2. Introduction
• 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. s
• 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. Objectives and Goals
• 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. Equipments:
• 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
• Sterile large-bore pipettes, 5 ml, 10 ml - Aluminum foil –
• Inverted microscope, such as Zeiss Axiovert 100
6. Procedure :
Preparation of Media:
o 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.
o 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. Treatment of Materials:
• 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 :
o 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.
o 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. Protocols:
• Callus Initiation and Maintenance :
o Collect the aseptically germinated seedlings when the cotyledons are
fully expanded and the epicotyls is beginning to emerge.
o 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.
o Excise the hypocotyl from each seedling, and cut them transversely
into two equal sections.
o Culture the hypocotyl sections on MS-CAR agar medium for callus
initiation.
9. a
o Seal the culture vessels. Place the cultures in the incubator for 4
weeks.
o Observe the cultures every week for callus induction and growth
patterns.
o 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. Schedule of Observations and Measurements:
• Callus Cultures:
o Observe the explants every week for callus formation.
o At the end of the first month, summarize the callusing response for
origin/location on the explants, frequency, morphology, and color.
o Summarize the results according to each genotype tested.
11. • Somatic Embryogenesis:
o Observe weekly. At the end of each monthly passage for callus, and
each weekly passage for cell suspensions, summarize the
embryogenic response.
o 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.
o Analyze the response according to genotype and medium treatment,
i.e., MS liquid vs. MS agar media, MS-CAR agar vs. MS agar
media.
12. Result:
• 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.
