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1. Presented by ‘ M.Phool Badshah ’

2.  Plant regeneration by somatic embryogenesis from
cultured cells was originally observed with carrot .
 In somatic embryogenesis, somatic cells develop by
division to form complete embryos analogous to
zygotic embryos.
 The bipolar structure of the somatic embryo
contains both shoot and root meristems.
 As the embryos develop, they progress through the
distinct structural steps of the globular, heart,
torpedo, cotyledonary, and mature stages.
 Somatic embryogenesis in carrot and many other
plant species is initiated in the same manner as the
production of callus.

3.  Induction of somatic embryogenesis in most
species requires a high concentration of auxin,
usually 2,4-D, in the culture medium.
 Cytokinin usually is not required for induction of
somatic embryogenesis, but certain mono cot
species do have a specific requirement for
cytokinin.
 The high concentration of auxin used for induction,
however, usually is inhibitory to development of
the somatic embryos into advanced stages.
 A hormone-free medium often is used for the
development of globular-staged somatic embryos
into plantlets.
 Somatic embryogenesis does require a different
medium, with no or lower concentrations of
hormones, for development of the embryogenic
cells into plantlets.

4.  To initiate and maintain callus cultures of carrot.
 To initiate and maintain cell suspension cultures of
carrot, and to observe the growth pattern of a cell
suspension culture.
 To observe the induction and development of somatic
embryos from cultured cells and callus of carrot.
 Plant Materials :
 Follow the protocol in Chapter 3 to initiate
aseptically germinated seedlings of Danvers carrot.
Other genotypes and wild carrot can be substituted, but
the response may vary from that described.
Alternatively, callus cultures can be purchased from
Carolina Biological Supply Company.

5.  Table-top centrifuge, such as Beckman TJ-6
 Sterile graduated conical centrifuge tubes, 15 ml
 Sterile petri dishes, 100 X 20 mm –
 Sterile Erlenmeyer flasks, 125 ml, capped with
aluminum foil –
 Gyratory shaker, such as Lab-Line 3590
 Sterile stainless steel mesh, 75-250 iJ-m (optional),
such as Cellector Tissue Sieve, VWR #62399-918,
with Fine Mesh Kit, VWR #62399-951
 Sterile large-bore pipettes, 5 ml, 10 ml -
Aluminum foil –
 Inverted microscope, such as Zeiss Axiovert 100

6.  Preparation of Media :
 Agar Media : Agar-solidified culture media should
be prepared in 100 X 20 mm petri dishes. Other
culture vessels can be substituted. Magenta boxes
are used for BSG medium to provide more room for
plantlet development.
 Liquid Culture Media : Liquid media used for cell
suspensions should be prepared in 125-ml
Erlenmeyer flasks. Only 15 ml of medium per flask
is used for suspension initiation, while 25 ml of
medium per flask is used for subcultures of
established cell lines.

7.  Carrot cultures do well in an incubator set at 25°C with
continuous light or 16-h light/8-h dark photoperiod,
15f.,lmolm-2 s- 1.
 A gyratory shaker should be located in a room with
similar environmental conditions, but the light intensity
can be lower.
 Design of Experiment :
 The experiments are designed to provide experience in
the initiation and maintenance of callus and cell
suspension cultures, recovery of callus from cell
suspensions, and the induction of somatic
embryogenesis from callus or cell suspensions.
 Two media are compared during the regeneration
experiment. A minimum of four replications should be
started, requiring at least four seedlings to initiate the
experiment.

8.  Callus Initiation and Maintenance :
 Collect the aseptically germinated seedlings when
the cotyledons are fully expanded and the epicotyl
is beginning to emerge. Usually this will occur
when the seedlings are between 7-14 days old.
Place each seedling on a sterile petri dish, one at a
time, to prepare explants as described below.
 Excise the hypocotyl from each seedling, and cut
them transversely into two equal sections.
 Culture the hypocotyl sections on MS-CAR agar
medium for callus initiation. Place a pair of
transverse sections from each seedling into each
culture vessel. Prepare at least four replicate
culture vessels.

9.  Seal the culture vessels. Place the cultures in the
incubator for 4 weeks.
 Observe the cultures every week for callus
induction and growth patterns.
 Excise small pieces of callus and subculture on
fresh medium of the same composition every
month in order to maintain callus stocks. Incubate
the cultures under the same conditions.

10.  Callus Cultures : Observe the explants every week for
callus formation. At the end of the first month,
summarize the callusing response for origin/location on
the explants, frequency, morphology, and color.
Summarize the results according to each genotype
tested.
 Somatic Embryogenesis : Observe weekly. At the end of
each monthly passage for callus, and each weekly
passage for cell suspensions, summarize the
embryogenic response. Calculate the frequency of
cultures expressing embryogenesis, number of somatic
embryos per gram fresh weight, and categorize the
embryos by stage of development, i.e., globular, heart,
torpedo, cotyledonary, complete plantlets. Analyze the
response according to genotype and medium treatment,
i.e., MS liquid vs. MS agar media, MS-CAR agar vs. MS
agar media.

11.  A high frequency of explants formed callus.
 Carrot cultures produced up to 50 germinating
somatic embryos/ml of packed cell volume or up to
100 germinating somatic embryos/g of callus.
 Somatic embryos developed into plantlets on the
hormone-free medium, but not on the auxin-
containing MSCAR medium where embryos were
arrested at early stages of development.
 Carrot somatic embryos at various stages of
development, from the globular through mature
stages,