Somatic cell hybridization allows genetic analysis using cell culture rather than sexual reproduction. It involves fusing somatic cells from two different species or tissues to form hybrid cells. Gene mapping can be done by selecting hybrids that retain specific genes as parental chromosomes are lost. Chromosomal rearrangements like deletions, duplications, and translocations also help map genes to specific chromosome regions. A case study describes using somatic cell selection in potato cultures with a herbicide to recover resistant variants with mutations in the AHAS gene.

1. SOMATIC CELL GENETICS
Term paper presentation
GP-501
SUBMITTEDTO:
Dr. D. Shivani
PROFESSOR
DEPT. OF GENETICS AND PLANT
BREEDING
SUBMITTED BY:
ANIL KUMAR
RAM/2020-68

2. CONTENTS
 Introduction
 Somatic cell hybridization
 Spontaneous and Induced fusion
 Gene mapping by Somatic cell hybridization
 Selection of Hybrids
 Gene mapping using chromosomal rearrangements
■ Deletion mapping
■ Duplication mapping
■ Translocation mapping
 Case study
 References

3. INTRODUCTION
■ The study of mechanism of inheritance in animals and plants by
using cells in culture. In such cells, chromosomes and genes can be
reshuffled by parasexual methods, rather than having to depend
upon the chromosome segregation and genetic recombination that
occur during the meiotic cell division preceding gamete formation
and sexual reproduction. Genetic analysis is concerned with the role
of genes and chromosomes in the development and function of
individuals and evolution of species.

4. SOMATIC CELL HYBRIDIZATION:
■ SOMATIC CELL HYBRIDIZATION 1st demonstrated by G. Barski et.al in
1960.
■ The technique of hybrid production through the fusion of somatic
cells under invitro condition, is called somatic hybridization.
■ It may be heterokaryon ( fusion of cells of two different species) or
homokaryon ( two parental cell come from two different species)

6. Spontaneous and Induced fusion
■ The frequency of spontaneous cell fusion is very low.
■ It can be increased either by addition of UV-inactivated
Sendai virus or the chemical polyethylene glycol.

7. Gene mapping by Somatic cell hybridization
CELL FUSION
SELECTION OF HYBRIDS
CHROMOSOME LOSS
CHROMOSOMAL MAPPING

8. CELL FUSION
■ Mouse-human somatic cell hybrids have been particularly useful
for mapping human genes.
■ The fusion is usually mediated chemically with polyethylene
glycol, which affect cell membrane or with inactivated virus.
■ Cell fusion 1st produce binucleate hybrid cell.
■ Cell fusion followed by nuclear fusion to produce uninucleate
hybrid cell.

10. Selection of Hybrids
■ Somatic cell hybridization experiments are done using selection procedure that
prevent the growth of parental cells.
■ The most commonly used selection medium is HAT medium (hypoxanthine-
aminopterin-thymidine).
■ In HAT medium, if one parental cell type is deficient for the enzyme thymidine
kinase (TK- ) and other parental cell type is deficient for enzyme hypoxanthine
phosphoribosyl transferase (HPRT-), the parental cell type will not grow.
■ Thus HAT medium select only hybrid cells.
■ The hybrid cell survive as long as they retain the human copy of TK+ allele.

12. Chromosomal loss & mapping
■ The mouse-human somatic cell hybrids have four important
features that make them especially useful for genetic analysis:-
1. The mouse and human chromosomes are easily distinguishable.
2. All mouse chromosomes are usually retained in hybrid cells but only
few human chromosomes are retained in hybrid colons.
3. Human chromosomes are eliminated at random during subsequent
mitotic division of hybrid cells.
4. Both sets of chromosomes, human and mouse are expressed in
hybrid cells.
■ Genes are shown to be located on specific human chromosome by
correlating the human gene product in hybrid cell colons.

14. Gene mapping using chromosomal
rearrangements
■ With the somatic cell technique, we can map a gene to specific region of a
chromosome if informative chromosome rearrangements are available.
Three types of rearrangement that is valuable in regionalizing a gene locus
are translocation, deletion & duplication.
■ Deletions and duplications have been extraordinarily useful in locating
genes on the cytological maps of Drosophila chromosome.

15. DELETION MAPPING
■ The basic principle in deletion mapping is that a deletion that uncovers a
recessive mutation must lack a wild type copy of the mutant gene
■ This fact localizes that gene is within the boundaries of deletion.
■ This approach has been most thoroughly developed in Drosophila genetics,
where the large, banded polytene chromosomes make it possible to define
deletion and duplication accurately.
■ As an example of cytogenetic mapping, let’s consider ways of localizing the X-
linked white gene of Drosophila, a wild-type copy of which is required for
pigmentation in the eyes.

16. CONT…
■ If the white gene has been deleted from the Df chromosome, then the w/Df
heterozygotes will not be able to make eye pigment.
■ The eyes of the w/Df heterozygotes will therefore be white (the mutant
phenotype). If, however, the white gene has not been deleted from the Df
chromosome, then the w/Df heterozygotes will have a functional white
gene somewhere on that chromosome, and their eyes will be red (the wild
phenotype).
■ By looking at the eyes of the w/Df heterozygotes, we can therefore
determine whether or not a specific deficiency has deleted the white gene.
If it has, white must be located within the boundaries of that deficiency.

19. DUPLICATION MAPPING
■ We can also use duplications to determine the cytological locations of
genes. The procedure is similar to the one using deletions, except that
we look for a duplication that masks the phenotype of a recessive
mutation.
■ The basic principle in duplication mapping is that a duplication that
covers a recessive mutation must contain a wild type copy of the
mutant gene.
■ This fact localizes that gene within the boundaries of the duplication

20.  Only one of these duplications, Dp2, masks the white mutation; thus a wild-
type copy of white must be present within it.

21. TRANSLOCATION MAPPING
■ Consider an example in which human hybrid cell line were developed from a patient with
a reciprocal translocation between X chromosome and chromosome 14.
■ Long arm of X chromosome was translocated on the tip of chromosome 14 and small
piece of chromosome 14 was translocated into X chromosome.
■ Only those hybrids survived on HAT medium which has chromosome 14 with long arm of
X attached.
■ Since they survived on HAT medium, they must be HPRT + ,and since the HPRT gene is
known to be x linked, the HPRT gene must map to the long arm of x chromosome.
■ Two other x linked genes were also expressed in these hybrids: PGK and G6PD. These loci
must also map to the long arm of x chromosome.

22. CASE STUDY
Name of Journal: BMC biotechnology
NAAS Score: 8.30
Received: 21 March 2017 Accepted: 1 June 2017
Published: 6 June 2017

23. BACKGROUND
 Somatic cell selection in plants allows the recovery of spontaneous
mutants from cell cultures.
 When coupled with the regeneration of plants it allows an effective
approach for the recovery of novel traits in plants.
 This study undertook somatic cell selection in the potato (Solanum
tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide,
chlorsulfuron, as a positive selection agent.

24. RESULTS
 Following 5 days’ exposure of potato cell suspension cultures to 20 μg/l
chlorsulfuron, rescue selection recovered rare potato cell colonies at a
frequency of approximately one event in 2.7 × 105 of plated cells.
 Plants that were regenerated from these cell colonies retained resistance to
chlorsulfuron and two variants were confirmed to have different independent
point mutations in the acetohydroxyacid synthase (AHAS) gene.
 The two independent point mutations recovered were assembled into a
chimeric gene and binary vector for Agrobacterium-mediated transformation of
wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene
conferred chlorsulfuron resistance in the resulting transgenic plants

25. A. The arrow indicates a rare potato cell colony rescued from cell suspension culture
growing on media containing 20 μg/l chlorsulfuron (each Petri dish was seeded
with approximately 5 × 104 cells)
B. An in vitro potato plant regenerated from a cell colony with resistance to
chlorsulfuron
A B
Somatic cell selection and regeneration of chlorsulfuron resistance
in potato

26. Conclusions
Somatic cell selection in potato using the sulfonylurea herbicide,
chlorsulfuron, recovered resistant variants attributed to mutational events
in the AHAS gene. The mutant AHAS (acetohydroxyacid synthase) genes
recovered are therefore good candidates as selectable marker genes for
intragenic transformation of potato.

27. REFERENCES
1. Barrell, P
., Latimer, J., Baldwin, S., Thompson, M., Jacobs, J., & Conner, A. (2017).
Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of
point mutations in the acetohydroxyacid synthase gene. BMC Biotechnology, 17(1).
doi: 10.1186/s12896-017-0371-4
2. Gardner, E., Simmons, M., & Snustad, D. (2010). Principles of genetics (pp. 183-186).
New Delhi: Wiley India.
3. sarkar. (2021). Cell cell hybridization or somatic cell hybridization. Retrieved 6 March
2021, from https://www.slideshare.net/Subhradeepsarkar/cell-cell-hybridization-or-
somatic-cell-hybridization
4. Snustad, D., Simmons, M., Jenkins, J., Crow, J., & Price, H. (1997). Principles of
genetics (pp. 166-172). Chichester: John Wiley.
5. Somatic Cell Genetics. (2021). Retrieved 6 March 2021, from
https://www.ncbi.nlm.nih.gov/books/NBK216404/