CHAPTER 3

MATERIALS AND METHODS

3.1 Plant Materials

Leaves and stems are excised from in vitro Pereksia bleo plant for this research study.
In order to ensure there is enough plant, sub cultured Pereksia bleo in bottle jar as
shown in Figure 3.1 by using full strength MS medium without any plant growth
regulators. For sub culture, tip of the shoot and root were cut and insert onto the
culture medium with aseptic techniques.

Figure 3.1: Sub-cultured P. bleo from roots and shoots and cultured in a culture
bottle.

3.2 Medium Preparation

Full strength Murashige and Skoog (MS) medium (Murashige and Skoog1962 ) were
used and prepared according to the formulation in Appendix A. MS basal medium
consists of macronutrients, micronutrients, iron sources (FeNaEDTA), vitamins listed
in Appendix A and distilled water. All these liquids and solutions are measured by
volumetric flask to ensure the accuracy and consistency in this research. Sucrose was
used as carbon source and 3% (w/v) of sucrose was used. Next, various types of auxin
and cytokinin were added into dissolved sucrose solution to create 2.0 mg/L
concentration for each set of plant growth regulator respectively. The pH of the
medium was adjusted to 5.75 0.01 with 0.1 M of HCL or NaOH by using the pH
meter (Mettler Toledo, China). 0.8 % (w/v) of agar powder was added into the
medium as gelling agent. 121 C and 15 psi pressure are adjusted on autoclave
machine to sterilize the medium in 15 minutes. After autoclaved, medium was poured
onto Petri dish (Grenier) and solidify the medium in laminar air flow hood.

3.3 Callus induction

Leaf and stem explants from Pereksia bleo were used in this study. Approximate 5mm
X 5mm size of leaf and 5mm length of stem were cut. In this study, there are five
types of auxin , two types of cytokinin and full strength basal MS medium to study
the effects of plant growth regulators on callus induction. Five types of auxins are 2,4dichlorophenoxyacetic

acid (2,4-D), IAA (Indole-3-acetic

aicd), NAA (1-

Napthaleneacetic acid),3,6-dichloro-2-pyridinecarboxylic acid (Dicamba) and 4-

Amino-3,5,6-trichloro-2-pyridinecarboxylic

acid

(Picloram).

Meanwhile,

N6-

furfuryladenine (kinetin) and 6-Benzylaminopurine (BAP) are the cytokines used in

this study, Single concentration (2mg/L) of these plant growth regulators used and fell
strength basal MS medium act as control.

Each replicate or Petri dish was cultured 4 explants onto medium and four replicates
were required to test on each type of medium. There are 8 types of medium including
MS medium without plant growth regulator. Moreover, two sets of experiment
environment were required to be done where the cultures were incubated under light
and dark conditions. In order to obtain an unbiased and consistent result, two batch of
experiment for light and dark conditions are needed. Altogether 512 leaf explants and
512 explants were used in this study. For light incubation, cultures were kept at 25 1
C under fluorescent light for 16 hours. Meanwhile, cultures were kept inside a box to
avoid light and kept at 25 1 C.

Observations and results were recorded by weekly basis. For observation, callus
morphologies, the colour of callus, the degree of callusing, days required for callus
initiation and the percentage of callus induction were recorded. The percentage of
callus induction as calculated using the formula as follows:

3.4 Statistical Analysis

SPSS (Version 16.0) was used for statistical analysis. One way ANOVA and Turkeys
Honestly Significant Difference (HSD) test at 5 % of significance (p<0.05) were used
to determine the significant differences between means of the parameters that
recorded during observation.