Report practical 1 Petunia Tissue Culture

Student Number 32391471 - Marcelo Vieira Da Silva

Abstract:

Introduction
Plant tissue culture can be initiated in vitro by placing explants onto
a growth medium under sterile and controlled conditions. Endogenous
growth regulators or growth regulating compounds added to the medium
can stimulate the metabolism of cells, and they begin active division. In
this process, cell differentiation and specialisation, which may have been
occurring in the intact plant, are reversed, and the explant originates a
new tissue composed of undifferentiated and totipotent cell types (George
et al., 2008). Some endogenous substances have a regulatory, instead of
a nutritional function in plant growth and development. There are
basically two classes of compounds that stimulate plant growth and
development: plant hormones (endogenous) and plant growth regulators
(synthetic), which are generally active at very low dosages.
Auxins, abscisic acid, cytokinin, ethylene, and gibberellins are
commonly recognized as the five main classes of endogenous plant
hormones. Auxins, cytokinin, and the interactions of auxin-cytokinin are
usually considered the most important hormones because they regulate
growth and organized development in plant tissue and organ cultures
(Gaspar et al, 1996).

Auxins concentrations in plant tissue culture

generally range from 0.01 to 10mg/L. and according to the concentrations

they may regulate several process leading to organogenesis such as, and
induction

of

embryogenesis.

Cytokinins

concentration range of 0.1-10 mg/L.

are

generally

used

at

When added in appropriate

concentrations they may regulate cell division, stimulate auxiliary and

adventitious shoot proliferation, regulate differentiation, inhibit root
formation, activate RNA synthesis, and stimulate protein and enzyme
activity (Ponmurugan et al, 2005).
In these experiment was analysed if the plant growth substances
promote cell redifferentiation and development in explants of Petunia
leaves when cultured under several different treatment containing

different combinations proportions of plant growth auxin:cytokinin and

different amount of sugar source.
Materials and methods: this is from the lab book but must be written in
paragraphs with subheadings for sections preparation of plant material,
experiment 1, experiment 2, experiment 3

Results:
Current year class data
Experiment 1 Effect of auxin
After two weeks in culture the explants became slightly rough in
texture, indicating callus formation in some of the numbered media
(image 1), but none of them developed shoots or roots, all material in the
media seemed to be dead, presenting a brownish colour. For the
experiment 1, the Murashige and Skoog basal medium containing different
levels of auxin did not show any effect of concentration for development
of shoots or roots (Fig. 1). The higher mean value for callus weight in
experiment 1 was 0.67g where the auxin concentration was 10uM (Fig. 2).

Figure 2. Effects of Auxin on Callus weight

Experiment 2 Effect of sugar

The effect of sugar on tissues redifferentiation was not possible,

because none of the explants survived the treatment.

Figure 3. Effects of the Interactions Auxin-Cytokinin on Tissue

Development
SD
SE
SD
SE
No.
Mean Callu Callu Mean Shoo Shoo plan
NAA:B
No.
Callus s
s
Shoot t
t
ts
AP
Root
Weigh Weig Weig Weigh Weig Weig with
Ratio
s
t (g)
ht
ht
t (g)
ht
ht
shoo
(g)
(g)
(g)
(g)
ts
0.057 0.020
0.000 0.000
11
0.036
6
4
0.000
0
0
0
0
0.091 0.032
0.000 0.000
12
0.070
7
4
0.000
0
0
0
0
0.110 0.039
0.000 0.000
13
0.136
2
0
0.000
0
0
0
5
0.314 0.111
0.269 0.095
14
0.658
2
1
0.149
2
2
9
3
0.375 0.132
0.361 0.127
15
0.683
3
7
0.192
4
8
8
4
0.615 0.217
0.381 0.134
16
0.930
6
7
0.203
5
9
6
0
Experiment 3 Relationship between auxin and cytokinin
The best combination of auxin and cytokinin proportion was
observed in ___________ (Fig. 3).

Last year class data

Experiment 1 Effect of auxin
After two weeks in culture the explants became slightly rough in
texture, indicating callus formation in some of the numbered media
(image 1), and others included shoot (image 2) and root formation (image
3). The Murashige and Skoog basal medium containing different levels of
auxin showed that the effect of concentration is more pronounced when in
small amounts (Fig. 1).
Figure 1. Effects of different dosages of Auxin on Callus
Development in the Medium Containing 0.5 uM of BAP

2-4 D
(M)

Mean
Callus

SD Callus
Weight (g)

SE Callus
Weight (g)

0
1
10
50
100

weight
(g)
0.2906
0.6591
0.6698
0.4018
0.2308

0.2086
0.1786
0.4091
0.3455
0.1218

0.0695
0.0565
0.1294
0.1093
0.0385

Experiment 2 Effect of sugar

The effective proportion of sugar was ___________, those medium
that contained ______levels of sugar presented the mean callus weight
equal to (Fig. 2).
Figure 2. Relationship between Sugar Concentration
and Tissue Development
SD
SE
no.
Sucrose
Mean
Mean
Mean
Mean
plants
gl-
callus
callus
callus
shoot
with
weight weight weight weight
shoots
0 0.3870
0.1876
0.0593
0.0000
0
10 1.1067
0.7184
0.2272
0.5638
19
20 1.4545
1.3053
0.4128
0.7624
24
40 0.7656
0.3575
0.1131
0.6488
17
60 0.4062
0.2232
0.0706
0.0590
6

Experiment 3 Relationship between auxin and cytokinin

the best combination of auxin and cytokinin proportion was
___________ (Fig. 3).

NAA:B
AP
(uM)
0:0
0.5:0
5.0:0
0:2.5
0.5:2.5
5.0:2.5

Figure 3. Auxin:Cytokinin Ratio and Organogenesis

SD
Mean
SD
SE
Mean
No.
Mean
Callus
Callus
Callus
Shoot
Plants
shoot
weight Weight Weight Weight
with
Weight
(g)
(g)
(g)
(g)
Shoots
(g)
0.177
0.086
0.0272
0.0000
0.0000
0
0.168
0.069
0.0217
0.0000
0.0000
0
0.179
0.119
0.0376
0.0000
0.0000
0
0.291
0.093
0.0295
0.0088
0.0247
3
1.036
0.898
0.2839
0.9385
1.2489
18
1.542
0.805
0.2547
0.5650
0.4555
31

No.
Roots
0
0
0
8
0
0

Discussion: The first paragraph gives a summary of your results. In

subsequent paragraphs write about the importance of plant hormones in
tissue culture also include a paragraph on the possible reasons for the
poor results in this years data. Do not refer to figures or tables in the
discussion
References:
Dodds, J.H. and Roberts, L.W. 1985. Experiments in Plant Tissue Culture.
9780521315166.

https://books.google.com.au/books?id=-XlXQchGe5wC.

Cambridge University Press 2 ed.

Ponmurugan, P., and Kumar, Suresh K.. Applications of Plant Tissue
Culture. Daryaganj, IND: New Age International, 2012. Accessed April 20,
2015. ProQuest ebrary.
Gaspar, T., Kevers, C.,Penel, C., Greppin, H., Reid, D.M., Thorpe, A. T. 1996.
Plant Hormones and Plant Growth Regulators in Plant Tissue Culture. In
Vitro Cellular & Developmental Biology. Plant Vol. 32, No. 4 (Oct. Dec.,
1996), pp. 272-289
Edwin F. George, Michael A. Hall, Geert-Jan De Klerk. 2008. Plant Propagation by
Tissue Culture. Chapter 1 Plant Tissue Culture Procedure Background. Pp. Springer

The growth regulator requirements for most callus cultures are auxin and
cytokinin. Auxins, as a class of compounds that stimulate shoot cell elongation,
resemble IAA in their spectrum of activity. Cytokinins, which promote cell division
in plant tissues under certain bioassay conditions, regulate growth and
development in the same manner as kinetin. Auxin-cytokinin supplements are
instrumental in the regulation of cell division, cell elongation, cell differentiation,
and organ formation (Dodds and Roberts, 1985).