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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
iv
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.
vi
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
vii
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
viii
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
ix
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.
x
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.
xi
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
xvii
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
xix
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
xx
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
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
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