Sun Wan Fong.pdf

IN VITRO INDUCTION OF POLYPLOIDY IN Nepenthes gracilis

SUN WAN FONG

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA KUALA LUMPUR

2008

IN VITRO INDUCTION OF POLYPLOIDY IN Nepenthes gracilis

SUN WAN FONG

DISSERTATION SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF MASTER OF SCIENCE

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA KUALA LUMPUR

2008

ABTRACT

The present study aims to determine the 2C nuclear DNA content of Nepenthes

gracilis via flow cytometry as well as to investigate in vitro polyploidy induction in this

plant. The 2C DNA content of N. gracilis was found to be 1.60 ± 0.02 pg based on Glycine

max cv. Polanka (2C = 2.50 pg) as the internal standard. Polyploidy induction was

conducted by treating nodal segments (explants) from in vitro-grown plants with liquid

solution of colchicine (1.25, 2.5, 5 and 10 mM) and oryzalin (20, 40, 60 and 80 µM) for

different durations (24, 48, 72 and 96 hours). The first visible effect of polyploidization

treatments was delayed growth of shoot buds and aberrant morphology of the treated

explants. Flow cytometry was used to screen for tetraploids. Tetraploids were obtained

from two treatments: 10 mM colchicine for 72 hours and 40 µM oryzalin for 24 hours.

Overall, the percentage of tetraploid induction was low: 1.39% and 0.70% in colchicine and

oryzalin treatments, respectively. However, oryzalin treatments produced significantly

more mixoploids than colchicine treatments as inferred by Duncan’s Multiple Range Test at

P≤0.05, where oryzalin and colchicine treatments produced 79.86% and 45.14%

mixoploids, respectively. Stomata studies (stomata length, stomata width and stomata

frequency) demonstrated a significant difference between diploid and tetraploid plants, as

inferred by two-sample t-test at P≤0.05. The stomata size (length and width) of tetraploids

were significantly larger than those of diploids, while the stomata frequency was lower in

tetraploids than diploids. Root induction medium (WPM + 0.5 mg/l IBA + 0.3% gelrite)

was formulated to induce suitable roots (thick with obvious root tip regions) to facilitate

root tip squashing for chromosome studies. The chromosomes of N. gracilis were found to

be small in size but in large number (near to 80). Chromosome studies also revealed the

chimeric nature of the tetraploid plants produced.

ii

ABSTRAK

Kajian ini bertujuan menentukan kandungan DNA nucleus 2C Nepenthes gracilis

melalui analisis ‘flow cytometry’ dan mengkaji penginduksian poliploidi secara in vitro

dalam pokok ini. Kandungan DNA 2C N. gracilis didapati 1.60 ± 0.02 pg berdasarkan

Glycine max cv. Polanka (2C = 2.50 pg) sebagai piawai dalaman. Penginduksian poliploidi

dilakukan dengan merawat bahagian nodal (eksplan) dari anak pokok kultur tisu dengan

larutan colchicine (1.25, 2.5, 5, 10 mM) dan oryzalin (20, 40, 60, 80 µM) untuk tempoh

masa yang berlainan (24, 48, 72 dan 96 jam). Tanda ketara selepas rawatan adalah

pertumbuhan eksplan yang terbantut dan morfologi yang tidak normal. Tetraploid

ditentukan melalui analisis ‘flow cytometry’. Tetraploid diperoleh dari dua rawatan, iaitu

rawatan dengan 10 mM colchicine selama 72 jam serta 40 µM oryzalin selama 24 jam.

Secara keseluruhan, peratus induksi poliploidi adalah rendah, iaitu 1.39% dari rawatan

colchicine dan 0.70% dari rawatan oryzalin. Rawatan oryzalin menunjukkan perbezaan

yang ketara dalam penghasilan mixoploid berbanding dengan rawatan colchicine

berdasarkan ‘Duncan’s Multiple Range Test’ (P≤0.05), di mana rawatan oryzalin

menghasilkan 79.86% mixoploid manakala rawatan colchicine menghasilkan 45.14%

mixoploid. Kajian stomata (panjang, lebar dan bilangan) telah menunjukkan perbezaan

yang ketara antara diploid dan tetraploid berdasarkan ‘two-sample t-test’ (P≤0.05).

Didapati saiz stomata (panjang dan lebar) tetraploid lebih besar berbanding diploid

manakala bilangan stomata tetraploid adalah kurang daripada diploid. Medium pengakaran

diformulasi untuk merangsang pembentukan akar yang sesuai (iaitu tebal dengan ‘root tip

regions’ yang jelas) untuk kajian kromosom melalui teknik ‘root tip squashing’. Didapati

bahawa bilangan kromosom N. gracilis banyak (mendekati 80) serta bersaiz kecil. Kajian

kromosom telah mendapati bahawa tetraploid yang diperolehi merupakan ‘chimera’.

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ACKNOWLEDGEMENTS

First and foremost, I would like to thank FRIM and UM for their financial support

(through FRIM’s Research Grant and UM’s IPPP grant). Secondly, I would like to express

my greatest gratitude to Dr. Kodiswaran and Dr. Ng Ching Ching for guiding me

technically and academically throughout the completion of this study.

Besides, I would like to thank Dr. Maria for her advice and permission to use the

facilities in Cytogenetic Lab, MPOB, especially flow cytometer. Also, the technical support

from the members in Cytogenetic Lab, MPOB was highly appreciated. Not forgotten also

my colleagues in FRIM (especially Tissue Culture Lab and Genetic Lab) and friends who

had involved directly or indirectly in this study. I would also like to extend my gratitude to

Dr. Rasip (from FRIM) for his assistance in data analysis as well as his useful comment.

Last but not least, many thanks to my ever-supportive, considerate and beloved

family members (my parents, sister, brothers and Bok Hui).

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TABLE OF CONTENTS

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER I: INTRODUCTION

PAGE

1.1 The Nepenthes plant 1

1.1.1 The structure of the plant 2-4

1.1.2 Nepenthes gracilis Korth 4-6

1.2 Plant breeding 6-7

1.2.1 Polyploidization 7-9

1.2.1.1 Polyploidization approach 9-10

1.2.1.2 Plant tissue culture 11-13

1.2.1.3 Mitotic inhibitors 14-15

-Colchicine

-Oryzalin

1.3 Methods in detecting polyploidy 16

1.3.1 Chromosome studies 16-17

1.3.2 Flow cytometry (FCM) 17-20

1.3.3 Other methods 21

1.4 Nuclear DNA C-value 21-23

1.5 Scope and objectives of study 24

v

CHAPTER II: MATERIALS AND METHODS

PAGE

2.1 Nuclear DNA C-value determination 25-27

2.2 In vitro polyploidy induction 28-33

2.3 Identification of polyploid plants 34-39

2.3.1 Growth and morphology observation 34

2.3.2 Polyploidy screening via flow cytometry 34

2.3.3 Stomata studies 35


-Roots induction experiment

-Root tip squashes preparation

CHAPTER III: RESULTS AND DATA ANALYSIS

PAGE


3.2 Identification of polyploid plants

3.2.1 Growth and morphology observation 42-47

3.2.2 Polyploidy screening via flow cytometry 47-55

3.2.3 Stomata studies 56-57



-Chromosomes of Nepenthes gracilis

3.2.5 Morphology of diploid and tetraploid plants 62

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CHAPTER IV: DISCUSSION

PAGE



4.2.1 Growth and morphology observation 66-67

4.2.2 Polyploidy screening via flow cytometry 67-75

-Flow cytometric profiles

-Polyploid plants production

4.2.3 Stomata studies 75-76



-Chromosomes of Nepenthes gracilis

4.3 Future work and suggestions 80-81

CHAPTER V: CONCLUSION 82

REFERENCES 83-92

APPENDICES 93-95

vii

LIST OF TABLES

Table Page

Table 1.1 Macro- and micro-elements important for plant nutrition and

their physiological functions.

Table 1.2 Fluorochromes frequently used to label nucleic acid for

FCM analysis.

Table 2.1 Mitotic inhibitor treatment solution preparation.

Table 2.2 Components of one liter shoot proliferation medium.

Table 2.3 Polyploidization treatments with various combinations of

mitotic inhibitor’s concentrations and treatment durations.

Table 2.4 Control treatments.

Table 2.5 Rooting medium based on either WPM or MS medium.

Table 3.1 Results on analysis of variances (ANOVA) for the effects

of colchicine on number of shoot bud formed.

Table 3.2 Results on analysis of variances (ANOVA) for the effects

of oryzalin on number of shoot bud formed.

Table 3.3 Effects of colchicine concentration and treatment duration

on number of shoot bud formation.

Table 3.4 Effects of oryzalin concentration and treatment duration on

number of shoot bud formation.

Table 3.5 Results on analysis of variances (ANOVA) for mixoploids

production.

Table 3.6 Results on analysis of variances (ANOVA) for tetraploids

production.

Table 3.7 Influences of colchicine concentration on mixoploids

production.

Table 3.8 Influences of colchicine treatment duration on mixoploids

production.

Table 3.9 Influences of oryzalin concentration on mixoploids

production.

Table 3.10 Influences of oryzalin treatment duration on mixoploids

production.

13

20

29

30

32

33

37

44

44

45

46

51

52

52

52

53

54

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Table

Page

Table 3.11 Mixoploids production in colchicine and oryzalin

treatments.

Table 3.12 Stomata studies of diploid and tetraploid plants of

N. gracilis.

55

56

ix

LIST OF FIGURES

Figure

Page

Figure 1.1 Basic structure of Nepenthes pitcher.

Figure 1.2 Lower and upper pitcher of Nepenthes gracilis.

Figure 1.3 Chemical structure of colchicine.

Figure 1.4 Chemical structure of oryzalin.

Figure 2.1 Diagram of FCM analysis procedures.

Figure 2.2 Diagram of polyploidization procedures and post-

treatment evaluations.

Figure 2.3 Measurement of stomata length and stomata width.

Figure 3.1 Representative DNA histogram of relative fluorescence

intensity obtained from PI-stained leaves nuclei of

N. gracilis and G. max (standard)

Figure 3.2 Plant showing normal and aberrant morphology.

Figure 3.3 Representative DNA histogram of relative fluorescence

intensity obtained from PI-stained nuclei isolated from

leaves of diploid, mixoploid and tetraploid plants of

N. gracilis.

Figure 3.4 Percentage of diploid, mixoploid and tetraploid plants

produced after treating in vitro-derived nodal segments of

N. gracilis with various combinations of colchicine

concentrations and treatment durations.

Figure 3.5 Percentage of diploid, mixoploid and tetraploid plants

produced after treating in vitro-derived nodal segments of

Nepenthes gracilis with various combinations of oryzalin

concentrations and treatment durations.

Figure 3.6 Percentage of diploids, mixoploids and tetraploids

produced by colchicine and oryzalin treatments.

3

5

14

15

27

33

35

41

47

48

49

50

54

x

Figure

Page

Figure 3.7 Stomata cells from abaxial leaf imprints of diploids,

tetraploids and mixoploids.

Figure 3.8 Rooting percentage of N. gracilis in medium formulation

based on WPM.

Figure 3.9 Rooting percentage of N. gracilis in medium formulation

based on MS medium.

Figure 3.10 Roots induction by different PGR.

Figure 3.11 Well-spread chromosomes in diploid cell.

Figure 3.12 Morphology of diploids and tetraploids.

57

59

60

60

61

62

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LIST OF ABBREVIATIONS ANOVA Analysis of variances

BAP 6-benzylaminopurine

CCD charge-coupled device

cv. cultivar

CV coefficient of variation

DAPI 4’,6-diamidino-2-phenylindole

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid

FCM flow cytometry

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IBA indole butyric acid

MOPS 3-(N-morpholino)propanesulfonic acid

MS Murashige & Skoog

NAA naphthalene acetic acid

pg picogram

PGR plant growth regulator

PI propidium iodide

psi pounds per square inch

PVP polyvinylpyrrolidone

RNase Ribonuclease

rpm resolution per minute

SD Standard deviation

TRIS tris(hydroxymethyl)aminomethane

WPM Woody Plant Medium

xii

Chapter I: Introduction _________________________________________________________________________

1.1 The Nepenthes plant

The Nepenthes are a plant genus in the family Nepenthaceae that comprises over

100 species. They are carnivorous plants found in tropical forests ranging from South China,

Indonesia, Malaysia and the Philippines; westward to Madagascar and the Seychelles;

southward to Australia and New Caledonia; and northward to India and Sri Lanka. The

majority of Nepenthes are highland or mountain plants, which prefer habitats with both

high humidity and rainfall. Only 30 percent of the species are found in lowland areas where

the days are hot and the nights are warm (D’amato, 1998).

Biological taxonomy

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Caryophyllales

Family : Nepenthaceae

Genus : Nepenthes

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1.1.1 The structure of the plant

Nepenthes plants are vines with thin stems and long, narrow leaves. The stem may

attain lengths of over 20 m, but most tend to be 2-5 m on mature plants. The forms of the

stem vary from cylindrical, winged to square in cross-section. Thickness of the stem, on the

other hand, varies from a few milimetres in N.gracilis to over three centimetres in some

plants of N. bicalcarata. The leaves of Nepenthes plants vary greatly in shape, colour,

texture and size. The size can range from five centimetres long in the smallest species to

one metre in the largest (Clarke, 1997).

Nepenthes’s uniqueness is attributed to its pitcher (which is the true leaf). The

pitcher is supported by a tendril loop to the leaf petiole. It is the natural cup used by the

plant to capture organisms like insects. For example, the predominant inhabitants or preys

found in the pitchers of N. gracilis are ants (Beaver, 1979). Most Nepenthes species

produce two types of pitchers. Young stems produce short, squat pitchers called terrestrial

or lower pitchers. On climbing stems, narrower, funnel-shaped pitchers called aerial or

upper pitchers are produced. Although Nepenthes pitchers share the same basic structure,

they vary in size, shape and color. This variation in pitcher morphology provides useful

guides in identifying different species of Nepenthes (Clarke, 1997).

The pitcher comprises of four principle units: the lid, the peristome, the upper waxy

zone and the lower glandular zone. The lower pitcher may have additional two fringed

wings at the front, which run from the peristome to the base of the pitcher. The wings are

2

Chapter I: Introduction _________________________________________________________________________ thought to guide the crawling insects to the pitcher entrance (Clarke, 1997). Figure 1.1

illustrates the basic structure of Nepenthes pitcher.

Lid

Peristome

Tendril

Lower zone

Upper zone

(b) Lower pitcher (a) Upper pitcher

Wings

Figure 1.1: Basic structure of Nepenthes pitcher (adapted from Clarke, 1997).

At the top of the pitcher is the lid. The lid comes in a variety of shape and color,

which assists in attracting insects to the pitcher. The second function of the lid is to cover

the mouth, in order to prevent dilution of pitcher contents by the rainwater. Below the lid is

the mouth of the pitcher. The hardened tissue which runs around the mouth is called

peristome. The main function of the peristome is to prevent captured prey from escaping

from the pitcher. In addition, it may play a role in luring potential prey items to enter the

pitcher (Clarke, 1997). The pitcher itself can be divided into two distinct zones: waxy and

digestive zones. The waxy zone is covered with minute wax plates, which break off when

an insect crawls over them, increasing the chances that it will fall into the fluid below. The

digestive zone, on the other hand, is covered with glands that will secrete acid and digestive

3

Chapter I: Introduction _________________________________________________________________________ enzymes to breakdown the prey. Examples of enzymes are ribonuclease, lipase, esterase,

acid phosphatase and protease (Slack, 1979).

The stems, leaves, tendrils and pitchers of the plants go through some quite

remarkable changes as the plants grow. For example, young plants produce tightly packed

rosettes of leaves and pitchers on very short stems. Usually three to four years after

germination, the stem begins to elongate, with the distance between the nodes increasing

dramatically. Young climbing stem tends to produce larger leaves (exceeding one meter in

length in some species). As the stem grows taller, the size of the leaves decreases. The

tendrils of climbing stems are quite different from those of the rosettes. Tendrils of the

rosette plants tend to be straight whereas climbing stems often has curl in the middle of

tendrils (Clarke, 1997).

1.1.2 Nepenthes gracilis Korth

Nepenthes gracilis Korth or commonly known as pitcher plant (locally known as

“periuk kera”), is one of the common pitcher plant found in Malaysia. The natural

distribution of N. gracilis includes Thailand, Sumatra, Peninsular Malaysia, Singapore,

Borneo and Sulawesi. It is found in lowland peat-swamp forest, kerangas, podsol heath

scrub, swamp edge or disturbed areas at sea level reaching 800 m (Cheek & Jebb, 2001).

4


The plant is a terrestrial climber reaching 5 meters tall. The climbing stems are

triangular (1.5-5 mm in diameter) with 2.5-9 cm long internodes. Climbing stems have

rounded corners with 2 wings (1-3.5 mm broad) (Cheek & Jebb, 2001). The leaves are thin-

leathery, gloosy, decurrent, 10-25 cm long and 2 cm broad. The lower pitchers are ovoid in

shape and up to 5 cm in height, whereas the upper pitchers are tubular in shape with 5-15

cm in height (Roger, 1984). The pitchers are green, red or green-mottled with red in color.

The influorescence is a raceme with one flowered pedicels. Flowers had been described as

white, green, light red or brown in color. The seeds are fusiform and papillate at the centre

(Cheek & Jebb, 2001). Figures 1.2 shows the lower and upper pitcher of N. gracilis.

(b) (a)

Figure 1.2: Lower (a) and upper (b) pitcher of Nepenthes gracilis.

5


Grown in the wild, this species attracts not much attention. It grows like a creeper

along the ground and up onto other trees. Its pitcher is greenish in color and pigmented

pitcher can only be found occasionally. When this plant is cultivated in the nursery, the

plants are usually grown from single shoot cultivation with frequent pruning. Unlike other

Nepenthes species, the pitchers of N. gracilis are not particularly impressive and lack of

unusual morphological features. In view of the commonness demonstrated by this species,

there is room for improvement in this species via plant breeding in order to create new

variety with enhanced features.

1.2 Plant breeding

Traditionally, plant breeding is achieved through selection where the individuals

with desirable traits are selected and propagated. Increased knowledge in the pollination

process and reproduction biology (e.g. self-pollination, cross pollination or asexual

reproduction) has brought to the manipulation of plant reproduction for the development of

new plant varieties. This can be achieved by crossing closely or distantly related species

(interbreeding) to produce interspecific hybrids, or crossing among the same species

(inbreeding) to produce intraspecific hybrids (Kuckuck et al. 1985).

Breeding can also be done by generating genetic diversity within a species by

artificial induction of mutations. This is achieved by subjecting plant materials to ionizing

radiation (e.g. X rays, gamma rays) or chemical mutagen (e.g. ethane methyl sulfonate,

EMS) to produce mutants with different traits (North, 1979). Inducing chromosomal

doubling (polyploidization) through the application of mitotic inhibitors such as colchicine

6

Chapter I: Introduction _________________________________________________________________________ had been demonstrated too (Hancock, 1997), and this technique will be used as a plant

breeding tool to induce polyploidy in N. gracilis in this study.

1.2.1 Polyploidization

Most natural plant species are made up of diploid cells formed by combination of

two gametes (each contains half the number of chromosomes of a somatic cell). Polyploidy

is a condition of having more than the typical two sets of chromosomes (North, 1979).

In general, polyploidy results in significant cell enlargement. The increase in cell

size may be reflected in changes in shape and texture of organs (Stebbins, 1971). For

example, larger leaves, stems and roots, increased width-to-length ratio of leaves, larger

and more heavily textured flowers, thicker twigs, lower seed production and larger fruits

(Eigsti & Dustin, 1954; North, 1979; Gao et al. 1996; Kehr, 1996; Hancock, 1997; Stanys

et al. 2006). These expected changes brought by polyploidization had been utilized in plant

breeding and improvement programs.

For example, polyploidy manipulation is particularly advantageous for ornamental

plants and food crops. Polyploidy was induced in phalaenopsis orchids in an attempt to

achieve new floral characteristics such as size, form and color (Griesbach, 1981).

Polyploidy breeding in gaining increment in flower size was done on Rhododendron

hybrids (Väinölä, 2000), pomegranate (Shao et al. 2003), Mecardonia tenella (Escandón et

al. 2007) and Phlox subulata (Zhang et al. 2008) to improve their ornamental value. Thao

et al. (2003) induced tetraploids in ornamental Alocasia to get variation in leaf shape, as

7

Chapter I: Introduction _________________________________________________________________________ this plant is cultivated for their dramatic foliages that are extremely variable in size, shape

and color.

In polyploid plants, fruit size often increases and seed productivity decreases.

Stanys et al. (2006) reported tetraploid Japanese quince fruit with decreased seed

production and increased fruit flesh proportion, which are desirable properties for food

processing.

Increasing the chromosome numbers can sometimes enhance the expression and

concentration of certain secondary metabolites and defense chemicals. This is especially

useful to improve the production of natural plant products (Dhawan & Lavania, 1996). In

Salvia miltiorrhiza (a traditional Chinese medicinal plant), Gao et al. (1996) reported

higher contents of tanshinones in tetraploid plants than that in their diploid counterparts.

Polyploidization was done in Scutellaria baicalensis (Gao et al. 2002) too, to increase

baicalin production.

In addition, genome doubling provides a wider germplasm base for breeding studies.

For example, production of tetraploids for use in breeding work with natural tetraploids.

This approach was used by Cohen & Yao (1996) to overcome hybridization barriers in

Zantedeschia (calla lily) species. Rose et al. (2000) induced tetraploidy in Buddleia

globosa to facilitate introgression of yellow flower color into Buddleia davidii, which is a

natural tetraploid. Some polyploidization works focus on establishing homozygous lines.

This is achieved by producing haploid plants via microspore culture, followed by

chromosome doubling to obtain doubled haploid plants. This method was used in maize

8

Chapter I: Introduction _________________________________________________________________________ (Wan et al. 1991; Barnabás et al. 1999), apple (Bouvier et al. 1994) and wheat (Hansen &

Andersen, 1998) breeding programs as spontaneous chromosome doubling among plants

derived from microspores is relatively low or does not occur at all.

Another application of polyploidization in plant breeding would be producing

sterile triploids by crossing diploids with induced tetraploids. In triploids, chromosome

pairing during meiosis is irregular and gametes with different number of chromosomes are

produced, thus resulting in decreased fertility. In mulberries breeding, triploids are superior

to diploids with respect to leaf nutrition, genetic adaptability and resistance to

environmental stress. Hence, works were done on the induction of superior tetraploid in

mulberry for hybridization with diploids (Chakraborti et al. 1998). Wu & Mooney (2002)

induced autotetraploid Citrus which can be used for subsequent interploid hybridization

with diploid parents to obtain triploid progenies, which are seedless. Petersen et al. (2003)

produced tetraploid Miscanthus sinensis to cross with their diploid counterparts in pursuit

of sterile triploids as a mean to prevent seed dispersal to the natural environment. On the

other hand, Liu et al. (2007) induced chromosome doubling in Platanus acerifolia to

produce tetraploid plants that can be used in breeding programs aimed at producing low-

fertility triploid lines.

1.2.1.1 Polyploidization Approach

In the past, induction of polyploidy artificially has had a difficult and tenuous start.

Traditionally, induction was done by in vivo treatment of shoots, smaller axillary or sub-

9

Chapter I: Introduction _________________________________________________________________________ axillary meristems, seeds or seedlings with colchicine solution (Zhang et al. 2008).

However, high frequency of chimeras and low production efficiency of polyploid plants are

often associated with this method. When inducing tetraploidy in Platanus acerifolia under

in vivo conditions, Liu et al. (2007) observed a low tetraploid formation rate.

The occurrence of chimera in in vivo treatment might be due to varying

environmental conditions (Sikdar & Jolly, 1994). Moreover, the treatment is uneconomical

due to greater consumption of colchicine during the treatment. Madon et al. (2005)

suggested the use of in vitro cultures to induce chromosome doubling after obtaining low

number of tetraploids from treating germinated oil palm seeds with colchicine and oryzalin.

On the other hand, in vitro induction was found to increase efficiency and decrease

occurrence of chimera (Shao et al. 2003). According to Chakraborti et al. (1998), in vitro

culture environment provides controlled temperature and light regime which might favour

synchronized cell divisions, thus lessening the occurrence of mixoploids. In addition,

monitoring of polyploid production can be easier by using in vitro system. Once detected,

the polyploid plant can be propagated via tissue culture system again.

In vitro induction of tetraploids was first achieved in tobacco by Murashige and

Nakano (1966) which paved the way for other plant species. The success for any in vitro

approach depends very much on the existence of a reliable regeneration system, based

either on organogenesis or embryogenesis (Carvalho et al. 2005). Once the in vitro

regeneration system (based on plant tissue culture) was established, in vitro polyploidy

induction is feasible for the plant.

10


1.2.1.2 Plant tissue culture

Plant cells are different from animal cells in having the ability to regenerate into a

whole organism from any part of the plant (e.g. leaves, stems, shoot tips, root tips, pollen

grains) if given the correct stimuli (Hartmann & Kester, 1975). This ability to maintain

genetic and regenerative potential is called totipotency. Plant tissue culture involves the

identifying of the correct stimuli to manifest this totipotency under aseptic and controlled

environment (in vitro). When cultured in vitro, both chemical and physical needs of the

plant cells have to be taken cared of. The chemical needs are met by providing essential

nutrients in the growth medium, whereas the physical needs are met by creating a suitable

external environment (Adrian, 2003).

In general, the growth medium should supply essential elements for proper growth

and development. Table 1.1 summarizes the lists and functions of each macro- and micro-

elements. Carbon source (in the form of sugar such as sucrose, glucose and fructose) is

necessary to satisfy the energy demands of the cultures due to insufficient photosynthetic

activity taking place (Pierik, 1987). Sometimes, organic supplements such as amino acids

(e.g. arginine, asparagine, glutamine, glycine) and vitamins (e.g. thiamine, pyridoxine,

pantothenic acid, myo-inositol) are included in the medium. Amino acids are added to

augment the nitrogen supply while added vitamins would supplement the insufficient

supply of vitamins synthesized by the plant itself (Endress, 1994).

11


Also, plant growth regulators are included in the medium to direct the

developmental pathway of the plant cells. Cytokinins and auxins are two main classes of

growth regulators that are of special importance in plant tissue culture. Auxins are a class of

compounds that stimulate shoot cell elongation and adventitious roots formation while

inhibiting bud formation. In addition, auxins play a role in embryogenesis. Cytokinins, on

the other hand, stimulate bud proliferation, initiate callus formation and inhibit rooting

(Dodds & Roberts, 1995). Examples of auxins are indole acetic acid (IAA), indole butyric

acid (IBA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid (2,4-D).

Examples of cytokinins are zeatin, 2-isopentenyladenine (2-iP), kinetin and 6-

benzylaminopurine (BAP). IAA and zeatin are naturally occurring hormones while the

others are synthetic growth regulators (Gamborg & Shyluk, 1981).

Besides chemical factors, physical factors such as temperature, light, relative

humidity and pH are equally important for the growth of in vitro plants. Generally, nutrient

medium’s pH ranges from 5.0-6.0 is suitable for in vitro growth of explants. pH below 5.0

does not allow satisfactory gelling of agar whereas pH higher than 6.0 give a fairly hard

medium (Chawla, 2000). The plant cultures are usually kept at temperature range of 24-

26ºC with 14-16 hours photoperiod for optimal growth and development (Pierik, 1987).

12


Table 1.1: Macro- and micro-elements important for plant nutrition and their physiological functions.

Elements Function(s)

MACROELEMENTS

Nitrogen (N)

Used for the synthesis of complex organic molecules such as proteins, nucleic acids and some coenzymes.

Phosphorus (P) Formation of ‘high-energy’ phosphate compounds such as adenosine-5’-triphosphate (ATP) & adenosine diphosphate (ADP).

Potassium (K) Regulates the osmotic pressure of the cytoplasm. Enzyme cofactor.

Calcium (Ca) Serves in cell wall synthesis and the regulation of cell membrane function.

Sulphur (S) Used for the synthesis of some amino acids, proteins and enzymes.

Magnesium (Mg)

MICROELEMENTS

Component of chlorophyll. Enzyme cofactor.

Iron (Fe) Component of cytochromes.

Manganese (Mn) Involves in photosynthesis. Enzyme cofactor.

Cobalt (Co) Important in nitrogen fixation.

Boron (B) Involves in the lignification of the cell wall and differentiation of the xylem. Influences calsium ion (Ca2+) utilization.

Copper (Cu) Enzyme cofactor.

Zinc (Zn) Enzyme cofactor.

Molybdenum (Mo) Nitrogen fixation and nitrate reduction.

(adapted from Raven et al. 1986)

13


1.2.1.3 Mitotic inhibitors

Artificial polyploidization is achieved using mitotic inhibitors. Mitotic inhibitors are

also known as microtubule depolymerizing agents. Microtubules are subcellular structures

built up of subunits of protein called tubulin. They play a role in mitosis, cell-plate

formation and the growth of cell wall (Raven et al. 1986). Drug such as colchicine and

antimicrotubule herbicides (e.g. oryzalin, trifluralin, pronamide and amiprophos-methyl)

are mitotic inhibitors that have potent activities towards microtubules (Wan et al. 1991).

Colchicine

Figure 1.3: Chemical structure of colchicine (adapted from Morejohn et al. 1987).

Colchicine is an alkaloid extracted from the corm and seeds of Colchicum autumnae,

an autumn-flowering crocus like plant (Eigsti & Dustin, 1954). The chemical structure of

colchicine is illustrated in Figure 1.3. Colchicine disrupts mitosis by preventing spindle

fibers (composed of arrays of microtubules) formation, thereby inhibits the polar migration

of chromosome at anaphase. As a result, mitosis is not completed although chromosomes

have divided, and the cell will gain an extra set of chromosomes (North, 1979).

14


Colchicine has been successfully applied to induce polyploids in African violet

(Espino & Vazquez, 1981), banana (Van Duren et al. 1996), wheat (Hansen & Andersen,

1998), Brachiaria brizantha (Pinheiro et al. 2000), pomegranate (Shao et al. 2003) and

Phlox subulata L. (Zhang et al. 2008).

Oryzalin

Figure 1.4: Chemical structure of oryzalin (adapted from Morejohn et al. 1987).

Oryzalin is a dinitroaniline herbicide. The chemical structure of oryzalin is

illustrated in Figure 1.4. Oryzalin binds strongly to plant tubulins to form tubulin-oryzalin

complex. This complex is incapable of polymerizing into microtubules and therefore,

disrupts cell division (Morejohn et al. 1987). The mode of action is similar to colchicine

but colchicine has lower binding activities in plant tubulins (Doležel et al. 1994a). Doležel

et al. 2004 reported that plants treated with micromolar concentration of oryzalin gave

similar response as plants treated with milimolar concentration of colchicine.

The efficient use of oryzalin in inducing polyploidy was shown in apple (Bouvier et

al. 1994), Rhododendron hybrids (Väinölä, 2000), Alocasia (Thao et al. 2003), annatto

(Carvalho et al. 2005), Rosa rugosa (Allum et al. 2007) and cork oak (Pintos et al. 2007).

15


1.3 Methods in detecting polyploidy

After polyploidization, the doubled chromosomes will retain in one cell, resulting in

the increase in chromosome number as well as the nuclear DNA content. Several

approaches can be employed in detecting the increment. The classic and straight forward

method would be by studying and counting the chromosomes. The doubling of nuclear

DNA content, on the other hand, can be rapidly screened via flow cytometric analysis. As

cell enlargement is common after polyploidization, measurement of cell size (i.e. stomata

and pollen grains) is of considerable application. Physical observations can be done too, as

polyplodization often causes side effect such as stunted growth and aberrant morphology.

1.3.1 Chromosome studies

Chromosomes carry the hereditary information that control and direct the activities

within a cell. A group of cells combine into tissue, then organs and finally the entire

functional plant. As the plant grows, more cells are made and in making more cells, the

chromosomes undergo division. However, fewer cells continue to divide as the plant

matures. Instead, most of the cells become specialized for certain functions, such as leaf for

photosynthesis and tuber for storage. Cells that continue to divide usually occur in specific

meristematic regions, such as stem tips, root tips and buds (Uno et al. 2001).

16


Cells from these regions are often used for cell division and karyotype study of a

particular plant. Karyotype refers to the characterization of the chromosomal complement

of an organism, including number, size and morphology of the chromosomes (Adrian,

1979). To conduct chromosome studies, these actively dividing cells are subjected to the

following sequential treatments: pretreatment, fixation, hydrolysis and staining (Clark,

1996).

Pretreatment aims to arrest the cells at metaphase by using ice water or spindle-

inhibiting drugs (e.g. colchicine or 8-hydroxyquinoline), prior to fixation. Fixation step

serves to preserve tissue morphology and to minimize loss of nucleic acids. There are two

types of fixative: cross-linking fixatives (e.g. glutaraldehyde and formaldehyde) or protein-

precipitating fixatives (e.g. ethanol mixed with acetic acid in 3:1 ratio) (Leitch et al. 1994).

Fixation is followed by hydrolysis with acid hydrochloric (usually 1 N HCl) before

staining with dye such as aceto-carmine and Feulgen. The dye will give darker stain to the

heterochromatin region of the chromosomes, due to tighter packing of DNA in that region

(Adrian, 1979), thus, facilitating the visualization of chromosomes.

1.3.2 Flow cytometry (FCM)

In plant genomes studies, flow cytometry is a relatively quick, convenience and

reliable tool for various applications ranging from basic research to breeding uses. Flow

17

Chapter I: Introduction _________________________________________________________________________ cytometry can be used for the following purposes: (i) nuclear genome size determination

(in pictogram or base pair unit after conversion), which can be achieved by measuring the

nuclei of sample in comparison to a reference standard, (ii) cell cycle analysis to study cell

populations in G1, S and G2 phases, (iii) ploidy level determination after interploidy crosses,

haploidization and polyploidization treatment, and (iv) chromosome sorting based on

morphological or fluorescent characteristics (Galbraith et al. 1997; Eeckhaut et al. 2005).

A flow cytometer is an instrument which can quantify the fluorescence from DNA-

specific stain. Briefly, this method involves treating samples of nuclei suspensions with a

DNA-specific fluorochrome (e.g. propidium iodide or DAPI) and measuring their relative

fluorescence intensity. This is accomplished by passing the stained nuclei suspensions

through a narrow but intense beam of light. The beam of light is scattered by the passing

nuclei. At the same time, fluorochrome will absorb the illuminating light (a process called

excitation) to produce its characteristic fluorescent output (i.e. emitted light). The scattered

light and fluorescence will be collected and directed into different detectors through

integrated series of lenses, mirror and filters. The detectors will convert the light into

electrical pulses. These pulses are then fed to an amplifier and converter to give the final

signal in digitized form that can be stored in a computer memory (Price & Johnston, 1996).

In conducting FCM analysis, the specimen must be in the form of single cell

suspension. Nuclei can be isolated either by mechanical chopping of tissues or by lysis of

protoplasts. In chopping method, nuclei are released into a nuclei isolation buffer by

mechanical homogenization of a small amount of fresh plant tissue (Doležel, 1991). Leaf

tissue is frequently used, although in specific applications, particular tissues have to be used

18

Chapter I: Introduction _________________________________________________________________________ such as pollen, embryos and endosperm (Eeckhaut et al. 2005). The functions of isolation

buffer include facilitating the release of sufficient quantity of nuclei free of cytoplasm,

preserving the integrity of nuclei, protecting DNA against endonucleases and facilitating

DNA staining (Doležel, 1997).

The six most popular isolation buffer are Galbraith’s buffer, LBO1, Arumuganathan

and Earle, Marie’s nuclear isolation buffer, Otto buffer and Tris-MgCl2 buffer. Unlike the

rest, only the method of nuclei preparation using Otto buffer consists of separate nuclear

isolation and staining steps. The nuclei are released into the Otto I buffer and staining is

performed in a mixture of Otto I and Otto II buffer (Doležel & Bartoš, 2005).

The chemical component of each buffer varies, and may include chemical which

stabilizes the nuclear chromatin or homogenizes chromatin structure (e.g. magnesium ion,

spermine, citric acid), chelating agent (e.g. EDTA, sodium citrate) to bind divalent cations,

organic buffers (e.g. TRIS, MOPS, HEPES) to stabilize the pH of solutions, non-ionic

detergents (e.g. Triton X-100, Tween 20) to facilitate nuclear release from the cytoplasm,

and reducing agents (e.g. β-mercaptoethanol, dithiothreitol, polyvinylpyrrolidone) to

counteract the interference of phenolic compounds with DNA staining (Doležel, 1991;

Doležel et al. 1994a; Doležel & Bartoš, 2005). However, there is no single isolation buffer

which works well with all species. Hence, continuous improvements and modifications of

buffer composition are important to identify the most suitable buffer for a particular plant

species.

19


The DNA of isolated nuclei will be stained with fluorochrome (known as

fluorescent stain or dye). Considering the mode by which the fluorochrome bound to DNA,

there are two types of fluorescent dye. Ethidium bromide (EB) and propidium iodide (PI)

are DNA intercalator dyes that intercalate with double-stranded DNA, and their binding

does not seem to be affected by DNA base composition. However, the samples should be

treated with ribonuclease prior to analysis because this type of dye binds to double-stranded

RNA too (Doležel & Bartoš, 2005). The second type of fluorochrome is highly specific for

double-stranded DNA and shows a base preference. Examples are Hoechst 33258, 4’,6-

diamidino-2-phenylindole (DAPI), mithramycin and chromomycin A3 (Doležel et al. 1992).

Table 1.2 summarizes the fluorochromes frequently used to label nucleic acids and their

binding mode.

Table 1.2: Fluorochromes frequently used to label nucleic acid for FCM analysis.

Wavelength (nm) Fluorochrome Primary binding

mode Excitation Emission

Ethidium bromide Intercalation 525 605

Propidium iodide Intercalation 535 620

Hoechst33258 AT-rich regions 345 460

DAPI AT-rich regions 360 460

Chromomycin A3 GC-rich regions 445 570

Mithramycin GC-rich regions 445 575

(adapted from Doležel, 1997)

20


1.3.3 Other methods

Quantitative observations on cell characteristics such as measurement of cell size,

have considerable application in detecting polyploidy. The most favorable materials are the

guard cells of stomata. Their size and number per unit leaf area have been in general use for

investigating ploidy change (Sybenga, 1992). Guard cells of stomata are different from

other epidermal cells in having chloroplasts. The chloroplast number in the epidermal guard

cells could be used as an alternative approach to detect polyploidy, as the chloroplast

number is genetically correlated with the ploidy level of a plant (Qin & Rotino, 1995;

Compton et al. 1999). Besides that, measurement of pollen grain diameter was being used

as an indicator of ploidy level too (North, 1979).

1.4 Nuclear DNA C-value

Cell contains DNA which plays central role in heredity. Early attempts to estimate

DNA amounts in cell nuclei were followed by the introduction of the term ‘C-value’ which

refers to as constancy of DNA amount per organism. ‘C-value’ was coined by Swift (1950)

as the DNA content of an unreplicated haploid chromosome complement (n). For instance,

cells in G1 phase are cells having 2C nuclear DNA content. The DNA content doubles to

4C during S phase where duplication of nuclear genome occurs. This is followed by G2

phase where the DNA content is maintained at the 4C level. During mitosis, the cell divides

and two daughter cells are formed each with 2C DNA content (Doležel, 1991).

21


C-value is useful in many biological fields like in modern molecular practice in

estimating the genome size. Knowledge of genome size can later be applied in ecology,

phytogeography and systematic study. Thus, data relating to the C-value of plants should be

readily available for reference (Bennet et al. 2000).

In view of the importance of C-value, the first Angiosperm Genome Size Workshop

and Discussion meeting sponsored by Annals of Botany held at the Royal Botanic Gardens,

Kew, UK, in 1997 highlighted the need to improve the taxonomic coverage for plant DNA

C-values of angiosperms especially at familial level. A second Plant Genome Size

Workshop held in 2003 targeted 75% familial coverage by 2009. In 2005, the angiosperm

familial representation had increased to approximately 50% (Hanson et al. 2005).

The 2C DNA content of an organism can be determined either by analyzing DNA

extracted from a large number of cells or by analyzing individual nuclei. Chemical analysis

and analysis of reassociation kinetics represent the first approach. The first approach is

imprecise and provide somewhat questionable estimation of DNA content, because the

number of nuclei present in the source tissue is uncertained and the source tissue may

contain cells at different phases of cell cycle. The second approach of analyzing individual

nuclei offers higher precision but is technically more complex. Feulgen

microspectrophotometry and flow cytometry represent examples of the second approach

(Hardie et al. 2002; Doležel & Bartoš, 2005).

22


Flow cytometry analyzes relative fluorescence intensity and hence relative DNA

content. The absolute DNA amount of an unknown sample can only be determined in

comparison with a reference standard. Comparison can be done by internal or external

standardization. Internal standardization involves simultaneous analysis of isolated nuclei

of sample and standard, whereas external standardization involves separate analysis of

nuclei of sample and standard (Doležel & Bartoš, 2005).

In general, the standard should be genetically stable with constant genome size,

easy to use and easily obtained. Johnston et al. (1999) recommended that the standard used

should have DNA values close to, but not overlapping the 2C and 4C peaks of the target

species. A range of standards is needed to cover the range of genome size variation

observed in plants (Doležel, 1997).

Some of the plant species used for DNA standardization are Lycopersicon

esculentum cv. Stupicke (2C = 1.96 pg; Leal et al. 2006), Glycine max cv. Polanka (2C =

2.50 pg; Doležel et al. 1994b; Lysák et al. 1999; Madon et al. 2008), Petunia hybrida (2C

= 2.85 pg; Marie & Brown 1993; Rival et al. 1997), Zea mays CE-777 line (2C = 5.43 pg;

Thiem & Śliwińska 2003) and Hordeum vulgare (2C = 11.12 pg; Obrien et al. 1996).

23


1.5 Scope and objectives of study

In this study, polyploidization technique will be used to induce chromosome

doubling in N. gracilis to bring about different phenotypic feature (e.g. larger pitcher,

higher intensity of pigmentation) in the plant, in order to create new varieties with

enhanced characteristic.

This study consisted of several scopes. First of all, the 2C nuclear DNA content of

N. gracilis will be determined using FCM analysis (based on Glycine max cv. Polanka as

internal standard). Following that, induction of polyploidy will be carried out by treating

uniform, clonal materials from tissue culture-derived plantlets with mitotic inhibitors such

as colchicine and oryzalin. After polyploidization treatment, the growth and morphology of

the treated explants will be assessed. Treated explants will be subjected to ploidy analysis

via FCM. Stomata and chromosome studies will be carried out to compare diploid and

tetraploid plants. A root induction experiment will be carried out to facilitate the

chromosome studies.

Following this study, two main objectives are to be addressed.

a) Determination of 2C DNA content of N. gracilis via FCM analysis.

b) Development of polyploidization regime for N. gracilis to obtain tetraploids.

24


25


26

Chapter II: Materials and methods _________________________________________________________________________

2.1 Nuclear DNA C-value determination Buffers and chemicals preparation

• Modified Otto I buffer

1.92 g citric acid (R&M, UK), 2 g PVP 40 (Sigma, USA) and 0.5 ml Triton X-100

(Sigma, USA) were dissolved in distilled water to 100 ml. The pH of the solution

was approximately 2-3. The solution was filtered through Whatman (grade number

1) filter paper and stored at 4°C.

• Otto II buffer

5.68 g of sodium phosphate (Ajax, Australia) was dissolved in 100 ml distilled

water. The pH of the solution was approximately 8-9. The solution was filtered

through Whatman (grade number 1) filter paper and stored at room temperature.

• RNase

RNase (100 mg/ml) was prepared by dissolving 2 g of RNase (Sigma, USA) in 20

ml of sterile distilled water. To make RNase (1 mg/ml), 10 ml of RNase (100 mg/ml)

was diluted with 990 ml sterile distilled water. RNase (1 mg/ml) solution was

dispensed into 1.5 ml Eppendorf tube and kept at -20ºC before use.

• Propidium iodide (PI)

PI (25 mg/ml) was prepared by dissolving 1.25 g of PI powder (Sigma, USA) in 50

ml of sterile distilled water. To make PI (1 mg/ml), 40 ml of PI (25 mg/ml) was

25


diluted with 960 ml sterile distilled water. The PI (1 mg/ml) solution was dispensed

into 1.5 ml Eppendorf tube and kept at -20ºC before use.

Methods

The DNA C-value of N. gracilis was determined following a two-step FCM

analysis procedure (Otto, 1990; Doležel & Godhe, 1995). Young leaves were excised from

proliferating shoots of tissue culture-derived N. gracilis in a laminar air-flow cabinet. The

leaves were co-chopped with a similar amount of leaves from Glycine max cv. Polanka (a

known standard with 2C DNA content = 2.5 pg) in 1 ml cold modified Otto I buffer.

Chopping was done for 2-3 minutes with a sharp razor blade in a plastic Petri dish. The

homogenate was filtered through 70 µm Syringe Filcons (Becton Dickinson, USA) using a

clean syringe into an Eppendorf tube to remove cell fragments and large debris. The filtrate

was centrifuged at 1400 rpm for 5 minutes to collect the nuclei (pellet). After centrifugation,

the supernatant was removed until about 100 µl of the liquid was left above the pellet.

Following that, 100 µl of modified Otto I buffer was added and the nuclei were

resuspended by gentle shaking.

Following one hour incubation at room temperature, 1 ml of Otto II buffer with

RNase (50 µg/ml) and PI (50 µg/ml) was added to the nuclei suspension. FCM

measurements were performed using FACS Calibur flow cytometer (Becton Dickinson,

USA) equipped with an argon ion laser. The waste tank of the flow cytometer was emptied

while the sheath tank was filled with sheath fluid (Becton Dickinson, USA) before running

the analysis.

26


Prior to analysis, the gain of the flow cytometer was adjusted so that the peak

corresponding to G1 nuclei isolated from leaves of N. gracilis and G. max (standard) was

localized on approximately channel 200 and 320, respectively. This setting was then used

for subsequent ploidy analysis in Section 2.3.2. Relative fluorescence intensity (or relative

DNA content) of at least 5,000 events were captured before analyzing using CellQuest

software (version 3.3). The absolute 2C DNA value (in picogram unit) of N. gracilis was

calculated based on the following formula:

Chopping of leaf tissues in 1 ml cold modified Otto I buffer

Collection of nucleivia centifugation

Removal of debris by filtration

Remove supernatant, add 100 µl modified Otto 1,

resuspend and incubate for 1 hour

Addition of 1 ml Otto II bufferFlow cytometry

Chopping of leaf tissues in 1 ml cold modified Otto I buffer

Collection of nucleivia centifugation

Removal of debris by filtration

Remove supernatant, add 100 µl modified Otto 1,

resuspend and incubate for 1 hour

Addition of 1 ml Otto II bufferFlow cytometry

Relative DNA content of G. max

Relative DNA content of N. gracilis

x 2.50 pg 2C DNA content of N. gracilis =

Leaves from five plants were analyzed where one leaf was taken from each plant on

six different days for FCM analysis. The 2C value was determined based on the mean value

of all the measurements made. Figure 2.1 summarizes the various steps in the FCM

analysis.

Figure 2.1: Diagram of FCM analysis procedures.

27


2.2 In vitro polyploidy induction

Chemicals, treatment solutions and medium preparation

• Mitotic inhibitor stock solution preparation

100 mM stock solution of colchicine was prepared by dissolving 0.8 g colchicine

(FW 399.44, Sigma, USA) in 20 ml distilled water. The solution was filter sterilized

using a sterile 0.2 µm nylon syringe filter (Sartorius, Germany). On the other hand,

10 mM oryzalin was prepared by dissolving 0.0173 g oryzalin (FW 346.36, Sigma,

USA) in 95%(v/v) ethanol before adding distilled water to 5 ml. (Note: mask and

doubled glove were worn during preparation).

• Mitotic inhibitor treatment solution preparation

The colchicine treatment solution was prepared by diluting different volume of

colchicine stock solution with sterile distilled water to make up to the final

concentration required. Oryzalin treatment solution was prepared by diluting

different volume of oryzalin stock solution with distilled water before autoclaving

(Table 2.1).

28


Table 2.1: Mitotic inhibitor treatment solution preparation.

Mitotic

inhibitor

Final concentration Volume of stock solution used

(µl) to make up 10 ml treatment

solution

Colchicine 1.25 mM

2.5 mM

5 mM

10 mM

125

250

500

1,000

Oryzalin 20 µM

40 µM

60 µM

80 µM

20

40

60

80

• Shoot proliferation medium preparation

Shoot proliferation medium was made up from half-strength Murashige and Skoog

(1962) medium (see Appendix A), caster sugar (filtered cane sugar), myo-inositol,

glutamine, asparagines, arginine and 6-benzylaminopurine (BAP). The pH of the

medium was adjusted to 5.7 using 0.1 N NaOH and/or 0.1 N HCl, prior to the

addition of gelling agent. Medium was sterilized by autoclaving at 121ºC and 15 psi

(or 105 kPa) for 15 minutes. Table 2.2 lists the amount of each component in one

liter shoot proliferation medium for N. gracilis. All chemicals used, unless specified,

were tissue culture grade chemicals from Sigma, USA.

29


Table 2.2: Components of one liter shoot proliferation medium.

1. Half-strength Murashige & Skoog (1962) medium

10x macro 50 ml

100x micro 5 ml

1000x vitamin 0.5 ml

2. Carbon source

Caster sugar 20 g

3. Organic supplements

Myo-inositol 0.1 g

Glutamine 0.1 g

Asparagines 0.1 g

Arginine 0.1 g

4. Plant growth regulator

BAP 1 mg

5. Gelling agent

Gelrite (Duchefa, Netherlands) 1.5 g

Bacto agar (Becton Dickinson, USA) 4 g

Methods

A procedure was established by combining and modifying the polyploidization

approaches used by Rose et al. (2000), Shao et al. (2003), Thao et al. (2003) and Stanyls et

al. (2006). Healthy shoot cultures of N. gracilis (from Tissue Culture Laboratory, FRIM)

were cut into uniform leafless nodal segments (size about 2 cm) and used for the

polyploidization treatments. The polyploidization treatments consisted of four

30

Chapter II: Materials and methods _________________________________________________________________________ concentrations of colchicine (1.25, 2.5, 5 and 10 mM ) or oryzalin (20, 40, 60 and 80 µM)

in combination with four treatment durations (24, 48, 72 and 96 hours), as shown in Table

2.3. Each treatment consisted of three replications and each replication consisted of three

explants.

Treatments were carried out by immersing the explants in 50 ml conical flasks with

the respective treatment solutions. The flasks were agitated on a rotary shaker (Orbit shaker,

Lab-line) at 100 rpm to ensure adequate contact of explants with treatment solutions.

Colchicine treatments were conducted in continuous darkness due to its light sensitive

nature (done by completely wrapping the flasks with aluminium foil) whereas oryzalin

treatments were conducted under light condition (16-hour photoperiod). Both treatments

were done at ambient growth room temperature (26 ± 2ºC). Control treatments consisted of

sterile distilled water only were conducted under both continuous darkness and light

condition (16-hour photoperiod) at different time points (Table 2.4).

When the duration of treatment was completed, explants were rinsed three times

with sterile distilled water. Explants were slightly trimmed to remove damaged tissues and

cultured onto shoot proliferation medium. The cultures were incubated under ambient

growth room condition (temperature of 26 ± 2 º C, relative humidity below 50% and 16-

hour photoperiod). The polyploidization steps are shown in Figure 2.2. Post-treatment

evaluations were done when sufficient plant materials can be obtained.

31


Table 2.3: Polyploidization treatments with various combinations of mitotic inhibitor’s concentrations and treatment durations.

Mitotic inhibitor Concentration Treatment duration (hours)

1.25 mM

24 48 72 96

2.5 mM

24 48 72 96

5 mM

24 48 72 96

Colchicine

10 mM 24 48 72 96

20 µM

24 48 72 96

40 µM

24 48 72 96

60 µM

24 48 72 96

Oryzalin

80 µM 24 48 72 96

32


Table 2.4: Control treatments.

Condition Treatment duration (hours)

Dark

24 48 72 96

Light

24 48 72 96

Nodal segments Treatment solution (colchicine or oryzalin)

Agitation at 100 rpm under dark (colchicine) or light (oryzalin) condition

Cultures incubation under ambient

growth room condition

Regenerated plants

Physical observation

Flow cytometry

Stomata studies

Chromosome studies

Nodal segments Treatment solution (colchicine or oryzalin)

Agitation at 100 rpm under dark (colchicine) or light (oryzalin) condition

Cultures incubation under ambient

growth room condition

Regenerated plants

Physical observation

Flow cytometry

Stomata studies

Chromosome studies

Figure 2.2: Diagram of polyploidization procedures and post-treatment evaluations.

33



2.3.1 Growth and morphology observation

The treated explants were assessed for sign of shoot proliferation after every four

weeks in culture. Number of axillary shoot buds formed was counted after eight weeks.

ANOVA (SAS, version 6.2) was conducted on the collected data. The significance of

differences among the means was made by Duncan’s Multiple Range Test at P≤0.05. The

morphology of the treated explants was being observed too.

2.3.2 Polyploidy screening via flow cytometry

The same two-step FCM analysis procedure and setting used in Section 2.1 (Otto,

1990; Doležel & Godhe, 1995) was followed. Newly-emerging leaves were excised from

the first shoot from nodal explants and used for analysis. Leaves of G. max were chopped

separately for use as external standard. External standard was run after every 10

measurements of samples to rule out mechanical error. Based on the FCM output, explants

were grouped as diploids (with only 2C peak), mixoploids (with both 2C and 4C peak) and

tetraploids (with only 4C peak). ANOVA (SAS, version 6.2) was conducted on the

collected data. The significance of differences among the means was made by Duncan’s

Multiple Range Test at P≤0.05.

34


2.3.3 Stomata studies

30 diploids, 30 mixoploids and 3 tetraploids (due to the low number of tetraploids

obtained from this present study) were used for stomata studies. Two leaves from each

plant were sampled. A small area on the abaxial leaf surfaces was smeared with nail polish.

After the nail polish solution dried, the nail polish impression was removed using a strip of

transparent adhesive cellulose tape (Loytape). The abaxial epidermis imprints will form on

the tape. The tape was placed on a microscope slide and observed under light microscope.

Images were captured using a JVC 3-CCD color video camera connected to a Zeiss

Axioplot microscope via a computer. The determination of the stomata frequency, stomata

length and stomata width was carried out using images viewed under magnification of

10x10. Ten microscopic fields (260 µm x 260 µm) for each slide were randomly sampled to

determine stomata frequency. In each field, ten stomata were randomly chosen to determine

the stomata length and width using the Zeiss KS100 3.0 software.

The stomata size measurement was made as shown in Figure 2.3. For significant

test of differences between diploids and tetraploids, three tetraploids and three

representatives of diploids were selected. Significant test of differences was done by using

two-sample t-test (Minitab, version 12.1) at P≤0.05.

(b)

(a)

Figure 2.3: Measurement of (a) stomata length and (b) stomata width.

35

Chapter II: Materials and methods _________________________________________________________________________ 2.3.4 Chromosome studies

Roots induction experiment

Media preparation

Different types of roots induction medium were prepared by using various

combinations of basal medium (WPM, MS), gelling agents (gelrite, bacto agar) and plant

growth regulators (IBA, NAA), as shown in Table 2.5. Besides these components, 3% (w/v)

caster sugar, 0.1 g/l each of myo-inositol, glutamine, asparagines and arginine were

included in the medium. The pH of the medium was adjusted to 5.7 using 0.1 N NaOH

and/or 0.1 N HCl, prior to addition of gelling agent. Medium was sterilized by autoclaving

at 121ºC and 15 psi (or 105 kPa) for 15 minutes.

Methods

In vitro shoot cultures of N. gracilis were selected and cultured onto the rooting

medium (15 shoot cultures for each medium). The cultures were incubated under ambient

growth room condition. Observation was done every four weeks and rooting percentage

was determined after 12 weeks. The quality of the roots (e.g. thick, thin, fragile, etc) was

also assessed. The selected rooting medium was used to induce roots from diploid,

mixoploid and tetraploid plants for chromosome studies.

36


Table 2.5: Rooting medium based on either WPM or MS medium.

Plant growth regulator (mg/l) Gelling agent Treatment

IBA NAA 0.3% gelrite 0.15% gelrite +

0.40% bacto agar

1 - - + -

2 - - - +

3 0.5 - + -

4 1.0 - + -

5 2.0 - + -

6 3.0 - + -

7 0.5 - - +

8 1.0 - - +

9 2.0 - - +

10 3.0 - - +

11 - 0.5 + -

12 - 1.0 + -

13 - 2.0 + -

14 - 3.0 + -

15 - 0.5 - +

16 - 1.0 - +

17 - 2.0 - +

18 - 3.0 - +

Key: + presence

- absence

37


Root tip squashes preparation

Chemicals preparation

• 8-hydroxyquinoline stock solution (2 mM)

0.058 g of 8-hydroxyquinoline (FW 145.16, Sigma, USA) was dissolved in 200 ml

distilled water (with heat and stirring). Preparation was done in fume hood.

• Carnoy’s solution I

1 ml of glacial acetic acid (Merck, Germany) was added to 3 ml of absolute ethanol

(HmbG, Germany). Preparation was done in fume hood.

• Acid hydrochloric stock solution

83 ml of commercial concentrated HCl (Merck, Germany) was diluted with distilled

water to make 1 liter of 1 M HCl. Preparation was done in fume hood.

• Feulgen stain solution (Schiff’s reagent)

5 g of Fuchsin Basic (Sigma, USA) and 9.5 g of sodium meta-bisulfite (Sigma,

USA) was added to 500 ml HCl (0.15 M) and stirred for two hours. After adding 2

g of activated charcoal (Sigma, USA), the solution was agitated for a few minutes.

Preparation was done in fume hood. Decolorized solution was filtered using conical

funnel with Whatman (grade number 1) filter paper. If the solution is yellow,

additional activated charcoal was added and refiltered.

38


Methods

Squashing and slide preparation method was modified from Kondo (1969) and

Thao et al. (2003). Root tips from 30 diploids, 30 mixoploids and 3 tetraploids (as there

were only three tetraploids obtained from this study) were used as the source for

chromosome studies. The root tips (1-2 cm) were harvested from (in vitro) grown plants in

the morning (9-11 am) with a sterile scalpel and rinsed in distilled water to remove traces

of agar. Root tips were pretreated with 2 mM 8-hydroxyquinoline for 2-3 hours at room

temperature. Pretreated root tips were then fixed in freshly prepared Carnoy’s solution I

and stored at 4°C until use (within one week for optimal results).

Before squashing was done, the root tips were hydrolyzed in 1 N HCl at 60°C for 8

minutes. After hydrolysis, root tips were rinsed with distilled water and stained with

Feulgen staining solution in the dark at room temperature for 1 hour. Then, stained root tips

were squashed in 45% acetic acid. When making root tip squashes, root tip region was

removed and gently tapped with a blunt-end needle to disperse the cells. A clean coverslip

was then applied to the material in a minimal amount of liquid. Cells were spread by

applying pressure (using thumb or tweezer end) on the coverslip layered with filter paper.

The filter paper will absorb the excess acetic acid. The edges of coverslip were sealed with

nail polish to prevent drying. Slides were observed under microscope. Photographs of

chromosome spread were taken using (i) COHU High Performance CCD camera via Leica

DMRA microscope supported by Leica Fluorescence Acquisition & Processing Software

Version 1.20, at a magnification of 10x20 or 10x40 or (ii) JVC 3-CCD color video camera

connected to a Zeiss Axioplot microscope supported by Zeiss KS100 3.0 software at a

magnification of 10x40 or 10x100.

39


40

Chapter III: Results and data analysis _________________________________________________________________________

3.1 Nuclear DNA C-value determination

The 2C DNA content of N. gracilis was found to be 1.60 ± 0.02 pg, based on G.

max cv. Polanka (2C = 2.50 pg) as the internal standard. Figure 3.1 shows a representative

DNA histogram of relative fluorescence intensity. The DNA histogram displays the number

of nuclei counted as a function of relative fluorescence intensity resulting from the flow

cytometric (FCM) analysis of nuclei suspensions (of N. gracilis and G. max) stained with

propidium iodide (PI).

The DNA histogram was obtained after counting at least 5,000 nuclei. In most plant

research using flow cytometry, 5,000 nuclei (Petersen et al. 2003; Hanson et al. 2005) to

10,000 nuclei (Stanys et al. 2006) were usually counted and analyzed. In the DNA

histogram obtained (Figure 3.1), only two distinctive peaks were detected, one for N.

gracilis (Peak A) and the other for G. max (Peak B). Each peak represents the G1 phase

cells depicting the 2C DNA value. No apparent G2 phase peak (for 4C DNA value) was

observed.

The relative fluorescence intensity reflects the relative DNA content, and is usually

given as channel number. Higher channel number represents greater amount of DNA (Price

& Johnston, 1996). The 2C peak of N. gracilis and G. max was localized at approximately

channel 200 and 320, respectively. This indicates that the DNA content of G. max is greater

than N. gracilis. The coefficient of variation (CV) values obtained throughout the analysis

were within 1 to 5%. This low CV values was indicated by the sharp and symmetrical peak.

40


Peak B Channel 320

Peak A Channel 200

Figure 3.1: Representative DNA histogram of relative fluorescence intensity obtained from PI-stained leaves nuclei of N. gracilis and G. max (standard). Peak A and Peak B shows the G1 phase cells of N. gracilis and G. max, respectively.

41




In this study, all treated explants survived, indicating that the designed

polyploidization treatments did not have fatal effects on the explants. Sprouting of shoot

buds could be seen after eight weeks in culture. Comparison between different

polyploidization treatments on the number of shoot bud formation was accomplished by

analysis of variance (ANOVA) followed by Duncan’s multiple range test (SAS, version

6.2). Tables 3.1 and 3.2 show the results of ANOVA for the effects of colchicine and

oryzalin on number of shoot bud formation, respectively. The ANOVA showed significant

differences in the number of shoot bud formed among different concentration, treatment

duration and treatment combination at P ≤ 0.05. The null hypothesis of equal means was

rejected and thus, a multiple comparison test to compare each treatment mean with every

other treatment mean was conducted subsequently using Duncan’s Multiple Range Test.

The respective results for colchicine and oryzalin treatment after Duncan grouping are

shown in Table 3.3 and 3.4.

42


According to Table 3.3, exposure to higher concentration of colchicine for longer

duration, the mean number of shoots formed decreased significantly. The average number

of shoots formed in colchicine-treated explants ranged from 3.7 (96 h-10 mM treatment) to

5.3 (24 h- 2.5 mM and 48 h- 2.5 mM treatment). In the case of oryzalin too, harsher

treatments (higher concentrations and longer treatment durations) produced less shoots,

where the mean number of shoots formed were 1.9 (96 h- 80 µM treatment) to 5.2 (24 h- 20

µM treatment) (Table 3.4).

From the results, it is apparent that oryzalin is more detrimental towards the

explants compared to colchicine. For example, an increase in treatment duration from 24 to

96 hours resulted in a decrease in mean shoot formation from 4.1 to 1.9 (in 80 µM oryzalin

treatment) (Table 3.4). On the other hand, an increase in treatment duration from 24 to 96

hours resulted in only a slight decrease in mean shoot formation from 4.7 to 3.7 (in 10 mM

colchicine treatment) (Table 3.3).

43


Table 3.1: Results on analysis of variances (ANOVA) for the effects of colchicine on number of shoot bud formation.

Source Degree of

freedom

ANOVA sum

of square

Mean

Square

F value Probability

> F

Replication 2 3.478 1.739 1.43 0.2426

Concentration 4 53.856 13.464 11.07 0.0001

Treatment

duration

3 16.994 5.665 4.66 0.0038

Treatment

combination

19 74.328 3.911 3.22 0.0001

Error 151 183.672 1.216

Table 3.2: Results on analysis of variances (ANOVA) for the effects of oryzalin on number of shoot bud formation.

Source Degree of

freedom

ANOVA sum

of square

Mean

Square

F value Probability

> F

Replication 2 1.81 0.91 8.96 0.0002

Concentration 4 209.63 52.41 518.36 0.0001

Treatment

duration

3 70.18 23.39 231.37 0.0001

Treatment

combination

19 300.36 15.81 156.36 0.0001

Error 151 15.27 0.10

44

Chapter III: Results and data analysis _________________________________________________________________________ Table 3.3: Effects of colchicine concentration and treatment duration on number of shoot bud formation.

Treatment duration (hours)

Concentration (mM)

N Mean ± SD*

0 9 6.1 ± 0.8A

1.25 9 5.2 ± 0.8ABCD

2.50 9 5.3 ± 0.7ABCD

5.00 9 4.9 ± 0.2ABCDE

24

10.00 9 4.7 ± 0.6BCDEF

0 9 6.1 ± 0.2A

1.25 9 5.1 ± 0.5ABCDE

2.50 9 5.3 ± 1.0ABCD

5.00 9 4.8 ± 0.5BCDEF

48

10.00 9 4.7 ± 0.9BCDEF

0 9 5.7 ± 0.7AB

1.25 9 5.2 ± 0.9ABCD

2.50 9 4.7 ± 1.0BCDEF

5.00 9 4.3 ± 0.6CDEF

72

10.00 9 4.1 ± 1.0DEF

0 9 5.4 ± 0.5ABC

1.25 9 5.0 ± 0.3ABCDE

2.50 9 4.4 ± 0.2BCDEF

5.00 9 3.9 ± 0.7EF

96

10.00 9 3.7 ± 0.7F

* Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (Duncan’s multiple range test, P≤0.05).

45

Chapter III: Results and data analysis _________________________________________________________________________ Table 3.4: Effects of oryzalin concentration and treatment duration on number of shoot bud formation.

Treatment duration (hours)

Concentration (µM)

N Mean ± SD*

0 9 6.7 ± 0.3A

20 9 5.2 ± 2.2C

40 9 4.4 ± 0.7DE

60 9 4.4 ± 0.7DE

24

80 9 4.1 ± 0.5E

0 9 6.2 ± 0.2B

20 9 4.6 ± 2.1D

40 9 4.2 ± 0.4E

60 9 4.3 ± 0.6DE

48

80 9 4.1 ± 1.3E

0 9 6.2 ± 0.7B

20 9 3.6 ± 0.5FG

40 9 3.3 ± 0.3GH

60 9 3.3 ± 0.3GH

72

80 9 3.1 ± 0.4H

0 9 6.1 ± 0.7B

20 9 3.8 ± 0.8F

40 9 3.1 ± 1.5H

60 9 2.1 ± 1.1I

96

80 9 1.9 ± 0.7I

*Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (Duncan’s multiple range test, P≤0.05).

46


Aberrant morphology was also being observed among treated explants. Physically

normal and aberrant plants are shown in Figure 3.2. The stem and leaf of the normal plant

were distinguishable but the aberrant plant tended to have coarser texture of leaves on the

main shoot but with normal auxiallary shoots.

(b)

Auxiallary shoot

Axillary shoot

Aberrant leaf

Auxiallary shoot

(a)

Axillary shoot

Normal leaf

Figure 3.2: Plant showing (a) normal and (b) aberrant morphology (bar= 1 cm).


All treated explants were subjected to FCM analysis. Individual explants were

classified as diploid, mixoploid or tetraploid plants according to the FCM profiles (i.e.

DNA histogram of relative fluorescence intensity) generated. Representative histograms are

shown in Figure 3.3.

47


Diploids containing 2C DNA showed Peak 1 at approximately channel 200 (Note:

as previously determined in Section 3.1, the peak localized at approximately channel 200

represents the 2C peak of N. gracilis). Tetraploids with 4C DNA showed histogram with

Peak 2 at approximately channel 400. Mixoploids possess both 2C and 4C DNA nuclei and

showed histogram with Peak 1 and Peak 2.

Peak 2

(c) (b)

Peak 2

Peak 1

(a)

Peak 1

Figure 3.3: Representative DNA histogram of relative fluorescence intensity obtained from PI-stained nuclei isolated from leaves of (a) diploid, (b) mixoploid and (c) tetraploid plants of N. gracilis.

48


The number of diploid, mixoploid and tetraploid plants produced from each

replication in each treatment were pooled and presented in percentage. Figures 3.4 and 3.5

show the percentage of diploids, mixoploids and tetraploids produced from the colchicine

and oryzalin treatments, respectively. Obviously, only treatments with 10 mM colchicine

for 72 hours and 40 µM oryzalin for 24 hours were successful in producing tetraploid plants.

All controls (0 mM colchicine and 0 µM oryzalin) showed histogram with 2C peak only

following FCM analysis, and were classified as unaffected diploid plants.

0% 20% 40% 60% 80% 100%

01.252.5

510

01.252.5

510

01.252.5

510

01.252.5

510

Col

chic

ine

conc

entr

atio

n (m

M)

Percentage (%)

Diploid Mixoploid Tetraploid

96 h

72 h

48 h

24 h

Figure 3.4: Percentage of diploid, mixoploid and tetraploid plants produced after treating in vitro-derived nodal segments of N. gracilis with various combinations of colchicine concentrations and treatment durations.

49


0% 20% 40% 60% 80% 100%

020406080

020406080

020406080

020406080

Ory

zalin

con

cent

ratio

n (u

M)

Percentage (%)

Diploid Mixoploid Tetraploid

96 h

72 h

48 h

24 h

Figure 3.5: Percentage of diploid, mixoploid and tetraploid plants produced after treating in vitro-derived nodal segments of N. gracilis with various combinations of oryzalin concentrations and treatment durations.

In colchicine treatments, a pattern in mixoploids induction was evident, where, as

the colchicine concentration increased, the production of mixoploids also increased (Figure

3.4). This is particularly obvious when longer treatment duration was used. For example, in

the 72 hours treatments, increasing the colchicine concentration from 1.25 mM to 10 mM

resulted in an increase in mixoploids production from 33.3% to 66.7%. The increment was

more apparent in the 96 hours treatments, i.e., from 44.4% to 100%. A 100% mixoploids

production was seen from explants treated with 5 and 10 mM colchicine for 96 hours (the

longest treatment duration).

50


From the ANOVA, significant differences were shown in mixoploids production

(Table 3.5). On the other hand, no significant difference was shown in tetraploids

production (Table 3.6). Hence, subsequent Duncan’s Multiple Range Test (P ≤ 0.05) was

conducted on mixoploids production only. The influences of colchicine concentration and

treatment duration on mixoploids production are shown in Table 3.7 and 3.8, respectively.

Both factors had influences on the production of mixoploids and were more apparent in the

treatment with 10 mM colchicine; and treatment duration for 72 and 96 hours. This

indicates that ploidy changes occurred in this species at much higher concentration of

colchicine with longer treatment duration.

Table 3.5: Results on analysis on variances (ANOVA) for mixoploids production.

Source Degree of

freedom

ANOVA sum

of square

Mean

Square

F value Probability

> F

Replication 2 1.800 0.900 4.41 0.0145

Mitotic

inhibitor

1 20.833 20.833 102.06 0.0001

Concentration 8 104.667 13.083 64.09 0.0001

Treatment

duration

3 15.267 5.089 24.93 0.0001

Error 105 21.433 0.204

51


Table 3.6: Results on analysis on variances (ANOVA) for tetraploids production.

Source Degree of

freedom

ANOVA sum

of square

Mean

Square

F value Probability

> F

Replication 2 0.050 0.025 1.08 0.3438

Mitotic

inhibitor

1 0.008 0.008 0.36 0.5500

Concentration 8 0.342 0.043 1.84 0.0771

Treatment

duration

3 0.092 0.031 1.32 0.2723

Error 105 2.433 0.023

Table 3.7: Influences of colchicine concentration on mixoploids production.

Colchicine concentration (mM) N Mean ± SD*

10 12 2.0 ± 1.0A

5 12 1.5 ± 1.0AB

2.5 12 1.1 ± 0.7BC

1.25 12 0.8 ± 0.7C

0 12 0.0 ± 0.0D


Table 3.8: Influences of colchicine treatment duration on mixoploids production.

Treatment duration (hours) N Mean ± SD*

96 15 1.8 ± 1.2A

72 15 1.2 ± 0.9B

48 15 0.7 ± 0.8C

24 15 0.6 ± 0.7C


52


Figure 3.5 shows that the induction of mixoploids in oryzalin treatments was

unpredictable and did not exhibit any trend, where the production of mixoploids fluctuated

among the treatments. Four treatments (i.e. 48 h- 60 µM, 72 h- 20 µM, 96 h- 60 µM and 96

h- 80 µM) produced 100% mixoploids. The rest of the treatment, produced considerably

high percentage of mixoploids (≥ 50%).

The influences of oryzalin concentration and treatment duration on mixoploids

production are shown in Table 3.9 and 3.10, respectively. Obviously, the effect of oryzalin

on mixoploids production was not significantly different among the four concentrations (20,

40, 60, 80 µM) but was significantly different between 24 and 96 hours treatment duration.

It appeared that oryzalin concentration as low as 20 µM can markedly abrupt mitosis.

Meanwhile, concentration above 20 µM can still induce ploidy changes without

compromising the survivability of the explants (as all explants survived). This indicates

that ploidy changes in this species can occur at low oryzalin concentration.

Table 3.9: Influences of oryzalin concentration on mixoploids production.

Oryzalin concentration (µM) N Mean ± SD*

80 12 2.2 ± 0.8A

60 12 2.6 ± 0.7A

40 12 2.3 ± 0.6A

20 12 2.6 ± 0.7A

0 12 0.0 ± 0.0B

*Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (Duncan’s multiple range test, P≤0.05)

53


Table 3.10: Influences of oryzalin treatment duration on mixoploids production.

Treatment duration (hours) N Mean ± SD*

96 15 2.3 ± 1.2A

72 15 2.0 ± 1.2BA

48 15 1.8 ± 1.2BA

24 15 1.6 ± 1.1B

*Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (Duncan’s multiple range test, P≤0.05)

Overall, colchicine and oryzalin treatments produced 45.14% (65/144) and 79.86%

(115/144) of mixoploids, respectively (Figure 3.6). According to two sample t-test

(P≤0.05), significant different was found between colchicine and oryzalin treatments in

mixoploids production (Table 3.11).

Figure 3.6: Percentage of diploids, mixoploids and tetraploids produced by (a) colchicine and (b) oryzalin treatments.

53.47%45.14%

1.39%

Diploids Mixoploids Tetraploids

19.44%

79.86%

0.70%

Diploids Mixoploids Tetraploids

(b) (a)

54


Table 3.11: Mixoploids production in colchicine and oryzalin treatments.

Chemical N Mean ± SD

Oryzalin 60 1.9 ± 1.2A

Colchicine 60 1.1 ± 1.0B

Means ± standard deviation followed by different letters (A or B) are significantly different at 95% confidence level (P≤0.05) by two sample t-test (Minitab, version 12.1).

In tetraploidy induction, colchicine and oryzalin treatments produced 1.39% (2/144)

and 0.70% (1/144) of tetraploids, respectively (Figure 3.6). No significant difference was

observed between colchicine and oryzalin treatments in tetraploids induction (Table 3.6) as

only two particular treatments produced tetraploids and the number of tetraploids produced

is low. However, two tetraploids were derived from the same colchicine treatment (in

different replication) suggesting that this treatment may induce tetraploids effectively (see

Appendix B).

55

Chapter III: Results and data analysis _________________________________________________________________________ 3.2.3 Stomata studies

The diploid, mixoploid and tetraploid plants produced were subjected to stomata

studies. Table 3.12 summarizes the stomata length, width and frequency obtained from

analyzing the abaxial leaf imprints of diploid and tetraploid plants. On the average, diploids

and tetraploids have a mean (stomata) length of 31.42 ± 1.53 µm and 45.39 ± 2.74 µm,

respectively. The stomata length of diploids and tetraploids was significantly different as

inferred by the two-sample t-test at P≤0.05 (Minitab, version 12.1). Similarly, the stomata

width too, was significantly different between diploids and tetraploids. The mean (stomata)

width of diploid and tetraploid plants is 21.97 ± 1.38 µm and 35.10 ± 3.73 µm, respectively.

The stomata frequency between the diploids (413.71 ± 50.22 mm-2) and tetraploids (346.15

± 59.81 mm-2) was also significantly different.

Table 3.12: Stomata studies of diploid and tetraploid plants of N. gracilis.

Ploidy level Stomata length

(µm)

Stomata width

(µm)

Stomata frequency

(mm-2)

Diploid 31.42 ± 1.53A 21.97 ± 1.38A 413.71 ± 50.22A

Tetraploid 45.39 ± 2.74B 35.10 ± 3.73B 346.15 ± 59.81B

Values represent mean ± standard deviation. 600 measurements were made to determine stomata length and width whereas stomata frequency was based on measurements made on 60 microscopic fields. Mean ± standard deviation followed by different letters (A or B) are significantly different at 95% confidence level (P≤0.05) by two sample t-test (Minitab, version 12.1).

56


Figure 3.7 shows the stomata cells (structures) of diploids, tetraploids and

mixoploids. The size of stomata cells between diploids and tetraploids appeared remarkably

different. Another observation from this study was the deformed stomata shape seen in

tetraploids. Normal diploid stomata cells were elliptical in shape (Figure 3.7a) whereas the

deformed stomata of tetraploids exhibited circular or undefined shape (Figure 3.7b).

Mixoploids, on the other hand, showed both diploid and tetraploid stomata cells (Figure

3.7c).

(a) (b) (c)

Figure 3.7: Stomata cells from abaxial leaf imprints of (a) diploids, (b) tetraploids and (c) mixoploids (bar= 50 µm).

57




Roots induction experiment was conducted to produce suitable root materials for

preparation of root-tip squashes from diploid, mixoploid and tetraploid plants. Figures 3.8

and 3.9 show the rooting percentage (based on the number of rooted plants but not the

number of roots formed) in N. gracilis using WPM- and MS-based medium, respectively.

In WPM-based medium, the rooting percentage increased when the concentration of

plant growth regulator (PGR) increased, regardless of the type of PGR (IBA or NAA) and

gelling agent (gelrite or gelrite + bacto agar) used (Figure 3.8). MS-based medium, on the

other hand, supported considerably higher rooting percentage, only in the presence of high

levels of PGR (e.g. 3.0mg/l IBA or NAA) with gelrite (Figure 3.9). Thus, WPM-based

medium was more suitable for roots induction in N. gracilis compared to MS-based

medium.

WPM medium with gelrite showed roots induction (only a few roots) in the absence

of PGR (0.0 mg/l of IBA or NAA) (Figure 3.8). With the incorporation of PGR, number of

roots formed was greatly increased and could not be counted. Different types of root were

formed when different PGR was used. IBA induced thick dark roots in moderate quantity

(Figure 3.10a) whereas NAA promoted numerous thin roots (Figure 3.10b). The latter

58

Chapter III: Results and data analysis _________________________________________________________________________ (numerous thin roots) were fragile, easily breakable and were not suitable for root tip

squashes preparation.

Therefore, WPM is the preferred basal medium for roots induction, when combined

with gelrite and IBA which produced suitable qualities of thick roots with obvious root tip

regions (in sufficient amounts) for root tip squashing. IBA (0.5 mg/l) was chosen even

though higher levels of IBA induce higher rooting percentage, due to economical reason.

0

10

20

30

40

50

60

70

80

90

100

IBA+ gelrite IBA+ gelrite& bacto agar

NAA+ gelrite NAA+gelrite&

bacto agar

Combinations of different PGR and gelling agent

Roo

ting

perc

enta

ge (%

)

0.0 mg/l0.5 mg/l1.0 mg/l2.0 mg/l3.0 mg/l

Figure 3.8: Rooting percentage of N. gracilis in medium formulation based on WPM.

59


0

10

20

30

40

50

60

70

80

90

100

IBA+ gelrite IBA+ gelrite& bacto agar

NAA+gelrite

NAA+gelrite&

bacto agar

Combinations of different PGR and gelling agent

Roo

ting

perc

enta

ge (%

)

0.0 mg/l0.5 mg/l1.0 mg/l2.0 mg/l3.0 mg/l

Figure 3.9: Rooting percentage of N. gracilis in medium formulation based on MS medium (b)

(a)

Figure 3.10: Roots induction by different PGR. (a) IBA induced thick and stronger roots and (b) NAA induced thin and fragile roots (bar= 1 cm).

60

Chapter III: Results and data analysis _________________________________________________________________________ Chromosomes of Nepenthes gracilis

The exact number of chromosome was difficult to determine because the

chromosomes were not well-separated in most cases. Also, pieces of the meristem

epithelium cells and unwanted debris were also found in most slides. Sometimes,

overlapping of cell debris with chromosome spread made chromosome counting difficult.

The number of chromosome counted was near to 80. Figure 3.11 shows a considerably

good chromosome spread at metaphase in diploid cell.

Figure 3.11: Well-spread chromosomes in diploid cell (bar= 20 µm).

Chromosome studies were done on mixoploid and tetraploid plants as well. As the

chromosome number was large and the size was small, it was difficult to distinguish

between the diploid and tetraploid chromosomes through chromosomes counting. Since

well-spread chromosomes were rarely obtained, observation by estimation was made based

on clump of chromosome spread. Most of the root tip squashes prepared from mixoploids

61

Chapter III: Results and data analysis _________________________________________________________________________ showed diploid chromosome spreads. In a few of the root tip squashes, both diploid and

tetraploid chromosome spreads were observed.

Given the low number of tetraploids obtained in this study, only a few root tip

squashes were made from tetraploid plants. Unfortunately, no tetraploid chromosome

spread was noticed in any of the root tip squashes. Surprisingly, normal diploid

chromosome spreads were found in some cells, indicating the possibility of chimerism in

these plants, where tetraploid cells were found in leaf tissues (as determined via FCM

analysis and stomata studies) while roots showed diploid cells.

3.2.5 Morphology of diploid and tetraploid plants

Figure 3.12 shows the unaffected diploids and tetraploids. Tetraploids have bigger

leaf than the diploids, and the texture of tetraploids leaves was slightly coarser than

diploids. Pitcher size comparison was not possible because there was no pitcher developed

as yet. At present, the tetraploids are being micropropagated using shoot proliferation

medium. The stability of the ploidy level of the tetraploids will be further assessed via

FCM analysis in future.

(b) (a)

Figure 3.12: Morphology of (a) diploids and (b) tetraploids.

62


63


64

Chapter IV: Discussion _________________________________________________________________________

4.1 Nuclear DNA C-value determination

The nuclear DNA content of N. gracilis was determined via FCM analysis using

nuclei suspension extracted from leaves of in vitro-grown plants and stained with

propidium iodide (PI). It is generally recommended that PI should be used as the

fluorochromes for nuclear DNA content estimation due to its insensitivity to base

composition (Doležel et al. 1992). The use of base-specific fluorochromes such as DAPI

should be avoided because the base content of the reference standard and sample might

differ. This is especially true in plants where overall molar AT or GC content is not

constant (Shapiro, 1976). In a study to evaluate the accuracy in measuring plant nuclear

DNA using PI and DAPI, PI-based flow cytometry was found to produce consistent results

from different laboratories, and the values were close to those obtained using Feulgen

microspectrophotometry. DAPI-based flow cytometry on the other hand, showed some

discrepancies (Johnston et al. 1999).

The type of material used will also affect the quality of the FCM output. Leal et al.

(2006) detected some differences in the quality of histograms generated from in vitro- and

ex vitro-derived leaves. They found that leaves from in vitro-grown plants presented peaks

with lower CV compared to ex vitro-grown (field-grown) plants. CV is the variance in

distributions shown by FCM measurement. Ideally, the generated peaks should be

symmetrical and with low CV. The CV value usually ranges from 1-10% (Doležel, 1991).

Galbraith et al. (1997) suggested that CV values less than 5% as the acceptance criterion.

63


Besides, field-grown leaves tend to produce higher amount of background noise in

the FCM output. This may be related to chlorophyll autofluorescence, presence of cytosolic

compounds (such as tannins) in higher concentrations in leaves of field-grown plants and

also the existence of trichomes in leaves of some cultivars (Leal et al. 2006). Tannins are

common phenolic compound frequently accumulated in various tissues of plants (Loureiro

et al. 2006). Noirot et al. (2000) suggested that these cytosolic compounds affect dye (i.e.

PI) accessibility to DNA, and thus interfere with the FCM analysis.

Initial attempt for nuclear DNA content determination was made by using young

leaves from field-grown N. gracilis but the FCM output was not usable due to very high

amount of background noise which interfered with the peak, even though 2% of

polyvinylpyrrolidone (or PVP, a tannin-complexing agent) was included in the extraction

buffer. However, young leaves from field-grown G. max did not exhibit this significant

problem. Hence, in vitro-derived leaves of N. gracilis is a better choice for FCM analysis

alongside field-grown leaves of G. max as internal standard (for nuclear DNA content

determination).

Internal standardization was used because it eliminates errors due to machine

instability and variability in sample preparation (Doležel, 1997). G. max was a suitable

standard plant as its DNA values were close to but not overlapping with the 2C and 4C

peaks of N. gracilis. In this study, cell nuclei from leaves of both N. gracilis and G. max

(2C = 2.50 pg) were analyzed simultaneously via flow cytometry and the absolute 2C DNA

value for N. gracilis was found to be 1.60 ± 0.02 pg.

64


According to Leitch et al. (1998), a 1C DNA value less or equal to1.4 pg is

considered small. The 1C DNA value of N. gracilis (approximately 0.80 pg) is smaller than

1.4 pg, indicating a small DNA C-value. Apart from Sarracenia flava (4C = 17.38 pg) and

Drosophyllum lusitanicum (4C = 60 pg), other carnivorous plants tend to exhibit small

DNA C-value. For example, Drosera species (range from 4C = 1.2 to 3.8 pg), Byblis

liniflora (4C = 3.55 pg), Pinguicula primuliflora (4C = 2.73 pg), Cephalotus follicularis

(4C = 2.55 pg) and Nepenthes pervillei (4C = 1.12 pg) have 1C DNA value lower than 1.4

pg as well. The nuclear DNA content (4C DNA value) of the above mentioned species

were determined using Feulgen microspectrophotometry by measuring cell nuclei at the

mid-prophase of mitosis (4C), taken from actively-growing root tip meristems (Hanson et

al. 2001; Hanson et al. 2005).

Most of the carnivorous plants have small DNA C-value and thus small DNA

content in order to adapt to their generally nutrient-poor living habitats (Hanson et al.

2005). Hanson et al. (2001) proposed that plants growing in nutrient-poor environments

minimize their genome sizes by reducing redundant phosphorus-rich nucleic acid such as

non-essential repeated DNA sequences.

65




In this study, all explants (nodal segments) treated with liquid colchicine (1.25, 2.5,

5 and 10 mM ) or oryzalin (20, 40, 60 and 80 µM) for 24 to 96 hours survived and retained

their growth. The 100% survival rate of explants in this present study might be explained

by the ability of the explants (of N. gracilis) to tolerate the polyploidization regimes with

colchicine or oryzalin. Although no fatality was observed, treated explants showed

decreased number of shoots formation. Also, aberrant-morphology plants were being

observed among treated explants.

Delayed sprouting and growth of shoot buds in colchicine-treated explants has been

reported by Pryor & Frazier (1968), Cohen & Yao (1996) and Carvalho et al. (2005). The

slow growth may be due to the physiological disturbance caused by colchicine, resulting in

reduced cell division (Eigsti & Dustin, 1954). Dinitroaniline herbicides such as oryzalin

will also interfere with cell division and cause abnormal shoot growth (Floyd & Alden,

1981), as shown in this study.

Besides that, Liu et al. (2007) reported physically aberrant plants (i.e. distorted

leaves) in which the first 1-2 leaves of colchicine-treated Platanus acerifolia seedlings

were morphologically abnormal (e.g. wrinkled), but subsequent leaves appeared normal.

This was also seen in Astragalus membranaceus where in most cases, the first few leaves

66

Chapter IV: Discussion _________________________________________________________________________ of tetraploids or mixoploids had a distorted appearance, but the subsequent leaves appeared

normal (Chen & Gao, 2007). Besides colchicine treatments, this study also revealed

physically deformed plants from oryzalin treatments. Hence, initial slow growth of shoot

buds (resulting in less number of shoot buds formation) and aberrant morphology are the

first visible effect of the polyploidization treatments.


Flow cytometric profiles

FCM is a rapid method for ploidy screening after polyploidy induction. The

extraction and staining buffer used in this study was suitable as it produced acceptable

peaks with 1-5% of CV values. The addition of 2% PVP (Section 4.1) in the Otto I

extraction buffer had significantly lessened the broadness of the peaks as well as lowering

the CV values.

Background noise (large number of signals) was noticed at the lower channel

number (Figure 3.3). Similar observations were found in several other plant species, such

as Rhododendron hybrids (Väinölä, 2000), Mecardonia tenella (Escandόn et al. 2007),

Platanus acerifolia (Liu et al. 2007), and Rosa (Khosravi et al. 2008) in their FCM profiles.

This problem is thought to be attributed by broken cells during extraction and/or non-

specific staining of other cell constituents (Emshwiller, 2002). This background noise was

considered acceptable as it did not interfere with the peak. Variation in peak position in

67

Chapter IV: Discussion _________________________________________________________________________ FCM analysis can also occur due to instrumental error. This error was minimized by

running standard after every ten sample measurements in order to correct the voltage of the

photomultiplier (Eeckhaut et al. 2005), which was practiced in this present study.

Plant nuclei may produce two peaks in a FCM profile, representing the different

phases of cell division, that is the G1 (consists of 2C cells) and G2 (consists of 4C cells)

phases, where the cell population in G2 phase is relatively small (Galbraith et al. 1983). In

Cyclamen persicum, the FCM analysis of zygotic embryos resulted in a 2C and a lower 4C

peak (Schmidt et al. 2006; Borchert et al. 2007). When analyzing in vitro leaves of Citrus

(Wu & Mooney, 2002) and Astragalus membranaceus (Chen & Gao, 2007), a small

percentage of nuclei at 4C peak representing the G2 phase nuclei was observed in the FCM

profiles.

Nevertheless, after polyploidization treatment, 4C cells may be produced. The 4C

cells from the induced polyploids might be interfered by the G2 phase cells from the

differentiated plant tissues and FCM analysis cannot distinguish G2 phase cells from these

4C polyploid cells (Galbraith et al. 1997). However, as all the controls (untreated plants)

produced histogram (Figure 3.3a) with only a 2C peak after FCM analysis (Figures 3.4 and

3.5), it could then be proposed that the occurrence of the 4C peak in the histograms

generated from this present study was attributed to the effect of mitotic inhibitors

(colchicine or oryzalin), which disrupted mitosis and resulted in cells with doubled

chromosome (i.e. 4C cells).

68


The absence of 4C peak (attributable to G2 phase cells) in FCM profiles was not

uncommon and it was dependent on the type of tissues used. De Laat et al. (1987) stated

that the distribution of cells (G1 and G2 phase) are dependent on the physiological state of

the material but was rather constant when similar explant sources were used. Lodhi &

Reisch (1995) and Lima et al. (2003) reported the absence of 4C peak when leaves (not in

vitro) of Vitis species were used for FCM analysis. The former described this obscurity of

4C peak to the developmental stage of leaves, and proposed that leaf cells are most likely to

be in G1 phase.

When using in vitro-derived leaves, Thao et al. (2003), Escandόn et al. (2007) and

Rubuluza et al. (2007) revealed that only a single peak corresponding to the G1 phase was

found in the DNA histogram of Alocasia, Mecardonia tenella and Colophospermum

mopane, respectively. This was in accordance with the observation made by Doležel et al.

(1994a) and Doležel (1997) which claimed that in vitro cultures often show low frequency

of mitotic cells and most young leaves produce a single peak in the FCM profiles. Hence, it

could then be suggested that when tissue culture-derived young leaves were used for FCM

analysis, a single 2C peak corresponding to cells in G1 phase may be obtained. Furthermore,

the materials used for the FCM analysis in this study were standardized (i.e. newly-

emerging leaves), hence, the results would be rather constant as suggested by De Laat et al.

(1987).

69


Polyploid plants production

In comparison to several other studies on different plant species using similar

approaches (i.e. explants and polyploidization regime), the results from this study was not

encouraging as only 1.39% and 0.70% tetraploids were obtained after treating nodal

explants with colchicine and oryzalin solution, respectively.

Different explants had been used for in vitro polyploids induction. Chromosome

doubling had been obtained in various plant species by treating in vitro materials (tissues)

of nodal segments (Rose et al. 2000; Väinölä, 2000; Shao et al. 2003; Stanys et al. 2006;

Escandón et al. 2007), shoot tips (Adaniya & Shirai, 2001; Thao et al. 2003; Gu et al.

2005), embryogenic callus (Chen & Goeden-Kallemeyn, 1979; Gao et al. 2002; Wu &

Mooney, 2002; Yang et al. 2006), leaves (Espino & Vazquez, 1981), zygotic embryos

(Ruiz & Vazquez, 1982), protoplasts (Tamura et al. 1996) and microspore cultures (Hansen

& Andersen, 1998) with mitotic inhibitors such as colchicine and oryzalin.

However, different explants might show different chromosome doubling efficiency.

For example, Kermami et al. (2003) demonstrated that tetraploidy could be induced more

rapidly in nodal sections than in shoot tips when using oryzalin as the mitotic inhibitor.

They explained this finding by suggesting that oryzalin reached the meristem mainly via

the cut surfaces, as there are two cut surfaces in the nodal segments and only a single cut

surface in the shoot tip explants.

70


When treating shoot tips of Alocasia with colchicine and oryzalin solution, Thao et

al. (2003) obtained 4.52% and 6.79% tetraploids, respectively. Adaniya & Shirai (2001)

produced 3.88% tetraploids when treating ginger shoot tips with colchicine in liquid MS

medium. Gu et al. (2005) obtained 2.6% tetraploids only in Zizyphus jujuba by treating

shoot tips in liquid MS medium containing colchicine. On the other hand, Escandόn et al.

(2007) produced 53.97% of tetraploids by treating nodal segments of Mecardonia tenella (a

herbaceous ornamental plants) with colchicine solution whereas Rose et al. (2000) obtained

65.52% of tetraploids in Buddleia globosa (a flowering shrub) by treating nodal explants

with colchicine solution. Although this present work was conducted using nodal explants,

the tetraploids production rate was fairly low.

In general, 0.25 to 25 mM colchicine is usually incorporated in liquid treatment

solution for the duration of 12 to 96 hours depending on the type of explants used (Rose et

al. 2000; Thao et al. 2003; Gu et al. 2005; Chen & Gao, 2007; Rubuluza et al. 2007). On

the other hand, explants were exposed to 0.13 to 1 mM colchicine for 10 to 30 days when

semisolid treatment systems were used (Gao et al. 2002; Shao et al. 2003; Carvalho et al.

2005; Zhang et al. 2008).

Oryzalin, on the other hand, can be used effectively at much lower concentration,

i.e. as low as 0.3 µM oryzalin was used to obtain in vitro chromosome doubling in Brassica

napus microspore culture (Hansen & Andersen, 1996); and as high as 150 µM oryzalin was

used in polyploidization of Rhododendron hybrids (Väinölä, 2000). However, 2 to 50 µM

oryzalin is commonly used in both liquid and semisolid treatment system (Chauvin et al.

71

Chapter IV: Discussion _________________________________________________________________________ 2003; Carvalho et al. 2005; Stanys et al. 2006). Similar to colchicine treatment, exposure

period ranged from hours and days when liquid and semisolid condition was applied,

respectively.

In this study, 1.25 to 10 mM of liquid colchicine and 20 to 80 µM of liquid oryzalin

were used for treatments conducted for 24 to 96 hours. Following that, only treatment with

10 mM colchicine for 72 hours and treatment with 40 µM oryzalin for 24 hours produced

tetraploids. Again, although the common polyploidization regime was practiced, low

tetraploids production rate was obtained and this might be attributed to the following

factors.

Azhar (2000) suggested that the efficiency in tetraploids production was dependent

on the time required for the cell cycle completion in a particular tissues or plants. Hence,

the polyploidization treatment should take place at the right time. As different plant species

might exhibit different sensitivity towards the level of mitotic inhibitor applied, the right

concentration has to be figured out. Thus, only certain treatment will produce tetraploids

and the optimum dose-time relation demonstrated by different species will differ.

Also, the growth of tetraploid cells might have been suppressed by the fast dividing

normal diploid cells, as suggested by Stanys et al. (2006) in their work on Japanese quine.

It is well known that a population of cell shows variations among themselves, where cells

which have adapted best to the growing environment coupled with a short doubling time

will predominate and take over the entire cell populations gradually (Gamborg & Shyluk,

1981). Polyploid cells often exhibit reduced number of cell division during growth and

72

Chapter IV: Discussion _________________________________________________________________________ development (Lewis, 1980). Hence, when explants treated with mitotic inhibitor grow

under favorable culture condition, the induced tetraploid cells with a low rate of cell

division may be easily replaced by original diploid cells with a relatively higher rate of cell

division during meristem organization (Adaniya & Shirai, 2001).

This is especially true when not all cells are affected and polyploidized in the first

place when multicellular tissues (such as nodal segment) are used. In multicellular tissues,

only a portion of the cells or cell layers may be affected, while the other portion remains

diploid (Pryor & Frasier, 1968) due to differential uptake of mitotic inhibitor by different

layer of cells (Tambong, 1998). This might be the reason why mixoploids and chimeric

tetraploids were obtained in this study. The mixoploids obtained in this study will also be

propagated the same way as the chimeric tetraploids (by isolating the stem to the leaf that is

affected) since Stanys et al. (2006) managed to regenerate tetraploid shoots from

mixoploids following subsequent subcultivations.

A recent finding from Khosravi et al. (2008) suggested that chromosome doubling

is genotype dependent, where they had induced polyploidy in Rosa with different ploidy

levels (i.e. diploid, triploid and tetraploid). From their study, they discovered that plants

with lower ploidy level have higher tendency towards chromosome doubling and no

chromosome doubling was induced in tetraploid genotype.

As the ploidy level of Nepenthes is uncertain, we cannot exclude the possibility that

Nepenthes are already plants with high ploidy level since this genus exhibits small

chromosome size in large number. Heubl & Wistuba (1997) had proposed the assumption

73

Chapter IV: Discussion _________________________________________________________________________ that Nepenthaceae are palaeopolyploids with the basic number of x = 20 (where 2n = 80)

based on the phylogenetic background of Nepenthaceae. If this is the case, N. gracilis

might be originally a tetraploid. Polyploidization work on this species, then, will be less

promising and difficult. This may explain the low rate of tetraploids production in this

study. Also, it may not be appropriate to use the term ‘tetraploid’ to elucidate the doubled

chromosome products obtained in this study. However, the diploid assumption will be

made in this study and the term ‘tetraploid’ will be used to represent the doubled

chromosome products so that the readers can follow the whole discussions.

In view of the low rate of tetraploids induction where only 1.39% and 0.70% of

tetraploids were derived from colchicine and oryzalin treatment, respectively, no concrete

conclusion could be drawn as to which mitotic inhibitor was more efficient in producing

tetraploids (as inferred by Duncan’s Multiple Range Test at P≤0.05).

However, influences of both mitotic inhibitors on mixoploids production were

assessed since it was an indication of diploids to tetraploids conversion. Duncan’s Multiple

Range Test (P≤0.05) showed that mixoploids production in colchicine treatment had

significantly increased with increased colchicine concentration and treatment duration

whereas mixoploids production in oryzalin treatment did not show significant increase

among the oryzalin concentration tested. Hence, it was proposed that ploidy changes

occurred in this plant species at much higher concentration of colchicine but readily

happened at low oryzalin concentration.

74


Also, as oryzalin treatments (79.86%) produced significantly higher percentage of

mixoploids than colchicine treatments (45.14%), this indicated that oryzalin was much

more effective in inducing ploidy changes, though not fully to tetraploids. This also showed

that µM concentration of oryzalin was more efficient than mM concentration of colchicine

to induce ploidy changes. Immunocytochemical studies have shown that oryzalin has

higher affinity for plant tubulins than colchicine (Morejohn et al. 1987). Hence, low

concentration of oryzalin can markedly abrupt mitosis by interfering with the structure of

mitotic spindle.

Comparison of colchicine and oryzalin was done in several polyplodization works

by other researchers using various plant species. Higher chromosome doubling efficiencies

shown by oryzalin, as compared to colchicine, were reported in potato (Ramulu et al.1991),

maize (Wan et al. 1991), apple (Bouvier et al. 1994), Rhododendron hybrids (Väinölä,

2000), Alocasia (Thao et al. 2003), annatto (Carvalho et al. 2005) and cork oak (Pintos et

al. 2007).

4.2.3 Stomata studies

Stomata size and frequency are widely used as a selection criterion in determining

ploidy level. It was successfully used in discriminating plants with different ploidy levels

(as a result of hybridization or polyploidization) in Bromus inermis (Tan & Dunn, 1973),

Actinidia deliciosa (Przywara et al. 1988), Zantedeschia (Cohen & Yao, 1996), cocoyam

75

Chapter IV: Discussion _________________________________________________________________________ (Tambong et al. 1998), black wattle (Beck et al. 2003), annatto (Carvalho et al. 2005),

Japanese quince (Stanys et al. 2006), Astragalus membranaceus (Chen & Gao, 2007) and

Platanus acerifolia (Liu et al. 2007).

Using tissue culture-derived materials for stomata studies has an advantage where

variation was minima as the plants are grown under controlled environment (Blanke &

Belcher, 1989). In order to avoid rapid dehydration via transpiration upon transfer of tissue

culture-derived leaves from the culture vessel to the external environment, only a few

samples were handled at one time. Abaxial (lower leaf surface) instead of adaxial (upper

leaf surface) leaf imprints were analyzed because the abaxial part of the leaf contains more

stomata (Azhar, 2000).

In this study, a positive relationship was found between ploidy level and stomata

size (length and width). Stomata frequency of diploid and tetraploid plants, however, was

inversely associated. This positive relationship between ploidy level and stomata size plus

the inverse relationship between ploidy level and stomata frequency, were also reported in

mulberry (Chakraborti et al. 1998), Zizyphus jujuba (Gu et al. 2005) and grapevine (Yang

et al. 2006). Stomata studies of mixoploids revealed both diploid and tetraploid stomata

cells on the same leaf. Thao et al. (2003) had also reported that their mixoploid Alocasia

plants had stomata cells with size similar to either diploids or tetraploids.

76




The ability to obtain good chromosome spread to perform cytological techniques

such as chromosome counting, chromosome banding and fluorescent in situ hybridization

(FISH) is very much dependent on the number and morphology of the roots which contain

the metaphase cells. The species N. gracilis tends to have very small and delicate roots.

Upon harvesting, most of the root tips which contain meristematic tissue are often lost.

Furthermore, the root tips are very small, making root tip squashes preparation extremely

difficult.

In order to obtain suitable root materials to facilitate chromosomes examination, in

vitro root induction was conducted for this species. Similar approach was successfully used

by Tatum et al. (2005) to produce adequate number of larger and stronger intact root tips in

weedy Amaranthus species (which also has thin and delicate roots), for use in mitotic

chromosome analysis.

The results from this study showed that WPM was better than MS for inducing root

formation in N. gracilis. WPM is generally lower in salt content compared to MS medium,

and some plants do prefer low mineral salts for improved root formation (Sun &

Kandasamy, 2005). The results showed that WPM supplemented with IBA (0.5 mg/l) and

77

Chapter IV: Discussion _________________________________________________________________________ solidified using gelrite can produce thick roots with obvious root tip regions in sufficient

quantity for root tip squashes preparation.

Chromosomes of Nepenthes gracilis

To the best of my knowledge, this study is the first attempt to investigate the

chromosomes of N. gracilis using in vitro-grown roots. Using in vitro-derived roots for

chromosome studies is convenient and effective in comparison to field-grown materials as

suggested by Gao et al. (1996). In addition, the seeds of N. gracilis are short-lived (Garrard,

1955; Lim & Prakash, 1973) and may require as long as 6 months to germinate (Green,

1967). Thus, obtaining root materials from germinated seedlings is difficult.

From this study, the number of chromosome was found to be near to 80. In 1969,

Kondo reported that the chromosome number of N. gracilis to be 78 whereas later in 1997,

Heubl & Wistuba claimed that the chromosome number of N. gracilis to be 80. Despite the

slight difference, both studies showed that N. gracilis has large chromosome number (i.e.

near to 80), as shown in this present study too.

This study also showed that the chromosomes of N. gracilis are very small and

difficult to distinguish in terms of size and structure. Furthermore, only a few mitotic cells

are visible in a single root tip. Hence, a few root tips from the same explants were pooled

for chromosome preparation. The difficulty in conducting microscopic cytological analysis

on species with large number of small chromosomes was faced by other researchers too, for

example, in Dioscorea alata L. (Egesi et al. 2002), Rubus (Thiem & Śliwińska, 2003) and

78

Chapter IV: Discussion _________________________________________________________________________ Vitis vinifera (Leal et al. 2006). Hence, these researchers chose to employ flow cytometry

in their work on ploidy level analysis.

In this study, the chimeric nature of the putative tetraploids was disclosed after

conducting chromosome studies on tetraploid plants via root tip squashing technique. It

was found that the root tips of tetraploids contained diploid chromosome spreads.

Chromosome studies were used as a method to detect chromosome doubling after

polyploidization in species such as onion (Song et al. 1997); Buddleia globosa (Rose et al.

2000), Scutellaria baicalensis (Gao et al. 2002) and Rosa (Zlesak et al. 2005). In most

cases, chromosome studies (of root tips) served as the determinative method in confirming

ploidy level. As demonstrated in this study, chromosome studies are important to check

whether the putative tetraploids are indeed solid tetraploids (which are the ideal outcome

from polyploidization work). Nevertheless, the chimeric tetraploids obtained can still be

raised as tetraploids by isolating and propagating the tetraploid shoot from the chimeral

plant as suggested by Pryor & Frasier (1968) and Adaniya et al. (2001). In addition, Cohen

& Yao (1996) had postulated that the initial chimeric polyploid shoots would produce

diploid and tetraploid shoots after several subculturing cycles.

79


4.3 Future work and suggestions

FCM analysis greatly facilitated the screening of ploidy level after polyploidization

treatments. Using FCM analysis, plants could be rapidly and accurately screened for altered

ploidy level at the in vitro stage. Stomata studies can be used to confirm ploidy changes

after FCM analysis. Chromosome studies (i.e. chromosome counting) is difficult in this

plant species which has large number of chromosome with small size. Nevertheless,

chromosome studies are necessary as a determinative method to differentiate chimeric from

solid tetraploid, since this study revealed that the induced tetraploids are chimeric plants

following chromosome studies. Hence, it was suggested that ploidy changes in different

tissues (e.g. leaves, roots, pollen grains) should be analyzed after polyploidization work.

Despite the low rate of tetraploids induction in this study, production of two

tetraploids in 72 h- 10 mM colchicine treatment (in different replication) showed that this

treatment can be potentially used to produce tetraploid plants in N. gracilis. The 24 h- 40

µM oryzalin treatment (which produced one tetraploid) can also be used as the dose-time

relation for inducing tetraploids in this plant species. As oryzalin is less toxic to human,

cheaper to apply and capable to induce significantly more ploidy changes than colchicine, I

would recommend the use of oryzalin to induce polyploidy. However, further modification

and optimization of the polyploidization treatments are needed to increase the tetraploids

induction rate.

80


For example, modification of the treatment solution by including DMSO may

improve polyploidization by facilitating penetration of mitotic inhibitor through the cell

walls. In addition, undifferentiated explants type such as embryogenic callus cultures can

be used (provided the somatic embryogenesis process was established) to produce solid

tetraploids because such tissues when converted into mass of polyploid cells, will

differentiate to produce large quantities of non-chimeric tetraploid plants. Or else, other

breeding technique should be sought after to create new varieties in this species, for

example, hybridization. Artificial hybridization of lowland species (e.g. N. gracilis) with

highland species (e.g. N. sanguinea, N. marfarlanei, N. ramispina) can be investigated

since the natural hybridization of both species is impossible due to differ in habitat. Also,

the ploidy level of the genus Nepenthes needed further confirmation to facilitate future

studies on this plant.

Following this study, the chimeric tetraploids and mixoploids obtained will be

propagated by isolating and culturing the shoots which carried the tetraploid or mixoploids

leaves (as inferred by FCM analysis) in hoping to get solid tetraploids. Subsequently, the

ploidy level of the propagated plants will be assessed via FCM analysis again. The

tetraploids will be raised together with diploids for further morphological investigation (e.g.

size of pitcher, intensity of pigmentation, etc).

81


82


83


84


85


86

Chapter V: Conclusion _________________________________________________________________________

82

The use of in vitro-derived leaves coupled with inclusion of 2% PVP (in Otto I

extraction buffer) is suitable for nuclear DNA content determination via FCM analysis in

this plant, as it produced distinct peak with low CV value. The 2C DNA content of N.

gracilis was determined as 1.60 ± 0.02 pg based on G. max (2C = 2.50 pg) as the internal

standard. The results from the polyploidization treatments showed that nodal segments of N.

gracilis can tolerate treatments (24 to 96 hours) with colchicine and oryzalin solutions up to

10 mM and 80 µM, respectively. Among the combinations of different concentrations and

treatment durations, two treatments (72 h- 10 mM colchicine and 24 h- 40 µM oryzalin

treatments) produced tetraploids (following ploidy screening via FCM). The tetraploids

were found to be chimeric following chromosome studies. Comparison between colchicine

and oryzalin in mixoploids production showed that oryzalin was more capable to induce

ploidy changes. Significant ploidy changes occurred in this species at the highest colchicine

concentration (10 mM) applied but readily happened at the lowest oryzalin concentration

(20 µM) applied. Longer treatment duration to both colchicine and oryzalin had

significantly increased the mixoploids production. The chimeric tetraploids and mixoploids

obtained from this study will be propagated and the ploidy level of the propagated plants

will be analyzed via FCM again.

References _________________________________________________________________________

83

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Appendices _________________________________________________________________________

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Appendix A

Components of Murashige & Skoog (1962) medium Macro stock solutions (10x)

Macro elements milligram per liter gram per liter for 10x concentration

CaCl2 332.02 3.32 KH2PO4 170.00 1.70 KNO3 1900.00 19.00 MgSO4 180.54 1.80 NH4NO3 1650.00 16.50 Micro stock solutions (100x)

Micro elements milligram per liter gram per liter for 100x concentration

CoCl2.6H2O 0.025 0.0025 CuSO4.5H2O 0.025 0.0025 FeNaEDTA 36.70 3.6700 H3BO3 6.20 0.6200 KI 0.83 0.0830 MnSO4.H2O 16.90 1.6900 Na2MoO4.2H2O 0.25 0.0250 ZnSO4.7H2O 8.60 0.8600 Vitamin stock solutions (1000x)

Vitamins milligram per liter gram per liter for 1000x concentration

Glycine 2.00 2.00 Nicotinic acid 0.50 0.50 Pyridoxine HCl 0.50 0.50 Thiamine HCl 0.10 0.10

Appendices ____________________________________________________________________________________________________________

94

Appendix B

Production of diploids, mixoploids and tetraploids in different replications of colchicine treatments. Rep 1 Rep 2 Rep 3

Concentration (mM) Hours Diploid Mixoploid Tetraploid Diploid Mixoploid Tetraploid Diploid Mixoploid Tetraploid

0 24 3 0 0 3 0 0 3 0 0 48 3 0 0 3 0 0 3 0 0 72 3 0 0 3 0 0 3 0 0 96 3 0 0 3 0 0 3 0 0

1.25 24 3 0 0 2 1 0 3 0 0 48 2 1 0 2 1 0 3 0 0 72 2 1 0 1 2 0 3 0 0 96 2 1 0 1 2 0 2 1 0

2.5 24 2 1 0 2 1 0 3 0 0 48 3 0 0 2 1 0 2 1 0 72 1 2 0 2 1 0 2 1 0 96 1 2 0 2 1 0 1 2 0 5 24 2 1 0 2 1 0 3 0 0 48 2 1 0 2 1 0 3 0 0 72 1 2 0 1 2 0 2 1 0 96 0 3 0 0 3 0 0 3 0

10 24 1 2 0 1 2 0 3 0 0 48 2 1 0 0 3 0 2 1 0 72 1 1 1 0 2 1 0 3 0 96 0 3 0 0 3 0 0 3 0

Appendices ____________________________________________________________________________________________________________

95

Production of diploids, mixoploids and tetraploids in different replications of oryzalin treatments.

Rep 1 Rep 2 Rep 3 Concentration

(µM) Hours Diploid Mixoploid Tetraploid Diploid Mixoploid Tetraploid Diploid Mixoploid Tetraploid 0 24 3 0 0 3 0 0 3 0 0 48 3 0 0 3 0 0 3 0 0 72 3 0 0 3 0 0 3 0 0 96 3 0 0 3 0 0 3 0 0

20 24 2 1 0 1 2 0 0 3 0 48 1 2 0 0 3 0 0 3 0 72 0 3 0 0 3 0 0 3 0 96 1 2 0 0 3 0 0 3 0

40 24 0 2 1 1 2 0 0 3 0 48 2 1 0 1 2 0 2 1 0 72 0 3 0 0 3 0 1 2 0 96 0 3 0 0 3 0 1 2 0

60 24 1 2 0 1 2 0 2 1 0 48 0 3 0 0 3 0 0 3 0 72 0 3 0 0 3 0 1 2 0 96 0 3 0 0 3 0 0 3 0

80 24 0 3 0 2 1 0 1 2 0 48 0 3 0 2 1 0 1 2 0 72 2 1 0 1 2 0 1 2 0 96 0 3 0 0 3 0 0 3 0

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