This document describes procedures for plant regeneration through callus culture using tobacco plants. It involves initiating callus cultures from tobacco explant tissue, observing the growth pattern of callus cultures over multiple phases, and inducing shoot and root organogenesis from the callus to regenerate tobacco plants. The objectives are to initiate and maintain callus cultures from tobacco explants, create cell suspension cultures, and induce organ development from callus to regenerate whole plants. Standard procedures are provided for callus initiation, maintenance, measurement of growth curves, and regeneration of shoots and roots. Results showed high rates of callus formation and organogenesis from explants and callus on optimal media formulations.
2. Introduction
• Callus formation from explant tissue involves the
development of progressively more random planes of cell
division, less frequent specialization of cells, and loss of
organized structures .tobacco plant is used.
• There are five phases of callus growth :
• A lag phase, where cells prepare to divide.
• An exponential phase, where the rate of cell division is highest
• A linear phase, where cell division slows but the rate of cell
expansion increases.
• A deceleration phase, where the rates of cell division and
elongation decrease.
• A stationary phase, where the number and size of cells remain
constant.
3. Objectives and Goals
• • To initiate callus cultures from explant tissue of tobacco, and
to observe the growth pattern of a callus culture.
• • To initiate cell suspension cultures of tobacco, and to
recover callus from a cell suspension culture.
• • To observe the induction of de novo shoot and root
organogenesis from tobacco callus, to compare this system to
adventitious organogenesis from tobacco explant tissue, and
to regenerate tobacco plants from callus.
• Plant Materials :
• callus cultures of tobacco can be purchased from Carolina
Biological Supply Company.
4. Equipment :
• Sterile petri dishes, 100 X 20 mm
• Electronic balance with enclosed weigh area, such as Mettler
AE200
• 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
• Oven, such as VWR 1350FD
• Inverted microscope, such as Zeiss Axiovert 100
5. Procedures :
• 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 MS medium to
provide more room for shoot or plantlet development in the
protocols for Callus Initiation and Maintenance, Step 3, and
Induction of Shoot Organogenesis, Step 4. Use petri dishes for
MS medium in the protocol for Induction of Shoot
Organogenesis.
• 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
suspensions.
6. a
• Treatment of Materials :
• Tobacco cultures do well in an incubator set at 25°C with
continuous light or 16-h light/8-h dark photoperiod, 15/-
tmolm-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 culture, and the induction
of shoots or roots from callus compared to intact leaf explant
tissue. The experiment will compare two explant types for the
initiation of callus cultures, and eight culture media for
morphogenetic response. A minimum of four replications is
sufficient, requiring a total of 32 seedlings to initiate the callus
cultures and 8 -16 shoot cultures to initiate the adventitious
regeneration comparison.
7. Protocols :
• 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 in steps 2-4.
• Excise the two cotyledons from half of the seedlings. Culture them
abaxial side up, or upside down, on MS-Tl medium for callus
initiation.
• Excise the shoot apex from the seedlings, half without cotyledons
(from step 2) and half with cotyledons, and insert the stem base
into MS medium. Use two shoots per vessel, one with and one
without cotyledons. This will establish stock shoot cultures for
future use. Subculture onto fresh MS medium on a monthly basis.
• Excise the hypocotyl section from the decapitated seedlings.
Culture them on MS-Tl medium for callus initiation.
8. a
• Callus Growth Curves :
• Use established callus stocks to follow the growth pattern for
an extended passage cycle. A total of 24 callus pieces of
similar size are needed.
• Measure the initial fresh weight (zero time) of three replicate
callus pieces in the laminar flow hood. Place each callus piece
inside a pre-weighed, sterile petri dish to weigh the callus.
Determine the fresh weight of the callus after subtracting the
weight of the petri dish.
• Schedule of Observations and Measurements :
• Callus Cultures :Observe the explants weekly for callus
formation. At the end of the first monthly passage, summarize
the callus response according to origin of callus in the
different explants, callus morphology, and color. Observe and
record fresh weight and dry weight growth of established
callus on designated days or weeks according to the protocol
given. Plot the growth curves over the extended passage.
9. Results :
• Callus formation from tobacco explants approached 100%
frequency.
• A sigmoidal pattern of callus growth was observed.
• The cotyledon explants usually responded faster than hypocotyl
explants.
• Shoot regeneration frequencies from tobacco cultures approached
100% on optimal media such as MS-T2 and MS-TS.
• The individual media supported either shoot organogenesis, root
organogenesis, callus growth.
• Organogenesis from callus took longer to initiate and to produce
normal shoots compared to adventitious shoot formation occurring
directly from leaf explants.
• Root regeneration frequencies from tobacco cultures approached
100% on optimal media such as MS-T3.
