UNIVERSITI PUTRA MALAYSIA

IMPROVEMENT OF IN VITRO REGENERATION OF BRASSICA OLERACEA SUBSP. ITALICA CV. GREEN MARVEL THROUGH

ORGANOGENESIS

SEYED ALI RAVANFAR

FP 2009 13

IMPROVEMENT OF IN VITRO REGENERATION OF BRASSICA

OLERACEA SUBSP. ITALICA CV. GREEN MARVEL THROUGH

ORGANOGENESIS

By

SEYED ALI RAVANFAR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirement for the Degree of Master of Science

2009

In The Name of AllAH, the Most Gracious and the Most Merciful

Specially Dedicated

To

My beloved wife Shaghayegh

My parents Seyed Naser and Zarintaj

 

 

 

 

 

 

  ii

Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Agriculture Science

IMPROVEMENT OF IN VITRO REGENERATION OF BRASSICA OLERACEA SUBSP. ITALICA CV. GREEN MARVEL THROUGH

ORGANOGENESIS

By

SEYED ALI RAVANFAR

April 2009

Chairman : Associate Professor Maheran Bt Abd. Aziz, PhD 

Faculty : Agriculture

Broccoli (Brassica oleracea subsp. italica) cv. Green Marvel is an important

vegetable crop grown in Cameron Highlands, Malaysia. Being an F1 hybrid crop,

selfing will result in segregation in the F2 generation. In Malaysia, the production of

hybrid seeds of cv. Green Marvel cannot be carried out due to the absence of parental

stock material for hybridization. Therefore, the development of in vitro regeneration

system is necessary to produce uniform and true-to-type plants of the cultivar.

Currently, in vitro regeneration of other broccoli cultivars have been carried out but

none has been reported on the cultivar Green Marvel.

This study was carried out with the main objective of establishing a protocol for

plant regeneration of broccoli cv. Green Marvel through multiple shoot formation

from hypocotyl, shoot tip and cotyledon explants, followed by rooting and

  iii

acclimatisation. All experiments were conducted and arranged in a Randomised

Complete Block Design (RCBD).

In the experiment on multiple shoot formation from hypocotyl explants, the

concentrations of 6-benzylaminopurine (BAP) alone or in combination with α-

naphthaleneacetic acid (NAA) or indol-3-butyric acid (IBA) were assessed. In

treatments containing only BAP, 3 mg/l of the plant growth regulator produced the

highest percentage of explant with shoots (96.7%) and the highest mean number of

shoots per explant (6.03). In treatments containing BAP and NAA, the highest

percentage of explant with shoots (93.3%) occurred on 1 and 3 mg/l BAP while the

highest mean number of shoots (6.0) was obtained on 5 mg/l BAP. In treatments

containing BAP and IBA, BAP alone enhanced shoot multiplication. The highest

percentage of hypocotyl explant with shoots (100%) and highest mean number of

shoots produced per explant (6.33) occurred on 3 mg/l BAP. Therefore, 3 mg/l BAP

was considered the most suitable for adventitious shoot formation from hypocotyl

explants of broccoli cv. Green Marvel.

In the experiment on shoot multiplication from shoot tip explants, the concentrations

of BAP either alone or in combination with NAA or IBA were assessed. The result

showed that BAP at 5 mg/l produced the most number of shoots than any

combinations of BAP with NAA or IBA. In treatments containing BAP and NAA,

the highest percentage of explants with shoots (100%) and the highest mean number

of shoots per explant (3.76) were on 5 mg/l BAP. In treatments containing BAP and

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IBA,, the highest percentage of shoot tip explants with shoots (96.7%) and the highest

mean number of shoots produced per explant (2.83) also occurred on 5 mg/l BAP.

BAP at 5 mg/l BAP was therefore the optimum hormonal treatment for shoot

proliferation from shoot tip explants of broccoli cv. Green Marvel.

In the experiment on multiple shoot formation from cotyledon explant using different

combinations of BAP and NAA, the highest percentage of explant producing shoots

(53.3%) and the highest mean number of shoots produced per explant (0.43)

occurred on treatment combination consisting of 3 mg/l BAP and 1 mg/l NAA. The

treatment combination consisting of 3 mg/l BAP and 1 mg/l NAA is therefore the

most suitable for high frequency of shoot regeneration from cotyledon explant of

broccoli cv. Green Marvel.

In the experiment on rooting of shoots using different concentrations of auxins, the

highest percentage of shoots with roots (100%) and the highest mean number of roots

produced per explant (6.5) occurred on 0.2 mg/l IBA, but did not differ significantly

compared to MSO medium. Meanwhile the highest mean length of root (2.46 cm)

was attained on hormone-free MS medium (MSO). Thus, MSO medium was

considered the most suitable medium for rooting of broccoli cv. Green Marvel

shoots.

In the acclimatisation study, various types of potting mixture were assessed. The

highest percentage of survival (83.3%) occurred on medium containing sand+ soil

  v

(1:1) while the maximum root length (4.37 cm) and the highest plant height (7.87

cm) were attained in potting medium containing peatmoss+ perlite+ vermiculite

(3:1:1). The mixture of peatmoss+ perlite+ vermiculite (3:1:1) was therefore

considered the most suitable potting medium for broccoli cv. Green Marvel. Plantlets

were well rooted and ready for field transfer after four weeks of acclimatisation.

The study on multiple shoot formation concluded that BAP alone at 3 and 5 mg/l

were more effective in inducing shoot proliferation on the hypocotyl and shoot tip

explants of broccoli cv. Green Marvel, respectively, while BAP in combination with

NAA was required for effective shoot formation from the cotyledon explants. The

study on rooting showed that MSO was most suitable for high percentage of root

formation, mean number of roots produced per explant and root length attained. In

the acclimatisation study, the most suitable potting mixture consisted of peatmoss+

perlite+ vermiculite.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains Pertanian

PEMBAIKAN REGENERASI IN VITRO BRASSICA OLERACEA SUBSP. ITALICA CV. GREEN MARVEL MELALUI ORGANOGENESIS

Oleh

SEYED ALI RAVANFAR

April 2009

Pengerusi: Professor Madya Maheran Bt. Abd. Aziz, PhD

Fakulti: Pertanian

Brokoli (Brassica oleracea subsp. italica) cv. Green Marvel adalah tanaman sayuran

yang penting di Cameron Highlands, Malaysia. Oleh kerana tanaman ini jenis hibrid

F1, kacukan sendiri akan menghasilkan segregasi di peringkat generasi F2. Di

Malaysia, pengeluaran biji benih hibrid cv. Green Marvel tidak dapat dilakukan

kerana ketiadaan bahan stok induk untuk penghibridan. Oleh itu, pembentukan

sistem regenerasi in vitro diperlukan untuk pengeluaran anak pokok cv. Green

Marvel yang sekata dan tulen. Pada masa ini, regenerasi in vitro kultivar brokoli

yang lain telah dijalankan tetapi belum ada laporan mengenai brokoli cv. Green

Marvel.

Kajian ini dijalankan dengan objektif utama untuk menghasilkan suatu protokol

regenerasi untuk cv. Green Marvel melalui pembentukan pucuk berganda daripada

hipokotil, tunas hujung, dan eksplan kotiledon, diikuti dengan pengakaran dan

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aklimatisasi. Kesemua eksperimen dilakukan dan diatur menggunakan Rekabentuk

Blok Penuh Berawak (RCBD).

Dalam eksperimen pembentukan pucuk berganda daripada eksplan hipokotil,

kepekatan 6-benzilaminopurin (BAP) secara sendiri atau gabungan dengan asid α-

naftalenasetik (NAA) atau asid indol-3-butirik (IBA) telah dikaji. Dalam rawatan

yang mengandungi BAP sahaja, 3 mg/l bahan pengawalatur tersebut menghasilkan

peratus eksplan membentuk pucuk yang tertinggi (96.7%) dan min bilangan pucuk

per eksplan yang tertinggi (6.03). Bagi rawatan yang mengandungi BAP dan NAA,

peratus eksplan membentuk pucuk yang tertinggi (93.3%) berlaku pada rawatan 1

dan 3 mg/l BAP, manakala min bilangan pucuk per eksplan yang tertinggi (6.0)

adalah dari rawatan dengan 5 mg/l BAP. Bagi rawatan yang mengandungi BAP dan

IBA, rawatan BAP bersendiri meningkatkan penggandaan tunas. Peratus eksplan

membentuk pucuk yang tertinggi (100%) dan min bilangan pucuk per eksplan

tertinggi (6.33) berlaku pada rawatan dengan 3 mg/l BAP. Oleh itu, 3 mg/l BAP

dianggap yang paling sesuai untuk pembentukan pucuk adventisius daripada eksplan

hipokotil brokoli cv. Green Marvel.

Dalam eksperimen pembentukan pucuk berganda daripada eksplan tunas hujung,

kepekatan BAP sama ada dibekalkan secara sendiri atau gabungan dengan NAA atau

IBA telah dikaji. Hasil kajian menunjukkan BAP pada kepekatan 5 mg/l

menghasilkan pucuk yang paling banyak berbanding gabungan BAP dengan NAA

atau IBA. Bagi rawatan yang mengandungi BAP dan NAA, peratus eksplan

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membentuk pucuk tertinggi (100%) dan min bilangan pucuk per eksplan tertinggi

(3.76) adalah pada rawatan dengan 5 mg/l BAP. Bagi rawatan yang mengandungi

BAP dan IBA, peratus eksplan membentuk pucuk tertinggi (96.7%) dan min

bilangan pucuk per eksplan tertinggi (2.88) juga terhasil pada rawatan dengan 5 mg/l

BAP. BAP pada kepekatan 5 mg/l adalah rawatan hormon yang optimum untuk

proliferasi pucuk daripada eksplan tunas hujung brokoli cv. Green Marvel.

Dalam eksperimen pembentukan pucuk berganda daripada eksplan kotiledon

menggunakan gabungan BAP dan NAA yang berbeza, peratus eksplan membentuk

pucuk tertinggi (53.3%) dan min bilangan pucuk per eksplan tertinggi (0.43) berlaku

pada kombinasi rawatan yang mengandungi 3 mg/l BAP dan 1 mg/l NAA. Rawatan

kombinasi 3 mg/l BAP dan 1 mg/l NAA adalah yang paling sesuai untuk regenerasi

yang tinggi daripada eksplan kotiledon brokoli cv. Green Marvel.

Dalam eksperimen pengakaran pucuk pada kepekatan auksin yang berbeza, peratus

pucuk berakar tertinggi (100%) dan min bilangan akar per pucuk tertinggi (6.5)

berlaku pada 0.2 mg/l IBA, tetapi tidak berbeza dengan signifikan berbanding

medium MSO. Manakala purata panjang akar tertinggi (2.46 cm) dihasilkan pada

medium MS tanpa hormon (MSO). Maka, medium MSO dianggap yang paling

sesuai untuk pengakaran pucuk brokoli cv. Green Marvel.

Dalam kajian aklimatisasi, pelbagai jenis medium pemasuan telah dikaji. Peratus

kebolehan hidup tumbuhan tertinggi (83.3%) berlaku pada medium yang

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mengandungi pasir+tanah (1:1), manakala panjang maksimum akar (4.37 cm) dan

ketinggian tumbuhan yang tertinggi (7.87 cm) dihasilkan pada medium pemasuan

yang mengandungi peatmoss+perlit+vermikulit (3:1:1). Medium yang mengandungi

peatmoss+perlit+vermikulit (3:1:1) dianggap sebagai medium pemasuan yang paling

sesuai. Anak pokok berakar dengan baik dan sedia untuk dialih ke lapangan setelah

empat minggu aklimatisasi.

Daripada kajian ke atas pembentukan pucuk berganda dapat disimpulkan bahawa

BAP pada 3 dan 5 mg/l adalah lebih efektif dalam meningkatkan proliferasi pucuk

masing masing pada eksplan hipokotil dan tunas hujung brokoli, manakala

kombinasi BAP dan NAA diperlukan untuk pembentukan pucuk yang berkesan

daripada eksplan kotiledon. Kajian tentang pengakaran menunjukkan medium MSO

adalah paling sesuai untuk menghasilkan peratus pengakaran, min bilangan akar per

eksplan serta panjang akar tertinggi. Bagi kajian aklimatisasi, campuran pemasuan

paling sesuai terdiri daripada campuran peatmoss+perlit+vermikulit.

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ACKNOWLEDGMENTS

IN THE NAME OF ALLAH

Thanks and Praise is due to Allah who gave me strength and determination to

complete my study. I would like to express my gratitude and sincere thanks to those

who have helped me in preparing and conducting the research and finishing this

thesis. Therefore, it pleases me to express my deep gratitude to them.

I would like to thank the chairman of my supervisory committee, Associate Professor

Dr. Maheran Abdul Aziz, for her supervision, kind guidance and advice in the

completion of the thesis. I appreciate her patience and sincere approach to motivate,

help, advice and guide me to finish my study. Thanks are also extended to my

committee member, Associate Professor Dr. Mihdzar Abdul Kadir, and I am grateful

to Mr. Azmi Abdul Rashid, for his continuous help and suggestion while I was

carrying out my project. The experience and knowledge, gained has resulted in a

significant and positive effect on my work.

Not forgetting my dearest friends, especially Arash Raafat, Hossein Torabi,

Mohammad Javadi, Fatemeh Hadadi and Hossein Kamaladini. Thanks for your

support and encouragement in times of need.

Last but not least, thanks to my beloved wife Shaghayegh, my father Naser, my

mother Zarin and my brothers Roham and Mohammad. I owe you every thing.

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I certify that a Thesis Examination Committee has met on 13 April 2009 to conduct the final examination of Seyed Ali Ravanfar on his thesis entitled “Improvement of In Vitro Regeneration of Brassica oleracea Subs. Italica Cv. Green Marvel Through Organogenesis” in accordance with the Universities and University College Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1988. The Committee recommends that the student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Halimi Mohd Saud, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Saleh Kadzimin, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal examiner) Uma Rani Sinniah, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal examiner) Ahmad Tarmizi Hashim, PhD Malaysian Advanced Biotechnology and Breeding Center Biology Division Malaysian Palm Oil Board (External Examiner)  

BUJANG KIM HUAT, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 29 May 2009

  xii

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfillment for the degree of Master of Agricultural Science. The members of the Supervisory committee are as follows:

Maheran Abdul Aziz, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Mihdzar Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

____________________________

HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date: 8 June 2009

 

 

 

 

  xiii

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

____________________

Seyed Ali Ravanfar

Date:

 

 

 

 

 

 

 

 

 

 

 

  xiv

TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK vii ACKNOWLEDGMENTS xi APPROVAL xii DECLARATION xiv LIST OF FIGURES xviii LIST OF PLATES xx LIST OF ABBREVIATIONS xxi

CHAPTER

1 INTRODUCTION 1 Objectives 4

2 LITERATURE REVIEW 5 Botany of Broccoli 5 In Vitro Plant Regeneration for Colonal Propagation 6 Stages of Micropropagation 7 Organogenesis 8 Types of Organogenesis 9 Direct Organogenesis 10 Adventitious Shoot Formation 10 Hypocotyl Culture 11 Shoot tip Culture 13 Cotyledon Culture 14 Shoot Multiplication 15 Factors Affecting Shoot Multiplication and Adventitious Shoot 16 Formation Age of Explant 16 Choice of Nutrient Medium 17 Plant Growth Regulators 18 Sugar 23 Physical Environment of Culture 24 Rooting 25 Acclimatisation 26

3 MATERIALS AND METHODS 28 Seed Sterilization and Germination 28 Explant Preparation and Culture 28 Elongation of Shoot and Rooting 29 Transfer of Plantlets to Potting Mixture and Acclimatization 29 Basic Medium 30

  xv

Experiments 30 Experiments 1: Effect of BAP on Multiple Shoot Formation from Hypocotyl explants 30 Experiments 2: Effect of Different Concentrations of BAP in Combination with NAA on Multiple Shoot Formation from Hypocotyl explants 31 Experiments 3: Effect of Different Concentrations of BAP in Combination with IBA on Multiple Shoot Formation from Hypocotyl explants 31 Experiments 4: Effect of BAP and NAA on Multiple Shoot Formation from Shoot tip explants 31 Experiments 5: Effect of BAP and IBA on Multiple Shoot Formation from Shoot tip explants 32 Experiments 6: Effect of BAP and NAA on Multiple Shoot Formation from Cotyledonary explants 32 Experiments 7: Effect of TDZ and IBA on Multiple Shoot Formation from Cotyledonary explants 32 Experiments 8: Effect of NAA, IBA and IAA on Rooting of Shoots 33 Experiments 9: Effect of Different Potting Media on Ex Vitro Performance 33 Experimental Design and Statistical Analysis 33

4 RESULTS AND DISCUSSION 34 Experiments 1: Effect of BAP on Multiple Shoot Formation from Hypocotyl explants 34 Experiment 2: Effect of Different Concentrations of BAP Either Alone or in Combination with NAA on Shoot Formation from Hypocotyl Explants 38 Experiment 3: Effect of Different Concentrations of BAP Either Alone or in Combination with IBA on Shoot Formation from Hypocotyl Explants 40 Discussion on Multiple Shoot Formation From Hypocotyls Explant 42 Experiments 4: Effect of BAP and NAA on Multiple Shoot Formation from Shoot tip Explants 45 Experiments 5: Effect of BAP and IBA on Multiple Shoot Formation from Shoot tip Explants 48 Discussion on Multiple Shoot Formation From Shoot tip Explant 50 Experiments 6: Effect of BAP and NAA on Multiple Shoot Formation from Cotyledonary Explants 54 Experiments 7: Effect of TDZ and IBA on Multiple Shoot Formation from Cotyledonary Explants 57 Discussion on Multiple Shoot Formation From Cotyledon Explant 57 Experiments 8: Effect of NAA, IBA and IAA on Rooting of

  xvi

Broccoli Shoots 59 Discussion on Rooting 63 Experiments 9: Effect of Potting Media on Ex Vitro Performance of Plantlets 65 Discussion on Acclimatization and Transfer of Plantlets to Soil 68

5 SUMMARY, CONCLUSION AND RECOMMENDATIONS 70 FOR FUTURE RESEARCH

REFERENCES 73 APPENDICES 83 BIODATA OF STUDENT 96

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LIST OF FIGURES

Figure Page

1 Effect of BAP on percentage of hypocotyl explants forming shoots after eight weeks of culture

35

2 Effect of BAP on mean number of shoots formed per hypocotyl explant after eight weeks of culture

35

3 Effect of different concentrations of BAP in combination with NAA on percentage of hypocotyl explants with shoots after eight weeks of culture

38

4 Effect of different concentrations of BAP in combination with NAA on mean number of shoots formed per hypocotyl explant after eight weeks of culture

39

5 Effect of different concentrations of BAP in combination with IBA on percentage of hypocotyl explants forming shoots after eight weeks of culture

40

6 Effect of different concentrations of BAP in combination with IBA on mean number of shoots formed per hypocotyl explant after eight weeks of culture

41

7 Effect of different concentrations of BAP with NAA on percentage of shoot tip explants producing shoots after eight weeks of culture

46

8 Effect of different concentrations of BAP with NAA on mean number of shoots produced per shoot tip explant after eight weeks of culture

46

9 Effect of different concentrations of BAP with IBA on percentage of shoot tip explants producing shoots after eight weeks of culture

49

10 Effect of different concentrations of BAP with IBA on mean number of shoots produced per shoot tip explant after eight weeks of culture

49

  xviii

11 Effect of different concentrations of BAP with NAA on percentage of cotyledon explants producing shoots after eight weeks of culture

55

12 Effect of different concentrations of BAP with NAA on mean number of shoots per cotyledon explant after eight weeks of culture

55

13 Effect of different concentrations of auxins (NAA, IBA, and IAA) on percentage of shoots producing roots after four weeks of culture

60

14 Effect of different concentrations of auxins (NAA, IBA, and IAA) on mean number of roots produced per explant after four weeks of culture

60

15 Effect of different concentrations of auxins (NAA, IBA, and IAA) on root length (cm) attained after four weeks of culture

61

16 Percentage of plant survival after four weeks of transfer to different potting media

66

17 Effect of growth media on root length attained after four weeks of acclimatization

66

18 Effect of growth media on plant height (cm) attained after four weeks of acclimatization

67

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LIST OF PLATES

Plate Page

1

Shoot formation from hypocotyl explants of broccoli cv. Green Marvel on 3 mg/l BAP, (A) after one week, (B) after four weeks and (C) proliferation of shoots after eight weeks of culture

36

2

Multiple shoot formation from hypocotyl explants of broccoli cv. Green Marvel on different concentrations of BAP

37

3 Shoot and callus formation from hypocotyl explants on MS medium containing 5 mg/l BAP and 0.05 mg/l NAA after eight weeks of culture

39

4 Shoot and callus formation from hypocotyl explants placed on MS medium containing 5 mg/l BAP and 0.1 mg/l IBA after eight weeks of culture

41

5 Multiple shoot formation from shoot tip explants of broccoli cv. Green Marvel

47

6 Shoot and callus formation on concentration of (A) 5 mg/l BAP with 0.1 mg/l IBA, and (B) 5 mg/l BAP with 0.5 mg/l IBA after eight weeks of culture

50

7 Multiple shoot formation from cotyledon explants of broccoli cv. Green Marvel

56

8 Root formation on different concentrations of NAA, IBA and IAA from shoots of broccoli cv. Green Marvel

62

9 Plant survival after four weeks of acclimatization 67

10

A survived broccoli cv. Green Marvel plant after eight weeks in medium containing peatmoss+ perlite+ vermiculite

68

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LIST OF ABBREVIATIONS

ANOVA analysis of variance

BAP 6-benzylaminopurine

BA N6-benzyladenine

cm centimeter

cv cultivar

2, 4-D 2, 4-dichlorophenoxyacetic acid

2-ip γ, γ-(dimethylallyl) aminopurine

α level of significance

DNMRT Duncan׳s New Multiple Range Test

e.g. exempli gratia (for example)

et al. et alia

etc. et cetera

F1 First filial generation

g gram

g/l gram per litre

  xxi

H hour (s)

IAA indol-3-acetic acid

IBA indol-3-butyric acid

KIN kinetin

KNO3 potassium nitrate

L liter

M molar

mg milligram

mg/l milligram per liter

ml milliliter

mm millimeter

µ mol m-² s-¹ micromole per meter square per second

µM micromolar, 10³־ mM

MS Murashige and Skoog

NAA α-naphthalene acetic acid

NO3 nitrate

PGR Plant Growth Regulator

  xxii

  xxiii

pH -log (H+)

RCBD Randomized Complete Block Design

sp. species

TDZ thidiazuron

var variety

CHAPTER 1

INTRODUCTION

Broccoli, (Brassica oleracea subsp. italica), a cultivar of wild cabbage, originated

along the northern and western coasts of the Mediterranean Sea. It is a hardy

vegetable which develops best during the cool seasons of the year. When broccoli

plants of most varieties are properly grown and harvested, they can yield over an

extended period of time, because the side heads develop after the large, central head

is removed. Two crops per year (spring and fall) may be grown. New heat tolerant

varieties allow broccoli to be produced in all but the hottest parts of the season

(Wolford and Banks, 2000).

Other cultivar groups of Brassica oleracea include: cabbage (Capitata Group),

cauliflower (Botrytis Group) kale and collard greens (Acephala Group), kohlrabi

(Gongylodes Group), and brussels sprouts (Gemmifera Group). Chinese broccoli

(Alboglabra Group) is also a cultivar group of Brassica oleracea. The edible part of

the broccoli plant is the tender stem and unopened flower buds. They are a good

source of vitamin A, D, C, calcium, and riboflavin or B2. It is usually boiled or

steamed, but may be eaten raw and has become popular as a raw vegetable

(Diindolylmethane Information Resource Center, 2007).

Broccoli is high in soluble fiber with potent anti-cancer properties which include

diindolylmethane and selenium. The 3' diindolylmethane found in broccoli is a potent