MOHD SHAMSHADL-2012-A-91-M

OVERCOMING THE BARRIERS OF ALIEN

GENE TRANSFER

INTRODUCTION

TYPES OF BARRIER

PRE-FERTILIZATION BARRIERI. Failure of pollen germination on stigmaII. Slow pollen tube growth III. Pollen tube unable to reach the styleIV. Arresting of pollen tube in style , ovary

and ovuleV. Failure to obtain sexual hybridsVI. Differences in ploidy level

POST-FERTILIZATION BARRIERI. Hybrid inviability and weakness, embryo

abortionII. Embryo abortion at very young stagesIII. Chromosome elimination IV. Hybrid sterilityV. Hybrid breakdownVI. Lack of recombination

Technique for overcoming barrier to alien gene transfer

Pre-fertilization barrier Technique to overcome

Failure of pollen germination 1. Mechanical removal of pistil followed by pollination of the exposed end of the style

2. Use of recognition pollen

Slow pollen tube growth 1. Use of recognition pollen 2. In vitro fertilization 3. Use of growth hormones and

immunosuppressant's Pollen tube is unable to reach the style

Shortening the style

Arresting of pollen tube in the style, ovary and ovule

In vitro fertilization

Failure to obtained sexual hybrids Protoplast fusion

Differences in ploidy level 1. Chromosomal doubling of species or species hybrid before hybridization with recipient species

2. Bridging species technique

Post fertilization barrier Technique to overcome

Hybrid in-viability and weakness (i) Embryo abortion Embryo rescue (ii) Embryo abortion at very young stages

1. Ovary culture2. Ovule culture 3. In vitro fertilization

(iii) Lethality of F1 hybrids 1. Reciprocal crosses 2. Grafting of hybrids3. Regenerating plant from callus

(iv) Chromosome elimination 1. Altering genomic ratios of two species 2. Inducing chromosomal exchanges before on set of

eliminationHybrid sterility 1. Chromosomal doubling (amphipoild production)

2. Back crossing Hybrid break down Growing larger F2 population

Lack of recombination 1. Inducing choromosomal exchange through tissue culture

2. By irradiation3. Homoelogous recombination through genetic

manupulation of chromosomal pairing system

TECHNIQUES FOR OVERCOMING PRE-FERTILIZATION BARRIER

Manipulation of the chromosome numberMany difficulties arise when hybridization is attempted between species with different ploidy level e.g wheat, cotton, potato, tobacco, oat, Brassica napus and B. junceaChromosome doubling in either of parents helps overcome such barrier Example

o PotatoSolanum chacoense (chromsomal doubling) XS.tuberosum by livermore and johnstone 1940

o Barely Hordeum chilense X H. vulgare by Martin 1982

BY USE OF BRIDGING SPECIES TECHNIQUE

Crossing between two species with same or different ploidy levels is difficult third species are used .

Helpful in wheat, Tobacco, lettuce Sears (1956)

Aegilops umbellulata 2n= 14

Triticum astivum2n=42

No viable seed

Use of third species T. dicoccoides (2n= 28) as bridging species

SHORTENING THE STYLE Several species that differ widely in stylar length are

difficult to hybridize .Style of one species is to long Example - Corn zea mays (30 cm) X Tripsacum dactyloides (2 cm)To overcome this shortening the style of maize plantLess than 2 cm each of ear treat as set some seeds by

Mangelsdorf and Reeves 1939

USE OF RECOGNITION MENTOR POLLEN

Pollen grains unable to germinate on the stigma of other plants

Use incompatible mixed with killed maternal pollen grains germination of incompatible pollen grains is obtained.

Pollen wall contain extracellular protein which are released on the stigma after pollination.

Used by Knox et al 1972 in Populus

USE OF GROWTH HORMONES AN IMMUNOSUPPRESSANTS

Various growth harmones have been found to stimulate pollen tube growth and embryo development .

Prolong receptivity of stigma and prevent early abscission of pollen flower

Use of 75 ppm gibberellic acid (GA3 ) to the maternal plant 1 or 2 days before and after pollination for producing interspecific hybrid and intergenic hybrid

IN VITRO FERTILIZATION Any manipulation of excised maternal and paternal

tissue to accomplished pollen tube penetration to the embryo sac to accomplish fertilization is referred to as in vitro fertilization .

Whole gynoecia are excised and placed on medium for 24 -48 hr followed by dusting of pollen on the stigma

Kanta et al. 1962 first try invitro pollination in Papaver somniferum.

SOMATIC HYBRIDIZATIONDevelopment of hybrid plants through the fusion of somatic protoplasts of two different plant species/ varieties is called somatic hybridization

Somatic hybridization technique

1. Isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants

PROTOPALST FUSIONProtoplast It is cell without cell wall created by

degrading the cell wall using enzymes

Cell wall

plasma membrane

Production of protoplast by enzymatic treatment (enzyme aredepicted above arrow) osmoticum (enzyme are depicted belowarrow) to stabilized the protoplast

N Npectinase 0.1-1% cellulase 1-2 %

500- 800 m mol/l serbitol 50-100 m mol/l CaCl2

Isolation of protoplast (Separation of protoplast form plant tissue)

1. Mechanical method

2. Enzymatic method

Mechanical method

Plant tissue

Release of protoplasm

Cell plasmolysis

Microscope observation of cells

Cutting cell wall with knife

Collection of protoplast

Plasmolysed cells Plasmolysed cells

Protoplast released

Release of isolated cells

Leaf sterilization removal of epidermis

ENZYMATIC METHODS

Isolation of protoplast

Pectinase + cellulase Pectinase

cellulase

Protoplast released

Protoplasm fusion ( Fusion of two different genome )

Spontaneous fusion Induced fusion

Intraspecific

Intergenric chemofus

ionMechanical fusion

Electric fusion

INDUCED PROTOPLAST FUSION Electro fusion : A high frequency of AC field is

applied between two electrodes immersed in the suspension of protoplast this induced charges on the protoplast and causes them to arrange themselves in lines between the electrodes. They are the subjected to high voltage discharge which causes them membranes to fuse where they are in contact.

Polyethylene glycol (PEG) causes agglutination of many types of small particles, including protoplasts which fuse when centrifuged in its presence.

A schematic representation of the 3 most successful protoplast fusion strategies.

PROTOPLASTS OF SPECIES(A)

PROTOPLAST OF SPECIES(B)

(SUSPENDED IN ENZYME MIXTURE)

HIGH Ca2 + , HIGH pH TREATMENT

PEG- INDUCED FUSION

ELECTROFUSION

Ca2+ 50 m mol 1-1

Ph 10.5,Temp.37OC

PEG 28-50% (MW 1,500-6,000)

LOW VOLTAGE15 -30 min PROTOPLAST AGGREGATION

30 min

WASHING MEDIUM Ph 9-10

Ca2+ 50 m mol 1 -1

PROTOPLAST CHAIN FORMED (DESIRED PROTOPLAST PAIR ALINGED WITH A MICRO –MANIPULATOR)

HIGH VOLTAGE (FEW MILLI-SECONDS)

PROTOPLAST FUSION

PROTOPLASTS (SPECIES A)

PROTOPLASTS (SPECIES B)

PRODUCTS THAT INVOLVE TWO PROTOPLASTS ARE DEPICTED HERE

FUSOGEN TREATMENT

A B A + A

B +B

B +A

UNFUSED PROTOPLASTS

HOMOKARYONSHETEROKARYON

USEFUL ONE

Possible Result of Fusion of Two Genetically Different Protoplasts 

Fusion

heterokaryon

Cybrid Cybrid Hybrid Hybrid

= Nucleus

= Mitochondria

= Chloroplast

Identification and Selection of somatic hybrid cells

Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to- Pigmentation Cytoplasmic markers

Fluorochromes like FITC (Fluoroscein Isothiocyanate) and RITC (Rhodamine Isothiocyanate) are used for labelling of hybrid cells.

Presence of chloroplast Nuclear staining

Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain

TECHNIQUES FOR OVERCOMING POST-FERTILIZATION BARRIER

Hybrid inviability and weaknes Abortion embryo is most common among post fertilization barrier Different stages of development depending upon the genomic

relationship between two speciesEmbryo rescue : 

When embryos fails to develop due to endosperm degeneration, embryo culture is used to recover hybrid plants; this is called hybrid rescue.

ExampleH. vulgare x Secale cereale. Embryo rescue generally used to overcome endosperm degeneration.

EMBRYO IMPLANTATIONUsed when embryo abortion starts at very young stages of development.Endosperm of female parent serve as nurse tissue for the hybrid embryo Normal endosperm from the female parent is placed on the surface of nutrient media but not direct contact to hybrid embryos. Sometimes the endosperm extract is added to the nutrient media

OVARY CULTURE Ovary culture is often used either for in vitro pollination and

fertilization or for embryo rescue, when the embryo culture and ovule culture either fail, or are not feasible due to very small ovules. Interspecific hybrids, using ovary culture, have successfully been obtained in several genera including Brassica (B. campestris x B. oleracea).

This technique is consist of excising ovaries 2-15 days after pollination depending upon the cross combination and culturing them on nutrient medium. The calyx, corolla, and stamen are removed. Before culturing the tip of distal part of the pedicel is cut off and ovary is implanted with the cut end inserted in to the medium. After embryo become visible they are excised aseptically on nutrient media followed by embryo rescue.

OVULE CULTURE

For a variety of difficult interspecific / intergeneric crosses involving members of the families Malvaceae, Fabaceae, Cruciferae, Solanaceae, etc., ovules after fertilization have been successfully cultured to obtain mature embryos /seeds.Depending upon, when the embryo aborts, the ovules have to be excised any time from soon after fertilization to almost developed fruits, which may sometimes be lost due to premature abscission.However, ovule culture is mainly tried only in those cases, where embryo aborts very early, and embryo culture is not possible due to difficulty of its excision at a very early stage. In some cases, the medium may need to be supplemented with some fruit/Vegetable juice to accelerate initial growth.Used affecting growth of zygote in early stages of development

Contd………

Ovaries are Harvested 1-12 days after pollination Surface are sterilized and cut open with sterilized scalpel. Fertilized ovule is scooped out placed as evenly as possible on nutrient mediumE.g. cotton

GRAFTING HYBRIDSGrafting of hybrid on the normal plant for successful production of hybrids

RECIPROCAL CROSSES Reciprocal differences in wide crosses are also vary common and can be due to chromosomal imbalance in the endosperm, the role of the sperm nucleus in differential endosperm development or the alteration of endosperm development by pollen through the effect of antipodal cells, which are assumed to supply nutrients during early endosperm development.If disharmony between the genome of one species and cytoplasm of another species is a cause of a fertilization barrier ,reciprocal crosses is successful in recovery of hybrids.

ALTERATION GENOMIC RATIOChromosome elimination sometime occur in wide crosses.In Hordeum vulgare H. bulbosumIn wheat maize crosses May be avoid by altering the ratios of parental genome Hordeum vulgare (diploid ) H. bulbosum (tetraploid)

No chromosomal elimination in the zygote (kasha 1974)Followed by mutagenic treatment of fertilized egg cell

could be used to induced chromosomal exchanges before the onset of chromosome elimination

CHROMOSOME DOUBLING OVERCOMES STERILITY OF F1 HYBRID

Crosses betweenTriticum aestivum X Secale cerealsPennisetum glacum X P.setaceum Wild Oryza spp. X Oryza spp. Etc

Lead to sterile F1 hybridsDoubling the chromosome by use of colchicine result formation

of restitution nuclei produces amphidiploids in which pairing occur between respective diploid genome present in F1 hybrid.

E.g. Triticale

Triticale Production

Durum wheat2n=28 AABB

Rye 2n=14 RR

Haploid embryo n= 21 ABR

Haploid plantlet n= 21 ABR

Embryo rescue

Chromosomal doubling

Primary hexaploid Triticale2n= 42 AABBRR

 “WIDE’’ CROSSING OF WHEAT AND RYE REQUIRES EMBRYO RESCUE AND CHEMICAL TREATMENTS TO DOUBLE THE NO. OF CHROMOSOMES TRITICAL

Triticale

BACKCROSSING Brar D S and Khush G S (1997) Alien introgression in rice. Plant Molecular Biology 35–47

HYBRID BREAKDOWN

F1 hybrids are fertile Recombination in F2 or later generation are lethal,

weaker and are gradually eliminated ,resulting in only parental types.

Cause Centromeric affinityCryptic structural hybridity Gene substitutionUnfavorable nuclear-cytoplasm interactions

E. g . Cotton

LIMITED RECOMBINATION Tissue culture of wide crosses Irradiation-induced chromosomal translocationManipulation of chromosome pairing

FUTURE STRATEGY FOR ALIEN GENE INTROGRESSION

Advanced backcross –QTL strategyLooking for gene based on molecular mapsRecombination DNA technologyDouble haploids

ADVACED BACKCROSSED-QTL STRATEGY

Productivity enhancing gene / QTLs are introgressed inoat from Avena sterilis (Frey et al 1983) in tomato fromLycopersicon pimpinellifolium and L. parviflorum(Tanksley et al 1996, Fulton et al 2000) and in chick peafrom Cicer reticulatum ( Singh et al 2005)

RECOMBINATION DNA TECHNOLOGY

Plant genetic transformation technique such as Agrobacterium-mediated transformation and direct gene delivery system (biolistics) allow the precise transfer of genes from any organism into either plant nucleus or chloroplast genome. Many isolated plant genes are now being transferred between sexually incompatible plant species.

CONCLUSION Wild species are an important reservoir of useful genetic variability for traits of economic importance to diseases and insects, tolerance for abiotic stresses, improved quality characteristics and new source of male sterility. There are several pre and post-fertilization barrier that hinder the transfer of useful alien gene into crop plant. The techniques of embryo rescue, ovary and ovule culture , invitro fertilization, protoplast fusion, use of growth hormones, chromosomal doubling , induced chromosomal exchange and recent approch Markered assisted selection (MAS), recombination DNA technology and Advaced backcrossed-QTL strategy wider the scope of introgression of alien gene in cultivated plant not only from plant kingdom but also from animal kingdom.