Plant Biotechnology

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. Carrot (Daucus carota) is an important member of the Apiaceae family. It is a cold-season crop that has carotene, a type of Vitamin A. Modified roots of carrot is the most widely used part as food but all parts of carrot plant are equally valuable. Carrot roots are used for cooking. Besides the food value, different parts of carrot can be used for different medicinal purposes. Carrot roots are used as refrigerant and seeds as aromatic, stimulant, and carminative. They are useful in the kidney diseases, in dropsy, nervine tonic, aphrodisiac, and given in uterine pain. 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 Treatment I: MS medium + NAA (4 mg/L) Changes DAY 3 DAY 6 Contamination 0% 0% Colour changes Morphological changes No changes Remarks Treatment II: MS medium + BAP (4 mg/L) Changes DAY 3 DAY 6 Contamination 0% 5% Colour changes Morphological changes No changes 4% shoot; Remarks No changes

DAY 9 DAY 12 DAY 15 1% 3% 7% No changes One become green; others no changes 5% root; 5% callus 10% root; 11% callus 18% root; 15% callus Medium lose its integrity Medium exhausted

DAY 9 10%

DAY 12 15%

DAY 15 22%

No changes 5% shoot; 5% callus 7% shoot; 6% callus Medium exhausted; fungal growth

Treatment III: MS medium + NAA (4 mg/L) + BAP (4 mg/L) Changes DAY 3 DAY 6 DAY 9 DAY 12 DAY 15 Contamination 0% 0% 0% 5% 11% Colour changes No changes Morphological changes 5% callus; 5% root; 10% callus 5% root; 21% callus; No changes Remarks Medium lose its integrity Medium exhausted Treatment IV: MS basal medium (control) Changes DAY 3 Contamination 5% Colour changes Morphological changes Remarks

DAY 9 DAY 12 10% 23% No changes No changes Medium become cloudy, substantial bacterial growth observed

DAY 6 10%

DAY 15 30%

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Plant Biotechnology

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: Treatment I: MS medium + NAA (4 mg/L) Contamination of fungus or bacteria was low in this treatment. All carrot cuttings do not exhibit any colour changes except for one that turns green (callus formed). Auxin (NAA) is a plant growth regulator that induces rooting. On Day 9, 5% root formation was observed and increased steadily up to 18% on Day 15. Callus was formed on 15% of the carrot cuttings at the end of the experiment. Solidified culture media starts to “crack” as a result of media moisture being used and the media is being depleted. Imbibitions for growth occurs, thus it makes the medium dry and lose its integrity. Treatment II: MS medium + BAP (4 mg/L) Contamination was recorded on 22% of carrot cuttings. Fungus was identified as the culprit. There were no visible colour changes on the explant along 15 days of observation. BAP (cytokinin) induces shoots formation. Shoots starts developing on Day 6 (4%), reaching just 7% during the end of the experiment. 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. Treatment III: MS medium + NAA (4 mg/L) + BAP (4 mg/L) An intermediate ratio of BAP (cytokinin) and NAA (auxin) concentration favours callus formation. Callus was first observed on Day 9 (5%), while at the same time medium starts to lose its integrity due to upsurge consumption of nutrients by growing explant. Callus was formed on 21% of the carrot cuttings on the final day of observation. Only 5% of root formation was recorded during Day 12 and Day 15. No shoot formation was recorded. We speculated that if observation was extended for several days more, calluses will differentiate into shoots and roots. Contamination occurs on Day 12 at 5% and then increased to 11%. This may due to improper techniques of asepticity and sterilisation withheld throughout the sterilisation, excision and culture process. The utensils used may have been contaminated or the sterilisation of the explants may be a cause. Turbulence

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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. Treatment IV: MS basal medium (control) Contamination of fungus is visible from Day 3 and escalated to 10% in Day 9 before reaching its peak at 30% on Day 15. Substantial bacterial growth due to improper aseptic practices resulted in cloudy culture media making it not feasible to support growth and development of the explant. The tissues of carrot were not being able to form callus as the plant growth regulators are absent in the MS medium. Plant growth regulator is essential for adequate growth and development of almost all plant explant as they activate sites for differentiation. Absence of these hormones will retard the growth and development of the explant unless the hormones are present naturally in the explant. 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. Dahleen, L. S. 1999. Donor-plant Environment Effects on Regeneration from Barley Embryoderived Callus. Crop Science. 39(3): 682-685. Dahleen, L. S. and Bregitzer, P. 2002. An Improved Media System for High Regeneration Rates from Barley Immature Embryo-derived Callus Cultures of Commercial Cultivars. Crop Science. 42(2): 934-938. Dodds, J. H. and Roberts, L. W. 1995. Experiments in Plant Tissue Culture. Cambridge University Press, Cambridge, UK.

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