Instituto Tecnolgico de Estudios Superiores de Monterrey

Campus Puebla
Biotechnology department
BT-3000.1 Tissue culture laboratory
Practice # 4. Callus generation using Carrots (Daucus carota) as explants

Professor: Dra. Hera Andrade

Group #1 Team #1

Team members:
Ana Paola Valladares Garca

A01327039

Natalia Asuncin Marcial Alfaro

A01326800

Laura Gabriela Rodrguez Barroso

A00398602

Luis Enrique Flores Barragn

A01094159

Due date: September, 19th 2016

Practice #4. Callus generation using Carrots (Daucus carota) as explants

Introduction
Daucus Carota L. best known as carrot, is one of the most important vegetables
representing 3% of world-wide vegetable production. Of commercial interest are
plant lines with high yield and high sugar for producing fuel alcohol or sugar, and
high resistance to diseases, insects and nematodes. (Sturm, n.d.). Wild carrot is a
member of the carrot family (Apiaceae), which includes parsnip, parsley, fennel and
angelica. The orange color of cultivated carrots is due to a high concentration of
beta-carotene, a precursor of vitamin A, which is important for growth and
development. (KEW, n.d.).
As a first step in the experimental plant tissue culture, induction of callus formation
from primary explants is necessary. Callus tissue means an unorganized proliferative
mass of cells produced from isolated plant cells, tissues or organs when grown
aseptically on artificial nutrient medium in glass vials under controlled experimental
conditions (Kumar, 2016).
There are three important things that should be accomplished when initiating a callus
culture, preparing the plant material in aseptic conditions, the selection of a suitable
nutrient medium and the incubation of the culture under controlled conditions. In this
practice, generation of carrot callus will be accomplished.
Objective
Obtain experience in the aseptic techniques and callus induction from explants of
plants cultivated in the garden (carrot).
Materials and reactives

Biological material
o 3 carrots

Solutions
o Sodium hypochlorite 10%
o Tween 20
o Etanol 70%

Sterilized magnetic stirrer

sterilized tweezers

sterilized scalpel

Sterilized 100 ml beakers

and 1 of 250 ml

Chronometer

Sterilized Petri dish

Adherent plastic

Laminar flow cabinet

MS Medium

Preparation of the medium MS for the induction of callus

For the preparation of 130 ml of medium; In an Erlenmeyer distillated water up to
80% of its final volume was added; 4.4g/L of medium Murashige & Skoog was
weighed and placed it in the Erlenmeyer. Then in the following order this
complements were added:

30g/L of sucrose. 5ml/L of stock solution of kinetin

(0.5ppm). 10 ml/L de solucin stock 2, 4-D (1 ppm final). After this the PH of the
solution was adjusted

5.7+-/-0.1 using HCL or NaOH. Then the volume was

completed and finally 6g/L of agar was added. Then the sterilization was done during
15 minutes at 121C and 15 psi.
Callus induction procedure
First the carrots were washed with water and soap. And removed cover with a
peeler. After this the carrots were cut into 8 slices of 0.5 cm wide. Then they were
washed with water and soap for 5 minutes. Then washes until all the soap was
removed. From this point on, the worked was done inside the chamber.
Then the slices were cut into cubes of 0.5 cm side, in the sterile petri dish and placed
in the ethanol 70% solution for 3 minutes, in constant agitation. Then they were
washed with water during 1 minute and placed them in chloride 10% and tween 20
during 7 minutes in constant agitation. Then 3 times they were washed

with

distillated water in different beakers. And the cubes were placed in a petri dish
sterile, and the damaged ends from the explant were eliminated. After this 4 cubes
per petri dish were placed. The total of boxes were 8. 4 petri dishes were incubated
in conditions of light for 16:8 hours in 25C. And 4 petri dishes in darkness conditions
at the same temperature (the ones in darkness were wrapped with aluminium
paper). Every 2 days (for 21 days) the petri dishes were observed and notes of the
changes were taken. Also the condensed water from the petri dishes was eliminated
into a beaker in the chamber.
Results

The first day that we registrated the data is also the day of realization of the practice,
during the weekends the laboratory was closed so the development in the day 2, 3, 9
and 10 have no changes. In the next figure (graphic 1), it can be observed the
changes in the development of cambium of carrot in percentage in the 21 days under
observation.
Graphic 1. Percentage of development of the vascular cambium of carrot.

Graphic 2. Type of contamination observed in the 21 days.

As seen in the graphic 2 and in the next images, the principal contaminant in the
culture of vascular cambium was fungus in the which occurs more frequently on
presence of dark, the presence of oxidation was presented in the seventh day and

and stop to appear on the 18th day when cover all the samples, as seen in the

image

3.

The contamination that was presented in the

culture

it

was retired from the incubator with the purpose

to

repeated lectures and possibilities to expand

the

avoid

contamination.
Image 1. Contamination of fungus

presented

on the 4th(a) and 10th(b) day of observation

in absence

of light.
a)

b)

Image 2. Contamination of fungus presented on 4th(a) and 17th(c) day on

presence of light.

a)

b)

Image 3. Presence of oxidation on 7th(a), 10th(b) and 18th(c) day of

observation on presence of light.

a)

b)

c)

Due to the contaminations presented in the experimentation we need to compare the

results with other team (team number 4), but they also have the results of the
contaminations with the following results (graphic 3).
Graphic 3. Percentage of development of the vascular cambium- team 4.

Graphic 4. Type of cont}amination for the team 4.

The results were the same for both teams.

Discussion
The main problem for this experiment was the contamination rate observed for the
21

days.

This

contamination

was

caused

by

fungus

according

to

the

white/orange/yellowish and slimy and fuzzy appearance of the culture, and could be
originated from the lack of aseptic conditions during the procedure. However, there
are other factors that could originated the contamination. First, contamination that
results from improperly sterilized tissue will generally arise from the explant and be
located in the medium adjacent to the explant like in this case as seen in image 1
and 2, so we can conclude that the contamination was originated for the quality of
the carrots. If the contamination is due to poor technique generally will appear over
the entire agar surface (Smith, 2013). So the contamination depends on the season
of the year where plant material is being grown (greenhouse, field) and location of
the explants on the source plant, plant material from the field like in this case is often
more contaminated as compared to greenhouse or growth chamber plant material.
Plant material growing in the soil (roots, tubers, bulbs) is usually harder to clean than
aerial plant material. Bulbs can sometimes be freed of systemic fungal contamination
by giving the bulb a heat treatment in a hot water bath for 1 h at 54 C. (Smith,
2013) Some tissues are more difficult than others for establishing clean cultures, for
that the concentration of chlorine bleach and the time of the treatment is important

(Smith,2013) but comparing with the other team's results this was not our main
problem.
One factor that could be the determinant to obtain callus is the selection of the
explant, because is dependent on species from which explants were obtained
together with the composition of the culture medium, the best explant to do a callus
induction must be a young age carrot because it increases the rate of successfully
callus induction (Quiros, 2010); the age of the carrot selected for this experiment
must cause the fail obtained.
As we could not test the dark factor to induce callus because of the contamination, in
the bibliography checked it says that usually, after 4 weeks in culture the explants
incubated on medium with 2, 4-D will form a substantial callus in this condition. Also
the medium exposure to the air may also have contributed to oxidation reactions that
formed toxins that could have affected the growth of the plant material. (Quiros,
2010).
Conclusions
By this experiment, it can be concluded that the explants were not the appropriate for
the induction of callus in D. carota because of its age and also the place where they
came from was not the ideal to avoid endogenous contamination. Also the MS
medium supplemented with kinetin(0.5 ppm) and 2,4-D(1 ppm) were not the ideal
conditions for the formation of the callus. The presence of light affected the
appearance of the oxidation that also affects the formation of the callus. So the
opportunity areas to improve this experiment are the age, the origin of the mother
plant and the aseptic conditions.
Bibliography
Kew. (n.d.). Daucus carota (wild carrot). September 14th, 2016, de Royal
Botanic

Gardens

Sitio

web:

http://www.kew.org/science-conservation/plants-

fungi/daucus-carota-wild-carrot
Kumar, S. (2016). Callus Culture: History, Principles and Significance | Plant
Tissue Culture. September 14th, 2016, de Biology Discussion Sitio web:
http://www.biologydiscussion.com/plant-tissues/callus-culture/callus-culture-historyprinciples-and-significance-plant-tissue-culture/14597

Quiros, C. F. (2010). Apiaceae: Daucus carota var. sativum, carrot. Retrieved

September

19,

2016

fromhttp://www.plantsciences.ucdavis.edu/vc221/carrot/Carrot.htm
Smith, R. (2013). Plant Tissue Culture: Techniques and experiments. 3 rd
edition. Elsevier. EUA
Strum, A., Hardegger, M. and Guo-Qing, T. (2016). Transformation and
regeneration

of

carrot.

1st

ed.

[ebook]

Available

at:

https://www.alice.cnptia.embrapa.br/alice/bitstream/doc/483386/1/Callusinductin.pdf
[Accessed 14 Sep. 2016].