In vitro regeneration of prospective cabbage Brassica oleracea L.

(Broccoli)
a∗ b c Department of Botany and Microbiology, A.V.V.M Sri pushpam col-

lege (Autonomous), Poondi, 613 503Thanjavur.Dt, Tamil nadu.India.

d Department of Botany, Goverment Arts College Kumbakonam.612 001.Tiru-

varur.Dt, Tamil nadu, India.

e Department of Botany, M.R. Goverment Arts College Mannargudi 614

001. Tiruvarur.Dt, Tamil nadu, India.

Corresponding author Email: rameshbiotech1986@gmail.com
Conduct number : 9585678449
Abstracts
Brassica oleracea L. var. italic commonly called Broccoli is a horticulture
plant and belongs to the family of Brassicaceae. Thus a diet rich in broccoli
plays a role in the prevention of chronic diseases, such as cardiovascular and
carcinogenic and breast and prostate cancer. In vitro rapid propagation was
deliberated from shoot tip node and flower buds explants of Brassica oleraceae
L. The explants were cultured on (MS medium supplemented with B5 vita-
mins (Murashige and skoog 1962). In various concentrations of cytokinins and
Auxins ranging from 0.1 mg to 3.0 mg combinations of BAP and KN was good
response from shoot tip nodal and flower buds. Highest number of callus in-
duced in the concentrations of 2.5 mg BAP+2 mg NAA. Multiple shoot was
noticed in 2.5 mg BAP and 1.5 mg NAA and 2 mg IBA. The present study en-
ables the large scale production of Brassica oleraceae L.using in vitro conditions
and disease free plants.
Key Words: Brassica oleraceae, In vitro propagation, flower buds explants,
growth regulators, (BAP, KN, NAA, IAA, IBA), Medicinal uses.

Introduction
Brassica oleracea L. var. italic commonly called Broccoli is a horticulture
plant and belongs to the family of Brassicaceae.(Bhalla et al 1998). Which origi-
nated from Italy at 2000 years ago and is related to the cabbage and cauliflower,
is a very important vegetable crop. It is well known for its high vitamin and
calcium content. It is also used as medicinal plant. The plant has been re-
ported containing sulforaphane which function as antioxidant. (Zhang et al.,
1992: Henzl et al 2000)
Broccoli is high in vitamin C and dietary fibre. H also contains multiple
nutrients with potent anti-cancer properties, such as diindolymethane (DIM)
and small amounts of selenium. DIM is a potent modulator of the innate im-
mune response system with anti – viral, anti bacterial and anti-cancer activity.
(Arona et al 1997, 1998.) Broccoli also contains the compound glucoraphanin,
an excellent source of indole-3-carbinol, a chemical DNA repair in cells and
appears to block the growth of cancer cells. Broccoli (Brassica oleracea L.) has
been marketed as a health promoting food because it naturally has high con-
tent of bioactive phytochemicals such as glucosinolates. Phenolic compounds,
vitamin C’ and mineral nutrients. (Michal et al., 1988, Chen et al 2004.,) Thus
a diet rich in broccoli plays a role in the prevention of chronic diseases, such

1
as cardiovascular and carcinogenic and breast and prostate cancer(Smith et al
2009 Bartolo 1989 ).
Materials and methods
Plant collection and Sterilization of explants:
Brassica oleracea L. var (Broccoli) was collected from Ooty, Kodaikkanal (Dt)
Tamilnadu, India during the month January 2015. The explants such as florets
and stigma were collected from in vivo plant flowerheads. The explants were
then prewashed 10% perecent (W/V) of (Bavistain) methyl 1-3 benzimidizole
carbonate solution and washed thoroughly in running tap water. The explants
were subsequently and disinfection surface sterilized with 0.12% Hgcl2 (mer-
curic chloride) solution (George and sherrington 1984) for 3-5 minutes and
washed 2-3 times in sterile distilled water. Hgcl2 was very penetrating that
it destroyed the microorganism present in most tissues of the explants. The
surface sterilized explants were trimmed gently with help of sterile surgical
blade. (Hall et al 1993).
Culture medium:-
Nutritional support must be essential for optimal growth of tissue by in
vitro. The nutrient media basically consists of inorganic nutrients. Carbon-
source, vitamins, ironsource, aminoacids and natural supplements. All the
stock solution were prepared and stored in Amber – sterilized coloured bottles
and preserved in a refrigerator at 40 C. In present study Ms (Murashige and
skoog, 1962) medium supplemented of different concentrations of growth reg-
ulators were added. Benzyl amino purine (BAP) kinetin (KIN) 2, 4-Dichlorophenary
acidic acid (2,4-D), NAA, IBA were (0.5 to 2.5mg). Multiple shoot was noticed
BAP, NAA, IBA (0.1 to 3.5mg) were used. Required sucrose and other organic
supplements were added. The final volume was made up with distilled water.
To the above said media 0.8% (W/V) sugar and Hi-media (agar) was added.
PH was adjusted to 5.8 – 5.9 with either 0.1 N NaOH or 0.1 N Hcl using a PH
meter Bensen 1995. .
Results
Plant tissues normally grow in an organized fusion in which specific cell
types differentiate from non specialised meristematic cells Plant developmen-
tal processes can be modified by culture in vitro in a suitable nutrients medium
and with the application of growth regulators. The interactions of the main
growth regulators can be used but at the simplest level anxins can cause cell
enlargement and division cytokinins cause cell division and shoot develop-
ment .

2
 The explants such as flowerbud for micropropagation were selected from
the same. The result showed that shoot formation was generated from direct
organogenesis of explants were detected in all treatment. After 15 days growth
of callus was observed. The MS medium was supplemented with different
concentration of hormones NAA, BAP, KN, IBA combination of it initiates basal
callus and shoot proliferation.
Callus induction from flowerbud explants:
The requirement of various plant growth regulators for inducing callus and
differentiation from flowerbud explants. The explants began to after 15 days
of culturing and callus proliferation occurred. The concentration of hormone
BAP, KN, NAA, large amount of callus induction from MS media at 4 weeks
(Table 1)
Table 1. Effect of various concentration of BAP, KN, NAA in callus induction
on MS media from flowerbud explants after 4 weeks of culture.

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 Growth regula- Culture showing Basal callus
tors (mg/L) response (%)
BAP KN NAA
0.5 - 0.5 55 +
1.0 - 1.0 65 +
1.5 - 1.5 72 ++
2.0 - 2.0 85 +++
2.5 - 2.5 95 ++++
- 0.5 0.5 70 +
- 1.0 1.0 72 +++
- 1.5 1.5 80 +++
- 2.0 2.0 90 +++
- 2.5 2.5 92 +++
0.5 0.5 0.5 80 ++
1.0 1.0 1.0 82 ++
1.5 1.5 1.5 95 +++
2.0 2.0 2.0 81 +++
2.5 2.5 2.5 90 +++
Callus induction: + poor ++ moderate +++ high response.
Table 2. Effect of various concentration of BAP, IBA, KN in shoot initiation on
MS medium from flowerbud explants after 4 weeks of culture
MS Medium Culture Mean Mean shoot Basal cal-
Growth regula- showing shoot/ length (cm) lus
tors (mg/L) response explants
(%)
BAP IBA KN
0.5 0.5 - 60 3 4.5 +++
1.0 1.0 - 70 4 4.8 +++
1.5 1.5 - 80 5 4.2 ++
2.0 2.0 - 75 4 5.1 +++
2.5 2.5 - 95 6 5.0 +++
- 0.5 0.5 55 2 2.5 ++
- 1.0 1.0 65 4 4.0 +++
- 1.5 1.5 80 3 4.2 +++
- 2.0 2.0 90 5 5.0 +++
- 2.5 2.5 85 4 5.1 +++
0.5 0.5 0.5 65 3 5.1 +++
1.0 1.0 1.0 75 2 4.9 +++
1.5 1.5 1.5 90 5 4.2 +++
2.0 2.0 2.0 82 4 5.2 ++
2.5 2.5 2.5 90 5 5.0 ++
Shoot induction have been indifferent grades viz.,

1. No response + poor ++ moderate +++ high

Table 3 Effect of various concentration of BAP, NAA, IBA in shoot multiplica-

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tion on MS medium from flowerbud explants after 4 weeks of culture.
MS Medium Culture Mean Mean shoot Basal cal-
Growth regula- showing shoot/ length lus
tors (mg/L) response explants
(%)
BAP NAA IBA
0.5 0.5 - 60 10 09.95 -
1.0 1.0 - 65 12 11.05 -
1.5 1.5 - 72 12 11.05 -
2.0 2.0 - 85 14 13.45 -
2.5 2.5 - 92 14 13.45 -
0.5 - 0.5 70 13 12.75 -
1.0 - 1.0 75 12 11.05 -
1.5 - 1.5 82 10 09.95 -
2.0 - 2.0 90 15 13.75 -
2.5 - 2.5 95 14 13.80 -
0.5 0.5 0.5 80 13 12.75 -
1.0 1.0 1.0 85 12 13.45 -
1.5 1.5 1.5 75 10 09.95 -
2.0 2.0 2.0 90 15 13.75 -
2.5 2.5 2.5 95 14 13.80 -
Shoot induction have been indifferent grades viz.,

1. No response + poor ++ moderate +++ high

Shoot initiation:
The highest frequency of shoots (Table 2) from flowerbud explants were
observed in MS media containing ( 0.5 mg to 2.5 mg) BAP, NAA, IBA, KN
were used. The maximum shoot regeneration frequency i.e 95% on MS basal
medium supplemented with 2.5mg/L BAP, NAA 2.5mg/L and IBA 2.0mg/L
respectively (Table 2). The shoot regenerated shoots obtained from both the
explants. Shoot multiplication and elongation took place on the same medium.
Shoot multiplication from flowerbud explants:
A large number of lateral shoots were recorded on the explants cultivated
at media many authers emphasize the influence of proper cytokinin and auxin
combination on the formation of shoots in in vitro conditions (Lazzeri and
Dunwell 1986, Msikita and Skirvin, 1989). Among different concentration of(
BAP 0.5 to 2.5mg/L) used. IBA at 1.5mg/L has given 85% growth of shoot with
a mean shoot length of 4.2cm (Table 3 and fig). Different concentration BAP
NAA IBA are used. The maximum amount of multiple shoot was observed at
2.5mg/L with 85% of response with mean shoot length 4.0cm (Table 3). The
highest number of shoots from flower bud explants were observed by on 45
days it has given 15 shoots per explants.
Discussion
The main ediple parts of broccoli are ediple sprouts and florets named the
inflorescence (Olga et al 2009, Cristea et al 2012). Broccoli is known as crown

5
Jewel of Nutrition since possess the nutrients namely vitamins, minerals, sec-
ondary metabolites and fibre proclaiming its exceptional health benefits. The
breakdown products of the sulfur containing glucose inolatesisothio cyanates
are the active principles in exhibiting the anticancer property at stage (Vasathi
et al 2009 Li et al 2009). For the present study, the results revealed direct
organogenesis of flowerbud explants there was callus formation and the dura-
tion of shoot initiation on B. oleracea L. fromflowerbud explants were 15-20
days this results is agreement with finding in Broccoli Raghavan (1997).
For the present study, the results when sub cultured on 35th day 15 shoots
per explants were achieved (Table 3). The shoots were aggregated. The mean
shoot lengths was 13.80 measured. The use of BAP as a growth regulator 98
more effective than KN, Lazzeri and Dun well (1986).KN, BAP, played a role
on inducing shoot multiplication. It was suggested that the use of BAP as a
cytokinin and NAA as an auxin in an appropriate ratio. They investigated
that BAP induced more shoot produced. Many investigators examined that
uses ofAuxin such as KN, IBA, BAP was the optimum concentration for shoot
regeneraton (Guo et al 2001). In this study has presented 95% regeneration at
BAP 0.5 to 2.5mg KN 0.5mg to 2.5mg for initiation of shoot BAP, IBA, KN= 0.5
to 2.5 mg multiple shoot formaton in explants (Ravantar et al., 2009)
Conclusion
Brassica oleracea L. nutritional and medicinal plant. The in vitro regener-
ation protocol is an efficient means of ex-situ conservation of plant diversity.
We have demonstrated in this study the effects of growth regulators on mor-
phogenic response of broccoli cultivated in vitro. Appropriate combinations
and concentration of plant hormones result in higher yield of plant biomass
and mass propagation.
Reference
1. Buck, P.A. (1956) “origin and taxonomy of Broccoli” Economic Botany
10 (3 : 250 – 253 Retrieved 24 April 2012.
2. George E.F. and Sherrington, P.D. 1984 plant propagation by tissue cul-
ture. Exogenetical Limited, England.
3. Gwo JT, Lee HL, chiang SH, Lin, F1 chang, cy (2001) Antioxidant proper-
ties of the extracts from different parts of broccoli in Taiwan J Food Drug
Anal, 9, 96-101.
4. Henzi MX christey MC, Mcneil DL (2000) Factors that influence Agrobac-
terium rhizogenes – mediated transformation of Broccoli (Brassica Olera-
caa L. var, italic). Plant cell ref 19:994-999.
5. Hamill S.D, sharrock S.L. and smith M.K. 1993. Comparision of decon-
tamination methods used in initiation of banana tissue culture from field
collected sukers, plant cell tissue organ. Cult 33:343-346
6. Joon-Ho-Hwang and sang – Bin Lim “Antioxidant and Anticancer ac-
tivites of Broccoli by products from Different cultivars and maturity stages
at Harvest.

6
 7. Lazzeri P.A., Dunwell J.M. (1986). In vitro regeneration from seedling
organs of Brassica oleracea. var italic plenck CV. Green comet. 1 Effect of
plant growth regulators. Annals of Botany, 58 689
8. Murashige T. Skoog F (1962). A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol plant. 15(??) 473-497
9. Michacl G.K. Johes Neil Fish, Keith Lindsey 1988. Plant tissue culture,
methods in molecular biology, 4:499-517.
10. Msikita W., Skirvin R.M. (1989). In vitro regeneration from hypocotyls
and seedling cotyledons of tronchuda (Brassica Oleracea vartronchuda
Bailey) plant cell tissue organ culture 19(??) 159-165.
11. Olga N. campus-Baypoli, Dalia I 89 Nchez-Machado, Carolina Bueno-
solano Jose A, No N EZ-GasteLum, cuauhtemoc Reyes – Moreno and
Jaime Lo Pre-cervantes 2009. Biochemical composition and phytochemi-
cal properties of broccoli flours. Int J Food sci and Nutr, 60 (S4):163-173
12. Ravanta SA, Aziz M A. Kadir Rashid AA. Sirchi MHT, 2009. Plant regen-
eration of Brassica oleracea sub sp, italica (Broccoli) CV Green marvel as
affected by plant growth regulators. Afr J. Biotechnol 8(??): 2523-2528
13. Smith, powell (June 1999) “HGIC 1301 Broccoli” Clemson university Re-
trieved 25 August 2009.
14. Vasanthi H R, Mukherjee S and Das D K 2009. Potential Health Benefits
of Broccoli – A Chemico – Biological overview mini – Reviews in medicin
al chemistry 9:749-759

15. Zhang YS, Talalay P cho CG, posner G (1992), A major inducer of anticar-
cinogenic protective enzymes from Broccoli : Isolation and elucidation of
structure. Proc. Natl. Acad. Sci. USA. 89:2399-2403.
16. Anderson WC & Carstens JB (1977) Tissue culture propagation of broc-
coli, Brassica oleracea (Italica group), for use in F1 hybrid seed produc-
tion. J. Amer. Soc. Hort. Sci. 102: 69-73
17. Arora N, Yadav NR & Chowdhury JB (1996) Efficient plant regeneration
in cauliflower (Brassica oleracea var. botrytis). Cruciferae Newsl. 18: 26-
27.
18. Arora N, Yadav NR, Yadav RC, Chowdhury JB & Arora N (1997) Role of
IAA and BAP on plant regeneration in cultured cotyledons of cauliflower.
Cruciferae Newsl. 19: 41-42.
19. Bhalla PL & Smith N (1998) Agrobacterium mediated transformation of
Australian cultivars of cauliflowers, Brassica oleracea var. botrytis. Molec-
ular Breeding 4 (??): 531-541

7
20. Bartolo WCF, Macey MJK (1989) Cobalt requirement in tissue culture
of three species: Brassica oleracea L., Passiflora mellissima Bailey, and
Saintpaulia inoantha Wendl. J Hortic Sci 64: 643–647
21. Bensen EE (1995) Cryopreservation of Brassica species. In: Bajaj YPS
(ed) Biotechnology in agriculture and forestry, vol 32. Cryopreservation
of plant germplasm I. Springer, Berlin Heidelberg New York, pp 308–318
22. Chen LP, Zhang MF, Xiao QB, Wu JG, Hirata Y (2004) Plant regeneration
from hypocotyl protoplasts of red cabbage (Brassica oleracea) by using
nurse cultures. Plant Cell Tiss Org Cult 77:133–138
23. Cristea TO, Leonte C, Brezeanu C, Brezeanu M, Ambarus S, Calin M,
Prisecaru M (2012) Effect of AgNO3 on androgenesis of Brassica oler-
acea L. anthers cultivated in vitro. Afr J Biotechnol 11(??):13788–13795
24. Li X, Peng RH, Fan HQ, Xiong AS, Yao QH, Cheng ZM, Li Y (2005)Vitre-
oscilla hemoglobin overexpression increases submergence tol-erance in
cabbage. Plant Cell Rep 23:710–715
25. Ovesna J, Ptacek L, Opatrny Z (1993) Factors influencing the regenera-
26. tion capacity of oilseed rape and cauliflower in transformation
27. experiments. Biol Plant 35:107–112
28. P

1. Maheshwari P, Selvaraj G, Kovalchuk I (2011) Optimization of Brassica

2. napus (canola) explant regeneration for genetic transformation. Nat
3. Biotechnol 29:144–155
4. Maheshwari P, Selvaraj G, Kovalchuk I (2011) Optimization of Brassica
5. napus (canola) explant regeneration for genetic transformation. Nat
6. Biotechnol 29:144–155

1. Maheshwari P, Selvaraj G, Kovalchuk I (2011) Optimization of Brassica

2. napus (canola) explant regeneration for genetic transformation. Nat
3. Biotechnol 29:144–155
4. Maheshwari P, Selvaraj G, Kovalchuk I (2011) Optimization of Brassica
5. napus (canola) explant regeneration for genetic transformation. Nat
6. Biotechnol 29:144–155