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🧬 Somaclonal variation

Somaclonal variation.

This lesson explains the core ideas, methods, and exam-relevant applications for this topic in plant biotechnology. Focus on definitions, process steps, and practical uses for revision.

Somaclonal variation and crop improvement

Larkin and Scowcroft (1981) proposed the term somaclone to describe the plants

originating from any type of tissue culture. Genetic variation found to occur between

somaclones in plant tissue cultures was called somaclonal variation. This variation

includes aneuploids, sterile plants and morphological variants, sometimes involving traits

of economic importance in case of crop plants. The usefulness of variation was first

demonstrated through the recovery of disease resistant plants in potato (resistance

against late blight and early blight) and sugarcane (resistance against eye-spot disease, Fiji

disease and downy mildew)

Genetic variation - mutations or other changes in the DNA of the tissue those are

heritable. This is only transmitted to the next generation and is thus important for crop

improvement. Therefore it is necessary to study the transmission of variation to sexual

progeny to facilitate the estimation of its utility for improvement of a sexually propagated

crop. In several crops R0, R1 and R2 progenies were analyzed for genetic analyses and

3:1 segregation leading to the isolation of true breeding variants was observed.

Epigenetic variation - non-heritable phenotypic variation. Epigenetic changes can be

temporary and are ultimately reversible. However, they may also persist through the life of

the regenerated plant.

Physiological variation- temporary in response to stimulus and disappear when it is

removed.

Causes for variation

Changes of mother plant origin

Chimeral - rearrangement of tissue layers. Many horticultural plants are periclinal

chimeras, that is, the genetic composition of each concentric cell layer (LI, LII, LIII) in the

tunica of the meristematic tissues is different. These layers can be rearranged during rapid

cellular proliferation. Therefore, regenerated plants may contain a different chimeral

composition or may no longer be chimera at all. Cell variation also occurs if callus is

initiated from explants containing differentiated and matured tissues that have specialized

function.

Explant derived variation

The most stable cultures are obtained from meristematic tissue of a mature plant or

tissues of a very young organ of meristematic nature. Polyploid cells can give more

variability than diploids

Genetic changes arising in culture

Ploidy changes

Three phenomena that occur during mitosis lead to most changes in ploidy:

  • Endomitosis (sister chromatids separate within the nuclear membrane, but there is

no spindle formation nor cytoplasmic division)

  • Endoreduplication (chromosomes at interphase undergo extra duplications)

  • Spindle fusion (giving binucleate or multinucleate cells).

Gross structural rearrangements appear to be a major cause of somaclonal variation.

These involve large segments of chromosomes and so may affect several genes at a time.

  • Deletions (genes missing, for example 1,2,3,4 now 1,2,4)

  • Inversions (gene order altered, for example 1,2,3,4 now 1,3,2,4)

  • Duplications (1,2,3,4 now 1,2,2,3,4)

  • Translocations (whole chromosomal segments moved to a new location, for

example 1,2,3,4 now 1,2,3,4,A,B,C)

Transposable elements are segments of DNA that are mobile and can insert into coding

regions of genes, typically resulting in a lack of expression of the gene. The culture

environment may make the transposable elements more likely to excise and move.

Point mutations (the change of a single DNA base), if they take place within a coding

region of a gene and result in the alteration of an amino acid, can lead to somaclonal

variation. Point mutations are often spontaneous and are more difficult to detect. Note that

they result in single gene changes

Structural changes in the DNA sequence

Chromosomal rearrangements, point mutations, or transposition of transposable

elements can occur during culture. These changes can occur spontaneously or can be

induced with chemicals or radiation

DNA methylation: Most of the mutational events occasioned by tissue culture are directly

or indirectly related to alterations in the state of DNA methylation. A decrease in

methylation correlates with increased gene activity

Lack of nucleic acid precursors: Shortage of the precursor necessary for rapid nucleic

acid biosynthesis, which occurs in many tissue cultures

Growth regulators: One of the triggers of polyploidy in vitro is growth regulators; both

kinetin and 2,4-D have been implicated.

Composition of culture medium: The level of KNO3 influences the albino plants from

wheat cultures. Level of organic N2, chelating agents and other micro nutrients are other

factors.

Culture conditions: Temperature, method of culture

Effect of the genotype

Effects of the culture process itself (lengthy culture periods, growth and other aspects of

the culture medium may also affect the ploidy of the cultured cells. Medium that places

cells under nutrient limitation will favor the development of "abnormal" cells. Chromosomal

alterations, like ploidy changes, increase with increased lengths of culture. In mixed

populations of cells with different ploidy, diploid cells retain their organogenic potential

better than polyploid and aneuploid cells (probably due to an enhanced ability to form

meristems).

One common alteration seen in plants produced through tissue culture is rejuvenation,

especially in woody species. Rejuvenation may lead to changes in morphology, earlier

flowering, improved adventitious root formation, and/or increased vigour.

Isolation of somaclonal variants

Mutants for several traits can be far more easily isolated from cell cultures than from whole

plant populations. This is because a large number of cells, say 10 [6] -10 [9], can be easily and

effectively screened for mutant traits. Screening of as many plants would be very difficult,

ordinarily impossible. Mutants can be effectively selected for disease resistance,

improvement of nutritional quality, adaptation of plants to stress conditions, e.g., saline

soils, low temperature, toxic metals (e.g., aluminium), resistance to herbicides and to

increase the biosynthesis of plant products used for medicinal or industrial purposes. The

various approaches to the isolation of somaclonal variants can be grouped into two broad

categories: (i) screening and (ii) cell selection.

Screening

It involves the observation of a large number of cells or regenerated plants for the

detection of variant individuals. This approach is the only feasible technique for the

isolation of mutants for yield and yield traits. In general, R1 progeny (progeny of

regenerated, Ro, plants) are scored for the identification of variant plants, and their R2

progeny lines are evaluated for confirmation. Screening has been profitably and widely

employed for the isolation of cell clones that produce higher quantities of certain

biochemicals. Computer based automated cell sorting devices have also been used to

screen as many as 1000-2000 cells/second from which desirable variant cells were

automatically separated.

Cell selection

In the cell selection approach, a suitable selection pressure is applied which permits the

preferential survival/growth of variant cells only. Some examples of cell selection are,

selection of cells resistant to various toxins, herbicides, high salt concentration etc. When

the selection pressure allows only the mutant cells to survive or divide, it is called positive

selection. On the other hand, in the case of negative selection, the wild type cells divide

normally and therefore are killed by a counter selection agent, e.g., 5 BUdR or arsenate.

The mutant cells are unable to divide as a result of which they escape the counter

selection agent. These cells are subsequently rescued by removal of the counter selection

agent. Negative selection approach is utilized for the isolation of auxotrophic mutants.

The positive selection approach may be further subdivided into four categories: (i) direct

selection, (ii) rescue method, (iii) stepwise selection and (iv) double selection.

In direct selection, the cells resistant to the selection pressure survive and divide to form

colonies; the wild type cells are killed by the selection agent. This is the most common

selection method. It is used for the isolation of cells resistant to toxins (produced by

pathogens), herbicides, elevated salt concentration, antibiotics, amino acid analogues etc.

In the rescue method, the wild type cells are killed by the selection agent, while the variant

cells remain alive but, usually, do not divide due to the unfavourable environment. The

selection agent is then removed to recover the variant cells. This approach has been used

to recover low temperature and aluminium resistant variant cells.

The selection pressure, e.g., salt concentration, may be gradually increased from a

relatively low level to the cytotoxic level. The resistant clones isolated at each stage are

subjected to the higher selection pressure. Such a selection approach is called stepwise

selection. It may often favour gene amplification (which is unstable) or mutations in the

organelle DNA.

In some cases, it may be feasible to select for survival and/or growth on one hand and

some other feature reflecting resistance to the selection pressure on the other; this is

called double selection. An example of double selection is provided by the selection for

resistance to the antibiotic streptomycin, which inhibits chlorophyll development in

cultured cells. The selection was based on cell survival and colony formation in the

presence of streptomycin (one feature) as well as for the development of green colour in

these colonies (second feature; only green colonies were selected). This approach has

been used for the selection of cells resistant to the herbicide amitrole, 2, 4-D, tobacco

mosaic virus (TMV) and aluminium.

Selection of somaclonal variants on subjecting the cells to selection pressure

Selection Selection of cells in the presence of
Resistance to herbicide Herbicide
Resistance to salt Sodium chloride / Aluminium
Resistance to drought PEG / Mannitol
Resistance to frost Hydroxy proline resistant lines
Resistance to pathogens Pathotoxin / Culture filtrate

Crop improvement through somaclonal variation for desirable characters

Crop Characters modified
Sugarcane Diseases (eye spot, fiji virus, downy miledew, leaf scald)
Potato Tuber shape, maturity date, plant morphology, photperiod, leaf
colour, vigour, height, skin colour, Resistance to early and late blight
Rice Plant height, heading date, seed fertility, grain number and weight
Wheat Plant and ear morphology, awns, grain weight and yield, gliadin
proteins,amylase
Maize T toxin resistance, male fertility, mt DNA
Medicago sativa Multifoliate leaves, elongated petioles, growth, branch, no.of plants,
dry matter yield.
Tomato Leaf morphology, branching habit, fruit colour, pedicel, male fertility,
growth
Avena sativa Plant height, heading date, awns
Hordeum spp Plant height and tillering
Lolium hybrids Leaf size, flower, vigour, survival

Characterization of variants

Somaclonal variants isolated through cell selection are often unstable. The frequency of

stable variants may range from 8-62%, perhaps depending on the species and the

selection agent. Many selected clones fail to exhibit their resistance during further

screening or selection. Obviously these clones are susceptible and were misclassified as

resistant, called as escapes . Several clones lose their resistance to the selection agent

after a period of growth in the absence of selection pressure. Such clones are called

unstable variants and may result from changes in gene expression and from gene

amplification (increase in the number of copies of a gene per genome of the organism in

comparison to that naturally present). Some variant phenotypes are quite stable during

the cell culture phase, but they disappear when plants are regenerated from the variant

cultures, or when the regenerated plants reproduce sexually, in case they are expressed

in the regenerated plants. Such changes are known as epigenetic changes and are

attributed to stable changes in gene expression e.g., hormone habituation of cell cultures

and, possibly, cold resistance in Nicotiana sylvestris .

The remaining variants which stably express the variant phenotypes during the cell culture

as well the regenerated plant phases, and exhibit the transmission of these phenotypes

through the sexual reproduction cycle are called mutants. Only this category of variants

would find an application in crop improvement. These may represent true gene mutations

or some other types of changes. Usually, expected mendelian ratios are obtained in the Rl

progenies. But sometimes aberrant segregation ratios are encountered in Rl possibly due

to the chimeric nature of Ro plants, the involvement of some cytological anomalies like

aneuploidy, deletions etc., gene dosage effects etc.

Achievements

Over a dozen varieties have been developed through the exploitation of somaclonal

variation. ‘Ono’ variety of sugarcane is a Fiji disease resistant somaclone of the

susceptible cultivar ‘Pindar’. It was identified by screening of plants regenerated from

unselected calli. ‘Ono’ also shows yield advantage over ‘Pindar’ and has been cultivated to

a limited extent in Fiji. A sweet potato cultivar ‘Scarlet’ was selected from

shoot-tipculture-derived clones. ‘Scarlet’ is comparable to the parent cultivar in yield and

disease resistance, but shows darker and more stable skin colour, which is a desirable

quality trait. A geranium variety called ‘Velvet Rose’ is a somaclone of ‘Rober’s Lemon

Rose’. The new variety has twice the chromosome number of the parent variety. An alfalfa

variety called ‘Sigma’ is a polycross of selected somaclones.

In India, so far somaclonal variation is the only biotechnological approach to give a

commercial variety. A somaclonal variant of Citronella java, a medicinal plant, has been

released as ‘Bio-13’ for commercial cultivation by CIMAP (Central Institute for Medicinal

and Aromatic Plants), Lucknow. Bio-13 yields 37% more oil and 39% more citronellol than

the control varieties. A somaclonal variant of the B. juncea variety ‘Varuna’ has been

released for commercial cultivation as ‘Pusa Jai Kisan’. The new variety has bolder seeds

and some yield advantage over the parent variety Varuna.

Advantages

  • Somaclonal variations occur in rather high frequencies, which is a great advantage

over conventional mutagenesis.

  • Some ‘new’ alleles or even ‘new’ mutations may be isolated which were not available in

the germplasm or through mutagenesis, e.g., joint less pedicel mutant in tomato.

  • Use of somaclonal variation may reduce by two years the time required for the release

of new variety as compared to mutation breeding. This is because somaclonal

variations are usually free from undesirable features like sterility, while induced

mutations are generally associated with such defects, which necessitate one or two

backcrosses with the parent variety.

  • A very effective selection can be practised at the cell level for several traits, e.g.,

disease resistance etc. This approach effectively selects few desirable cells from

among millions with relatively small effort, time, cost and space requirements.

  • This is the only approach for the isolation of biochemical mutants, especially

auxotrophic mutants, in plants.

Limitations

  • The technique is applicable only to those species of cell cultures which regenerate

complete plants.

  • Selected cell lines often show reduced or no regeneration potential.

  • Many selected clones show undesirable features like reduced fertility, growth and even

overall performance.

Somaclonal variation represents a useful source of introducing genetic variations that

could be of value to plant breeders. Single gene mutation in the nuclear or organelle

genome may give the best available variety in vitro that has a specific improved character.

In this manner, somaclonal variation could be used to uncover new variants retaining all

the favourable characters along with an additional useful trait, such as resistance to

diseases or a herbicide. Various cell lines selected in vitro may then prove potentially

applicable to agriculture and industry.

Questions

  1. The term somaclone was proposed by …………

a). Larkin and Scowcroft b). Skoog c). Murashige d). None of the above

  1. Somaclonal variation includes …………

a). Aneuploids b). Sterile plants c). Morphological variants d). All the above

  1. The usefulness of somaclonal variation was first demonstrated through the

recovery of disease resistant plants in …………

a). Potato b). Sugarcane c). Both a and b d). None of the above

  1. Epigenetic variation includes …………

a). Non-heritable phenotypic variation b). Temporary c). Ultimately reversible d). All the above

  1. Ploidy changes occur due to ……. during mitosis

a). Endomitosis b). Endoreduplication c). Spindle fusion d). All the above

  1. Gross structural rearrangements viz., …………. are major cause of somaclonal

variation

a). Deletions and duplications b). Inversions c). Translocations d). All the above

  1. Ono is a somaclonal variety of ……….

a). Sugarcane b). Geranium c). Alfa alfa d). Citronella

  1. Scarlet is a somaclonal variety of ……….

a). Sugarcane b). Sweet potato c). Alfa alfa d). Citronella

  1. Velvet rose is a somaclonal variety of ……….

a). Sugarcane b). Geranium c). Alfa alfa d). Citronella

  1. Sigma is a somaclonal variety of ……….

a). Sugarcane b). Geranium c). Alfa alfa d). Citronella

  1. Bio 13 is a somaclonal variety of ……….

a). Sugarcane b). Geranium c). Brassica juncea d). Citronella

  1. Varuna is a somaclonal variety of ……….

a). Sugarcane b). Geranium c). Brassica juncea d). Citronella

  1. Limitation(s) of somaclonal variation is/are ……….

a). Applicable only to those species which regenerate complete plants out of cell cultures b). Reduced or no regeneration potential c). Undesirable features like reduced fertility, growth and even overall performance d). All the above

Summary Cheat Sheet

Quick Recall Points

  • Define key terms in one line and revise their use in plant biotechnology.
  • Memorize major steps, methods, and applications covered in this lesson.
  • Practice exam-style distinctions between related concepts and techniques.

Exam Traps

  • Do not confuse similar terms without checking context and biological level.
  • Revise process order carefully; sequence-based questions are common.
  • Link each method with its most likely application question.

References

1 source • [1]

[1]

Standard BSc Agriculture Plant Biotechnology notes

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