Protoplast Isolation and Culture

Outline

- Protoplast
- Sources
- Protoplast isolation
- Protoplast culture
- Environmental factors
 Protoplast

 cell without the cell wall

 coined by Hanstein(1880)
Cell Protoplast
 Sources
 Leaves, Stems, Roots, Flowers, Anthers &
Pollen
 Sources
 Callus, Cotyledons, Somatic embryos
 Protoplast preparation

cell wall cell contents - cytoplasm,

nucleus, chloroplasts,
mitochondria etc
Plasma lemma
(cell membrane)

incubate in medium of high

osmotic pressure containing
cellulase & pectinase

plasmalemma cell contents

(cell membrane) cytoplasm, nucleus,
chloroplasts,
mitochondria etc

PROTOPLAST
 Protoplast isolation

 Mechanical - Klercker(1892)

 Enzymatic - Cocking(1960)
 Mechanical method

Cells kept in suitable plasmolyticum

Cell wall is cut through a fine knife

Protoplasts are released from the cell wall

Suitable for
- Onion, Bulbs, Radish roots
Disadvantage
- Poor yield of protoplasts
- Viability is low
- Tedious and labour oriented
- Not for less vacuolated & meristamatic cells
 Enzymatic method
• Cell wall consitutes of
primary & secondary
cell wall & middle
lamellae
• Cellulose-Cellulase
• Secondary-
Hemicelluase
• Pectin-Pectinase
 Commercial enzyme preparations
Cellulases
1. Onozuka R10 Cellulase -Trichoderma viride
2. Cellulase -Trichoderma viride
3. Celulysin -Trichoderma viride
4. Meicelase- Trichoderma viride
5. Driselase- Irpex lacteus

pectinases

1. Macerase- Rhizopus arrhizus

2. Macerozyme R 10- Rhizopus arrhizus
3. Pectolyase Y23- Aspergillus japonicus
Hemicellulases
1. Helicase- Helix pomatia
2. Hemicellulase- Aspergillus niger
3. Rhozyme HP 150- Aspergillus niger
4. Hemicellulase H2125- Rhizopus sp.
 General method
Cells kept in cell wall degrading enzyme

Debris is filtered and/or centrifuged

Re-suspension the protoplasts in

suitable cultured media
Protoplast isolation-Enzymatic
method
PH & Temperature requirement

 PH - 4.7 to 6.0

 Temperature - 25° C to 30° C

 Duration - 30 minutes- 20hrs

 Methods

 Two step / Sequential method

 One step / Simultaneous method
Two step / Sequential method
• First step
-cells are treated with Mecearse to degrade pectin
• Second step
- cells are treated with Cellulase & Hemicellulase
One step / Simultaneous method
-Mixture of enzymes (Mecearse + Cellulase
Hemicellulase

-less labour intensive

 Osmaticum

• Non-ionic - Mannitol, Sorbitol

• Ionic - Potassium chloride,

Calcium chloride
Protoplast Culture
Protoplast Culture
 Viability and Plating density of
Protoplasts
 FDA (Fluorescein diacetate)

 Exclusion of Evans Blue dye by intact membranes.

 Presence of photosynthetic and respiratory activity.

 Pheno-safranin (0.1%).

 Protoplast density within a range of 1 x 104 to 1x105/ml

 Culture techniques
Several methods,
• Agar culture
• Liquid culture
- Liquid droplet method
- Hanging droplet method
• Feeder layer
• Co-culturing
 Agar culture

• Directly add Protoplast suspension

Merits
• Protoplasts remain in fixed position, so
that Protoplast clumping is avoided
• Protoplast immobilized in semi-solid media
gives rise to cell clones & allow accurate
determination of plating efficiency.
 Liquid culture
• Protoplast suspension transfer liquid medium.
Advantages
• Allow easy dilution and transfer
• Protoplast of some species are not divide in
agarified media
• Osmotic pressure of medium is effectively
reduced
• Density of the cells can be reduced after a few
days of culture
Liquid droplet method
• Pipette out 100-200µl from suspending
protoplast (in culture media)
• 5-7 droplets are placed per plastic petri
dish, sealed & then incubated
• Fresh media can added every 5-7 days
interval.
• Convenient for microscopic examination
Hanging droplet method
• Small drops (40-100µl)protoplast
suspension placed inner side of the lidof the
petri dish
• When the lid is applied to the petri dish,
the culture drops are hanging or suspended
from the lid
• Allows fewer protoplast per droplet
 Feeder layer
 Exposing cell suspension protoplast to X ray
dose

 Inhibited cell division but cells are metabolically

active

 Washed three times to remove toxic substances

due to irradiation and plated on soft agar.

 Protoplasts are plated over this feeder layer

 Co- culturing
 Protoplasts of two different species can
be co cultured to promote growth

 Metabolically active and dividing

protoplasts of two types are mixed in a
liquid medium and plated together to
facilitate cross feeding
 Cell wall regeneration
 Protoplasts culture start to regenerate a wall
from few hours to several days to complete.

 The first division is observed after 3-5 days. The

second division is observed within a week and
after another week aggregate of cells are
formed.

 After three weeks, colonies are visible.

 Environmental factors

• High light intensity inhibits protoplast

growth
• Temperature ranging between 20-28° C
• Culture PH is 5.5-5.9
SOMATIC HYBRIDS
 SOMATIC HYBRIDS

 Carried out under in vitro conditions.

 Fusion of isolated somatic protoplasts.
 Development of heterokaryons.
 Sex is altogether eliminated.
 Provides opportunity to construct hybrids
between taxonomically distant plant species .
 PRODUCTION OF SOMATIC HYBRIDS

 STEPS:
 Fusion of protoplasts
 Selection of hybrid cells
 Identification of hybrid plants
 Verification and characterisation of somatic hybrids
 PROTOPLAST FUSION

 Induced fusion method:

 Fusogen is added to fuse the cells.
 Spontaneous fusion method.
 Treatment with sodium nitrate:
 First reported by power et.al.
 Calcium ions at high pH.
 Polyethylene glycol method:
 Kao and michayluk and wallin et al.
 Electrofusion
 1971: Nagata & Takebe
(Japan)
Regeneration of from
protoplast fusion

1972: Carlson
From protoplast fusion

N. Glauca + N. langsdorffii
(2n= 24) (2n=18)

somatic hybrid
(2n= 24)
 MECHANISM OF FUSION

 Protoplast fusion consists of three main phases:

 Agglutination
 Plasma membrane fusion at localised sites.
 Formation of heterokaryons.
 IDENTIFICATION AND SELECTION OF HYBRID
CELLS.

 Based on observation of visual characters,genetic

complementation for recessive mutations and
physiological complementation.
 In complementation,fusion of two protoplasts each
carrying a different recessive marker ,will generate
a fusion product which is functionally restored since
each parent contributes a functional allele that
corrects the respective deficiency of the other
parent.
 CHLOROPHYLL DEFICIENCEY
COMPLEMENTATION.

 Two distinct homozygous recessive albino mutants

of Nicotiana tabacum were used.
 Chlorophyll deficient light sensitive varieties
sublethal and virescent were used.
 Melcher and labib(1974) first used this.
 After two months of incubation under high light
condition,green colonies were developed.
 AUXOTROPH COMPLEMENTATION

 other Reported by Glimelius et.al.(1978)

 Nitrate reductase deficient mutants of N.tabacum
could not be grown on nitrate as a sole nitrogen
source.
 But hybrids could regenerate shoots in nitrate medium.
 The lack of nitrate reductase activity causes an
absolute requirement for reduced nitrogen and is
caused by a deficiency either in the NR apoenzyme
(nia-type mutant) or in the molybdenum cofactor(cnx-
type mutant).
 Both regularly complement each on fusion .
 USE OF METABOLIC INHIBITORS

 Treatment with irreversible biochemical inhibitor

• Iodoacetate
• Diethylpyrocarbamate

 Parent protoplast unable to to reproduce

 Hybrid protoplasts continue to develop and yield
hybrid plants

• N. sylvestris and N. tabacum

• N. plumbaginifolia and N. tabacum
 USE OF VISUAL CHARACTERSTICS

 Most efficient but the most tedious method.

 Products of protoplast fusion are visually identified.
 After identification cells are mechanically isolated.
 FLUORESCENT LABELING:
 Protoplasts are laoded with different fluorescent
dyes prior to fusion.
 The fluorescent label of fusion products can be
recognised in a fluoroscence microscope.
 Products are separated when fusion mixtures are
run through a fluorescence activated cell sorter.
 VERIFICATION AND CHARACTERISATION
OF SOMATIC HYBRIDS

 Morphology
 Isozyme analysis
 Chromosomal constitution
 Genetic characterisation.
SOMATIC HYBRIDIZATION IN SOLANUM

J. P. Helgeson, Dept Plant Pathology, UW

Madison
USDA-ARS
 OBJECTIVES

• To “capture” disease resistance from wild potato

species
• To use segregating populations to identify plant
disease resistance genes and transfer resistance
into potato cultivars
 METHODS

• Isolate protoplasts (typically leaf mesophyll) from

two parental lines
• Fuse protoplasts either chemically (PEG) or using
electrofusion
• Cell membranes fuse forming one cell containing 2
nuclei
• On cell division nuclear material condenses together
and hybrids cells are formed that contain DNA from
both parental lines.
POTATO PLANT GROWING IN TEST
TUBE
Freshly isolated potato protoplasts
TWO PROTOPLAST READY TO FUSE
Fusion product begin to divide in nutrient
medium
Small shoot emerging from green calli
A fertile somatic hybrid
Somatic hybrid have all the chromosomes from
each
parent plants
 Some new disease resistances from
somatic
hybrids of Solanum spp

• Late blight resistance S. bulbocastanum + S. tuberosum

• Early blight resistance S. bulbocastanum + S. tuberosum

• Soft rot resistance S. brevidens + S. tuberosum

• Bacterial wilt resistance S. commersonii + S. tuberosum

• PVY resistance S. etuberosum + S. tuberosum

• PLRV resistance S. brevidens + S. tuberosum-S.

stenotomum
 RESEARCH ARTICLE

Rice biotechnology:
Somatic hybridisation for improved
salinity tolerance and xylem
colonisation by rhizobia
for endophytic nitrogen fixation

Edward C. Cocking
Plant Science Division, School of Biological
Sciences,
University of Nottingham (U.K.), Sep:1996
 Overall aim of research

 Somatic hybridisation of rice for improved salinity

tolerance.
 Xylem colonization of rice by rhizobia for endophytic
nitrogen fixation.
 Somatic hybrid plants were obtained by
electrofusion of rice.
 INTRODUCTION

 Breeding rice for improved salinity.

 Incorporating nitrogen fixation capacity.
 Soil salinity supresses the growth of rice
 Heavy loss of applied nitrogen fertilizers.
 Nitrate pollution of ground water.
 Production of intergeneric somatic hybrids of rice and
the highly salt tolerant species porteresia coarctata.
 Inoculation of rice with diazotrophs azorhizobium
caulinodans.
 SOMATIC HYBRIDS OF
Oryza sativa + Porteresia coarctata

 p.Coarctata is a halophyte closely related

taxonomically to O.sativa.
 It can withstand submergence in sea water for 10
hours per day(BAL AND DUTT,1986).
 Pre-zygotic incompatibilities in sexual hybridisation.
 Fusion of mesophyll protoplast of P.coactata and rice.
 Production of heterokaryons.
 Production of somatic hybrids with chromosome no.72.
 Containing full chromosome complements of of both.
 ENDOPHYTIC ESTABLISHMENT OF
Azorhizobium caulinodans IN RICE.

 Endophytic interactions of nitrogen fixing organism

with non legumes.
 Organism produces nitrogen fixing nodules on the
legume Sesbania rostrata.
 Colonizes the xylem of roots of this
legume(O’Callaghan et. al.,1997).
 Surface sterilised rice varieties;ADT36 and CR1009
were grown in test tube,inoculated with organism
and maintained in growth chamber.
 Presence of bacteria in the xylem was confirmed by
electron microscopy.