Assignment

Plant cell and tissue culture

Topic:
Somaclonal variation as breeding tool
Somatic hybridization

Submitted to:
Dr. Sajjad

Submitted by:
Khadija Tahira
BBCH-01103011
Bs-Biochemistry
8th semester

Dated: 2nd April, 2014

Somaclonal variation:
Plants regenerated in vitro from undifferentiated cells often exhibit some level of variation called
somaclonal variation. Somaclonal variation in plants is caused by increased mutation rates
mainly due to nucleotide substitutions and small indels during the in vitro regeneration process
and could be provoked by exogenous growth stimulators. Thereby, genetic modifications lead to
somaclonal variations in regenerated plantlets (Volodymyr et al., 2012).

History:
It has been known for over 25 years now that plant cell and tissue cultures undergo genetic
erosions and show changes of various types, especially in chromosome numbers and ploidy level
(Partanen 1963; DAmato 1965; Murashige and Nakano 1966). However, till the mid 1970s
such changes were considered undesirable and were therefore discarded because the main
emphasis was on clonal propagation and genetic stability of the cell cultures. Extensive studies
conducted during the last decade have shown that the cell cultures, especially on periodical
subculturing, undergo various morphological and genetic changes, i.e., polyploidy, aneuploidy,
chromosome breakage, deletions, translocations, gene amplification, inversions, mutations, etc.
(see Nagl 1972; Meins 1983; DAmato 1985(Y. P. S. Bajaj (1990)
Somaclonal selection utilizing tissue culture and directed mutagenesis is a rapid breeding
strategy that has the benefit of retaining many of the key attributes of the parental line in the
derived clones, unlike the genetic reassortment that occurs with traditional breeding sexual
crosses. This particularly has importance for the French fry processing sector which has highly
demands on varietal characteristics. Russet Burbank is over 130 years old and yet remains the
most commonly used variety for the French fry processing industry globally despite over a
century of breeding efforts. Somaclonal type selection methods have been used to improve
resistance to several important potato pathogens. In production of these variants the somaclonal
selection process targeted common scab resistance using the pathogens key pathogenicity
determinant, the toxin thaxtomin A, as a positive cell selection agent. The aim was to obtain
toxin-tolerant plants that express disease resistance. Strong and robust disease resistance was
obtained but not always in variants expressing toxin tolerance (Tamilarasan et al., 2013).

Somaclonal variation as a tool for crop improvement:

Somaclonal variation is a tool that can be used by plant breeders. The review examines where
this tool can be applied most effectively and the factors that limit or improve its chances of
success. The main factors that influence the variation generated from tissue culture are:
(1) The degree of departure from organized growth,
(2) The genotype,
(3) Growth regulators and
(4) Tissue source.
Despite an increasing understanding of how these factors work it is still not possible to predict
the outcome of a somaclonal breeding programme. New varieties have been produced by
somaclonal variation, but in a large number of cases improved variants have not been selected
because:
(1) The variation was all negative,
(2) Positive changes were also altered in negative ways,
(3) The changes were not novel, or
(4) The changes were not stable after selfing or crossing.
Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time,
it is also more universally applicable and does not require containment procedures. It has been
most successful in crops with limited genetic systems and/or narrow genetic bases, where it can
provide a rapid source of variability for crop improvement.(Angela karp 1995)

Somaclonal variation caused by in vitro culture is also called tissue culture-induced variation.
Somaclonal variation may involve chromosome number and structure, gene mutation, altered
sequence copy number, activation of transposable elements, somatic crossing-over, sister
chromatide exchange, DNA amplification and deletion, and change in methylation pattern.
Genotype and in vitro culture conditions (type of explants, medium, duration) influence the
occurrence and frequency of somaclonal variation (Paul et al., 2008).

Limitations to the Use of Somaclonal Variation in Crop Improvement:

Somaclonal variation in the major crop plants, rice, wheat, maize, barley, triticale, sugarcane,
potato and a few forage grasses is reviewed. Reported somaclonal variants include chlorophylldeficient plants, and those with changed morphology, single-gene mutations, polyploidy,
aneuploidy, chromosomal re-arrangements, modified yield, quality and disease resistance, and
occasionally novel variants not present in the natural gene pools. Somaclonal variation results
from both dominant and recessive mutations. The type and frequency of variants suggests that
somaclonal variation is akin to non-directed, random mutagenesis which generates a large
amount of unwanted variation. Consequently, most of somaclonal variation is either useless or of
limited use in direct varietal upgrading. However, somaclonal variants are easier to detect than
those in conventional mutagenesis. It is concluded that the development of in-vitro selection
procedures is essential to sieve out useful from useless variation to overcome the constraints of
somaclonal variation in breeding programs.(B. S. Ahloowalia 1986)
The origins of somaclonal variations are important for ensuring the validity of this research.
Generation of somaclonal variations is attributed to genetic and epigenetic modifications in
DNA. In particular, transposable elements (TEs) are one of the causes of genetic rearrangements
in invitro culture. Tissue culture is reported to activate silent TEs, resulting in somaclonal
variations (Mitsuru et al., 2011).
It is suggested that most somaclonal variations were derived from newly generated mutations
arising from the tissue culture process and not pre-existing mutations in the explants. Mutated
cell percentages remained low in the explants but increased rapidly during the tissue culture. In
recent years, molecular biological approaches have been utilized for detecting somaclonal
mutations, including amplified fragment length polymorphisms and intersimple sequence
repeats. These methods are preferable for detecting unidentified or unspecified mutations and
determining whether a mutation has occurred (Mitsuru et al., 2011).

Somatic hybridization:
Somatic hybridization in citrus: An effective tool to facilitate variety improvement:
Citrus somatic hybridization and hybridization via protoplast fusion has become an integral part
of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been
regenerated from more than 200 parental combinations, and several cybrid combinations have
also been produced. Applications of somatic hybridization to citrus scion improvement include
the production of quality tetraploid breeding parents that can be used in interploid crosses to
generate seedless triploids, and the direct production of triploids by haploid + diploid fusion.
Applications of somatic hybridization to citrus rootstock improvement include the production of
allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus
with sexually.(J. W. Grosser et al 2000).
Since Carlsons reports on the isolation of mutants and production of somatic hybrids from
tobacco tissue cultures, there has been interest in the development of somatic cell genetic
systems in plants. Increasing numbers of biochemically selectable markers have been reported in
the past ten years; protoplast fusion and plant regeneration have become useful tools for
generating interspecific and intergeneric hybrid plants. These techniques allow the combining of
genetic material from different somatic cells. In addition to permitting genetic exchange, an
efficient genetic system should provide opportunities for gene mapping, e.g., by somatic
recombination and chromosome elimination. Both somatic segregation and chromosome
instability have been observed in mammalian somatic hybrids. They also are associated with an
increase in ploidy level (Lazar et al .1981).
Plant regeneration and somatic embryogenesis through interspecific hybridization among
different Carica species were studied for the development of a papaya ringspot virus-resistant
variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv.
Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days
after pollination and the embryos were aborted at 150 days after pollination. The immature
hybrid embryos were used for plant regeneration and somatic embryogenesis (Latifah et al.,
2012).

There has been a lot of work to transfer potentially valuable disease-resistance genes from
diploid sexually incompatible Solanum species into the cultivated potato via somatic
hybridization. Potato protoplasts have been fused with a number of sexually incompatible wild
Solanum species (including S. hmidens, S. bulbocastanum, S. commersonii, S. polyadenium and
S. etuberosum), and many fertile somatic hybrid plants have been regenerated. Somatic hybrids
have been screened for disease resistances and these resistances are heritable (Mitchell et al.,
1995).

Refrences:

Voldodymyr, R., Radchuk, R., Pirko, Y., Vankova, R., Gaudinova, A., Korkhovoy, V., Yemets, A.,
Weber, H.,
Weschke, W., and Blume, Y. 2012. A somaclonal line SE7 of finger millet
(Eleusine coracana) exhibits modified cytokinin homeostasis and increased grain yield. Journal
of experimental botany 63:5497-5506.

Tamilarasan, T., Tegg, R., and Wilson, C. 2014. Resistance to Multiple Tuber Diseases
Expressed in Somaclonal Variants of the Potato Cultivar Russet Burbank. The scientific world
journal 2014: 8 pages.

Paul, S., Mohler, V., Wenzel, G., and Walter, B. 2008. Variation in DNA methylation patterns of
grapevine somaclones (Vitis vinifera L.). BMC Plant biology

Mitsuru, S., Hosokawa, M., Doi, M. 2011. Somaclonal Variation Is Induced De Novo via the
Tissue
Culture Process: A Study Quantifying Mutated Cells in Saintpaulia. Plos one. n. page.

Chuanen, Z., Wei, D., Lu, H.,, Jiajie, W., Li, J., Yang, T., Daying, Z., Zeng-Yu, W., Guangmin, X.
2012. Construction of Whole Genome Radiation Hybrid Panels and Map of Chromosome 5A of
Wheat Using Asymmetric Somatic Hybridization. Plos one. n. page.

Deval, P., J. Brian P., Paul, A., Farah, B., J. S. (Pat) Heslop-Harrison and Michael R. 2011.
Somatic hybrid plants of Nicotiana 3 sanderae (1) N. debneyi with fungal resistance to
Peronospora tabacina. Annals of Botany 108: 809819.
Md. Abul, A., Md. Golam, R., and Latifah, A., 2012. Plant Regeneration and Somatic
Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among
Different Carica Species. Int. J. Mol. Sci. 13:17065-17076.
J. Mitchell M, Susan and John P. 1995. Segregation and Recombination of Solanum bravidens
Synteny Groups in Progeny of Somatic Hybrids With S. tuberosum: Intragenomic Equals or
Exceeds Intergenomic Recombination. Genetics society of America 142: 1335-1348.
Y. P. S. Bajaj Biotechnology in Agriculture and Forestry Volume 11, 1990, pp 3-48
Angela karp Euphytica February 1995, Volume 85, Issue 1-3, pp 295-302
R. Bozorgipour, J.W. Snape Euphytica 04-1997, Volume 94, Issue 3, pp 335-340
B. S. Ahloowalia Advances in Agricultural Biotechnology Volume 20, 1986, pp 14-27
J. W. Grosser, P. Ollitrault, O. Olivares-Fuster In Vitro Cellular & Developmental Biology Plant NovemberDecember 2000, Volume 36, Issue 6, pp 434-449
Horst Binding, Reinhard Nehls Molecular and General Genetics MGG 17. VIII. 1978, Volume
164, Issue 2, pp 137-143
Jihong Liu, Xiaoyong Xu, Xiuxin Deng Plant Cell, Tissue and Organ Culture July 2005, Volume
82, Issue 1, pp 19-44
Deepak Pental, John D. Hamill, Andrew Pirrie, Edward C. Cocking Molecular and General
Genetics MGG March 1986, Volume 202, Issue 3, pp 342-347