In vitro culture Applications - unina.it · In vitro culture Applications •Protoplast • Clonal...

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


Explants choice


Peeling of explants


Pre-plasmolysis

Plasmolysis



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).


Explants choice


Peeling of explants


Pre-plasmolysis

Plasmolysis



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


Explants choice


Peeling of explants


Pre-plasmolysis

Plasmolysis



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 (2)

Protoplasti di pisello in sospensione

(Percoll 40%)


Explants choice


Peeling of explants


Pre-plasmolysis

Plasmolysis



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


Explants choice


Peeling of explants


Pre-plasmolysis

Plasmolysis



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

In vitro culture Applications - unina.it · In vitro culture Applications •Protoplast • Clonal...

Documents

Transcript of In vitro culture Applications - unina.it · In vitro culture Applications •Protoplast • Clonal...