This document discusses using protoplast fusion technology to generate tetraploid and triploid citrus plants for use in scion and rootstock breeding programs. It provides a brief overview of the history and development of protoplast fusion techniques. It then describes a simple protoplast fusion protocol that can be adapted for citrus or other plants. The main applications discussed are generating tetraploid somatic hybrids for use as parents to produce seedless triploid hybrids for fresh fruit varieties, and fusing complementary diploid rootstocks to produce tetraploid rootstocks for improved tree size control. Recent progress and successful examples of these applications in citrus breeding programs are also summarized.

1. TERM PAPER PRESENTATION
COURSE NO: GP 604
Protoplast isolation and culture- protoplast
fusion, different pathways of in vitro
morphogenesis
SUBMITTED BY
E.RAMBABU
RAD/16-27
MBB

2. Contents
• Protoplast isolation
• Protoplast fusion
• Protoplast culture
• Different morphological pathways

3. 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

4. One step or
simultaneous
Two step or
sequential

5. Protocal for protoplast isolation
(Cell and protoplast washing
media solution)

7. 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

8. 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

10. 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

13. Protoplast fusion (somatic hybridization)
• Mixing of protoplasts of two different genomes
• Fusion methods
a) Spontaneous fusion: callus cultures
b) Induced fusion methods: fusogen

14. Induced fusion methods
S.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

16. Cybrids
Nuclear fusion
No nuclear fusion

17. Chromosome number in somatic hybrids

19. DETECTION OF SOMATIC HYBRIDS

23. 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)

27. Different pathways of invitro morphogenesis

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

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

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

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

32. March 2011, Volume 104, Issue 3, pp 343–357
Protoplast 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.