Protoplast Isolation and CultureProtoplast Isolation and Culture

OutlineOutline

- Protoplast- Protoplast

- Sources- Sources

- Protoplast isolation- Protoplast isolation

- Protoplast culture- Protoplast culture

- Environmental factors- Environmental factors

ProtoplastProtoplast

cell without the cell wall coined by Hanstein(1880)

CellCell ProtoplastProtoplast

SourcesSources

Leaves, Stems, Roots, Flowers, Anthers & Pollen

SourcesSources

Callus, Cotyledons, Somatic embryos

Protoplast preparationProtoplast preparation

cell wall cell contents - cytoplasm, nucleus, chloroplasts,

mitochondria etcPlasma lemma

(cell membrane)

incubate in medium of high osmotic pressure containing

cellulase & pectinase

cell contentscytoplasm, nucleus,

chloroplasts, mitochondria etc

PROTOPLAST

plasmalemma(cell membrane)

Protoplast isolationProtoplast isolation

Mechanical - Klercker(1892)

Enzymatic - Cocking(1960)

Mechanical methodMechanical 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 methodEnzymatic method

• Cell wall consitutes of primary & secondary cell wall & middle lamellae

• Cellulose-Cellulase• Secondary-

Hemicelluase• Pectin-Pectinase

Commercial enzyme Commercial enzyme preparationspreparations

Cellulases1. Onozuka R10 Cellulase -Trichoderma viride2. Cellulase -Trichoderma viride3. Celulysin -Trichoderma viride4. Meicelase- Trichoderma viride5. Driselase- Irpex lacteus

pectinases

1. Macerase- Rhizopus arrhizus 2. Macerozyme R 10- Rhizopus arrhizus 3. Pectolyase Y23- Aspergillus japonicusHemicellulases1. Helicase- Helix pomatia2. Hemicellulase- Aspergillus niger3. Rhozyme HP 150- Aspergillus niger 4. Hemicellulase H2125- Rhizopus sp.

General methodGeneral 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 Protoplast isolation-Enzymatic methodmethod

PH & Temperature requirementPH & Temperature requirement

PH - 4.7 to 6.0

Temperature - 25° C to 30° C

Duration - 30 minutes- 20hrs

MethodsMethods

Two step / Sequential methodOne step / Simultaneous method

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

Hemicellulase

-less labour intensive

OsmaticumOsmaticum

• Non-ionic - Mannitol, Sorbitol

• Ionic - Potassium chloride, Calcium chloride

Protoplast CultureProtoplast Culture

Protoplast CultureProtoplast Culture

Viability and Plating density of Viability and Plating density of ProtoplastsProtoplasts

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 techniquesCulture techniques

Several methods,

• Agar culture

• Liquid culture

- Liquid droplet method

- Hanging droplet method

• Feeder layer

• Co-culturing

Agar cultureAgar 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 cultureLiquid 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 layerFeeder 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- culturingCo- 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 regenerationCell 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 factorsEnvironmental 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.