1. Effect Of Varying Concentration Of
Plant Growth Regulator IBA On
The In-Vitro Rooting Of
Cauliflower Microshoots
Miniproject
Cell and Tissue Culture
BSc (Hons) Biotechnology
University of Mauritius
Presented by:
Ritesh Bhagea, Rouksaar Buctowar, Christabelle Cécile,
Keshavi Ghoorbin, Huda Nazeer
Date: 22nd
April 2014
3. Introduction
Cauliflower:
● Vegetable from species Brassica
oleracea
● From family Brassicaceae
● Food crop: white curd (head)
consumed
● Has low saturated fat and cholesterol
● Contains proteins, vitamin K and C,
dietary fibres
● High carbohydrate content
4. Introduction
Cauliflower (ctnd):
● Cauliflower can be tissue-cultured
● Production of crop free of diseases
such as Rhizoctonia
● A mass production can be
achieved in vitro
● Acclimatisation from in vitro culture
to soil:
○ Rooting system:
■ Strong?
■ Development satisfactory?
● Investigation carried out using
plant growth regulators
5. Plant Growth Regulators - IBA
● PGRs have morphological effects
on cultured tissues
● Different concentrations = different
growth patterns
● Auxins and cytokinins
● Auxin : cytokinin ratio affects
root/shoot formation
● More auxin = more root formation
● IBA is an auxin
● Known as indole-3-butyric acid
● For this experiment, different
concentrations of IBA were used
6. Aims & Objectives
● To assess the effect of varying concentration of plant
growth regulator IBA on the rooting process of cauliflower
microshoots in-vitro by:
○ Preparing an MS medium whereby different concentrations of
IBA are added
○ Inoculating cauliflower microshoots using aseptic techniques
○ Observing the number of roots produced over a period of 5
weeks
● To identify the issues faced with/during the experiment and
consequently suggest ways of improving it
Hypothesis: Appropriate IBA concentration will induce root
formation
8. Methods
25ml of stock 1
+ 1.25ml of
stock 2,3 & 4
500ml volumetric
flask
+ distilled water
20g sucrose added
100ml removed for
control
400ml: 100ml in 4
labelled beakers
(200, 300, 400,
500) µl of a stock of
1mg/ml IBA
respectively
pH adjusted to 5.7 1.2g agar in each
dissolved in
microwave
poured in labelled jars
+ sealed with cap - 4
replicates for each
concentration
jars + equipments
autoclaved for
about 15 hours
after agar solidified:
aseptic inoculation of
microshoots in jars -
sealed + kept in growth
room for 5 weeks
13. Comments
● Number of roots increased due to the effect of auxin IBA
● Maximum number of roots was observed at IBA conc. of 0.4
mg/ml
● By week 5, it is observed that:
1. The control got only 12 roots - not many - because of no
IBA
2. IBA conc. of 0.2mg/ml and 0.3mg/ml both got 28 roots - the
double of control
3. IBA conc. of 0.4mg/ml got highest number of roots -
optimal concentration of IBA
4. IBA conc. of 0.5mg/ml got 36 roots (less than at 0.4mg/ml)
14. Discussion
● In 1994, Farooqi et al. conducted an experiment on
Rosa damascena Mill and studied the effect of IBA.
● They found an increasing trend in rooting percentage,
number of roots per cutting, length of the longest root
(cm), thickness of the root (cm) with increasing
concentration of IBA from 100 mg/l to 300 mg/l.
● In the absence of IBA, explants tend to produce fewer
roots
● In the presence of IBA, roots come out earlier and with
greater frequency
15. Discussion
● However, researchers believe that high
concentrations of auxin can cause damage to
the explant / cutting base
● Auxin can be effective to rooting cuttings in a
certain conc, depending on the crop and
cultivar
● Auxin will have an inhibition effect at higher
concentrations
● This is why, at IBA conc. 0.5 mg/ml - where it
was supposed to have higher no. of roots -
lesser were observed
16. Problems & Improvements
Problem #1
First try:
● concentration of IBA too high: (2,3,4,5) g/L + 0 g/L (control)
● resulted in brown media, except control
● all of the microshoots got ‘burnt’ & turned brown after 1 week,
except for control batch
Improvement #1
● repeated the experiment all over again (methods above)
● used lower IBA levels
17. Problems & Improvements
Problem #2
Small jars
● not enough room for roots to grow after a few
weeks
● some started growing outside the agar & onto the
sides of the jar
● shoot tips & leaves almost touching jar caps
○ in some jars, leaves withered & turned black &
ended up falling into the agar
Improvement #2
● could make use of larger & taller jars since
start
● or, could eventually subculture in larger &
taller jars
Roots
18. Problems & Improvements
Problem #3
Contamination (in 5 jars)
● aseptic techniques maybe not well
implemented
● each time we would handle the jars - bring in
more contamination
● jars in proximity to contaminated ones further
increases risk
● + rusted jar caps - might not have been
closed properly
Improvement #3:
● contaminated jars discarded ASAP
● ensure less people is near the laminar flow hood while
inoculating
Contamination
19. Problems & Improvements
Problem #4
Few replicates (5*4)
● by the end of the 5 weeks, we were left with a few
uncontaminated jars only (15)
● few replicates + we had to discard the contaminated ones
Improvement #4
● could have increased the
number of replicates - more
accurate results
20. Precautions
● Parafilms would wear out & tear
apart after a while - risk of
contamination
○ made sure they were periodically
replaced
● Limited moving the jars around
○ to avoid stressing the plants out
● Not same person inoculated a whole
batch
○ decreased risk of human errors
Torn parafilm
21. Conclusion
● Our experiment, based on using a PGR - IBA, to see whether
it has any effect on root formation yielded positive results.
● It was found that, generally, increasing the conc. of IBA also
increased the number of roots produced by cauliflower
microshoots.
○ Hypothesis confirmed
○ Optimal conc. was 0.4mg/ml
Overall, it was an interesting mini project where we applied our
acquired lab skills and theories learnt during Tuesday lectures in
order to make the project a successful one.
22. References
● http://nutritiondata.self.com/facts/vegetables-and-vegetable-
products/2390/2
● http://www.omafra.gov.on.ca/IPM/english/brassicas/diseases-and-
disorders/rhizoctonia.html
● http://3.bp.blogspot.com/-
LY3gwjyBTBA/Tc18HC8kduI/AAAAAAAAIyw/8A0heN_X_IA/s1600/IMG_8
703.JPG
● Farooqi, A.A., Shenoy, R. and Ramu, B.S. (1994) Influence of planting
material and growth regulators on the rooting of cutting of Rosa
damascena Mill. Indian Perfumer. 38, 133-143. Website:
http://www.sciencedomain.org/uploads/1375263160-4-Revised-
manuscript_version1.pdf [Date accessed: 20/04/2014]
● Amssa, M.and Qaddoury, A. (2004) Effect of exogenous indole butyric acid
on root formation and peroxidase and indole-3-acetic acid oxidase
activities and phenolic contents in date Palm offshoots. Bot. Bull. Acad. Sin
45, 127-131. Website:
http://ejournal.sinica.edu.tw/bbas/content/2004/2/Bot452-04.pdf - [Date
accessed: 20/04/2014]
