gp 604 ppt
TRANSCRIPT
![Page 1: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/1.jpg)
TERM PAPER PRESENTATIONCOURSE NO: GP 604
Protoplast isolation and culture- protoplast fusion, different pathways of in vitro morphogenesis
SUBMITTED BY E.RAMBABU
RAD/16-27MBB
![Page 2: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/2.jpg)
Contents
• Protoplast isolation
• Protoplast fusion
• Protoplast culture
• Different morphological pathways
![Page 3: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/3.jpg)
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
![Page 4: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/4.jpg)
One step or simultaneous
Two step or sequential
![Page 5: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/5.jpg)
Protocal for protoplast isolation
(Cell and protoplast washing media solution)
![Page 6: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/6.jpg)
![Page 7: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/7.jpg)
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
![Page 8: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/8.jpg)
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
![Page 9: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/9.jpg)
![Page 10: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/10.jpg)
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
![Page 11: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/11.jpg)
![Page 12: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/12.jpg)
![Page 13: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/13.jpg)
Protoplast fusion (somatic hybridization)
• Mixing of protoplasts of two different genomes
• Fusion methods
a) Spontaneous fusion: callus cultures
b) Induced fusion methods: fusogen
![Page 14: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/14.jpg)
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
![Page 15: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/15.jpg)
![Page 16: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/16.jpg)
Cybrids
Nuclear fusion
No nuclear fusion
![Page 17: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/17.jpg)
Chromosome number in somatic hybrids
![Page 18: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/18.jpg)
![Page 19: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/19.jpg)
DETECTION OF SOMATIC HYBRIDS
![Page 22: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/22.jpg)
![Page 23: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/23.jpg)
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)
![Page 24: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/24.jpg)
![Page 25: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/25.jpg)
![Page 26: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/26.jpg)
![Page 27: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/27.jpg)
Different pathways of invitro morphogenesis
![Page 28: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/28.jpg)
(C & D) Induction and regeneration of direct organogenesis(DOG)
![Page 29: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/29.jpg)
(E) Coinduction of direct shoot organogenesis and direct somatic embryogenesis pathways. (F) Organogenesis through callus. (IOG)
![Page 30: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/30.jpg)
(A) Simultaneous induction and regeneration of direct somatic embryos. (B) Regenerated and elongated shoots by direct somatic embryogenesis pathway.(DSEG)
![Page 31: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/31.jpg)
(G & H) Somatic embryogenesis through callus (ISEG)
![Page 32: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/32.jpg)
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.
![Page 33: gp 604 ppt](https://reader036.vdocument.in/reader036/viewer/2022062302/58eff22b1a28ab98538b4689/html5/thumbnails/33.jpg)