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In vitro culture Applications
• Protoplast
• Clonal plant propagation
• Virus-free plants
• Genetic modified plants
• Germoplasm bank
• Somatic fusion
• Synthetic seeds
What is a protoplast?
• The living cytoplasm of each cell, bounded
by the plasma membrane, constitutes the
protoplast.
• Removing cell walls releases large
populations of spherical, osmotically
fragile protoplasts (naked cells), where the
plasma membrane is the only barrier
between the cytoplasm and its immediate
external environment.
Uses of protoplast for
Obtaining transgenic plants
Obtaining somatic hybrids
gene expression, cell wall and
membrane permeability
studying
Protoplast culture steps
A) Protoplast isolation
B) Protoplast development
C) Growth, division and plant regeneration
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Protoplast isolation can be obtained by two procedures
1) MECHANICAL PROCEDURES involving slicing of plasmolysed
tissues, are now rarely employed for protoplast isolation, but are
useful with large cells and when limited (small) numbers of
protoplasts are required.
–Recently, this approach has been used successfully to isolate
protoplasts of the giant marine alga, Valonia utricularis, for patch
clamp analyses of their electrical properties, including physiological
changes of the plasma membrane induced by exposure of isolated
protoplasts to enzymes normally used to digest cell walls(Binder et al.,
2003).
Type and Physiological state of explant.
• The physiological status of the source tissue influences the release of viable protoplasts.
• A convenient and most suitable source of protoplasts is mesophyll tissue from fully expanded leaves of
young plants or new shoots
• Leaf tissue is popular because it allows the isolation of large number cells without killing the plamts
Features of leaf explants
Tabaccofoglia con tricomiTabaccofoglia con tricomi
Protoplast have been isolated from a variety of tissues and organs
• Petioles
• Shoot apices
• Roots
• Fruit
• Coleoptiles
• Hypocotyls
• Stem
• Embryos
• Microspore
• Callus
• Cell suspension
Peeling off epidermid
Epidermises removing is essential for allowing digestion enzyme
to acts on the underneath tissues of explants cell walls
How to remove epidermide:
Peeling Brushing
Peeling
Brushing
pennello a setole mediepennello a setole medie
carburo di silicio (80-120)carburo di silicio (80-120)
Cell walls
• Parete Primaria e Secondaria:Cellulosa ed Emicelluosa
• Lamella mediana: Pectina
The release of protoplast is very much dependent on the nature and composition of enzymes
Type of enzymes:
Cellulase: acts on primary and secondary of cell walls. Ozonuka
R10 generally used for wall degradation has been partially
purified from the molds of Trichoderma ressei
Pectinase : acts on middle lamella. The most frequently used is
macerozyme (macerase) which was derived from the fungus
Rhizopus
Source of enzymes
Enzima Provenienza
Cellulasi Trichoderma viride
Cellulisina Trichoderma viride
Driselasi Irpex lacteus
Emicellulasi Aspergillus niger
Elicasi Helix pomatia
Mecerasi Rhizopus arrhizus
Pectolisai Aspergillus japonicus
Zimoliasi Arthrobacter luteus
Cell wall composition
Monocotiledoni Dicotiledoni
Cellulosa X X
Emicellulose:
Xylogucani X X
Glucoarabinoxylani X
Pectine X X
Acidi Fenolici X
Glico-Proteine X X
Enzymes sterilization
The release of protoplast is very much dependent on the nature and
composition of enzymes
The enzymatic isolation of protoplast can be performed in
two different ways :
• Two-step or sequential method : pectinase first and cellulase
after
• One step or simultaneous method
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Pre-plasmolyses
Protoplast yield and viability can be further enhanced by slicing of
source (preplasmolysed) tissues, manual or enzymatic removal of
the epidermis, and conditioning of donor material or its culture
on media containing suitable osmotica (Davey et al., 2000a, 2004;
Power et al., 2004).
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Sterilization of substrates
The high concentration of carbohydrate in the plasmolyses,
suspension and culture protoplast medium do not allow to sterilize
them in autoclave. Therefore, the filter sterilization by filter
(0.2mm) is the best way
Plasmolysis
• Plasmolysis prior to enzymatic digestion of source tissues in salts
(Frearson et al., 1973) and/or a sugar alcohol solutions, such as 13%
(wt/vol) sorbitol as used for leaves of apricot (Ortin-Parraga and
Burgos, 2003), reduces cytoplasmic damage and spontaneous fusion
of protoplasts from adjacent cells.
• Addition of glycine to the enzyme mixture was essential in maximising
protoplast release from cotyledons and hypocotyls of Cucumis melo
and C. metuliferus, although the optimum concentration of glycine
depended on the species and cultivar (Sutiojono et al., 2002).
• Yields from cotyledons were optimised by a 4-day dark treatment
before enzyme digestion.
A typical plasmolyses medium composition
Macro- e micro-elementi di Gamborg-B5
Mannitolo 0,5 M
CaCl2 1 gl-1
Time: 30 minute - 1 hour
Macro- e micro-elementi di Gamborg-B5
Mannitolo 0,5 M
Cellulasi 15 gl-1
Macerozyme 3 gl-1
CaCl2 1 gl-1
Hormones usually a citokinin
Digestion time: 16 ore
pectyolase, only2-4 ore
A typical digestion medium composition
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Protoplast purification
The enzyme digested mixture obtained at this stage would contain,
sub-cellular debris, undigested cells broken protoplasts and healthy
protoplast.
This mixture is purified by a combination of filtration ,
centrifugation and washing
A. Filtration: solution containing protoplast is filtered through nylon
mesh (50-100 m)
B. Centrifugation: the filtered protoplast-enzyme solution is mixed
with a suitable volume of carbohydrate and centrifuge about 100Xg
for 7-10 min.
C. The protoplast bands is easily sucked off with a Pasteur pipette and
are washed thrice and finally suspended inthe culture medium
Medium composition for protoplast purification
• Macro- e micro-elementi di Gamborg-B5
• CaCl2 1 gl-1
• Carbohydrate : Saccarosio (0,5 M) or Percoll
Pea protoplasts in digestion medium
Percoll separation
Protoplasti di pisello (1)
Protoplasti di pisello in sospensione (Percoll 0%)
Protoplasti di pisello in sospensione (Percoll 10%)
Protoplasti di pisello in sospensione (Percoll 15%)
Protoplasti di pisello in sospensione (Percoll 30%)
Protoplasti di pisello (2)
Protoplasti di pisello in sospensione
(Percoll 40%)
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Protoplast viability and density
The most frequently used staining methods for assessing protoplast
viability are:
• FDA
• Phenosafranine staining
• Calcofluor white
The true test of protoplast viability is the ability of protoplasts to
undergo continued mitotic division and regenerate plants
A) Protoplast isolation: Procedure
Explants choice
Physiological state of tissue
Peeling of explants
Nature and composition of enzyme
Pre-plasmolysis
Plasmolysis
Protoplast harvesting
Estimation of protoplast density
Culture techniques
Protoplast have both maximum and minimum plating densities for growth.
• Published reports suggest that protoplast should be cultured
at a density of 5X 10 3 to10 6 cells/ml.
• The concentration of protoplasts in a given preparations can
be determined by the use of hemocytometer
Growth medium
• Protoplasts from different species and from different tissues of
the same species may vary in their nutritional requirements.
• Consequently, the optimum medium for long-term culture
must be determined empirically.
• Many media have been based on the MS (Murashige and
Skoog, 1962) and B5 (Gamborg et al., 1968) formulations, with
addition of an osmoticum, usually a non-metabolisable sugar
alcohol, such as mannitol, or the somewhat more soluble,
sorbitol.
Growth regulator
• The major growth regulators, auxins and cytokinins, are
normally essential for sustained protoplast growth, although
exceptions exist where only auxin is required, as carrot and A.
thaliana (Dovzhenko et al., 2003).
• In contrast, auxins and cytokinins are detrimental to growth in
citrus (Vardi et al., 1982).
• The growth requirements of protoplasts often change during
culture, necessitating modification of medium composition,
typically involving a reduction of the auxin concentration.
Growth regulator
• Phenylurea derivatives, such as N-(2- chloro-4-pyridyl)-NV-
phenylurea (Sasamoto et al., 2002), and brassinosteroids,
which are similar structurally to animal steroidal hormones (Oh
and Clouse, 1998), can promote division of protoplast-derived
cell
Protoplast development
• Cell wall formation: generally starts within few hours after
isolation and may take two or several days to complete the
process under suitable conditions.
• The protoplast lose their characteristic spherical shape.
• Newly sinthesiesed cell wall can be demonstrated by staining
with 0.1 % calcofluor white fluorescent stain.
Growth, division and plant regeneration
A universal protocols does not exists in term of medium
composition and physical parameters to maximise protoplast
growth.
Recent examples of the application of plant protoplast