GERF Bulletin of BiosciencesJune 2015, 6(1):5-10

Abstract

Introduction

*Corresponding author: jyotigenpro@gmail.com

Key words: Somaclonal variation, crop improvement, plant tissue culture, sugarcane, conventional breeding

© Malhotra et al., 2015; licensee Green Earth Research Foundation. This article distributed under terms of Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/4.0). Which permits unrestricted use, distribution, and reproduction in any medium, providedthe original author and source are credited.

Review Article Open Access

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ISSN: 2229-6433

Received: 31 December 2014 Accepted: 18 June 2015 Published online: 27 July 2015

Somaclonal Variation: A new dimension for sugarcane improvement

Jyoti Rastogi*1, Siddhant1, Parvesh Bubber2 and Brij Lal Sharma1

1Center for Sugarcane Biotechnology,Sugarcane Research Institute (UPCSR), Shahjahanpur- 242001, India

2Biochemistry Discipline, School of Sciences, IGNOU, New Delhi-110068, India

Plant tissue culture or micropropagation technique is the rapid method to multiply newly released cultivar in limitedtime. Crop improvement by conventional method in vegetatively propagated crops like sugarcane is very difficult due toits narrow genetic base and other limitations. Somaclonal variations are easily achieved in asexually propagated cropslike sugarcane and banana. Tissue culture derived variations are known as somaclonal variation. These variations playan important role in crop improvement program. Genetic variations are heritable in next generation and important forcrop improvement, epigenetic changes are temporary ultimately reversible. Mutation breeding is also very advantageousfor improving a cultivar. Somaclonal variants of sugarcane are available for several traits like drought, salt tolerance, redrot, eye spot disease, quality and quantity trait. Molecular marker techniques like RFLP, RAPD, AFLP and SSR etc. areregularly used preferentially over traditional phenotypic or cytological methods.

Sugarcane (Saccharum species complex), being the mostvaluable commercial crop of the world. It is the major sourceof sugar in the world and recently to produce ethanol, ahigh energy rich biofuel. The crop improvement in manycrops like sugarcane is very difficult and time takingbecause it has a complex poly-aneuploid, large genome sizeand long breeding cycle. With the advancement ofbiotechnological tools for the genetic improvement of manyeconomically important crops like have been highlightingby different researchers in past years. In the past decade

considerable progress has been made in understanding andmanipulating the sugarcane genome using various biotech-nological and cell biological approaches. Notable amongthem are the crop improvement through somaclonalvariation, creation of transgenic plants with improvedagronomic or other important traits, advances in genomicsand molecular markers, and progress in understanding themolecular aspects of sucrose transport and accumulation. Itis anticipated that the rapid advancements in emergingbiotechnology innovations would play a significant role inthe future sugarcane crop improvement programs and offermany new opportunities to develop it as a new generationindustrial crop. Plant tissue culture gave an excellentopportunity for the creation of genetic variability of clones

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with desired character in sugarcane (1,2,3). Somaclonalvariations are important tool to overcome the constrains ofconventional breeding (4). The accumulation of geneticvariability is an important aspect in plant breeding. Geneticvariability has been easily discernible in in vitro regener-ated somaclonal variants.

In vitro regenerated variants are known as Somaclonalvariation or sub-clonal variation (5,6,7) mainly depends onvarious modes of plant regeneration . Somaclonal variantshave been available in many horticultural traits, ornamentalplants and recently the selection of new variants of manycrops like sugarcane for disease resistance (8,9,10) andvarious agronomic characters like yield and quality (4). Theoccurrence of phenotypic instability is a major problem, whenour objective to produce the true copies of original plant(11). The occurrence of somaclonal variation in tissue cultureculture derived plants is an alternative method to sort outmany barriers of traditional breeding program. Sugarcane isa good candidate for such type of studies due to its complexpolyploidy. The use of variation was first established throughthe recovery of disease resistant plants in potato (resistanceagainst late blight and early blight) and sugarcane (resistanceagainst eye-spot disease, Fiji disease and downy mildew).

Somaclonal variation may be of two types 1- geneticvariations (may be occurred in somatic cells of the motherplant), 2- epigenetic (may be occurred during in vitro tissueculture practices).

Types of variations

Genetic variation

The variation occurs due to mutations or other changesin the DNA of the tissue are known as genetic variation.These are heritable in next generation and important for cropimprovement. Genetic changes behave as Mendelian traitsin crosses.

Epigenetic variation

Epigenetic changes are temporary ultimately reversible(plants ‘revert’ to normal phenotype) also known asdevelopmental variation. These are non-heritable phenotypicvariation. There are several factors like explants source, ageof the donar plant, genotype, number of sub-culturepassaging, in vitro culture environment, concentration ofplant growth regulators, medium composition etc are thecomponents that might be induce variability in vitro(12).Tissue culture system itself acts as a mutagenic systembecause cells experience traumatic experiences from isolation

and may re-programme during plant regeneration.Reprogramming or restructuring of events can create a widerange of epigenetic variation in newly regenerated plants(13).

Physiological variation

These variations are temporary in response to stimulusand disappear when it is removed.

Need of improved varieties and limitations ofconventional approaches

Plant breeders have exploited the germplasm resource todevelop new cultivars with desirable traits viz., high yield,diseases resistance and tolerance to many abiotic stresses(14). However enormity of growing world population, crisisof arable land and continual demand for newer improvedcultivars by using available natural and induced geneticdiversity. Conventional breeding takes approx 8-10 years todevelop and commercialize a new selected sugarcane cultivar(15, 9). Another major problem of sugarcane growers its slowmultiplication rate cannot fulfill the need of colossal demandof newly released cultivar. In vitro multiplication and mutationbreeding is an alternative method to broaden the area ofnovel cultivar. Due to limited availability of seed cane of anew variety at the time of its release, it further takes severalyears to cover the desired area for commercial cultivation,by the time the variety starts deteriorating due to biotic andabiotic stresses. Tissue culture derived variations could bevery useful in sugarcane crop improvement program for thedevelopment of new trait.

Potential role of somaclonal variation

Somaclonal variants of various agriculturally importanttraits have been studied also in sugarcane. The first in vitroscreened somaclone of commercial sugarcane for resistantto Fiji disease reported by Heinz, 1973 (16). Somaclonalvariations are highly useful in plant breeding program.Somaclonal variations are easily achieved in vegetativelypropagated crops like sugarcane. The potential role of naturalor induced genetic variation is a very essential componentof crop improvement program. Induced mutations have beenused in the improvement of major crops such as sugarcanewheat, rice, barley, cotton, peanuts and beans, which areseed propagated. Mutation breeding is very advantageousfor the vegetatively propagated plants like sugarcane, bywhich a single or few characters of an excellent cultivar canbe change. The crop improvement through somaclonalvariation was first reported by Heinz and Mee 1971 (17).

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Table1: Crop improvement through somaclonal variation in many crops included sugarcane

Crop Trait

Sugarcane Diseases (Eye spot, Red rot, Fiji virus, Downy miledew, Leaf scald resistant), drought & salt tolerance, improvement of qualitative and quantitative traits viz., sugar content, sugar recovery and cane yield.

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

Potato Tuber shape, maturity date, plant morphology, photoperiod, leaf colour, vigour, height, skin colour, Resistance to early and late blight

Tomato Leaf morphology, branching habit, fruit colour, pedicel, male fertility and growth

Studies on Somaclonal variation in sugarcane

Somaclonal variations has been usually reported by manyresearchers in tissue culture raised plants of sugarcane (18,19). Variations in morphology, chromosome number andenzymatic pattern in sugarcane plants derived from callushave been reported (17, 20, 21). These make up a basicconstraint for a breeding program because the more variablethe genetic resources used, the more opportunities to selectsuperior genotype. In sugarcane somaclonal variation havebeen exloited for the improvement of many economicallyimportant traits like salt tolerant, eye spot and red rotresistant. Patade et al (22) studied the effects of salt anddrought stresses on irradiated cells of sugarcane andobtained plants tolerant to higher salt stress. Gandonou etal (23) deliberate the effects of salt stress by exposing thecallus to a single level of 68 mM NaCl, and observed thatphysiological and biochemical indicators could play a crucialrole in salt tolerance. Salt (NaCl) tolerent sugarcane cultivarCP65-357 developed from callus culture (24). Wagih et al(25) developed eight drought tolerant variants fromembryogenic callus of sugarcane (Saccharum hybrids) andgrown in a greenhouse for further testing under water stress.They found improved tolerance to drought in amongst thesomaclonal variants for different areas of tropics and sub-tropics. Four salt tolerant somaclonal variants weredeveloped from embryogenic calli of sugarcane varietyCP48-103 (26). Clonal variation in combination with in vitromutagenesis and selection has been applied for the isolation

Detection of Somaclonal variants

Somaclonal variations can be detected easily bymorphological characteristics, such as cane height, leafmorphology, bud shape, number of milable cane, sugar

concentration etc. (31). Chromosomal abbreviation and ploidychanges are highlighted by cytogenetic analysis, includingchromosome counting under microscope / flow cytometry.Proteins and isozymes also have been used as markers forrecognizing somaclonal variants in many fruit species butthey are limited in their sensitivity. Cytological evaluation isnot often used and can be complicated to detect in numerouscrops.

With the advancement of molecular marker techniqueslike RFLP, RAPD, AFLP and SSR etc. are regularly usedpreferentially over traditional phenotypic or cytologicalmethods. Restriction Fragment Length Polymorphism (41)analysis is one of the first techniques widely used to detectvariation. Later, PCR-based techniques, Random AmplifiedPolymorphic DNA (RAPD), Arbitrarily Primed PolymeraseChain Reaction (AP-PCR), Amplified Fragment LengthPolymorphism (AFLP), Simple sequence repeat (SSR) aremost extensively used molecular techniques throughout theworld. Among these PCR based techniques, the RAPD isfaster, inexpensive, simple, less time consuming, most reliableand frequently used to detect genetic variability at DNAlevel (11, 42) in sugarcane and other crops. As in typicalPCR, where a pair of reverse and forward primer is used, inRAPD only single primer amplify the unknown site in thetarget genome.

Oropeza et al (43) identified somaclonal variants ofsugarcane resistant to sugarcane mosaic virus throughRAPD marker. The somaclones AT 626 and BT 627 wereselected by their resistant to SCMV and analyzed by RAPD.Genetic changes have been detected with the help of RAPDmarker in sugarcane during tissue culture. Simple SequenceRepeat (ISSR) technique is a new technique based on theamplification of regions between microsatellites. It is usingto check genetic instability at early stages in in vitro.

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Advantages

Somaclonal variations occur in high frequencies. Some changes can be novel and may not be

achieved by conventional breeding. In vitro screening reduces the time to isolation a

somaclone with desirable trait. Sometimes new desirable characters may be

occurred which were not available in the germplasm. It is a cheaper than other genetic engineering

methods

Limitations

These variations are not stable after selfing orcrossing.

The variations are unpredictable in nature anduncontrollable.

Selected cell lines often reduced their regenerationpotential.

Many selected clones show undesirable featureslike reduced fertility, growth and even overallperformance.

Strategies to overcome the constraints

1. The breeding objective should be simple andimprove one character at a time. If we required morethan one trait, stepwise improvement must bepossible.

2. An easy and efficient screening technique shouldbe needed to select a desired trait in somaclonalvariants.

3. Molecular markers and in vitro selection techniquesfor various diseases are very helpful in theidentification of valuable variants.

4. A comparative study of plants produced throughsomaclonal variation and conventionallypropagated should be tested in field trials beforecultivation.

Conclusion

With a continued cultivation of a sugarcane variety forover 15-20 years, tremendous deterioration occurs in varietyleading in significant losses in cane yield and sugar recovery.Loss of resistance against major diseases and pests in oldelite varieties are the main reasons of deterioration. If the oldexisting varieties are improved using biotechnological toolssuch as somaclonal variation, the cane and sugarproductivity will be increase significantly. It is more efficientand feasible technology for wider adoption in the field ofsugarcane improvement through biotechnological tools.

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