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LABORATORY EXERCISE
Root Culture
Introduction
Plant tissue culture is the practice of isolating plant tissue or cells and culturing it in vitro.
This means that it is done outside of the living organism in a manner that allows
manipulation and control of the environment that is aseptic. The part of the plant cultured
can be of any type of explants; root, shoot, leaf, flower, embryo, etc.
In this experiment, students will attempt to culture carrots. Carrots are roots that have
secondary phloem. Root culture is the culture of root tips of either primary or lateral roots.
Roots are indeterminate organs and can therefore grow indefinitely.
Objectives
To improve the proper procedures in sterilizing root for plant tissue culture.
Materials
Carrot; petri dishes filled with MS medium supplemented with NAA or/and BAP and control
treatment (without NAA or/and BAP) previously prepared; 20% commercial bleach solution
(Clorox); 70% alcohol; Tween 20; beakers; Bunsen burner; blade (size: 24).
Methodology
1. Carrot cuttings were washed under running water for 10 minutes to clean off surface
contaminants such as debris and dirt.
2. Meanwhile the following was prepared: 20% commercial bleach solution (20 mL of
Clorox added onto 80 mL of sterile distilled water) and 70% alcohol (70 mL absolute
alcohol added onto 30 mL sterile distilled water)
3. After washing of carrots is completed, they were cut into smaller cuttings and
submerged in commercial bleach solution with two drops of surfactant Tween 20 for 20
minutes. The solution was constantly agitated.
4. Carrot cuttings were then washed three times with sterile distilled water
5. The water was discarded and carrots were sprayed with 70% alcohol and left for a
minute.
6. Carrot cuttings then were cut into small thin slices and were inoculated on petri dishes
filled with MS medium supplemented with plant growth regulators (NAA or/and BAP)
and control treatment (without NAA or/and BAP) and sealed with parafilm.
7. Petri dishes were incubated in culture room under 16 hour photoperiods.
8. Cultures were observed every 3 days for a period of 15 days.
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Plant Biotechnology
Results
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Discussion
The initial growth of carrot tissue promotes callus formation. This is because the explant
used in this experiment is a tissue with well defined structures of xylem, phloem, cambium
and more. Direct regeneration into plantlets is impossible. Thus the explants will undergo a
phase that promotes callus formation and in some cases of prolonged period, shoot and
root may proliferate from the calluses. The similar reaction was observed in the carrot
explant culture. For the first few days, all the explants remain stagnant with no difference.
Nearing Day 12, most of the explants were dead and contaminated. The rescued explant
tissues of carrot showed signs of the cambium turning green and clump of cells were
noticed along the greenish area. As the observation period reaches Day 14, MS medium
with various concentrations of BAP and NAA showed different results which are discussed as
the following:
Both treatment demonstrated relatively low shoot and root formation. Prolonged period of
observation may have more roots and shoots formation percentage. Other probable factors
of low rooting in Treatment I are unsuitability of NAA (other types of auxin such as 2,4-D can
be used) or explants used is not suitable (carrot cuttings too thick). Smaller and thinner
explants may also help improving shoot formation in Treatment II. Proliferation of fungus in
Treatment II, resulting in medium exhaustion as early as on Day 9, which leads to its inability
to support the growth of carrot cuttings.
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Plant Biotechnology
caused in the laminar air flow has a greater effect of contamination as turbulent air lifts up
microorganisms especially spores on the plantar or on other surfaces. The explants that are
exposed to the turbulent air trap the spore and when cultured, the nutrition medium
provided favours the growth of fungus.
Conclusion
This experiment has allowed us to understand the proper procedures involved in sterilizing,
inoculating, and maintaining root culture (carrot culture) in vitro. We learned to familiarize
with the technique of root tissue culture of carrot explants. Testing with NAA and BAP and
MS basal medium was also observed along with the growth rates.
References
Barcelo, P., Rasco-Gaunt, S., Thorpe, C., and Lazzeri, P. 2001. Transformation and
Gene Expression. In Shewry, P.R., Lazzeri, P.A., and Edwards, K.J. (eds), Advances in
Botanical Research Volume 34: Biotechnology of Cereals. pp. 59-126. Academic
Press, London, UK.