See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6171548 Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: A comparative phenotypic, cy Article in Planta · December 2007 DOI: 10.1007/s00425-007-0583-2 · Source: PubMed CITATIONS READS 46 490 4 authors: Sanjeev Sharma Glenn Bryan James Hutton Institute James Hutton Institute 36 PUBLICATIONS 1,099 CITATIONS 200 PUBLICATIONS 5,263 CITATIONS SEE PROFILE SEE PROFILE Mark Owen Winfield Steve Millam University of Bristol 52 PUBLICATIONS 1,042 CITATIONS 43 PUBLICATIONS 2,142 CITATIONS SEE PROFILE SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately Available from: Sanjeev Sharma Retrieved on: 27 October 2016 Planta (2007) 226:1449–1458 DOI 10.1007/s00425-007-0583-2 O R I G I N A L A R T I CL E Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment Sanjeev Kumar Sharma · Glenn J Bryan · Mark O WinWeld · Steve Millam Received: 22 January 2007 / Accepted: 26 June 2007 / Published online: August 2007 © Springer-Verlag 2007 Abstract The stability, both genetic and phenotypic, of potato (Solanum tuberosum L.) cultivar Desiree plants derived from alternative propagation methodologies has been compared Plants obtained through three clonal propagation routes—axillary-bud-proliferation, microtuberisation and a novel somatic embryogenesis system, and through true potato seeds (TPS) produced by selWng were evaluated at three levels: gross phenotype and minituber yield, changes in ploidy (measured by Xow cytometry) and by molecular marker analysis [measured using AFLP (ampliWed fragment length polymorphism)] The clonally propagated plants exhibited no phenotypic variation while the TPS-derived plants showed obvious phenotypic segregation SigniWcant diVerences were observed with respect to minituber yield while average plant height, at the time of harvesting, was not signiWcantly diVerent among plants propagated through four diVerent routes None of the plant types varied with respect to gross genome constitution as assessed by Xow cytometry However, a very low level of AFLP marker proWle variation was seen amongst the somatic embryo (3 out of 451 bands) and microtuber (2 out of 451 bands) derived plants Intriguingly, only AFLP markers generated using methylation sensitive restriction enzymes were found to show polymorphism No polymorphism was observed in plants regenerated through axillary-bud-proliferation The low level of molecular variation observed could be signiWcant on a genome-wide scale, and is discussed in the context of possible methylation changes occurring during the process of somatic embryogenesis Keywords AFLP · Clonal propagation · Methylation · Polymorphism · Potato (Solanum tuberosum L.) · Somatic embryogenesis · Stability · Synthetic seeds Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid ABP Axillary bud shoot plants AFLP AmpliWed fragment length polymorphism DAPI 4Ј-6-Diamidino-2-phenylindole EMB Embling(s) ISSR Inter simple sequence repeat MS Murashige and Skoog (1962) MTP Microtuber plants NAA Naphthalene acetic acid TPS True potato seedling(s) RAPD Random ampliWed polymorphic DNA RFLP Restriction fragment length polymorphism Introduction S K Sharma · G J Bryan (&) · M O WinWeld · S Millam Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK e-mail: glenn.bryan@scri.ac.uk Present Address: S Millam Institute of Molecular Plant Sciences, Daniel Rutherford Building, The University of Edinburgh, The Kings Buildings, MayWeld Road, Edinburgh EH9 3JH, Scotland, UK Potato (Solanum tuberosum L.), the most important noncereal food crop in the world, is a polyploid outbreeder which maintains a high degree of heterozygosity Consequently, tetraploid commercial cultivars are propagated vegetatively in order to maintain phenotypic characteristics and economically important traits Generally, propagation is via tubers from the previous crop, but alternative clonal 123 1450 propagation methods have been developed to accommodate emerging economic production requirements Tissue culture techniques, mainly micropropagation, have substantially augmented the supply of planting material, and reduced the time required for the release of new cultivars from more than 10 to 3–5 years (Struik and Wiersema 1999) Clonal in vitro propagation methods not only maintain the uniformity among oVspring, but also preserve health status as the planting material has reduced exposure to soil-borne and other diseases Micropropagation techniques in potato are mainly employed to allow rapid mass propagation of nuclear stock material, usually in the form of microtubers and axillary bud shoot plants Minitubers produced in initial cycles of multiplication from the in vitro material increase the number of propagules in the Wrst year, and bulks up the prebasic seed required for large-scale production of basic seed tuber material, which initiates the seed production programme The multiplication rate in microtuber and axillary bud based micropropagation systems depends upon the number of pre-formed meristems present in explants during each multiplication cycle; inherently, these numbers are low rendering such systems comparatively resource ineYcient Somatic embryogenesis, however, oVers the possibility of a novel system for the large-scale multiplication of plant material and could be more economical because of the greater multiplication rates, exploiting the potential of every cell to regenerate into a complete plant However, in potato, somatic embryogenesis requires further research before it can become a feasible option (Naik et al 2000) Apart from the developmental aspects of somatic embryogenesis, successful Weld testing of established plants is crucial for the success of the system In addition to evaluating the survival potential and growth performance of plants derived from somatic embryogenesis, it is vital to monitor their uniformity The variation observed from vegetative propagation, either genetic or epigenetic in origin, is commonly referred to as somaclonal variation (Larkin and Scowcroft 1981) It is of fundamental importance that micropropagated plants, irrespective of their development through either organogenesis or embryogenesis, remain true-to-parental type Over the last two decades, the phenomenon of somaclonal variation has been extensively investigated using cytogenetic, biochemical and molecular methods (e.g Karp 1995; Vazquez 2001) Though the underlying mechanisms that generate somaclonal variation remain unclear, major contributory factors are thought to be disorganised meristematic growth, the genetic make-up (ploidy and genotype) of the stock material, the type and concentration of plant growth regulators in the culture medium and explant source (Karp 1991) At the molecular level, it has been postulated that the tissue culture-induced stress that cells undergo in the presence of plant growth 123 Planta (2007) 226:1449–1458 regulators could induce alteration(s) in sensitive regions of the plant genome (Sala et al 1999), which can be attributed to DNA methylation, ampliWcation or the activation of transposable elements (Brar and Jain 1998) In some instances, somaclonal variation has been exploited to obtain variant genotypes with new and desirable characteristics, such as for salt tolerance in potato (Tal 1996; Ochatt et al 1998) However, somaclonal variation is highly undesirable where clonal propagation is the goal (Cassells and Curry 2001) Until recently, measures of whether plants were true to parental type were rather coarse, being based on phenotypic observations and/or cytogenetic characterisation Recent developments in molecular genetic techniques have made it possible to ‘proWle’ the genome looking, directly or indirectly, for changes in DNA sequence Thus, in principle, single base changes and even methylation events can be observed and interpreted as instances of tissue-culture-induced somaclonal variation However, at its extreme limit, such an approach would involve sequencing the entire genome, an infeasible task Highly multiplex molecular marker techniques [e.g ampliWed fragment length polymorphism (AFLP)] are potential tools for the assessment of uniformity, notwithstanding the inherent limitations of any genome proWling methodology Somatic embryogenesis in potato oVers a potentially novel method for producing nuclear seed material as compared to the existing two other forms of micropropagation viz by production of microtubers and axillary bud shoot plants The year round production and utilisation of microtubers is hampered by their dormancy and constraints related to physiological age Axillary bud shoot plants, though without the constraints that are encountered with microtubers, are fragile and production, in common with microtubers, is labour intensive and requires large amounts of laboratory space In contrast, somatic embryos are free from dormancy and constraints related to physiological age, and require signiWcantly less manual labour and laboratory space Thus, encapsulated somatic embryos (synthetic seeds) hold the potential to overcome the disadvantages of conventional micropropagation systems, and combine the advantages of clonal and seed propagation systems However, in order to conWrm whether or not somatic embryogenesis is a reliable clonal method of propagation in potato, a Wdelity and yield assessment of plants regenerated through emblings is required Somatic embryogenesis has been previously reported in potato (de Garcia and Martinez 1995; Seabrook and Douglass 2001; JayaSree et al 2001; Vargas et al 2005; Sharma and Millam 2004), but post-embryogenesis events, including the assessment of uniformity, have not been studied in detail In this investigation, potato cv Desiree plants Planta (2007) 226:1449–1458 1451 obtained from four propagation methods—somatic embryogenesis, axillary bud proliferation, microtubers and true potato seeds (TPS) obtained by selWng—were tested for stability and uniformity using phenotypic, cytogenetic and molecular approaches Materials and methods Plant material In vitro cultures of S tuberosum L cultivar Desiree were obtained from the Scottish Agricultural Science Agency, Edinburgh, Scotland All of the cultures, unless otherwise stated, were maintained on basal MS (Murashige and Skoog 1962) medium and incubated under controlled environmental conditions of 19 § 1°C, 16/8 h light/dark cycles and 90 mol m¡2 s¡1 photon Xux density (400–700 nm) Development of potato somatic embryos was as described previously (Sharma and Millam 2004) For experimental plant material, 3-week-old emblings (embling—a synonym to seedling for a plant obtained via a somatic embryo) were obtained from encapsulated somatic embryos (Sharma 2006), axillary bud shoot plants were obtained from 3-week-old in vitro rooted single-node-cuttings multiplied by axillary bud proliferation, microtuber plants were produced by sprouting microtubers and seedling plants were obtained by germinating true potato seeds obtained by selWng cv Desiree plants TPS were highly uniform in their appearance in contrast to their obvious heterozygous genetic make-up For propagules following vegetative modes of propagation, care was taken to keep their size range (within their respective categories) as uniform as possible For emblings, only somatic embryos measuring between and mm were used for encapsulation, axillary bud shoot plants were generated using single-node-cuttings between 1.5 and 2.0 cm in length and microtuber plants were obtained using microtubers having 7–9 mm diameter Once all the plants were established for transplantation, no screening for any noticeable variation was performed in order to achieve a non-biased progression of plant growth and development Figure shows the four comparative categories of propagules used in the current study Plants obtained through the four propagation routes were established under glasshouse conditions in a compost mix of 24:2:1 (by weight) peat, sand and perlite supplemented with fertiliser and Celcote water retaining gel The propagules (75 plants) for each category were grown in randomised blocks Plants were inspected for any noticeable variation after 30, 60 and 90 days of transplantation Plant height measurements were taken at the time of haulm (foliage) cutting (110 days after transplantation) Progeny Fig A depiction of four comparative potato planting propagule types a Synthetic seeds (encapsulated somatic embryos) b Axillary bud shoot plants c Microtubers d True potato seeds Bar = mm (a, c), 10 mm (b, d) tubers (minitubers) were left in the pots for two more weeks after haulm cutting, following which the tubers were harvested and used for biometric analysis Cytogenetic analysis Representative leaf samples (obtained from apical buds only) for all four-plant propagation categories (15 plants each) were collected from their respective 45-day-old plants Small pieces of leaf material (»40 mg) were 123 1452 Planta (2007) 226:1449–1458 macerated, using a sharp razor blade, in ml of an ice-cold DAPI (4Ј-6-diamidino-2-phenylindole) based nuclear extraction buVer (Arumuganathan and Earle 1991) Following this, the buVer containing cell constituents and large tissue remnants was passed through 40 m nylon Wlters and, after 15 of incubation, the solution containing stained nuclei was analysed in a Xowcytometer (CyFlow ML, Partec, Germany) Together with each leaf sample an internal standard (»10 mg of Ilex crenata ‘Fastigiata’ leaf tissue, nuclear DNA content = 2.16 pg/ 2°C) was also included and its G0/G1 peak (2°C) was adjusted to around channel 215 set on a linear scale of Xuorescence intensity (FL2-DAPI) The axillary bud shoot plants were used as tetraploid (2n = 4x) controls for ploidy comparisons Histograms were constructed using Flomax version 2.4 d (Partec) software For each plant, DNA-ratios were obtained by dividing the mean of the dominant (G0/G1) peak of the potato sample by the mean of the G0/G1 peak of the internal standard and ANOVA was performed DNA extraction Genomic DNA from representative leaf samples (from every 1st node) of 45-day-old plants from all four-plant groups was extracted, according to manufacturer’s instructions, using DNeasy Plant Mini Kits (Qiagen, Hilden, Germany) DNA concentration of each plant sample was quantiWed using a Nanodrop® Microphotometer (Nanodrop Technologies, Wilmington, Delaware, USA) by measuring its absorbance at 260 nm Table Details of AFLP primer combinations used and the corresponding numbers of bands observed EMB, emblings; ABP, axillary bud shoot plants; MTP, microtuber plants; TPS, true potato seedlings Primer combination AFLP analysis AmpliWed fragment length polymorphism (AFLP) assays were performed using a modiWcation of the protocol of Vos et al (1995), as described in Bryan et al (2002) The 6-bp cutting restriction enzymes PstI and EcoRI were used in combination with the 4-bp cutting restriction enzyme MseI Table contains primer sequences used For separation of labelled fragments, a 3.5 l aliquot of each ampliWcation product was electrophoresed in 5% denaturing acrylamide gels which, after drying, were exposed to X-ray Wlm (Biomax MS, Kodak) Exposed X-ray Wlms were developed through an automatic X-ray Wlm processor (XO Graph Imaging Systems, Compact X4) and used for AFLP analysis Experimental design and statistical analysis Data storage and calculations were done using the Genstat statistical package (Payne et al 1993) The data were analysed by analysis of variance (ANOVA) and the individual group means were ranked by comparing their mean diVerences against LSD (least signiWcant diVerence) AFLP was performed using representative plants from emblings (EMB, 15 plants), axillary bud shoot plants (ABP, 15 plants), microtuber plants (MTP, plants) and true potato seedlings (TPS, or 15 plants) In order to facilitate the intra-group comparisons of plants and keep track of individual plants, the order of plant samples in their respective groups was kept constant across all gels irrespective of the relative position of diVerent plant groups, with respect to Primer sequence (5Ј to 3Ј) Total number of bandsa Number of polymorphic bands EMB ABP MTP TPS 67 1 12 66 0 16 94 19 82 0 22 76 0 23 PstI/MseI combination P12 GACTGCGTACATGCAG AC M38 GATGAGTCCTGAGTAA ACT P13 GACTGCGTACATGCAG AG M36 GATGAGTCCTGAGTAA ACC P14 GACTGCGTACATGCAG AT M32 GATGAGTCCTGAGTAA AAC P15 GACTGCGTACATGCAG CA M41 GATGAGTCCTGAGTAA AGG EcoRI/MseI combination a Size range ¡150 to 700 bp 123 E32 GACTGCGTACCAATTC AAC M51 GATGAGTCCTGAGTAA CCA E35 GACTGCGTACCAATTC ACA M48 GATGAGTCCTGAGTAA CAC Total 66 0 21 451 113 Planta (2007) 226:1449–1458 1453 each other Variation between samples was observed as the presence of polymorphic bands Results Phenotypic analysis and assessment of minituber yield No morphological diVerences were observed among clonally propagated plants (plants derived from axillary bud shoot plants, microtuber plants and emblings) and all tubers showed normal morphology (Fig 2a–c) Initially, embling derived plants showed uneven vegetative growth compared to plants derived from axillary bud shoot plants and microtuber plants However, over a period of 30 days, these diVerences in growth pattern among the emblings disappeared The seedling-derived plants were variable in growth habit and, additionally, showed delayed Xowering (Fig 2d) and maturity compared to the other plant types (Fig 2a–c) No signiWcant diVerences (P = 0.492) in average plant height, taken at the time of harvesting (110 days after transplantation), were observed between any of the Fig Overview showing fully grown plant populations, inXorescence and minitubers produced from potato cv Desiree plants derived from somatic embryogenesis (a), axillary bud proliferation (b), four-plant groups (Table 2) Nevertheless, as compared to seedling-derived plants, the clonally propagated plants were more uniform and displayed less variation both within and among individual plant groups As expected, seedlingderived plants were variable in height and ranged from much shorter (88 cm) to taller (153 cm) plants with an average height of 127.3 (SE 5.1) cm at the time of haulm destruction (110 days) The minitubers were harvested weeks after the cutting of the haulm and data on tuber number and weight were collected The mean tuber number per plant was signiWcantly diVerent (P < 0.001) among all plant types except microtuber plants versus seedling-derived plants The axillary bud shoot plants yielded the highest average number of tubers per plant followed by embling-derived plants while the average per plant tuber number of microtuber plants and seedling plants was signiWcantly lower (Table 2) While average tuber weight was signiWcantly higher (P < 0.003) in microtuber plants and embling-derived plants as compared to axillary bud shoot plants and seedling plants, the tuber yield per plant was signiWcantly higher (P < 0.001) in axillary bud shoot plants and embling-derived plants as microtubers (c), and Desiree-selfed seedlings (d) Bar = cm (for minitubers only) Table Biometric analysis of potato plants propagated through four diVerent propagation routes Plants obtained from Mean plant height (cm) Mean tuber number Mean tuber weight (gm) Tuber yield per plant (gm) Tuber skin colour Emblings 131.5a (1.4) 19.5b (1.1) 31.6a (1.6) 615.0a (31.6) Reddish Axillary bud shoots 132.2a (1.1) 24.1a (1.3) 26.8b (1.5) 645.5a (24.8) Reddish Microtubers a 135.4 (0.9) 14.8 (1.6) True potato seeds 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Zabeau M (19 95) AFLP—a new technique for DNA Wngerprinting Nucleic Acids Res 23:4407–4 414 Zoriniants SE, Nosov AV, Monforte-Gonzalez M, Mendes-Zeel M, Loyola-Vargas VM (2003) Variation of nuclear DNA content during somatic embryogenesis and plant regeneration of CoVea arabica L using cytophotometry Plant Sci 16 4 :14 1 14 6 ... liquid culture Plant Sci 94 :17 3 17 7 Payne RW, Lane PW, Digby PGN, Harding SA, Leech PK, Morgan GW, Todd AD, Thompson R, TunnicliVe Wilson G, Welham SJ, White RP (19 93) Genstat 5 Release 3: reference manual Oxford University Press, Oxford Phillips RL, Kaeppler SM, Olhoft P (19 94) Genetic instability of plant tissue cultures—breakdown of normal controls Proc Natl Acad Sci USA 91: 5222–5226 Ronchi VN, Giorgetti ... colour Emblings 13 1.5a (1. 4) 19 .5b (1. 1) 31. 6a (1. 6) 615 .0a ( 31. 6) Reddish Axillary bud shoots 13 2.2a (1. 1) 24.1a (1. 3) 26.8b (1. 5) 645.5a (24.8) Reddish Microtubers a 13 5.4 (0.9) 14 .8 (1. 6) True potato... seeds 12 7.3a (5 .1) 13 .5c (1. 1)