presentation in MPhil biotechnology first semester course: somatic cell genetic

2. Protoplast Fusion between Indonesian Citrus maxima and
Citrus reticulata: A Preliminary Report
Journal: Journal of Agricultural Science Received: May 27, 2016, Accepted: March 12, 2018
Presented to: Dr.Bushra Sadia (Associate Professor/CABB)
Centre of Agricultural Biochemistry & Biotechnology (CABB)
Presented by: Group No: 12
1. Zeshan Haider
2. Rabail Saeed
3. Mubeen Sajid
4. Atika Sana
5. Adil Shabeer

3. Authors:
Dr. Dyah Retno Wulandari
Affiliation
 Indonesian Institute of Science, Indonesia
 Plant Tissue Culture Laboratory
Skills & Expertise
 Plant Breeding
 Plant Tissue Culture
 Genetic Diversity
 Primer designing
Project
 Somatic cell manipulation of tropical plant
species

4. Cont.….
Dr. Agus Purwito
Affiliation
 Bogor Agricultural University, Indonesia
 Department of Agronomy and Horticulture
 Dean of Faculty of Agriculture
Skills & Expertise
 Transgenic plants
 Capsicum
Research Publications
 53

5. Contents
1. Abstract
2. Introduction
3. Material & Methods
4. Results & Discussion
5. CONCLUSION
i. Protoplast isolation
ii. Protoplast fusion by PEG (polyethylene glycol)
iii. Fused protoplast culture

6. 1. Abstract
 Protoplast fusion is a useful technique for Citrus genetic
improvement
 Produce triploid seedless Citrus
 New Citrus genetic combination
 Cells divide and form cell colonies in liquid medium

7. 2. Introduction
 Protoplast fusion process can be autotetraplod,
allotetraploid and multifusion
 Protoplast fusion overcome the species barrier
 Indonesian Ministry of Agricultural issued a decree it
was a superior technique to produce a seedless fruit
and improve the quality

8. Cont.….
Citrus maxima
 C. maxima has good quality in
rooting systems and resistant
to CTV
 Better physical qualities
 chemical qualities
 largest citrus fruit
 original natural citrus fruit
 monoembryogenic citrus

9. Cont.….
Citrus reticulata
 Good orange flesh and its ability to
form Somatic embryogenic callus line
 Contains Vit. A, B, and a high level of
Vit. C
 Powerful antioxidant that neutralizes
free radicals
 Prevents infections
 Great for the health of your skin

10. 3. Material & Methods
i. Protoplast isolation
 Source for Protoplast isolation
 Two kinds of enzyme solution composition
 Dilution of Stock solution of enzyme (1:3)
 Incubation of Tissue and enzyme solution
 Purification
 Centrifugation
 Washing with sucrose
 Isolate protoplast from interface layer
 Hemocytometer
 Yield of protoplast

11. Cont.….

12. ii. Protoplast fusion by PEG (polyethylene glycol)
 Preparation PEG solution
 Preparation of elution solution mixture (9:1)
 Suspend the Protoplast mixture (1:1) in solution
 Incubate at room temperature for 8-10 minutes
 Washing of fused protoplasts
 Suspend in culture medium
 Protoplast placed sealed petri dishes and incubated
in dark at 25°C - 27°C

13. iii. Fused protoplast culture
 Two different concentration of liquid medium BH3
 Fused protoplast was grown in petri dishes
 Protoplast culture incubated in dark
 Micro callus formation was induced by decreasing medium
osmolarity
 When callus reached at 0.5 to 1mm in diameter transferred
to basal solid medium containing BAP

14. 4. Results & Discussion
i. Protoplast isolation
A. Cell mesophyll protoplast from C. maxima

15. Cont.….
B. Embryogenic cell callus protoplast from C. reticulata

16. Cont.….
C. Protoplast diameter and yield from enzymatic degradation of leaves
c. maxima tissue and C. reticulata callus
Explants Cellulase RS Macerozime R10 Pectoyase Y23 Mean of PPTs (D) Range of PPTs (D) PPTs (D)
(%) (%) (%) (um) (um) (PPTs m/L)
Leaves 0.5 0.5 0.1 21.00 13.99 -27.07 5.02 x 106
Leaves 0.5 0.5 0 18.24 11.85-33.77 3.31 x 106
Callus 0.5 0.5 0.1 16.68 9.95-31.56 1.10 x 106
Callus 0.5 0.5 0 16.96 12.56-20.95 2.08 x 106
Remarks: PPTs = protoplast, D = Density, leaves from Citrus maxima , callus from Citrus reticulata

17. ii. Protoplast fusion by PEG (polyethylene glycol)
D. Homofusion between protoplasts of callus of C. Reticulata (A) and leaves
of C. maxima (B)

18. Cont.….
E. Hetrofusion(C) and multifusion (D) between protoplasts of callus of C. Reticulata and
leaves of C. maxima

19. iii. Fused protoplast culture
F. After protoplast fusion cells began to clump together (A)

20. Cont.….
G. Cybrids were formed as a result of cell fusion (B)

21. Cont.….
I. Two weeks after fusion, a colony of cells with about 400 μm long (A)

22. Cont.….
J. Cybrids cell starts divide (arrow) after two weeks of fusion (B) and after three
weeks(C)

23. Cont.….
K. Cell from callus homofusion was ready to divide (arrows) after three weeks of
fusion (D)

24. Cont.….
L. Microscopic observation of Protoplast culture ( C. maxima and C. reticulata )
fusion (cell colonies formation)

25. 5. CONCLUSION
 Isolation of protoplast from leaves successfully carried out by
combination of enzyme
 Protoplast fusion successfully carried out by PEG mediated
method
 Three type of fused protoplast obtained
 Growth phases (cell wall regeneration, cell division, colony)
 The cell colonies formed in liquid medium

26. References
o Protoplast Fusion between Indonesian Citrus maxima (Burm.) Merr.
and Citrus reticulata L.: A Preliminary Report
o J. W., & Gmitter, F. G. (1990). Protoplast fusion and citrus
improvement
o Induction of autotetraploids in pummelo (Citrus grandis L.Osbeck)
through colchicine treatment ofmeristematically active seeds in
vitro
o https://www.researchgate.net/
o https://en.wikipedia.org/wiki/Pomelo