture: 0 gray

(Gy), 5 Gy, 10 Gy, 20 Gy, 30 Gy, and 40 Gy. Instead of , Perlite© was used, as it contained all the nutrients needed for plant growth. The plants were exposed to adequate sunlight and were watered as often as needed. REVIEW OF RELATED LITERATURE

Mangosteen (Garcinia mangostana L.) is regarded as one of the four most delicious and best-flavored fruits in the world. It is not as commonly grown as rambutan in the Wet Tropics, but it is gaining popularity because of its high economic value. Its production, however, is limited, due to its strict climatic requirements, its short seed viability, absence of a rapid method of propagation, slow plant growth and delayed maturity or long juvenile phase.

The mangosteen tree is very slow-growing, erect, with a pyramidal crown; it attains 6 to 25 m in height and has dark-brown or nearly black, flaking bark, with the inner bark containing yellow, gummy, bitter latex. The evergreen, opposite, short-stalked leaves are either ovate-oblong or elliptic, leathery and thick, dark-green, slightly glossy above, and yellowish-green and dull beneath; its measurement is usually from 9 to 25 cm long, 4.5 to 10 cm wide, with a conspicuous, pale midrib; new leaves are rosy. Flowers, 4 to 5 cm wide, and fleshy, may be male or hermaphrodite on the same tree. The former are in clusters of 3 to 9 at the branch tips. The flowers are borne singly or in pairs at the end or on the terminal raceme on the outer canopy. Although perfect flowers are borne, they are effectively unisexual since is not produced at any stage. The flowers are large, creamy-yellow in color, and are tinged with red (Chaup, 1998).

The fruit, capped by the prominent calyx at the stem end and with 4 to 8 triangular, flat remnants of the stigma in a rosette at the apex, is round, dark-purple to red-purple, and is smooth externally; it ranges from 3.4 to 7.5 cm in diameter. The rind is

0.6 to 0.10 cm thick, its cross-section red, yet purplish-white on the inside. It contains bitter yellow latex and a purple, staining juice. There are 4 to 8 triangular segments of

4 snow-white, juicy, soft flesh. The fruit may be seedless or may have 1 to 5 fully developed seeds, ovoid-oblong, somewhat flattened, 2.5 cm long and 1.6 cm wide, which cling to the flesh (Morton, 1987). The flesh is slightly acid and mild to distinctly acid in and is acclaimed as exquisitely luscious and delicious.

The mangosteen is a very slow growing tree and the bearing age is determined by growth rate. Poor nutrition and inadequate irrigation may delay fruiting from 15 to 20 years instead of from 7 to 8 years. The flowers form at the terminal end of branchlets just inside the outer canopy. Although there are two main flowering periods, each tree flowers only for one of these periods. The spring flowering occurs in August or October and the second flowering in late January or early February. Harvesting is three months from flowering (Chaup, 1998). In the first stage of the mangosteen, the fruit will be pale yellow green. It will then change to blotchy pink, then to pinkish red. After that it will change to maroon red and then to dark maroon red and eventually will be violet black

(Faucon,2001).

Mangosteen cannot tolerate below 277.6 K nor above 388 K. It ordinarily requires high atmospheric humidity and an annual rainfall of at least 127 cm.

The tree is not adapted to limestone and grows best in deep, rich organic soil, specifically in sandy-loam. A spacing of 10.7-12 m is recommended when planting a mangosteen tree. Planting is preferably done at the beginning of the rainy season. Pits 1.2 m x 1.2 m x

1.3 m are prepared at least 30 days in advance, enriched with organic matter and topsoil and left to weather. The young tree is put in place very carefully so as not to injure the root, and is given a heavy watering. Partial shading with palm fronds or by other means should be maintained for 3 to 5 years. When growth begins, a shoot emerges from one end of the seed and a root from the other end. But this root is short-lived and is replaced by roots that instead develop at the of the shoot. The process of reproduction being vegetative, there is naturally little variation in the resulting trees and their fruits. Some of the seeds are polyembryonic, producing more than one shoot. The individual embryos can be separated before planting.

Inasmuch as the percentage of germination is directly related to the weight of the seed, only plump, fully developed seeds should be chosen for planting. Even these will lose viability in 5 days after removal from the fruit, though they are viable for 3 to 5 weeks in the fruit. Seeds packed in lightly dampened peat moss, sphagnum moss or coconut fiber in airtight containers have remained viable for 3 months. Only 22% germination has been realized in seeds packed in ground for 15 days. Soaking in water for 24 hours expedites and enhances the rate of germination. Generally, sprouting occurs in 20 to 22 days and is completed in 43 days.

Because of the long, delicate taproot and poor lateral root development, transplanting is notoriously difficult. It must not be attempted after the plants reach 60 cm; the depth of the taproot may exceed that height. There is greater seedling survival if seeds are planted directly in the nursery row than if first grown in containers and then transplanted to the nursery. The nursery soil should at least be 1 m deep. The young plants take 2 years or more to reach a height of 30 cm, when they can be taken up with a deep ball of earth and set out. Fruiting takes place in 10 years (Faucon, 2001).

Conventional vegetative propagation of the mangosteen is difficult. Various methods of grafting have failed. Cuttings and air-layers, with or without growth- promoting chemicals, usually fail to root or result in deformed, short-lived plants. Tissue culture is an innovative technique developed for propagation that has been proven effective in various types of plants. It is the process of supporting the growth and the development of isolated plant cells, tissues, or organs on an artificial, nutritive medium that is usually axenic, or free from all other forms of (Renfroe, 1998). It is considered a way of micropropagation, for it allows the production of large numbers of plants from small pieces of the parent plant, and in a relatively short period of time. In most cases, the explant - the piece placed on the medium - is taken from the shoot tip, leaf, lateral bud, and stem or root tissue, and the parent plant, which must be actively growing still, is not destroyed. Once the plant is introduced to the medium, proliferation of lateral buds and adventitious shoots, or the differentiation of shoots directly from the callus, results in an increase of the number of shoots available for rooting (Lineberger,

1998).

The method of tissue culture usually starts with the preparation of both the explant and the medium going to be used. The explant must undergo surface sterilization before it is placed on the sterile medium to remove any contaminants. Its first wash may be that of soap and water, and then of sodium hypochlorite and ethanol. Finally, it is rinsed with distilled water inside a laminar flow hood, to maintain its axenic condition

(Kyte and Liu, 1998). As for the medium, it may be presented to the explant in a or semi- state. This gelled agar will support the explant and keep it from being submerged in the medium, but would also allow the diffusion of the nutrients into the plant tissues (Renfroe, 1998).

Basically, a nutritive medium used for tissue culture contains macronutrients, such as , phosphorous, potassium, , sulfur and calcium, micronutrients or trace elements, such as and , vitamins, such as thiamine and riboflavin, , and . Amino , nucleic acid bases and other organic may also be included. If the medium does not contain phytohormones or other plant regulators, it is referred to as a basal medium.

Phytohormones and plant regulators are added to stimulate the growth and development of the explant. Of the five classes of phytohormones - auxins, cytokinins, gibberellins, abscissic acid, and - the first two are most frequently employed.

Generally, auxins promote root initiation, although they may inhibit root elongation and subsequent development, while cytokinins tend to support the development of new vegetative buds, and the opening and growth of the existing ones (Renfroe, 1998).

Benzyladenine is known for promoting callus growth, and for formation of shoots when balance of auxins and cytokinins is appropriate. The type of benzyladenine commonly used for tissue culture is 6-benzyladenine. Naphthalene acetic acid promotes root initiation and development, callus formation and regulates fruit development and growth.

Micropropagation offers several distinct advantages compared to the conventional techniques of propagation. A single explant can be made to multiply to several thousands in less than a year. Once established, the actively dividing cultures become continuous sources of microcuttings, which result in non-seasonal plant production.

But this relatively new way of propagation does not focus only on simple . Rather, it has been employed as a necessary process to serve as a new controlled variable in scientific researches. It can be used to accelerate the selection of superior plant species that deserve to be reproduced on a wide scale - most especially if they can resist stress, diseases, and pests. It can even serve as a medium for growing

8 plants with induced genetic mutations, either from somatic hybridization - the genetic fusions of plant protoplasts, which are single cells that have been stripped off their cell walls by enzymatic treatment, or from irradiation (Lineberger, 1998).

Gamma radiation is a form of electromagnetic radiation. It was first detected as emissions from natural radioactive substances such as uranium, radium, and thorium.

Gamma radiation does not carry any electric charge or mass. It is a penetrating radiation from nuclear processes. Gamma rays emitted travel with the speed of light, at 300,000 miles/second.

There are several different nuclear sources of gamma radiation. After emission of an alpha particle or beta particle from a parent nucleus, the daughter nucleus formed may have more energy than it would have in its normal state. The nucleus then de-excites by the emission of gamma rays, which carry the excitation energy. Gamma radiation is also produced in a nuclear reaction such as the combination of a neutron with a to form a deuteron. When a subatomic particle combines with its antiparticle, they annihilate each other and give rise to gamma radiation.

At present, gamma radiation is used by the industry to sterilize that may ruin a specific fruit. It is also used to inhibit fungal decay and the formation of mold and microbial contamination, so as to improve the shelf life of . It can either inhibit the growth of plants and or stimulate growth, the latter usually happening.

Plant embryos are greatly affected by irradiation depending on the species, the stage of the embryo at irradiation, and the criteria used to measure the effect. Moderate radiation of zygotes or early "proembryos" markedly reduces the number that will develop into mature plants. Many fail to develop into seeds and if seeds are formed, they do not germinate. Middle or late proembryos are somewhat less radiosensitive than the

earlier stages. Seeds are usually formed, but the incidence of nongermination is high. The plants that do form may have specific morphologic anomalies. Irradiation at a still later

stage, when the embryo is differentiating, often results in the formation of abnormalities

as the plant develops. The morphology of seeds is also affected by irradiation. Dormant

seeds are less radiosensitive than seeds with developing embryos. The highest exposures

result in the least growth in height. The water content of seeds has been shown to

markedly influence their radiosensitivity. Radiation has a minimum effect on both shoot

and root growth when exposed seeds are hydrated at about air-dry conditions. Above and below of this water content, growth is much more severely inhibited by irradiation.

Similarly, germination of seeds, survival of seedlings, and seed production of the plant

from irradiated seeds are all inhibited more when irradiated at low or high water contents

than when air-dried. Irradiated plants may show morphologic and histologic changes in

roots, stems, buds, leaves, or flowers. The type of response depends primarily on the

level and duration of exposure, on the species, age and physiological condition of the

plants, and on the environment during and after exposure. Root growth and formation of

new roots can be inhibited by suitable doses of irradiation. Moreover, in some species,

acute irradiation of local stem areas can cause the formation of new adventitious roots

above the irradiated area. It may be due to an imbalance of plant growth hormones

produced by the radiation. Stem swelling, dwarfing, fasciation, and excessive branching

of stems characteristically result from irradiation. The exposure of leaves to radiation

shows relatively little change other than thickening, coarseness or drying. Young leaves,

however, may show dwarfing, asymmetrical development or thickening of the leaf blade,

10 distorted venation, a change in texture or mosaic-like color changes. The exposure of flowers to radiation is generally retarded by radiation, although the flowering of some plants is stimulated by low doses. Fasciation or branching in the flowerhead of sunflowers, snapdragons, and other species has been described. Frequently, there is a modification in the form and number of the petals and stamens (Casarett, 1968).

Former studies regarding the growth of mangosteen in tissue culture used young purple leaves and seeds as explants. Callus obtained from culturing the leaves were treated with gamma radiation in the following doses: 0, 5, 10,20, and 40 Gy. After being cultured for three weeks, callus recover rate was checked. Significant decrement of recovery rate was found in the plants exposed to 20 and 40 Gy, when compared to the control plant (with zero exposure to radiation). Such high dosage levels also affected

stem growth and length and generated some abnormal characteristics in plants, those of

which include two apex leaves, serrate leaves, and adventitious branches (Prommee,

1997, cited by Lapade, 2003). Hormonal regulation of shoot bud formation in leaf

explants of mangosteen has been studied, and it was shown that shoot bud differentiation

was greatly enhanced by BA (benzyladenine), but selectively delayed by ethylene. IAA

( acetic acid) also showed an inhibitory effect on shoot bud differentiation

(Lakshmonan, etal, 1997, cited by Lapade, 2003).

Shoot proliferation has been achieved in mangosteen using seed explants.

Maximum mean number of shoots per explant (16.5) was obtained from cultures on

Murashige and Skoog (MS) medium enhanced with 40 mM 6-benzyladenine (BA) and

2.5 mM alpha-naphthaleneacetic acid (NAA), and kept at 30 °C under an 8-hour

photoperiod (Normah etal, 1995, cited by Lapade, 2003).

11 MS medium was the most suitable medium for embryogenesis, shoot proliferation and shoot elongation cultures. In embryogenesis cultures, multiple embryos were observed on seed explants cultured on MS medium and supplemented with benzyladenine and naphthalene acetic acid after 4-6 weeks of cultures. Eighty to a hundred embryos rose on the seed surface and the multiple embryos developed to multiple shoots after 2-3 weeks of culture, hi shoot proliferation cultures, the seed with

multiple shoots was cut into four explants before subculturing them on MS medium also

added with the NAA and BA. After 8 weeks of culture, the highest mean number of

shoots per explant was formed on this medium. In shoot elongation cultures, plants with a height ranging from 20-30 mm developed after four weeks. When buds were cultured in

MS medium for rooting, about 75% of the plants produced roots with length from 3-4

cm, and had 3-4 roots per bud after four weeks of culture. Micropropagation through MS

medium via embryogenesis was the best method for the production of mangosteen

planting materials (Minn and Thu, 2001, cited by Lapade, 2003).

12 METHODOLOGY

The first part of the experiment involved determining the optimal concentrations of naphthalene acetic acid (NAA) and benzyladenine (BA) in proportion to one another.

When established, the treatment for callus formation was used as yet another controlled variable in the second part of the experiment.

I. Optimization of Growth Regulators

Preparation of the medium to be used was done one week before the preparation of the seeds for tissue culture. This was done to ensure that the medium prepared was free

of any contaminants that may have arisen during preparation. The Murashige and Skoog

(MS) medium was chosen because of its proven effectiveness in tissue culture.

Three stocks solutions needed for the preparation of the medium, including micronutrients (SSII), nitrate (NH4NO3) solution and the vitamins, were prepared. The chemicals that were used in the preparation of stock solutions (Table 1)

were weighed. Each chemical was then dissolved separately in distilled water to ensure that no precipitate will form during storage. All of the dissolved chemicals of Stock

Solution II were mixed in a large graduated cylinder; the volume was completed to 500 ml by adding distilled water. The same process was done for the other stock solutions, with 500 ml and 250 ml as the final volume for the Vitamins Stock Solution and the

NH4NO3 solution, respectively. Each solution was then poured into a separate flask and

was covered with aluminum foil to avoid impurities. Flasks containing the stock solutions

were wrapped with paper to avoid photochemical reactions from occuring.

The group also made two stock solutions that were later used in the optimization

process. These included the NAA Stock Solution (20 ppm) and BA Stock Solution (20

13 ppm). The necessary amounts of NAA and BA were first weighed and each was dissolved separately in 0.05 ml of . Distilled water was added to both solutions until the volume reached 500 ml.

Table 1. Chemicals Used in the Preparation of Stock Solutions.

Chemical Name/Formula Mass of Chemical (g) Stock Solution II (Micronutrients) KI 0.0104

MnSO4. H20 0.2113 H3BO4 0.775

ZnSO4. 7 H20 0.1075

CuSO4. 5 H20 0.003

COCI2.6 H20 0.0003

(NH4)6Mo7O24.4 H20 0.0031 NHJNOJ Stock Solution.

NH4NO3 40 Vitamins Stock Solution Nicotinimide 0.015 Pyridoxine HC1 0.015 Thiamine HC1 0.06 BA Stock Solution Benzyladenine 0.01 NAA Stock Solution Naphthalene Acetic Acid 0.01

To prepare the medium, the other chemicals were weighed (Table 2). The chemicals for the macronutrients stock solution (Table 2) were dissolved separately in 7 ml of distilled water and poured into a small beaker. Distilled water was added up to a volume of 100 ml. Thirty-two (32) ml of the solution was poured into a one-liter beaker.

To prepare the potassium phosphate (KH2PO4) solution, 0.17 g of KH2PO4 was dissolved in 10 ml distilled water; only 8 ml was used in preparing the media. Thirty-two (32) ml of

SSII solution, eight (8) ml of NH4NO3 solution, and four (4) ml of the Vitamins stock

14 solution were mixed completely. Ascorbic acid, and sequestrene (Table 2), which were all dissolved separately in 3 ml of distilled water, were also added. Eighty (80) ml of coconut water was also added; the solution was stirred and distilled water was added until the volume reached 200 ml.

Table 2. Other Chemicals Used in the Preparation of MS Basal Medium.

Chemical Name/Formula Mass of Chemical (g) Stock Solution I (Macronutrients)

KNO3 3.22

CaNO3 1.75 KC1 1.128

MgSO4.7 H2O 0.926

KH2PO4 Stock Solution KH2PO4 0.17 Additives Ascorbic Acid 0.04 Glycine 0.016 Sequestrene 0.04

Four 250 ml Erlenmeyer Flasks were labeled as E, F, G and H. The 200 ml medium was divided among the 4 flasks, each flask having 50 ml of the medium. Each flask received the following amounts of BA and distilled water:

Table 3. Volume of BA and Distilled Water Added to Treatments.

Treatment Amount of BA (ml) Amount of Distilled Water (ml) E 0 70 F 20 50 G 40 30 H 60 10

Each flask was stirred completely. Sixteen 100 ml Erlenmeyer flasks were collected and labeled such that each treatment has four Erlenmeyer flasks each. Flask E

15 was divided among all the flasks with the label containing the letter E and each flask received 30 ml of the medium. The same was done to the other remaining flasks. Each flask received specific amounts of NAA and distilled water as shown in Table 4.

Table 4. Volume of NAA and Distilled Water Added to Treatments.

Flasks Amount of NAA (ml) Amount of Distilled Water (ml)

EI.FKGKH! 0 20

E2.F2.G2.H2 5 15

E3.F3.G3.H3 10 10 E^F^G^ H4 15 5

Each of the sixteen flasks had their pH adjusted to 5.6 to 5.7 using a pH meter.

Four-tenths (0.04) of a gram of agar was added to each of the sixteen flasks and each was set to boil until the agar had completely dissolved. The medium of one flask was dispensed into ten test tubes, each labeled like the flask where their medium came from.

Five (5) ml of the final medium was poured into each test tube and was sealed with a cotton plug. These were set to autoclave at 15 psi for 20 minutes at 394.15 K. The test tubes were cooled to allow the medium to gel and were left to stand one week before inoculation to see if the medium had any biological contaminants. The final sixteen treatments are shown in Table 5.

Ninety (90) seeds were collected from mangosteen fruits. A series of steps was done to prepare the explant for planting in the sterilized medium. The and hair were removed from the seed surface; the seeds were rinsed in a soap-water solution three times until no trace of soap was left. The seeds were sterilized by being submerged in 70% ethanol for 30 seconds and in 20% Chlorox© solution another for 25 minutes. This was 16 Table 5. The Sixteen Treatments of Growth Regulators for Optimization.

Treatment Concentration Concentration Treatment Concentration Concentration of BA (ppm) ofNAA(ppm) OfBA(ppm) of NAA(ppm) E, 0 0 Gi 4 0

E2 0 2 G2 4 2 E3 0 4 G3 4 4 E4 0 6 G4 4 6

Fi 2 0 H, 6 0

F2 2 2 H2 6 2 F3 2 4 H3 6 4 F4 2 6 H4 6 6

done to ensure that it will have the minimum amount of biological contaminants upon

inoculation. Seeds were drained of Chlorox© inside the laminar flow hood. The scalpel

and the forceps were also sterilized. During inoculation, the group used only half of a

seed per test tube. This was done because one seed is too big for one test tube and the

group wanted to ensure that the seed will grow properly in the medium. Using forceps,

the seeds were placed in a Petri dish and were halved using a scalpel. After inoculation,

the test tubes were stored in a room with cool temperature, high humidity and sufficient

light. The tissue cultures were observed after 51 days for the following morphological

characteristics: number of roots, plantlets and leaves formed, length of roots, stems and

leaves formed, and size of callus. The T-test was employed to determine the significant

difference between the manipulated treatments over the control. The treatment that best

induced callus formation was chosen for the next part of the experiment.

II. Tissue Culture of Irradiated Mangosteen Seeds (Callus Formation)

For the preparation of the basal medium for callus formation, the same

formulation as in Tables 1 and 2 were used. This time however, distilled water was added

17 to the solution until its volume reached 400 ml. This was divided into two flasks labeled

E3 and G4, which were the treatment concentrations that best induced callus formation in the seeds. In the first flask containing 200 ml of basal media, no BA and 80 ml NAA, together with 120 ml of distilled water, were added. This corresponded to a treatment combination of 0 ppm BA with 4 ppm NAA. In the second flask, 80 ml of BA, 120 ml of

NAA, and no distilled water was added. This corresponded to a treatment combination of

4 ppm BA with 6 ppm NAA. The treatments for callus formation are summarized in

Table 6. The pH was then adjusted to a value between 5.6 and 5.7, and 3.2 grams of agar was placed in each flask. After dispensing 5 ml from each flask to 72 test tubes labeled with the same treatment, the media was autoclaved at 15 psi for 20 minutes at 394.15 K.

Table 6. Treatment Combinations for Callus Formation.

Treatment Concentration Concentration Irradiation ofBA(ppm) of NAA (ppm) Dosage (Gy) E3-O 0 4 0 E3-5 0 4 5 E3-IO 0 4 10 E3-2O 0 4 20 E3-30 0 4 30 E3-4O 0 4 40 G4-0 4 6 0 G4-5 4 6 5 G4-IO 4 6 10 G4-2O 4 6 20 G4-30 4 6 30 G4-40 4 6 40

Seventy-two (72) seeds were gathered and were packaged according to the amount of gamma irradiation they received: 0 Gy, 5 Gy, 10 Gy, 20 Gy, 30 Gy and 40 Gy.

Each package contained 12 seeds, 6 for E3 and the other half for G4. The same process of

18 inoculation and storage as in Part I was also followed. Observations were made after 32 days.

III. In Vivo Planting of Irradiated Mangosteen Seeds

Three hundred and sixty seeds (360) were collected from the mangosteen fruits.

Twenty seeds were packaged into a clear plastic bag punched with holes. There were 3 sets of twenty seeds per treatment: 0 Gy, 5 Gy, 10 Gy, 20 Gy, 30 Gy, and 40 Gy. The seeds were exposed to the amount of irradiation designated according to their label. A rectangular plot (2.7 m x 1 m) with Perlite was sifted and the seeds were planted in blocks, as shown in Figures 1 to 3.

Figure 1. In Vivo Planting (Block 1).

The plants were watered as often as necessary. Observations were made every week two months after planting; the length of all the stems and leaves were

measured. The final gathering of data was done 71 days after planting.

19 4

1

•Mli * —4lf •

•• ••*••

Figure 2. /« F/vo Planting (Block 2).

Figure 3. /« F/vo Planting (Block 3).

20 The process flowchart for the whole methodology is as follows:

A. Optimization of Growth Regulators

Preparation of Stock Solutions J3- Preparation of MS medium J3- Inoculation 43. Observations made .a Best treatment for callus formation chosen

B. Tissue Culture of Irradiated Mangosteen Seeds (Callus Formation) Preparation of MS medium Irradiation of seeds

Inoculation

Observations made.

C. In Vivo Planting of Irradiated Mangosteen Seeds

Cleaning of seeds. 43- Irradiation of seeds 43- Planting of seeds XX Observations made

21 RESULTS AND DISCUSSION

The characteristics observed for the in vitro planting of mangosteen were the

following: the number of roots, stems and leaves, the length of roots, stems and leaves,

and the size of the callus. The T-test for two independent samples was the statistical test

used to determine the significance of the discrepancy between the variable and control

treatments; the level of significance used for all calculations was 0.05 (5%), because this

value as a basis of significance is neither too high nor too low. Graphs are presented to

further illustrate the trends and variation in values; these are representations of the tables

proceeding below.

Table 7. Averages Observed for the Optimization of Growth Regulators.

Treatment Size of Length of Length of Length of Number of Number of Number of Callus* Roots (cm) Stems (cm) Leaves (cm) Roots (cm) Stems (cm) Leaves (cm) E, 0.33 0.62 0.5 0.35 1.5 0.17 0.33 E2 0.26 0.6 2.2 1.27 0.4 0.4 1.2

E3 0.58 1.6 0.58 0.23 0.75 0.25 0.5

E4 0.43 0.7 2.7 0.81 1 0.71 1.71

F, 0.56 0.79 1.5 1.34 0.83 0.33 1.33

F2 0.44 1.2 0.37 0.73 1.33 0.33 0.33

F3 0.38 1.35 1.56 0.87 1 0.43 0.86

F4 0.28 1.27 1.7 0.91 1.5 0.5 0.67

G! 0.33 1.26 2.22 2.61 0.8 0.33 1.6

G2 0.4 0.8 0.93 0.93 1.4 0.4 1.6

G3 0.5 1.7 2.93 2.01 0.75 0.5 1

G4 0.67 0.79 1.86 1.25 2.4 0.4 0.8

Hi 0.22 1.33 0.47 0.2 1 0.33 0.67

H2 0.13 0.91 0.98 0.53 1.2 0.6 0.8

H3 0.53 0.74 3.88 1.63 1.4 0.8 0.8 H4 0.33 1.98 3.73 1.14 3.5 0.75 1.5 * The size of the callus was measured by the area of the seed that it covered.

22 Table 8. Averages Observed for Callus Formation.

Irradiation Size of Callus

Dosage (Gy) E3 G4 0 0.44 0.46 5 0.48 0.44 10 0.36 0.37 20 0.14 0.14 30 0.11 0.06 40 0.1 0.06

Table 9. Averages Observed for In Vivo Planting.

STEM LENGTH Amount of Gy Total Length (cm) Mean Length (cm) 0 360.5 6.01 5 352.8 5.88 10 344.4 5.74 20 303.85 5.06 30 225.2 3.75 40 131.3 2.19 LEAF LENGTH AMOUNT OF Gy Total Length (cm) Mean Length (cm) 0 296.52 4.94 5 322.72 5.38 10 388.20 6.47 20 279.16 4.65 30 205.14 3.42 40 114.24 1.90

For callus formation (Appendix A), one plus sign (+) meant a total covered area of minimal to one-third (1/3) of the seed; two plus signs (++) meant a total covered area of one-third (1/3) to two-thirds (2/3) of the seed, and three plus signs (+++) meant a total covered area of two-thirds (2/3) to the whole of the seed. The following treatments ranked the highest in callus formation (Figure 4): G4 (4 ppm BA with 6 ppm NAA) at an

23 average of 2/3 of the total area per replicate; E3 (0 ppm BA with 4 ppm NAA) at an average of 0.58 of the total area per replicate; and H3 (6 ppm BA with 4 ppm NAA) at an average of 0.53 of the total area per replicate. The T-test was not done because the values measured were ordinal. The treatments E3 and G4 were carried over to the second part of the experiment.

Figure 4. Average Area of Seed Covered with Callus of In Vitro Plants in Different Combinations of Growth Regulators

El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4

Growth Regulator Treatments

Figure 5. Average Root Length of In Vitro Plants in Different Combinations of Growth Regulators

2.5 2 i-i 1.5 -1 f- r-i 1 0.5 n fl n . 0 M 1 I fl-1 El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

For root length, the treatments with plants having the longest roots are H4 (6 ppm

BA with 6 ppm NAA), G3 (4 ppm BA with 4 ppm NAA) and E3 (0 ppm BA with 4 ppm

24 NAA), respectively. When the statistical test was performed, none of the three treatments

had a significant difference over the control Ei (0 ppm BA with 0 ppm NAA). The

calculated /-values (Appendix C) for E3, G3 and H4 were 1.48, 1.46 and 1.55,

respectively, all of which were lower than the tabular value of 2.396. Even so, H4 can still be used to help seeds develop plants with longer roots, which are advantageous because

they could anchor the plant more firmly in place and absorb more nutrients from the

medium to help the plant develop.

According to Table 7, the treatments with the plants having the longest stem

lengths (Figure 6) are that of G3 (4 ppm BA with 4 ppm NAA), H3 (6 ppm BA with 4

ppm NAA) and H4 (6 ppm BA and 6 ppm NAA). When the T-test was done, however,

the values of all three variable treatments had no significant difference over the control.

The measurements from H3 were not high enough; the calculated /-value for H3 was 2.14,

which was less than the tabular value of 2.262. With a difference of only 0.122 in the t-

values, it can be said that H3 can also be employed to develop plants with longer stems.

The significant difference from statistical calculations might not be large enough, but

definitely a difference of 3.5 cm in the length of the stem does, especially if the plant

continues to grow that way. Long stems will be more able to hold and support the plant

upright. Although short stems, when they develop into short trunks, make harvesting

easier, long stems are indicative of more productive plants.

Treatments capable of helping grow plants with long leaves included Gi (4 ppm

BA with 0 ppm NAA) and G3 (4 ppm BA with 4 ppm NAA), as seen in Figure 7.

However, when the T-test was done, both treatments' values have no significant

difference compared to that of Ei. Treatment Gi can nevertheless still be used to develop

25 plants with long leaves; long leaves are important because it helps the plant lose water more slowly, and because they can produce more food for the plant through photosynthesis.

Figure 6. Average Stem Length of In Vitro Plants in Different Combinations of Growth Regulators

I.I III I• I• El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Figure 7. Average Leaf Length of In Vitro Plants in Different Combinations of Growth Regulators

3 2.5 2 1.5 n - t-i I 0.5 n -fhfh nil El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Two treatments out of sixteen caused the formation of more roots: H4 (6 ppm BA with 6 ppm NAA) and G4 (4 ppm BA with 6 ppm NAA), with an average of 3.5 roots per plant and 2.4 roots per plant, respectively (Figure 8). These values have the greatest differences over the average 1 root per plant in Ei. Formation of more roots will also help in supporting the plant and supplying it with more nutrients.

All of the seeds that developed in all treatments had only one plantlet, or stem, each. However, among the sixteen treatments, E4 (0 ppm BA with 6 ppm NAA), H3 (6 26 ppm BA with 4 ppm NAA) and H4 (6 ppm BA with 6 ppm NAA) had the most number of stems (Figure 9). Out of the 5 replicates in H3,4 had stems, resulting in a percentage of

80%. Out of the 4 replicates in H4, 3 had stems, which is equivalent to 75%. Out of the 7 replicates in E4, 5 had stems, which is equivalent to 71.4%. All these percentages are high compared to that of Ei, which is only 16.67%. These percentages are the same for the percentage formation of stems in Figure 11.

Figure 8. Average Root Number of In Vitro Plants in Different Combinations of Growth Regulators

4 -] 11: 5 2.5- • 1 « 1.5 U 1 1 1 1 • 1 2 i- I • 1 1 fl M 1 11•1 ill lill 1 1 1 1 Illl III i°r El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Figure 9. Average Number of Stems of In Vitro Plants in Different Combinations of Growth Regulators

Z 0.8 jj 0.6 I °-4 l>0.2 lil nil nil 1 El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Among the sixteen treatments, E4, H3 and H4 had the most number of stems

(Figure 9). Out of the 5 replicates in H3, 4 had stems, resulting in a percentage of 80%.

27 Out of the 4 replicates in H4, 3 had stems, which is equivalent to 75%. Out of the 7 replicates in E4, 5 had stems, which is equivalent to 71.4%. All these percentages are high compared to that of Ei, which is only 16.67%.

Treatments Gi (4 ppm BA with 0 ppm NAA), G2 (4 ppm BA with 2 ppm NAA),

E4 (0 ppm BA with 6 ppm NAA) and H4 (6 ppm BA with 6 ppm NAA) produce the most number of leaves (Figure 10), at an average of 1.6 leaves per plant, 1.6 leaves per plant,

1.57 leaves per plant, and 1.5 leaves per plant, respectively. This is higher than the

average number of leaves per plant in Ei, which amounts to only 0.33. The number of

leaves are important because the more leaves, the more food is made available for the

plant.

Figure 10. Average Leaf Number of In Vitro Plants in Different Combinations of Growth Regulators - 1.3

1- n 0.5 n n [ 1 ]\W\ El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Figure 11 shows the percentage formation of the callus, roots, stems, and leaves

of the plantlets, regardless of length and number. Several treatments had replicates that

all developed callus, no matter how small the area it might have occupied. All of the G3

(4 ppm BA with 4 ppm NAA) replicates developed callus, but covered only an average of

0.33 of the area of the seed (Table 8).

28 It is also evident in Figure 11 that not all seeds that developed roots developed stems and leaves. In the case of Ei (0 ppm BA with 0 ppm NAA), over 60% of the replicates developed roots, but les than 20% formed stems and leaves. In H2 (6 ppm BA with 2 ppm NAA), out of the 80% that developed stems, only 75% developed roots and leaves. However, in E2 (0 ppm BA with 2 ppm NAA), all 40% of the replicates that grew into plantlets had complete morphological characteristics: roots, stems, and leaves.

Figure 11. Percentage Formation of the Parts of In Vitro Plants in Different Combinations of Growth Regulators

120 •B 100 § 80 • Callus n t) 60 It • Root Tl • Stem w> pi. In I 40 • Leaf n_ I! I 20 11 n 0 ill 1 1 11 El E2 E3 E4 Fl F2 F3 F4 Gl G2 G3 G4 HI H2 H3 H4 Growth Regulator Treatments

Figure 12. Size of Callus for Two Growth Regulator Treatments at Different Doses of Irradiation

a IE3 IG4

5 10 20 30 40 Dosage of Irradiation (Gy)

29 In the optimization of both the growth regulator treatment and seed irradiation dosage for callus formation, it is apparent that in both E3 (0 ppm BA with 4 ppm NAA) and G4 (4 ppm BA with 6 ppm NAA), the average area of the seed that the callus covered generally decreased as the level of irradiation increased. For E3 at 5 Gy, however, the area of the seed covered by the callus increased from 0.44 to 0.48 (Figure 12). A difference this small is normally negligible, but the extra callus could be used for further propagation of the plant.

Figure 13. Average Leaf and Stem Length of Mangosteen Exposed to Different Amounts of Irradiation

"^^- — -Leaf Length - Stem Length I

5 10 20 30 40 Amount of Irradiation Received (Gy)

In in vivo planting, the T-test was also performed to determine if there is a

significant difference between measurements of the plantlets of the seeds not exposed to radiation (0 Gy) and those exposed to radiation. For the stem length, plants exposed to both 5 Gy and 10 Gy did not render any significant change. At 20 Gy, there was a

significant difference between the average stem lengths of the plants exposed to 0 Gy and

20 Gy. The calculated t value was 2.32; the degree of freedom (df) equal to 118 was considered to be infinity (a), with its tabular value corresponding to 1.98. It was at this

dosage that the decrease in stem length was large enough to be significant.

30 At 30 Gy, the calculated / value was 6.28, which is many times greater than the tabular value. This suggests that the length of the stems is reduced even more greatly. At

40 Gy, the calculated t value was found to be at 11.9. It can then be inferred that as the amount of radiation received increases, the stem length decreases at an increasing rate.

As for the leaf length, measurements of plants exposed to amounts of 5 Gy and 20

Gy of radiation also had no significant difference when compared to the values of 0 Gy.

At 10 Gy, however, a calculated t of 5.88 signified a valuable discrepancy from the control variable. This time, however, it was an increase in the average leaf length, seen in

Figure 13 to be the highest point on the blue curve. At 30 Gy and 40 Gy, however, the significant difference suggested by the calculated t values 4.35 and 8.94, respectively, meant that the plants' average leaf length was diminishing at those points. These computations are consistent with Figure 13.

Micropropagation, or tissue culture, is the newer, faster way of propagating the plant chosen - in this case, mangosteen. One of its several benefits over the conventional way of propagation is that a plant with desirable characteristics (long stem and long leaves, for example) can be produced with the right combination of chemicals, and can easily be reproduced. Another advantage is that the nutrients absorbed by the plants are more complete, as they were controlled through measurements. These advantages defend the results discussed in the first part of this analysis: the different optimum concentrations presented could serve as protocols to produce the kind of mangosteen that is desired, which is deemed to be productive. The growth of plantlets on the culture media signified that tissue culture is, aside from being compatible with mangosteen, can be manipulated with the different growth regulators to produce even better plants.

31 The data presented from in vivo planting is also relevant because it supplies another factor, which is irradiation, which is capable of helping plants develop whatever the wanted characteristics are. Regarding the comparison between in vitro and in vivo propagation, the first plantlet was observed in the former 3 weeks after inoculation, while the first plantlet in the latter was seen 4 weeks after planting. Micropropagation is indeed the faster way to grow plants, although it requires a lot of practice and technique to lower contamination rates. The plantlets grown in vitro did not develop as much as those planted in vivo. This is probably because the seeds were subcultured only after every two months; because of this, they were not able to get the nutrients they needed.

The following combinations that best developed a specific morphological characteristic are summed up in Table 10.

Table 10. Summary of Optimum Treatments.

Characteristic Treatment Concentration Concentration ofBA(ppm) ofNAA(ppm) PARTI

stem length H3 6 4 leaflength G! 4 0 root length H4 6 6

stem number H3 6 4 leaf number H4 6 6 root number H4 6 6

callus G4 4 6 PART II

callus E3 (5 Gy) 0 4 PART III stem length none ...... leaf length 10 Gy — —

32 SUMMARY AND CONCLUSION

Tissue culture was once again shown to be applicable for propagation of mangosteen. The addition of growth regulators resulted in plants with more favorable morphological characteristics, such as longer roots, stems and leaves, more roots, stems and leaves, and a larger size of callus. The optimum concentrations for the said characteristics are H4 (6 ppm BA with 6 ppm NAA), H3 (6 ppm BA with 4 ppm NAA),

Gi (4 ppm BA with 0 ppm NAA), H4 (6 ppm BA with 6 ppm NAA), H3 (6 ppm BA with

4 ppm NAA), H4 (6 ppm BA with 6 ppm NAA), and G4 (4 ppm BA with 6 ppm NAA), respectively.

There are two ways to be able to produce plantlets with all the characteristics mentioned. One is to grow the seed in H4 (6 ppm BA with 6 ppm NAA), because this is the treatment combination that was found to promote growth and development in all plantlet morphological aspects. The other way is to first inoculate the seed in G4 (4 ppm

BA with 6 ppm NAA) where it can first develop callus. It can then be transferred into a

new medium with a different concentration combination of growth regulators for the

development of roots, stems and leaves, in that order. The concentrations H4 (6 ppm BA with 6 ppm NAA), H3 (6 ppm BA with 4 ppm NAA) and Gi (4 ppm BA with 0 ppm

NAA) can be used, respectively.

For callus formation, the best treatment was the growth concentration E3 (0 ppm

BA with 4 ppm NAA) with seeds exposed to 5 Gy. With a small difference compared to the seeds of E3 and G4 (4 ppm BA with 6 ppm NAA) that were not exposed to any

radiation, it might be impractical to use irradiation for the generation of more callus,

unless the seed must be propagated to large amounts.

33 Irradiation of the seeds before planting them in vivo, however, did not yield the results expected. As the amount of radiation received by the plants increased, the average length of their stems decreased in comparison to the control group, which received 0 Gy.

As for the leaf length, only at 10 Gy did the incremental difference become significant; at dosages higher than 10 Gy, the length of the leaves also deteriorated. The best dosage then for long stems and leaves is irradiation at 10 Gy. This is where leaves lengthened the most, and with significant difference over 0 Gy. Even if the stem length decreased at this point, its decrease was still not significant enough over 0 Gy.

The results of this research prove that the manipulation of three variables - the concentration of the growth regulators, the amount of radiation received by the seed, and the method of propagation - can be combined to produce a plant that can develop the desired morphological characteristics.

34 RECOMMENDATIONS

It is recommended that in doing micropropagation experiments, the number of replicates be increased. Aside from being a solution to high contamination rates, more replicates signify more reliable results. In in vitro planting, the seeds must be subcultured every two weeks even if the media has not yet dried up, to ensure an ample supply of nutrients for the plant.

To have values with positive significant differences over the control variable Ei, the group recommends that other concentrations and combinations of growth regulators be used. Research on the optimization of the growth regulator treatments and irradiation dosage combination for the other morphological characteristics is also suggested.

For in vivo, it is suggested that the plants be grown in a glass plot, so that the

length of the roots can be measured.

Lastly, researches on mangosteen must be performed and completed within

August to October, when the fruit is readily available at a low price.

35 BIBLIOGRAPHY

• Casarett, A. 1968. Radiation Biology. New Jersey. Prentice-Hall, Inc.

• Chaup, Patricia. 1998. http://www.dpi.qld.gov.an/horticulture/5447.html (18 July

2003).

• Faucon, Philippe. 2001. http://www.desert.tropicals.com/Plants/Guttiferae/

Garcinae_Mangostana.html (18 July 2003).

• Kyte, Lydiane (presenter) and Kathy Liu (host). 1998. Plant Tissue Culture

Protocol, http://www.accessexcellence.org/ TSN/ST/st2bgplantprot.html. (19 July

2003).

• Lapade, Avelina G. 2003. Effects of Gamma Radiation on Mangosteen. Quezon

City. Philippines. Philippine Nuclear Research Institute. Unpublished.

• Lineberger, Daniel R. 1998. Webmaster of Aggie Hortriculture. http://aggie_

horticulture, tamu.edu/tissucult/pltissue.html. (19 July 2003).

• Morton J, 1987. Fruits of Warm Climates. http://www.hort.purdue.

edu/newcrop/morton/ mangosteen.html. pp 301 - 304. (18 July 2003).

• Renfroe, Michael. 1998. Getting Started with Plant Tissue Culture.

http://www.jmu.edu/ biology/pctc/ tcstarthtm (15 July 2003).

36 APPENDIX A Complete Data Tables

Table 11. Complete Data for the Optimization of Growth Regulators.

Treatment Stem Leaves Root Callus length number length number length number Rank (1 to 3)* E,-l 3 1 2.1 2 1.2 2 0 Er2 0 0 0 0 0 0 2 E,-3 0 0 0 0 0.5 2 1 Ei-4 0 0 0 0 0 0 1 E,-5 0 0 0 0 0.8 1 1 E,-6 0 0 0 0 1.2 1 1

E2-l 0 0 0 0 0 0 1

E2-2 6.2 1 2.375 4 1.5 1 0

E2-3 0 0 0 0 0 0 1

E2-4 0 0 0 0 0 0 1

E2-5 4.8 1 3.95 2 1.5 1 1

E3-I 0 0 0 0 0.6 1 2 E3-2 0 0 0 0 0 0 3 E3-3 2.3 1 0.9 2 3 1 1 E3-4 0 0 0 0 2.8 1 1

E4-I 0.7 1 0.1 2 0.5 1 1 E4-2 0 0 0 0 0.2 1 3

E4-3 5.2 1 1.6 3 1.1 1 1 E4-4 0 0 0 0 0.7 2 1 E4-5 4.8 1 1.9 2 1.2 1 1 E4-6 1.2 1 1 1 0 0 1 E4-7 7 1 1.1 4 1.2 1 1

Fi-1 0 0 0 0 0 0 1 F,-2 5.2 1 1.325 4 1.35 2 1 F,-3 0 0 0 0 1.2 1 2 F,-4 3.8 1 1.35 4 1.6 1 1 F,-5 0 0 0 0 0.6 1 2 F1-6 0 0 0 0 0 0 3 * The ranking for callus from lowest to highest is 1 to 3, or one plus sign (+) to three plus signs (+++).

37 Table 11 (Continued)

Treatment Stem Leaves Root Callus length number length number length number Rank (1 to 3)

F2-l 0 0 0 0 1 1 1

F2-2 0 0 0 0 1.3 1 1

F2-3 1.1 1 2.2 1 1.3 2 2

F3-I 5.1 1 4.1 2 3.3 1 1 F3-2 0 0 0 0 2.7 1 2 F3-3 0 0 0 0 1 1 1 F3-4 0 0 0 0 0.5 1 1 F3-5 0 0 0 0.9 1 1 F3-6 2.6 1 1.6 2 1.05 2 1 F3-7 3.2 1 0.4 2 0 0 1

F4-I 0 0 0 0 1.167 3 1 F4-2 0 0 0 0 1.2 1 1 F4-3 2.6 1 1.65 2 0.7 2 0 F4-4 0 0 0 0 2.1 1 1 F4-5 2.5 1 0.4 1 1.2 2 1

F4-6 5.1 1 3.4 1 0 0 1

G,-l 0.6 1 1.25 2 0 0 1 Gi-2 0 0 0 0 0.3 1 1 Gi-3 5.5 1 2.95 2 2.6 1 1 Gi-4 0 0 0 0 2.1 1 1 Gi-5 5 1 8.85 4 1.3 1 1

G2-I 0 0 0 0 1.4 2 1

G2-2 3.6 1 1.325 4 0.4 1 1 G2-3 0 0 0 0 0.5 1 1

G2-4 0 0 0 0 0.6 1 2

G2-5 4.3 1 1.45 4 1.1 2 1

38 Table 11 (Continued)

Treatment Stem Leaves Root Callus length number length number length number Rank (1 to 3)

G3-l 0 0 0 0 1.1 1 2 G3-2 4.5 1 3.85 2 1.6 1 2 G3-4 0 0 0 0 0 0 1 G3-5 7.2 1 4.2 2 4.1 1 1

G4-I 4.2 1 2.8 2 1.07 7 1 G4-2 0 0 0 0 0.95 2 2

G4-3 0 0 0 0 0.3 1 3

G4-4 0 0 0 0 0 0 3 G4-5 5.1 1 3.45 2 0.85 2 1

H,-l 1.4 1 0.6 2 1.1 1 0 Hi-2 0 0 0 0 1.4 1 1

Hr3 0 0 0 0 1.5 1 1

H2-l 0 0 0 0 1.5 1 1

H2-2 0.4 1 0.1 2 0.9 2 1

H2-3 0.8 1 0 0 0 0 0

H2-4 0 0 0 0 1 1 0

H2-5 3.7 1 2.55 2 1.15 2 0

H3-I 0 0 0 0 0.8 5 3 H3-2 0.9 1 0 0 1.1 1 1 H3-3 5 1 1.1 1 1.8 1 1 H3-4 5.2 1 1.2 1 0 0 2 H3-5 8.3 1 5.85 2 0 0 1

H4-1 0 0 0 0 1.4 6 0 H4-2 4.1 1 1.2 1 0.9 1 2 H4-3 6.3 1 1.167 3 5.1 1 2 H4-4 4.5 1 2.2 2 0.53 6 0

39 Table 12. Complete Data for Callus Formation.

Treatment E3 G4 Treatment E3 G4 o. - 1 20, 0 0 O2 2 1 202 0 0

O3 2 2 203 0 0 O4 2 2 204 0 - O5 1 - 205 1 1 0 1 20 0 0 o6 6 O7 - 1 207 1 - - - 20 1 - o8 8 09 - 209 0 - O10 - - 2O,o 0 - On - 2 20,, 1 1 0,2 1 1 20,2 1 1

5, 3 - 30, 0 0

52 1 2 302 1 0 53 1 1 303 0 0

54 1 1 304 0 0 55 - 1 305 1 1 56 1 1 306 - 1

57 2 1 307 0 0 58 1 2 308 0 0 59 1 - 309 0 - 5,o 1 - 3O,o 1 0 5n 2 1 30,, - 0 5,2 2 2 3012 0 0

10i 1 - 40, 1 0

102 1 1 402 - 0

103 1 - 403 0 - 104 1 1 404 0 0 10s 2 1 405 1 0

106 - 1 406 0 0 107 1 1 407 0 1

108 1 1 408 0 0 109 1 1 409 0 0

10,0 1 - 4010 0 0 10,, 1 2 40,, 1 1

1O12 1 1 4012 - 0

40 Table 13. Complete Data for Stem Length in In Vivo Planting. OGy Replicate Mean Length(cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 7 21 6 41 7.5 2 6 22 5.6 42 7.3 3 6 23 5.8 43 7 4 6.5 24 5.5 44 8 5 6.2 25 5.7 45 8.5 6 8 26 5.3 46 0 7 0 27 5.5 47 8 8 6.5 28 5.6 48 6.2 9 7.5 29 5.3 49 8.5 10 8.1 30 6.2 50 8.2 11 4.2 31 0 51 8 12 0 32 6 52 5.2 13 6 33 6 53 6.8 14 6.2 34 5.9 54 8 15 7.2 35 5.5 55 5.6 16 9.3 36 5.4 56 2 17 7.4 37 5.6 57 5.3 18 8.5 38 5.8 58 7.2 19 7 39 5.4 59 7.4 20 4 40 4.6 60 7.5 5Gy 1 5 21 4.45 41 7.5 2 6.05 22 5.4 42 4.6 3 6 23 4 43 7.2 4 1.8 24 6.5 44 7.3 5 6 25 5.8 45 8.2 6 7.1 26 6 46 6.5 7 4.2 27 5.8 47 4.55 8 5.9 28 5.4 48 7.1 9 5.7 29 4.6 49 4 10 0 30 6.3 50 7.2 11 5 31 4.85 51 8.3 12 7.3 32 5.4 52 6.6 13 8.5 33 6.7 53 7 14 5.7 34 6.2 54 8.1 15 6 35 5 55 10.1 16 5 36 5.6 56 2 17 3.2 37 6.5 57 7.9 18 6.2 38 6 58 5.6 19 5.4 39 5.3 59 6.8 20 6.3 40 6.6 60 7.5

41 Table 13 (Continued) 10 Gy Replicate Mean Length(cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 8.05 21 6.95 41 6.3 2 5.93 22 5.48 42 6.8 3 5.89 23 5.98 43 6.5 4 6.08 24 5.43 44 6.8 5 5.78 25 5.89 45 6.65 6 5.68 26 5.67 46 6.81 7 7.85 27 6.35 47 6.33 8 6.35 28 6.73 48 6.32 9 7.3 29 6.15 49 7.5 10 6.24 30 6.8 50 7.81 11 6.05 31 6.45 51 6.35 12 6.13 32 6.83 52 7.5 13 7.1 33 5.58 53 7.65 14 6.3 34 6.83 54 7.94 15 7.1 35 6.97 55 6.89 16 5.58 36 6.2 56 5.88 17 5.75 37 6.23 57 5.37 18 6.4 38 6.1 58 6.58 19 5.38 39 6.63 59 5.89 20 5.89 40 6.68 60 7.57 20 Gy 1 3 21 5 41 8.3 2 0 22 5.7 42 3 3 4.6 23 0 43 6.9 4 6.7 24 6.3 44 6.6 5 6.3 25 6.4 45 4.7 6 5.8 26 6.3 46 4.7 7 6.2 27 6.4 47 7.8 8 6.1 28 5.9 48 0 9 4.6 29 3.65 49 4.3 10 0 30 5.4 50 6.1 11 5.4 31 7.3 51 6.7 12 7.1 32 0 52 5.6 13 0 33 6.5 53 6.3 14 5.7 34 5.5 54 6.5 15 5.8 35 5.5 55 8.6 16 0 36 3.3 56 6.8 17 4 37 2.1 57 8 18 5.6 38 7 58 0 19 6.4 39 7.1 59 8.4 20 5.1 40 3.2 60 7.6

42 Table 13 (Continued)

30 Gy Replicate Mean Length(cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 4 21 3.8 41 2.1 2 3.4 22 3.4 42 2.6 3 6 23 4.3 43 5.6 4 4.5 24 4.5 44 5.4 5 0 25 4.4 45 4.8 6 1.7 26 2.7 46 4.3 7 4.2 27 1.2 47 2.5 8 2.5 28 2.4 48 3.8 9 4 29 3.7 49 0 10 4.3 30 0 50 5.6 11 7.6 31 2.9 51 5.7 12 3.6 32 3 52 1.3 13 0 33 1.7 53 3.5 14 3.6 34 0 54 4.7 15 1.1 35 4.6 55 6 16 3.7 36 4.9 56 5.3 17 4.1 37 5.3 57 7.2 18 2.7 38 3.4 58 6.8 19 5.3 39 2.4 59 6.5 20 5.2 40 6.7 60 4.7 40 Gy 1 4.5 21 3 41 2.3 2 2.5 22 0 42 2.1 3 2 23 2.4 43 2.6 4 3.6 24 4.2 44 0 5 1 25 2.8 45 2.7 6 4.2 26 4 46 3.2 7 1.5 27 3.1 47 4 8 1.2 28 2.7 48 2.9 9 1.1 29 0.8 49 2.8 10 0 30 2.2 50 2 11 0 31 1.5 51 0 12 2.6 32 3 52 0.5 13 2 33 2.6 53 1 14 2.5 34 2 54 2.5 15 3.2 35 0 55 3.1 16 0 36 1.7 56 0 17 0.7 37 3 57 2.2 18 2.7 38 3 58 3.3 19 3.6 39 0 59 3.9 20 0 40 5 60 4.3 43 Table 14. Complete: Data foir Leaf Length in In Vivo Planting OGy Replicate Mean Length (cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 4.93 21 6.5 41 2.53 2 5.58 22 4.38 42 5.25 3 5.55 23 3.78 43 6.9 4 6.65 24 5.5 44 6.78 5 4.6 25 6.15 45 5.95 6 3.38 26 4.7 46 0 7 0 27 5.15 47 6.38 8 4.98 28 4.03 48 5.95 9 4.43 29 4.35 49 5.6 10 4.7 30 5.05 50 5.93 11 5.28 31 0 51 4.03 12 0 32 4.03 52 4.43 13 4.17 33 6.05 53 7.4 14 5.15 34 6.53 54 6.13 15 6.45 35 5.33 55 5.45 16 8.1 36 6.38 56 0 17 6.95 37 5.1 57 4.5 18 6.9 38 6.55 58 7.5 19 5.45 39 4.65 59 3.4 20 4.5 40 3.85 60 6.6 5Gy 1 3.8 21 5.9 41 6.65 2 4.67 22 4.98 42 4.1 3 5.53 23 4.18 43 5.7 4 2.7 24 6.9 44 4.5 5 6.15 25 6.33 45 7.25 6 6.45 26 7.8 46 4.13 7 4.83 27 4.58 47 3.33 8 5.15 28 3.13 48 4.5 9 6.08 29 3.14 49 4.6 10 0 30 7.15 50 7.45 11 4.63 31 5 51 7.8 12 5.83 32 6.3 52 5.48 13 5.13 33 5.95 53 5.58 14 6.77 34 3.4 54 6.1 15 6.5 35 6.7 55 7.35 16 5.93 36 6.08 56 0 17 4.38 37 7.3 57 7.13 18 6.7 38 4.58 58 5.15 19 4.35 39 6.8 59 5.88 20 6.6 40 6.23 60 5.43

44 Table 14 (Continued) 10 Gy Replicate Mean Length (cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 7.05 21 6.95 41 5.9 2 5.93 22 5.48 42 5.8 3 3 23 5.08 43 6.5 4 6.08 24 5.43 44 6.8 5 3.78 25 3.223 45 6.65 6 5.68 26 3.78 46 4.81 7 6.85 27 6.35 47 6.33 8 6.35 28 5.73 48 2.77 9 6.3 29 5.5 49 4.5 10 0 30 3.73 50 2.55 11 6.05 31 6.45 51 3.35 12 4.13 32 6.13 52 5.5 13 6.3 33 5.58 53 4.65 14 5.3 34 6.83 54 5.13 15 6 35 6 55 5.8 16 5.58 36 6.2 56 0.88 17 4.75 37 5.23 57 4.5 18 6.4 38 6.1 58 0 19 5.38 39 4.63 59 4.78 20 0 40 4.68 60 5.55 20 Gγ 1 4.55 21 6.1 41 4.03 2 5.45 22 6.9 42 3 3 0 23 0 43 6.38 4 6.63 24 6.05 44 5 5 5.55 25 4.62 45 4.3 6 0 26 6.75 46 5.05 7 5.38 27 6.4 47 5.98 8 6.65 28 4.63 48 0 9 7.1 29 6.1 49 6.8 10 6.05 30 4 50 5.47 11 5.63 31 5.35 51 4.38 12 0 32 0 52 6.35 13 5.1 33 5.35 53 3.55 14 3.9 34 7.1 54 4.85 15 5.93 35 6.1 55 5.9 16 4.35 36 3.54 56 5.45 17 5.9 37 1.75 57 6.05 18 5.8 38 6.6 58 0 19 6.33 39 7.1 59 4.53 20 0 40 3.35 60 4

45 Table 14 (Continued)

30 Gy Replicate Mean Length (cm) Replicate Mean Length (cm) Replicate Mean Length (cm) 1 2.5 21 5.1 41 0 2 6.1 22 0 42 2.7 3 4.03 23 3.9 43 4.7 4 5.3 24 3.4 44 4.76 5 0 25 4.45 45 5.2 6 2 26 1.7 46 4.35 7 5.13 27 1.3 47 3.6 8 0 28 3.9 48 3.15 9 2 29 3.65 49 0 10 1.1 30 0 50 4.98 11 5.5 31 3.35 51 5 12 2.05 32 2.08 52 5.03 13 0 33 0 53 3.18 14 5.2 34 1.1 54 5.75 15 1.2 35 3.85 55 4.23 16 4.85 36 5.45 56 6.53 17 3 37 5 57 5.63 18 3.5 38 3.78 58 5.43 19 2.6 39 1.65 59 5.15 20 3.25 40 6.8 60 6 40 Gy 1 5.1 21 2.95 41 1.87 2 1.2 22 0 42 0 3 0 23 1.35 43 2.5 4 2.9 24 4.9 44 0 5 0 25 1.7 45 2.5 6 5.35 26 2.85 46 1.65 7 0 27 2.77 47 5.75 8 0 28 3.2 48 3.1 9 0 29 0 49 3.8 10 0 30 5.1 50 0.9 11 0 31 0.6 51 0 12 2.9 32 4.3 52 0 13 1 33 4.7 53 0 14 0 34 0 54 3.7 15 5.05 35 0.75 55 0 16 0 36 1.25 56 1.7 17 0 37 5.15 57 0 18 2.9 38 1.2 58 1.35 19 4 39 0 59 2.8 20 0 40 5.15 60 4.3 46 APPENDIX B Statistical Test T-Test Calculations

The T-test for two independent samples was the statistical test used, because the values have a true zero point (ratio). In the tables, Xi and X2 are the two treatments being compared, S(Xi-X2) is the standard error of difference, T-calc is the calculated /-value, and T-tab is the tabular /-value.

A. In Vitro'. Root Length 2 2 2 2 Replicate E, E, E3 E3 G3 G3 ft. H4 1 1.2 1.44 0.6 0.36 1.1 1.21 1.4 1.96 2 0.5 0.25 0 0 1.6 2.56 0.9 0.81 3 0.8 0.64 3 9 0 0 5.1 26.01 4 1.2 1.44 2.8 7.84 4.1 16.81 0.53 0.28 5 0 0 - . - - - _ 6 0 0 ------Total 3.7 3.77 6.4 17.2 6.8 20.58 7.93 29.06 X! x2 SCXrXa) T-calc T-tab Conclusion Ei E3 0.66 1.48 2.396 No significant difference.

Ei G3 0.74 1.46 2.396 No significant difference. Ei H4 0.88 1.55 2.396 No significant difference.

Table 15. T-Test Calculations for In Vitro Root Length.

B. In Vitro: Stem Length 2 2 2 Replicate Ei E! G3 G3* H3 H3 H4 H4 1 3 9 0 0 0 0 0 0 2 0 0 4.5 20.25 0.9 0.81 4.1 16.81 3 0 0 0 0 5 25 6.3 39.69 4 0 0 7.2 51.84 5.2 27.04 4.5 20.25 5 0 0 - - 8.3 75.69 - - 6 0 0 - - - - - Total 3 9 11.7 72.09 19.4 128.54 14.9 76.75 X, x2 S(Xi-X2) T-calc T-tab Conclusion E, G3 1.54 1.58 2.396 No significant difference. Ei H3 1.58 2.14 2.262 No significant difference. E, H4 1.51 2.14 2.396 No significant difference.

Table 16. T-Test Calculations for In Vitro Stem Length. 47 c.In Vitro: Leaf Length 2 2 2 Replicate Ei E! G, G, G3 G3 1 2.1 4.41 1.25 1.56 0 0 2 0 0 2.95 8.7 3.85 14.82 3 0 0 0 0 0 0 4 0 0 0 0 4.2 17.64 5 0 0 8.85 78.32 . 6 0 0 - - • - Total 2.1 4.41 13.05 88.58 8.05 32.46 X, S(Xi-X ) T-calc T-tab Conclusion x2 2 Ei Gi 1.55 1.46 2.262 No significant difference.

E, G3 1.02 1.58 2.396 No significant difference.

Table 17. T-Test Calculations for In Vitro Leaf Length.

D. In Vivo: Stem Length x, x2 S(X!-X2) T-calc T-tab Conclusion OGy 5Gy 0.34 0.382 1.98 No significant difference. OGy 10 Gy 0.36 0.75 1.98 No significant difference. OGy 20 Gy 0.41 2.32 1.98 Negative significant difference. OGy 30 Gy 0.36 6.28 1.98 Negative significant difference. OGy 40 Gy 0.32 11.9 1.98 Negative significant difference.

Table 18. T-Test Calculations for In Vivo Stem Length.

E. In Vivo; Leaf Length Xi x2 S(XrX2) T-calc T-tab Conclusion OGy 5Gy 0.317 1.37 1.98 No significant difference.

OGy 10 Gy 0.26 L 5.88 1.98 Positive significant difference. OGy 20 Gy 0.35 0.83 1.98 No significant difference. OGy 30 Gy 0.348 4.35 1.98 Negative significant difference. OGy 40 Gy 0.343 8.94 1.98 Negative significant difference.

Table 19. T-Test Calculations for In Vivo Leaf Length.

48