TERM PAPER PRESENTATIONCOURSE NO: GP 604

Protoplast isolation and culture- protoplast fusion, different pathways of in vitro morphogenesis

SUBMITTED BY E.RAMBABU

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Contents

• Protoplast isolation

• Protoplast fusion

• Protoplast culture

• Different morphological pathways

Protoplast isolation

• Naked plant cell (Hanstein in 1880)

• By 1. Mechanical method 2. Enzymatic method

1. Mechanical method

• klercker (1892)

• Tissue is dissected and deplasmolyzed

Disadvantages:

1. Restricted to certain tissues (root tissues)

2. Yield and viability is low

3. Method is tedious and laborious

One step or simultaneous

Two step or sequential

Protocal for protoplast isolation

(Cell and protoplast washing media solution)

Factors affecting

1. Physiological state of tissue: mesophyll cells

2. Enzymes: cellulase (cellulase R10) purified from Tricoderma

viride and pectinase (macerozyme) from Rhizopus fungus.

3. Osmoticum: mannitol and sorbitol, potassium and calcium

chloride, glucose and sucrose

4. Culture medium: ratio of nutrients and pH (5.5 to 5.9)

5. Environmental factors

Methods for assessing protoplast viability

1. Fluorescein diacetate(FDA) staining method : accumulates

inside and fluoresce yellow green

2. Phenosafranine staining: specific for dead protoplasts that

turn red

3. Calcofluor white (CWC) staining: by detecting the onset of

cell wall formation

Protoplast culture techniques

1. Agar culture: fixed position and clumping is avoided

2. Liquid culture (it allows easy dilution and transfer, to

maintain osmotic pressure and to reduce density of cells)

a) liquid droplet method: easy examination

b) Hanging droplet method: allows culture of fewer protoplasts

c) Feeder layer: reduce the plating density

d) Co-culturing: combination of slow and fast growing

protoplasts

Protoplast fusion (somatic hybridization)

• Mixing of protoplasts of two different genomes

• Fusion methods

a) Spontaneous fusion: callus cultures

b) Induced fusion methods: fusogen

Induced fusion methodsS.no Scientist Method Description

1. Power et al. (1970) Treatment with sodium nitrate

Suspended in a mixture of 5.5% sodium nitrate in a 10% sucrose solution

2. Keller and melchers (1973)

Calcium ions at high pH

Solution of 0.5M + 0.05M CaCl2..2H2O

3. Kao and michayluk (1974) and wallin et al.

Polyethylene glycol method (PEG)

28-56% of PEG, high frequency heterokaryon formation,low cytotoxicity and non specific

4. Electrofusion AC and DC fields application, simple,quick and efficient

Cybrids

Nuclear fusion

No nuclear fusion

Chromosome number in somatic hybrids

DETECTION OF SOMATIC HYBRIDS

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Intergeneric hybrids

1. Raphanobrassica (R.sativus 2n=18 + B.oleracea 2n=18)

2. Erucobrassica (Eruca sativa 2n=22+ b.napus 2n=38)

3. Nicotiopersicon (N.tabacum 2n=24+ L.esculentum 2n=24)

4. Solanopersicon (S.tuberosam 2n=24 + L.esculentum 2n=24)

5. Daturotropa (D.innoxia 2n=48 + A.belladona 2n=24)

6. Oryzochola (O.sativa 2n=24 + E.oryzichola 2n=24)

7. Arabidobrassica (A.thaliana 2n=10 + B.campestris 2n=20)

Different pathways of invitro morphogenesis

(C & D) Induction and regeneration of direct organogenesis(DOG)

(E) Coinduction of direct shoot organogenesis and direct somatic embryogenesis pathways. (F) Organogenesis through callus. (IOG)

(A) Simultaneous induction and regeneration of direct somatic embryos. (B) Regenerated and elongated shoots by direct somatic embryogenesis pathway.(DSEG)

(G & H) Somatic embryogenesis through callus (ISEG)

March 2011, Volume 104, Issue 3, pp 343–357Protoplast fusion for production of tetraploids and triploids:

applications for scion and rootstock breeding in citrus• ABSTRACT:Protoplast fusion technology has been utilized in many crops to generate allotetraploid

somatic hybrids, and sometimes autotetraploids as a byproduct of the process. A brief history of this technology development is provided, along with a simple protocol developed for citrus, which can be easily adapted to other plants. Protoplast fusion has become a significant tool in ploidy manipulation that can be applied in various cultivar improvement schemes. In rare cases, a new somatic hybrid may have direct utility as an improved cultivar; however, the most important application of somatic hybridization is the building of novel germplasm as a source of elite breeding parents for various types of conventional crosses for both scion and rootstock improvement. Somatic hybridization is generating superior allotetraploid breeding parents for use in interploid crosses to generate seedless triploids. Seedlessness is a primary breeding objective for new fresh fruit citrus varieties, and several thousand triploid hybrids have been produced using somatic hybrids as the tetraploid parent. Protoplast fusion is also being utilized to produce somatic hybrids that combine complementary diploid rootstocks, which have shown good potential for tree size control. Tree size control has gained importance as a means of reducing harvesting costs, maximizing the efficiency of modern cold protection methodology, and facilitating the adaptation of new fruit production systems. Successful somatic hybridization in citrus rootstock improvement has enabled rootstock breeding at the tetraploid level via sexual hybridization, which can yield maximum genetic diversity in zygotic progeny upon which to impose selection for the many traits required in improved rootstock cultivars, including disease and insect resistance, broad adaptation, tree size control, and the ability to consistently produce high yields of quality fruit. Recent progress and successful examples of these applications are discussed. Finally, a discussion of the genetic potential of somatic hybrids as breeding parents, including meiotic behavior and inheritance is provided.