ORGANOGENESIS SOMATIC EMBRYO HYBRIDIZATION Presented by Neha Agrawal

ORGANOGENESIS & SOMATIC EMBRYO HYBRIDIZATION Presented by: Neha Agrawal m. sc. 3 rd sem

CONTENT v Introduction (organogenesis) v Factor affecting organogenesis v Advantages of organogenesis v Application of organogenesis v Somatic hybridization technique v Mechanical v enzymatic v Isolation of protoplast v Advantages of somatic hybridization v Limitation of hybridization v Difference between organogenesis and somatic hybridization

INTRODUCTION Introduction Organogenesis is a process of differentiation by which plant organ viz. roots, shoots, bud flower, stem, etc. are formed while adventitious refers to the development of organ or embryo from unusual points of origin of an organized explant where a preformed meristem is lacking. This process is much more common than somatic embryogenesis and has far more potential for mass clonal propagation of plants.

FACTORS AFFECTING ORGANOGENESISq Physical factor q Chemical factor

PHYSICAL FACTOR Intensity of light-the blue region of spectrum promotes shoot formation and red light induce rooting. The treatment of blue light followed by treatment of red light also stimulates the organogenesis phenomenon. Temperature-increase in temp. up to 33°c may be associated with rise in the growth of tobacco callus but for shoot-bud differentiation a lower temp. 18°c may be optimal. Electric stimulation- Bagga et. al 1985 suggested the role of phytochrome in shoot induction.

CHEMICAL FACTOR Cytokinin or auxin - cytokinin in the medium leads to the promotion of bud differentiation and development. Kinetin is 30, 000 times more potent than adenine. Phosphate-increase level of the phosphate in the medium is reported to counteract the inhibitory effect of auxin. Casein hydrolysate – casein hydrolysate or tyrosine also induces kinetin type bud formation even in the presence of higher level of IAA in the medium.

Advantages of organogenesis Cheap. Fast No process. variables. Less space. Easily scale.

APPLICATION OF ORGANOGENESIS Scale up easily. Variation Use is not produce. as a bioreactor. Protoplast Gene culture. transfer.

SOMATIC HYBRIDIZATION Development 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

Isolation of Protoplast (Separartion of protoplasts from plant tissue 1. Mechanical Method 2. Enzymatic Method

Mechanical Method Cells Plasmolysis Plant Tissue Microscope Observation of cells Cutting cell wall with knife Release of protoplasm Collection of protoplasm

Mechanical Method Used for vacuolated cells like onion bulb scale, radish and beet root tissues. Low yield of protoplast. Laborious and tedious process. Low protoplast viability.

Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed cells Pectinase +cellulase Protoplasm released Pectinase Release of isolated cells Protoplasm released cellulase Isolated Protoplasm

Enzymatic Method Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores. Mesophyll tissue - most suitable source. High yield of protoplast. Easy to perform. More protoplast viability.

SOMATIC HYBRIDIZATION The technique of hybrid production through the fusion of isolated somatic protoplast under in vitro conditions and subsequent development of their product (heterokaryon) to a hybrid plant is known as somatic hybridization. In somatic hybridization the nucleus and cytoplasm of both parents are fused in the hybrid cell. Sometimes; nuclear genome of only one parent but cytoplasmic genes (plastome) from both the parents are present in the fused hybrid, which known as cybrid or cytoplasmic hybrid. Somatic hybridization involves the following aspects: 1) Fusion of protoplasts. 2) Selection of hybrid cells. 3) Identification of hybrid plants.

Protoplast fusion It involves mixing of two different genomes and can be achieved by spontaneous or induced fusion method. q Spontaneous fusion : - I. cell fusion is natural process as is observed in case of egg fertilization. II. During the course of enzymatic degradation of cell walls, adjoining protoplasts may fuse to form homokaryocytes (homocaryons). III. These fused cells may sometimes contain high number of nuclei (2 -40). IV. The frequency of homocaryon formation was found to be high in protoplasts isolated from dividing cultured cells.

q. Induced fusion method : I. Isolated protoplasts can be fused by induction. II. There are several fusion inducing agents which are collectively referred to as fusogen. III. example: sodium nitrate , high glycol, polyvinyl alcohol, lysozyme, concavalina, electro fusion dextran and dextran sulphate , fatty acid and esters.

TREATMENT WITH SODIUM NITRATE Induced fusion by Na. NO 3 was first reported by power et al. (1970). Isolated protoplasts are exposed to a mixture of 5. 5% carried Na. NO 3 in 10% sucrose solution. Incubation is carried out for 5 minutes at 35 ⁰C. Centrifugation for 5 minutes at 200 x g High p. H and high sodium Calcium ion treatment : The different protoplast in one solution are together treated with condition like high calcium and high ph so that they fuse together. In some cases such extreme conditions has proved to be toxic to certain protoplasts.

Poly ethylene glycol treatment This has proved to be one of the most effective methods for protoplast fusion. The cells are treated with a concentration of around 30% poly ethylene glycol which binds to plasma membrane. This is treated with calcium solution which being cationic binds to PEG. During washing the PEG pulls out the plasma lemma leading to fusion of protoplasts in close proximately.

Electro fusion technique This process involves passing low voltage electric pulses in a solution of protoplast to be fused so that they line of for fusion. High voltage electric current which leads alteration of membrane the adjacent protoplast fuse.

SELECTION OF HYBRID CELL The protoplasts are actually involved in the fusion. After the fusion process, the protoplast population consists of heterogenous mixture of unfused chloroplast, homokaryons and heterokaryons. It is therefore necessary to select the hybrid cells (heterokaryons). The commonly used methods employed for the selection of hybrid cells are biochemical, visual and cytometric method.

Culture of the hybrid cells Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

Regeneration of hybrid plants Plants These are induced to regenerate from hybrid cells. hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

Cybrids The cytoplasmic hybrids where the nucleus is derived from only one parent and the cytoplasm is derived from both the parents are referred to as cybrids. The phenomenon of formation of cybrids regarded as cybridization. Normally cybrids are produced when protoplast from two pythogenetically distinct species are fused. Genetically are hybrids only for cytoplasmic traits.

Advantages of somatic hybridization Production of novel interspecific and intergenic hybrid. Pomato (Hybrid of potato and tomato). Production of fertile diploids and polyploids from sexually sterile haploids, triploids and aneuploids. Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters.

Limitations of Somatic hybridization Poor regeneration of hybrid plants. Non-viability of fused products. Not successful in all plants. Production of unfavorable hybrids. Lack of an efficient method for selection of hybrids. No confirmation of expression of particular trait in somatic hybrids.

Difference between organogenesis and somatic embryogenesis organogenesis Production of unipolar structure ( shoot or root primordium ). Vascular system connected to parent tissue. Organogenesis is a property of the somatic tissue. Organogenesis is a popular process. Somatic embryogenesis Production of bipolar structure ( shoot or root axes). Vascular system not connected to parent tissue. Somatic embryogenesis pertains to the reproductive tissue Somatic embryogenesis is a relatively less popular.

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