Exp 6: Preparation of seedling cultures and callus cultures

Exp 7: Embryo culture of plant tissue

Introduction
Plant tissue culture has been used in a variety of contexts including: organogenesis (Dodds and
Roberts, 1985); somatic embryogenesis (Dodds and Roberts, 1985); protoplast isolation and
culture (Boitino, 1981); effects of hormone balance on growth and morphogenesis of explants
(Mineo, 1990); an exercise to analyze growth (Cooper, et al., 1993); and to demonstrate the
genetic variation caused by plant tissue culture (Morgan and Marcotrigiano, 1987/1988). In the
experiment described here, we used plant tissue culture as a means to study cell differentiation
and the permanence of developmental fate in plant tissue. Moreover, we collected both
qualitative and quantitative results.
The first part of the experiment is to prepare healthy tissue that can be used to establish callus
cultures. This material must be sterile and so seedling preparation is done using aseptic
technique. While for second part of the experiment is to culture the callus to form new plantlet.

Figure 1: Tissue culture and totipotency

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The instructional objectives of this experiment were to:

introduce students to the technique of plant tissue culture;

permit students to become skilful in aseptic technique;

learn about cell differentiation and dedifferentiation (the loss of specialized

characteristics) in plant tissue;

carry out a long-term, team project; and collect and analyze a large data set.

Exp 6: Preparation of seedling cultures and callus cultures

Materials
15% silver nitrate solution
Carrot seeds
Sterile filter paper
Petri dish containing sterile standard medium
Methodology
1. To sterilize the carrot seeds, place them in a small beaker and cover them with a 15 percent
silver nitrate solution. Since carrot seeds are small, 10 cm3 of solution is adequate to sterilize 20
30 seeds.
2. Swirl the seed suspension for 15 sec and then quickly pour the mixture through a funnel lined
with a cone of filter paper.
3. Allow the filter to drain, open it. and let the seeds air dry for 2-4 hrs.
4. After the seeds have dried adequately, sprinkle 20-30 on to a Petri dish containing standard
culture medium (table 1 ).
5. Seal the edge of the Petri dish with parafilm and incubate in the light. The silver nitrate often
darkens the medium right around the seeds. Germination is not affected; it will commence 4-7
days after the seeds are sown.

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Preparation of callus cultures: general instructions

After the seeds have germinated, students can proceed to the next stage of the experiment, the
establishment of the callus culture. As with the preparation of seedlings, this procedure will
succeed only if sterile technique is used.
1. To initiate a callus culture, use sterile forceps to medium, and place it in an empty sterile
Petri dish.
2. With a sterile scalpel, separate leaf, stem, and root tissue. Cut the desired tissue into
sections 1-5 mm long.
3. Transfer the cut tissue sections, with sterile forceps, to callus initiation medium (table 1).
(Students are either provided with Petri dishes containing callus initiation medium or
with melted medium with which they pour their own plates.)
4. Put 4-5 tissue sections on each of 2-3 plates.
5. Record the length and width of all tissue pieces.
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6. Seal the edge of the Petri dishes with parafilm and incubate in the dark at 25-28C for 8
weeks.
Data collection and analysis
1. Measure the length and width of each tissue in millimetres. Use a ruler to make these
measurements but do not open the dishes. (Record this based on each tissue or explants)
2. Repeat these measurements weekly for 8 weeks. Keep a careful record.
Exp 7 Embryo culture of plant tissue
Preparation of callus sub cultures:
Subculture of callus onto fresh medium was carried out after an interval of 30 days. For shootbud regeneration and rooting, the callus was transferred onto MS medium after 2 months of
callus growth.
Transfer to soil
Rooted plants were removed from culture flasks, washed free of agar and transferred to small
plastic pots containing sterilized soil and organic manure (2 : 1 ratio) mixture and covered with
polythene bags. The pots were watered with liquid MS medium and acclimatized for 1520 days
in culture room conditions before transferring to field conditions.
Data collection and analysis
1. Measure shoot and root regeneration from calli of different ages, measure every 3 days for 2
weeks.

Reference:
Bipasha C. and Goswami B.C. (1999) Plantlet regeneration from long term callus cultures of
Citrus acida Roxb. And the uniformity of regenerated plants. Scientia Horticulturae 82 (159169).
Donna M. Bozzonea (1997). Using tissue culture to investigate plant cell differentiation and
Dedifferentiation. Journal of Biological Education, 31 (4).
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