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Industrial Crops and Products 32 (2010) 88–96

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Industrial Crops and Products

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n vitro induction of polyploidy by colchicine treatment of shoots and preliminaryharacterisation of induced polyploids in two Miscanthus species

. Głowacka ∗, S. Jezowski, Z. Kaczmareknstitute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszynska 34, 60-479 Poznan, Poland

r t i c l e i n f o

rticle history:eceived 21 December 2009eceived in revised form 5 March 2010ccepted 18 March 2010

eywords:olyploidisationulture in vitroetraploidexaploidiofuelsiscanthus

a b s t r a c t

Grasses from the genus Miscanthus are becoming a key renewable raw material for energy production. Theaim of this study was to find a suitable treatment combination that would effectively induce polyploidsusing in vitro shoots propagated from Miscanthus grass species. The experiments had a three-factorialarrangement (genotype, colchicine concentration, exposure time). In this study, combinations of fourcolchicine concentrations (156.5, 313, 626 and 1252 �M) and three exposure times (6, 18 and 24 h)were tested in diploid and triploid genotypes belonging to M. sinensis and M. x giganteus. A total of448 polyploids, as assessed by flow cytometry of regenerates, were obtained for all experiments. Theefficiency of polyploidisation in one of the tested genotypes reached 55% after 18 h of treatment with313 �M colchicine. Regardless of the genotype tested, the treatment with 313 �M colchicine for 18 hseemed to be the most effective combination (14.9%) for generating polyploidy and, together with the6 h treatment with 313 �M colchicine, was optimal for shoot survival. Stomatal length and pollen grain

diameter were identified as suitable parameters for the identification of putative Miscanthus polyploids.The beneficial impact of increased ploidy on agronomic traits was genotype-dependent. In four out of fivegenotypes tested, the stem diameter significantly increased in colchicine-induced polyploids comparedto corresponding controls. The tetraploid forms of two genotypes were found to have significantly greatervalues of tuft weight (dry matter), significantly greater stem diameter and, in the case of one genotype,even significantly taller stems when compared with controls. Thus, they might be valuable materials in

high

breeding efforts to create

. Introduction

Species of the genus Miscanthus are perennial giant grassesrom East Asia, and they were introduced to Europe from Japann 1935 (Greef and Deuter, 1993; Linde-Laursen, 1993). Becausef their C4 type of photosynthesis and highly efficient water use,hey are highly promising for biomass production (Lewandowski,006; Lewandowski and Schmidt, 2006) and will likely play an

mportant role in sustainable agriculture in the near future. Foriomass production, the most valuable species from the genusre the fertile diploid Miscanthus sinensis (Anderss.) (2n = 2x = 38)nd the sterile triploid Miscanthus x giganteus (GREEF et DEU.)

2n = 3x = 57), the latter being an interspecific allopolyploid hybridf the diploid M. sinensis and the tetraploid M. sacchariflorus (Linde-aursen, 1993). Approximate genome sizes of the diploid M. sinensisnd the triploid M. x giganteus are 5.2–5.3 and 6.8 Gbp, respec-

∗ Corresponding author at: Institute of Plant Genetics, Polish Academy of Sciences,uantitative Genetics, ul. Strzeszynska 34, 60-479 Poznan, Poland.el.: +48 061 65 50 256; fax: +48 061 65 50 301.

E-mail address: kglo@igr.poznan.pl (K. Głowacka).

926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2010.03.009

-yielding Miscanthus forms.© 2010 Elsevier B.V. All rights reserved.

tively (Rayburn et al., 2009). The majority of M. sinensis genotypesare diploids, although triploid intraspecific hybrids between adiploid and tetraploid M. sinensis can also occur, e.g., M. sinensis‘Goliath’.

In a narrow sense, polyploidisation is an event in which thechromosome number doubles. However, in a wider sense, it is animportant process in nature and plant breeding (deWet, 1980).Polyploidisation can result in larger and darker leaves, delays inflowering, larger inflorescences, prolongations of the floweringperiod, apomixes, larger fruits, and greater secondary metaboliteproduction and yield (Dhawan and Lavania, 1996; Gao et al., 1996;Gu et al., 2005; Predieri, 2001; Roy et al., 2001; Shao et al., 2003;Urwin et al., 2007). Additionally, interspecific hybrid fertility can berestored by polyploidisation (e.g., Nimura et al., 2006). Polyploidsexhibiting valuable new phenotypic traits can occupy new nichesand become important agriculturally and horticulturally. Numer-ous allopolyploid (Avena sativa, Triticum aestivum, x Triticosecale,

Saccharum officinarum, Solanum tuberosum) and autopolyploid (Fra-garia ananassa, Vitis spp., Coffea arabica, Nicotiana tabacum) cropspecies demonstrate polyploid characteristics that have estab-lished utility for humans (Chakraborti et al., 1998; Dewey, 1980;Gao et al., 2002; Zeven, 1980; Zhang et al., 2008).

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The induction of polyploids is a useful tool in plant breeding,s important characteristics such as enhanced biomass yield andesistance to both drought and low temperatures can be achievedhrough chromosome doubling. Hence, there is reason to believehat tetraploids of M. sinensis and hexaploids of M. x giganteus mayave better agronomical traits than their diploid and triploid forms.n efficient method for doubling chromosome number would bearticularly useful for the future production of triploid forms of M.inensis. Intraspecific triploid hybrids such as M. sinensis ‘Goliath’an exhibit biomass yields that are comparable to those obtainedor M. x giganteus.

This study is aimed at developing an effective polyploidisationystem in the Miscanthus genus using colchicine treatment of initro shoots. Ex vitro colchicine treatment of plants that are estab-ished in soil appears to be the cheapest and fastest method toolyploidisation in many species. However, in previous experi-ents with M. sinensis and M. x giganteus (Głowacka et al., 2009),

hat method showed low effectiveness. Hence, to enhance theffectiveness of polyploid induction, the method of in vitro shootolyploidisation was used in the current work.

The effects of varying colchicine concentration and exposureime on increases in ploidy in genotypes that differ in species andtarting ploidy were analysed. In this study, the basic method usedor ploidy estimation was flow cytometry, which is a quick, reli-ble and widely used technique for identifying ploidy in plantsBohanec, 2003; Dolezel et al., 1989; Urwin et al., 2007; Yangt al., 2006). The reliability of flow cytometry has been provedn cytological analyses (Gu et al., 2005; Yang et al., 2006; Zengt al., 2006). However, traits such as stomatal length and width,ollen grain diameter and spikelet length can be useful either in

dentification of putative polyploids in preliminary scanning ofbtained material or as additional evidence for their induction.ence, an additional aim of this study was the development oflternative methods to estimate ploidy in Miscanthus. We also car-ied out a preliminary evaluation of the polyploids obtained in fieldxperiments.

. Materials and methods

.1. Plant material

The experiments were conducted on two species: M. sinensis and. x giganteus. M. sinensis was represented by four genotypes: 930146010 (Ms10), 93 M0146026 (Ms11), 93 M0006004 (Ms16),

nd 92 M0088 (Ms19). M. x giganteus was represented by MG1.he M. sinensis genotypes have different origins: genotypes Ms10nd Ms11 are European hybrids, Ms16 originates from Japan, ands19 is a selected German form named ‘Goliath’. All of the M.

inensis genotypes that we chose were diploids, with the exceptionf the Ms19 genotype, which is a triploid artificial sterile hybridJørgensen and Muhs, 2001). All plant material was grown in soilnder unheated greenhouse conditions.

.2. Colchicine treatment

The experiment had a factorial arrangement with three fac-ors (genotype, colchicine concentration, and exposure time)nd was conducted on in vitro propagated shoots, which werebtained from calluses induced on inflorescence segments orig-nating from plants grown in a ground greenhouse. For callus

nduction, 1-mm segments of immature inflorescences (0.1–2.5 cmn length) were dissected and grown in the dark on a mod-fied MS medium (Murashige and Skoog, 1962) containingmg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/l 6-enzylaminopurine (BAP) (Głowacka et al., 2010). For shoot

nd Products 32 (2010) 88–96 89

regeneration, calluses were transferred into MS medium supple-mented with 2 mg/l BAP and cultured under 16-h photoperiodconditions (100 �mol m−2 s−1) at 26 ± 2 ◦C. The shoots were sub-cultured at least twice (every 4 weeks), and shoot clusters werethen divided into single axillary shoots, trimmed to 5 cm in lengthand placed in test tubes containing 5 ml of an aqueous, filter-sterilised solution of colchicine. In the experiment, we tested fourcolchicine concentrations (156.5, 313, 626 and 1252 �M) and threeexposure times (6, 18 and 24 h) in all combinations except for thelowest colchicine concentration (156.5 �M), for which the shortestexposure time (6 h) was not tested. Non-colchicine-treated shoots(0 �M/0 h) transferred directly into shoot regeneration mediumwere used as controls. Thirty shoots were used in each treatment.Colchicine treatments were given under 40–50 �mol m−2 s−1 lightconditions. After treatment, the remaining colchicine solution wasremoved using filter paper, and the shoots were washed threetimes with sterile water. All of the shoots were transferred into MSmedium supplemented with 2 mg/l BAP. The shoots were grown for12 weeks, subculturing every 4 weeks. During subculture, the shootclumps were subdivided into single shoots and counted. These datawere used to calculate the survival rate (the percentage of theshoots that survived) and the tillering rate of the shoots (definedas the ratio between the number of the multiplied shoots andthe total number of the treated shoots). The rates were estimatedfor the cultures in the 12th week after the colchicine treatment.For the root induction of single shoots, MS medium with 1 mg/l1-naphthylacetic acid (NAA) was used. Shoot cultures were regen-erated and maintained under light conditions (16 h photoperiod,100 �mol m−2 s−1) at 26 ± 2 ◦C. Regenerated plants were trans-planted into plastic boxes containing a 1:3 mixture of peat and sand,and they were kept in a greenhouse.

2.3. Ploidy determination

For determining the ploidy of regenerates, 2 cm segments offully developed leaves were collected and analysed using flowcytometry (the fluorescence intensity is proportional to the nuclearDNA content). All of the plants that successfully passed acclima-tisation to greenhouse conditions were analysed. Samples wereprepared according to Petersen et al. (2002) and analysed using aPartec PA flow cytometer. Collected data were used to calculate thepolyploidisation rates (the percentage of the detected polyploids inthe samples analysed).

To investigate methods of determining ploidy aside from flowcytometry, plants with known ploidy were examined in the secondgrowing season for stomatal length and width, pollen grain diam-eter and spikelet length. Both untreated plants of known ploidyand the colchicine-generated polyploids (first colchicine genera-tion – C0) were grown under greenhouse conditions and were insimilar developmental stages. For determination of the length andwidth of the stoma, the leaf segments were hand-scraped with arazor blade, leaving only the abaxial region of the epidermis. Afterwashing once, the epidermal segments were mounted on a micro-scope slide with a drop of distilled water, covered with a cover slipand examined under a light microscope with the help of an ocular-micrometer. The size of 240 stomata (2 ploidy level × 8 plants × 3leaves × 5 stomata) was measured for each genotype. For deter-mination of pollen grain diameter, the anthers were mounted ina 2% acetocarmine solution and examined under a light micro-scope with the help of an ocular-micrometer. A total of 480 pollengrain diameters (2 ploidy levels × 3 plants × 1 inflorescence × 8

anthers × 10 pollen grains) were measured for each genotype. Thespikelet lengths, defined as the length of the lower glume (with-out the distance of an awn), were estimated under a stereoscopicmicroscope using an ocular scale on spikelets dissected from themiddle part of inflorescences collected in late autumn. A total of 480

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pikelet lengths (2 ploidy levels × 8 plants × 3 inflorescences × 10pikelets) were measured for each genotype.

.4. Preliminary characterisation of polyploids

Field plots were established in May (2005) to characterise gener-ted polyploids. Because of an insufficient number of polyploids forull field experiments using each genotype (56 tetraploids in Ms11,8 hexaploids in Ms19 and 19 hexaploids in MG1) only small, exper-

mental plots could be prepared for preliminary characterisation ofenerated polyploids. The same plants as those planted in experi-ental plots were grown in the greenhouse until they reached the

econd growing season, when the stomatal and pollen grain diam-ter and the spikelet length were determined. As a consequencef these number limitations, plants derived from experiments onolyploidisation by colchicine treatment of shoots (described inhis work), initial explants and callus tissue (unpublished) andlants from culture in vitro (Głowacka et al., 2009) were used foronducting both field and greenhouse experiments, and the pre-iminary assumption has been made that neither experimentalonditions used to induce polyploidy nor part of the plant treatedlters the subsequent affects of polyploidy on plant characteristics.oth, derived from experiments plants of control ploidy levels andhe colchicine-generated polyploids (C0) were in similar develop-

ental stages. Each of the genotypes was represented by two fieldlots, with control and polyploid plants established in four rowsf 10 plants each. Light loamy soils of the 4th classification valueere used in the experiment. At the end of the third growing sea-

on, agronomic traits such as plant height, tillering (the number ofhoots measuring 20 cm above the ground), tuft diameter and stemiameter (measured 5 cm above ground level on producing stemsigher than 1 m) were measured. Each plot was divided into threearts (the front, middle and back), and 5 randomly chosen plantsrom each were measured. Additionally, in December, 15 plantsrom the central area of each plot were harvested. For determina-ion of the dry matter, the tuft weight biomass was dried to 21%

oisture content and measured.

.5. Statistical analysis

Altogether, 12 combinations of treatments were tested on fiveenotypes. Each combination was tested in three replicates on0 shoots from each genotype. The survival, shoot tillering andolyploidisation rates were analysed using the three-factor anal-sis of variance while the stomatal length and width, the pollenrain diameter, spikelet length, plant height, tillering, tuft diam-ter, stem diameter and tuft weight were analysed using thewo-factor analysis of variance (Gomez and Gomez, 1984). Beforetatistical analysis to normalise the distribution, the arcsine trans-ormation (the angular transformation given by y = arcsin (x1/2))f the survival and polyploidisation rates, and the square rootransformation (y = (x + 0.5)1/2) of the shoot tillering rate were cal-ulated. Differences were estimated and statistically verified usinghe Newman–Keuls test at a significance level of ˛ = 0.05. Contrastsere estimated and statistically verified using the F-test at signif-

cance levels of ˛ = 0.05 and ˛ = 0.01. To study the differences andnterrelations between the induced polyploids and control plants

ith respect to the stomatal length and width, the pollen grainiameter and spikelet length jointly (the variables), multivariatenalyses of variance (MANOVA) were performed (Morrison, 1976).

s a measure of the similarity of induced polyploids and controllants with respect to four traits, Mahalanobis distances were cal-ulated for plotting the objects with different ploidy on the plane,nd the graphic distribution of objects was made with help of thestatistical programme (R Development Core Team, 2006).

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3. Results

3.1. Polyploidisation effectiveness

Out of 1800 shoots that were treated, 524 (29.1%) survived.For colchicine-treated shoots, the survival rate ranged from 0 to63.3% (Table 1). The latter resulted from applying the 313 �Mcolchicine solution for 18 h to the Ms11 genotype. By contrast, incontrol combinations (non-colchicine-treated shoots), the survivalrate ranged from 56.7% (Ms19) to 86.7% (MG1). Twelve weeks afterthe colchicine treatment, 6422 shoots were counted, representing ashoot tillering rate of approximately 3.57 (0–21.37). In colchicine-treated combinations, the highest plant tillering rate (19.93) wasobserved in the Ms10 genotype after the application of 313 �Mcolchicine for 6 h. Analysis of the variance showed that the survivalrate depended significantly both on the genotype (P < 0.001) and onthe colchicine concentration × exposure time (P < 0.001). This wasalso true for the plant tillering rate (genotype P < 0.001; experi-mental combination P < 0.001). The Newman–Keuls comparison ofmean test showed that, in comparison with the non-colchicine-treated shoots all of the combinations of colchicine and exposuretimes tested significantly reduced both the number of shoots sur-viving as well as the tillering rates (data not shown). Amongthe combinations with colchicine that were studied, the 313 �Mcolchicine solution applied for 6 and 18 h showed the highest sur-vival rates (44.67% and 38.67%, respectively) and shoot tilleringrates (6.67 and 5.53, respectively). Comparisons of the mean valuesfor all combinations showed that lower colchicine concentrationsresulted in significantly higher numbers of shoots surviving andhigher tillering rates regardless of exposure time. The exceptionwas the treatment with 156.5 �M colchicine, which resulted in asignificantly lower number of shoots surviving and a lower tiller-ing rate than the treatment with 313 �M colchicine. Comparisonsof the mean values for all treatment combinations showed higher(but not in all cases significantly higher) numbers of shoots sur-viving and tillering rates for tests that used shorter exposure timesregardless of colchicine concentration. Recalculated tillering rateas the ratio between the number of multiplied shoots and thenumber of shoots surviving showed that for some colchicine treat-ments, shoots that survived had a higher tillering rate than theuntreated controls. Higher values of recalculated tillering rateswere obtained for the 18-h treatment of the Ms10 and Ms11genotypes with 626 �M colchicine and for the 18-h treatmentof Ms16 and MG1 with 313 �M colchicine compared with con-trols. For the Ms19 genotype colchicine treatments did not resultin higher values of recalculated tillering rates compared with thecontrol.

The nuclear DNA content was estimated using flow cytome-try. Fig. 1 shows four types of histograms: diploid (Fig. 1A) andtetraploid (Fig. 1B) for experiments with diploid starting geno-types, and triploid (Fig. 1C) and hexaploid (Fig. 1D) for experimentswith triploid starting genotypes. The flow cytometric histogramsshowed one dominant peak corresponding to cells in the G1 phase,indicating the ploidy of the sample. Controls containing 2C and3C DNA in the diploid and triploid genotypes, respectively, weredetermined by analysing leaf samples collected from vegetativepropagated plants that were also the donors of initiation explants(Fig. 1A and C). Flow cytometric analyses of 3884 plants showedthat 448 (11.5%) of them were polyploids (Table 1). Colchicine treat-ment generated 232, 56 and 93 tetraploids in Ms10, Ms11 andMs16, respectively, 48 hexaploids in the triploid genotype of M.

sinensis (Ms19) and 19 hexaploids in the triploid species M. x gigan-teus (MG1). Among the analysed leaves, no mixoploid was found.The polyploidisation effectiveness ranged from 0 to 55%. The high-est percentage of polyploids occurred after an 18-h treatment ofMs10 shoots using 313 �M colchicine.

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Table 1Influence of genotype and treatment combination on the effectiveness of shoot multiplication and polyploidisation.

Genotype Treatment combination Survival rate (%)a No. of shoot (12th week of culture) Shoot tillering rateb Polyploidisation rate (%)c

Ms10 18h156.5 23.3 158 5.27 1.4Ms10 24h156.5 10.0 21 0.70 37.5Ms10 6h313 60.0 598 19.93 24.1Ms10 18h313 20.0 138 4.60 55.0Ms10 24h313 16.7 92 3.07 0.0Ms10 6h626 23.3 60 2.00 37.9Ms10 18h626 3.3 35 1.17 12.5Ms10 24h626 6.7 5 0.17 0.0Ms10 6h1252 3.3 1 0.03 0.0Ms10 18h1252 3.3 13 0.43 0.0Ms10 24h1252 0.0 0 0.00 0.0Ms10 Control 73.3 641 21.37 –

Ms11 18h156.5 33.3 92 3.07 16.3Ms11 24h156.5 30.0 31 1.03 0.0Ms11 6h313 23.3 70 2.33 14.3Ms11 18h313 63.3 125 4.17 18.6Ms11 24h313 16.7 26 0.87 3.8Ms11 6h626 23.3 81 2.70 12.3Ms11 18h626 6.7 58 1.93 0.0Ms11 24h626 10.0 32 1.07 0.0Ms11 6h1252 33.3 46 1.53 8.7Ms11 18h1252 16.7 14 0.47 14.3Ms11 24h1252 3.3 6 0.20 0.0Ms11 Control 80.0 539 17.97 –

Ms16 18h156.5 16.7 50 1.67 0.0Ms16 24h156.5 23.3 60 2.00 16.7Ms16 6h313 33.3 120 4.00 6.7Ms16 18h313 33.3 288 9.60 18.4Ms16 24h313 26.7 109 3.63 8.3Ms16 6h626 16.7 43 1.43 11.6Ms16 18h626 20.0 57 1.90 0.0Ms16 24h626 16.7 63 2.10 12.7Ms16 6h1252 20.0 45 1.50 0.0Ms16 18h1252 6.7 17 0.57 0.0Ms16 24h1252 10.0 18 0.60 0.0Ms16 Control 83.3 481 16.03 –

Ms19 18h156.5 43.3 70 2.33 8.6Ms19 24h156.5 56.7 63 2.10 20.6Ms19 6h313 46.7 135 4.50 9.6Ms19 18h313 36.7 41 1.37 19.5Ms19 24h313 30.0 23 0.77 0.0Ms19 6h626 33.3 31 1.03 6.5Ms19 18h626 6.7 14 0.47 0.0Ms19 24h626 30.0 18 0.60 16.7Ms19 6h1252 26.7 11 0.37 0.0Ms19 18h1252 10.0 9 0.30 33.3Ms19 24h1252 0.0 0 0.00 0.0Ms19 Control 56.7 197 6.57 –

MG1 18h156.5 43.3 196 6.53 0.0MG1 24h156.5 46.7 96 3.20 0.0MG1 6h313 60.0 78 2.60 0.0MG1 18h313 40.0 238 7.93 0.0MG1 24h313 53.3 291 9.70 3.6MG1 6h626 33.3 56 1.87 4.3MG1 18h626 36.7 129 4.30 4.1MG1 24h626 23.3 68 2.27 0.0MG1 6h1252 33.3 22 0.73 0.0MG1 18h1252 23.3 45 1.50 7.1MG1 24h1252 0.0 0 0.00 0.0MG1 Control 86.7 358 11.93 –

Total 6422Mean 29.1 3.57 11.5

the tr

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a The percentage of the shoots that survived.b The ratio between the number of the multiplied shoots and the total number ofc The percentage of the detected polyploids in the samples analysed.

The analysis of variance showed that the effectiveness of poly-loidisation depended significantly on the combination (P < 0.001)f colchicine concentration and exposure time. The estimate ofenotype as the main effect on polyploidisation effectivenesshowed no significant impact (P = 0.1), and regardless of the com-

eated shoots.

bination tested, polyploidisation of all four M. sinensis genotypesthat were used had a similar effectiveness, ranging between 7.48%and 7.75%. This was significantly better than that observed for M.x giganteus (1.65%). Irrespective of the genotype, the highest poly-ploidisation was seen with four treatments: 24 h with 156.5 �M

92 K. Głowacka et al. / Industrial Crops and Products 32 (2010) 88–96

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ig. 1. Flow cytometric histograms for (A) control, diploid plant; (B) tetraploid planhe M. sinensis Ms19 genotypes: DAPI fluorescence of the nuclei (x-axis) versus the

olchicine, 6 and 18 h with 313 �M colchicine and 6 h with 626 �M

olchicine (Fig. 2). The rest of the tested combinations resulted inignificantly less polyploidisation. Interaction between genotypend the tested combination for the polyploidisation rate was sig-ificant (P < 0.01), as shown Table 1 (the last column).

ig. 2. The effects of various colchicine concentration and exposure time combina-ions on the polyploidisation rate, irrespective of the genotype.

e M. sinensis Ms10 genotype; (C) control, triploid plant and (D) hexaploid plant ofer of nuclei counted (y-axis).

3.2. Polyploid traits

The stomatal length and width, pollen grain diameter andspikelet length in induced polyploids were greater than in the con-trols (Fig. 3 and Table 2). Among the four traits that were analysed,stomatal width had the highest coefficient of variation (between3.01% and 14.45%). Comparisons of the mean stomatal length andwidth, diameter of pollen grains and spikelet length for polyploidsand control plants of the same genotype showed that the poly-ploids had statistically greater values for all traits (Table 3). To studythe differences between the induced polyploids and plants of thecontrol ploidy level with respect to stomatal size, diameter of thepollen grains and spikelet length jointly, a multivariate analysisof variance (MANOVA) and an analysis of canonical variates wereused (Fig. 4). With the reduction of a four-dimensional space (cre-ated by four traits) to a two-dimensional space (a plane), the loss ofinformation was equal to 2.7%. Mahalanobis distances were takenas the measure of similarity in polyploids and plants of controlploidy level with respect to the four traits. Visibly higher similaritieswere noticed between plants of control ploidy levels (Mahalanobisdistances from 5.4 to 6.1) and induced polyploids (Mahalanobisdistances from 3.1 to 8.6) independent of genotype than betweenplants of the same genotype but with different ploidys (e.g. Maha-lanobis distance between triploid and hexaploid MG1 plants wasequal to 20.2) (Fig. 4). Additionally, it can be seen that values ofanalysed traits are mainly affected by ploidy level and not by thegenotype of the plants.

Table 4 shows characteristics of plant height, tillering, tuftdiameter, stem diameter and weight of tuft (dry matter) in thecolchicine-induced polyploids and the control plants under thefield conditions. For three out of five analysed traits, induced poly-

ploids presented greater mean values. Statistical verification ofthe difference between means for polyploids and control plantsshowed that the response for chromosome number doubling var-ied widely for particular genotypes (Table 5). Only the tetraploidsof Ms16 were significantly taller than corresponding diploids. The

K. Głowacka et al. / Industrial Crops and Products 32 (2010) 88–96 93

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ig. 3. Stomata in M. sinensis: (A) diploid plant, (B) tetraploid plant; pollen grains in. sinensis; (F) spikelets in triploid and hexaploid M. x giganteus (bar represents 50

etraploid forms of Ms10, Ms11 and Ms16 and the hexaploid formf MG1 had significantly greater shoot diameters compared withhe corresponding control plants. In comparison with the control,etraploid plants of Ms11 and Ms16 had significantly greater tufteight of dry matter.

. Discussion

To develop an effective polyploidisation system, the effects ofolchicine concentration and exposure time on genotypes thatiffered in species and ploidy were examined. The efficiency ofolchicine-mediated polyploidisation of in vitro propagated shootseached up to 55%. Colchicine treatment at 313 �M for 18 hegardless of the genotype tested seemed to be the most effec-ive combination (14.9%) in polyploidy generation and was most

ffective for the Ms10 genotype (55%). Other effective combina-ions included 156.5 �M colchicine for 24 h (13.8%) and 626 �Molchicine for 6 h (12.3%). In a previous report on polyploid induc-ion in one M. sinensis genotype by the colchicine treatment ofhoots in vitro, Petersen et al. (2002) reported that the applica-

nensis: (C) diploid plant, (D) tetraploid plant; (E) spikelets in diploid and tetraploidnd arrow indicates stoma).

tion of colchicine (at 313, 626, 1252 and 2504 �M) for 18 and48 h resulted in only 5 tetraploids (2.9%) and 6 mixoploids (3.5%)having cells of diploid and tetraploid levels. In another study onnine genotypes of M. sinensis, Petersen et al. (2003) obtained lowpolyploidisation, ranging from 0 to 27.3% (mean 8.8%), after thetreatment of shoots in vitro with 156 �M colchicine for 5 h. In ourstudy, we were able to enhance the polyploidisation rate (over 69%)by testing low colchicine concentrations with long exposure timesand high colchicine concentrations with short exposure times.Well-balanced experimental conditions are likely to be crucial inavoiding either toxic levels of colchicine in the majority of cellsbefore the chromosome number doubles or insufficient exposuretimes to low colchicine concentrations failing to promote regener-ation of polyploid shoots. Similar conclusions have been reportedin studies on polyploid induction in both Rhododendron (Väinölä,

2000) and pomegranate (Punica granatum) (Shao et al., 2003).

In previous experiments on Miscanthus polyploidisation, DMSOwas added to the colchicine solution to facilitate colchicine uptake(Petersen et al., 2002, 2003; Głowacka et al., 2009). While, Petersenet al. (2002, 2003) found no clear effect of DMSO, the experiments

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Table 2Characteristics of the polyploids obtained and the control plants, as determined by the stomatal length (stl) and width (stw), the pollen grain diameter (pgd) and the spikeletlength (spl).

Trait Genotype

Ms10 Ms11 Ms16 Ms19 MG1

2× 4× 2× 4× 2× 4× 3× 6× 3× 6×stl (�m)

x 29.0 40.7 26.9 36.4 26.9 38.4 30.8 44.0 34.0 46.3Sd ±0.8 ±1.5 ±1.0 ±1.6 ±0.8 ±2.3 ±1.2 ±1.0 ±2.2 ±2.0Vs 2.8 3.7 3.8 4.3 2.9 5.9 3.9 2.3 6.3 4.4

stw (�m)x 20.6 26.5 18.8 25.0 21.6 28.1 20.3 31.0 19.6 26.1Sd ±1.1 ±3.8 ±1.1 ±2.6 ±0.7 ±2.3 ±2.2 ±2.3 ±1.5 ±1.6Vs 5.5 14.5 5.8 10.3 3.0 8.3 10.9 7.3 7.7 6.0

pgd (�m)x 39.6 48.8 32.9 48.8 38.2 49.2 35.9 53.0 34.6 42.9Sd ±0.7 ±0.8 ±0.8 ±0.6 ±1.2 ±1.1 ±0.4 ±0.4 ±0.5 ±0.8Vs 1.8 1.7 2.3 1.1 3.3 2.2 1.2 0.7 1.3 1.9

spl (mm)x 4.9 6.2 4.9 6.2 4.2 5.1 5.5 6.9 4.6 5.8Sd ±0.08 ±0.24 ±0.12 ±0.12 ±0.19 ±0.17 ±0.28 ±0.05 ±0.07 ±0.25Vs 1.6 3.8 2.6 2.0 4.5 3.3 5.1 0.7 1.6 4.4

x: the arithmetic mean; Sd: standard deviation; Vs: coefficient of variation (%).

Table 3Estimate of the comparison of the stomatal length (stl) and width (stw), the pollen grain diameter (pgd) and the spikelet length (spl) between the polyploids obtained andthe control plants.

Compared polyploids and controls Comparison valuea

stl stw pgd spl

Ms10 (4×)–Ms10 (2×) 11.70** 5.85** 9.20** 1.29**

Ms11 (4×)–Ms11 (2×) 9.51** 6.21** 15.88** 1.28**

Ms16 (4×)–Ms16 (2×) 11.55** 6.43** 10.92** 0.98**

ally ve

cctcp

TCs

x

Ms19 (6×)–Ms19 (3×) 13.20**

G1 (6×)–G1 (3×) 12.23**

a Difference between means for compared polyploids and control plants statistic** Significant at the level of ˛ = 0.01.

onducted by Głowacka et al. (2009) showed that application ofolchicine supplemented with DMSO caused a significant reduc-ion in plant survival and tillering. Additionally, supplementing theolchicine solution with DMSO gave a significantly higher poly-loidisation rate only at a high colchicine concentration (1252 �M).

able 4haracteristics of plant height, tillering, tuft diameter, stem diameter and tuft weight ineason.

Trait

Ms10 Ms11

2× 4× 2× 4× 2×Plant height (cm)

x 179.2 139.2 197.1 190.0 196.3Sd ±8.3 ±10.4 ±6.4 ±2.2 ±3.4Vs 4.7 7.5 3.3 1.1 1.7

Tillering (no.)x 37.4 21.8 74.2 58.4 53.2Sd ±4.7 ±4.0 ±3.3 ±3.5 ±20.0Vs 12.5 18.5 4.5 6.0 37.6

Tuft diameter (cm)x 29.6 29.2 33.6 30.3 42.2Sd ±2.7 ±1.6 ±2.2 ±1.0 ±2.3Vs 9.3 5.6 6.4 3.3 5.4

Stem diameter (mm)x 6.3 8.1 5.0 7.0 7.4Sd ±0.6 ±0.3 ±0.3 ±0.3 ±0.3Vs 8.9 3.5 5.1 3.6 3.6

Tuft weight (kg)x 0.8 0.5 1.0 1.3 0.8Sd ±0.02 ±0.12 ±0.04 ±0.14 ±0.08Vs 2.9 24.7 3.9 10.6 9.8

¯ : the arithmetic mean; Sd: standard deviation; Vs: coefficient of variation (%).

10.71** 17.13** 1.41**

6.46** 8.37** 1.25**

rified by the F-test.

Accordingly, in the present experiments, DMSO was not added tothe colchicine solution.

In our results, it is noteworthy that no mixoploids were detectedin any of the plants examined. Petersen et al. (2003) reported from0% to 22.7% mixoploids in their study on the induction of polyploids

the polyploids obtained and in the control plants at the end of the third growing

Genotype

Ms16 Ms19 MG1

4× 3× 6× 3× 6×

215.6 202.1 92.1 288.0 256.7±4.7 ±10.6 ±4.4 ±3.9 ±6.4

2.2 5.3 4.8 1.4 2.5

36.3 47.5 24.8 23.7 19.8±5.0 ±3.0 ±2.0 ±2.5 ±6.413.8 6.4 8.0 10.4 32.5

41.4 24.3 14.1 55.7 53.5±1.8 ±3.2 ±0.8 ±1.9 ±7.8

1.0 12.9 5.7 3.4 14.6

8.15 5.5 4.1 10.4 12.6±0.4 ±0.3 ±0.7 ±0.5 ±0.2

5.0 5.1 16.9 5.1 1.9

1.0 0.9 0.1 1.7 1.0±0.15 ±0.07 ±0.01 ±0.03 ±0.1214.2 7.3 9.1 1.8 11.2

K. Głowacka et al. / Industrial Crops and Products 32 (2010) 88–96 95

Table 5Estimate of the comparison of the plant height, tillering, tuft diameter, stem diameter and tuft weight between the polyploids obtained and the control plants.

Compared polyploids and controls Comparison valuea

Plant height Tillering Tuft diameter Stem diameter Tuft weight

Ms10 (4×)–Ms10 (2×) −40.00** −15.61* −0.42 1.75** −0.32**

Ms11 (4×)–Ms11 (2×) −7.08 −15.75* −3.25 1.99** 0.30**

Ms16 (4×)–Ms16 (2×) 19.30** −16.91* −0.75 0.77* 0.24**

Ms19 (6×)–Ms19 (3×) −110.00** −22.75** −10.17** −1.33** −0.82**

** ** **

ally ve

ictimfipatld

pdpPpelnelp

Ftto

G1 (6×)–G1 (3×) −31.33 −3.92

a Difference between means for compared polyploids and control plants statistic* Significant at the level of ˛ = 0.05.

** Significant at the level of ˛ = 0.01.

n M. sinensis of different genotypes by treatment with 156 �Molchicine for 5 h. Not only experimental conditions but also geno-ype seems to influence the induction of mixoploids. Additionally,n the case of ploidy level, the developmental stage of examined

aterial may affect results. Mixoploid tissue is likely to be morerequent in leaves collected directly from plants in test tubes thann samples of plants established in a greenhouse. In our work, com-ared with the experiments of Petersen et al. (2002, 2003), whonalysed M. sinensis shoots collected directly from in vitro cultures,he delay in the examination of plants (2 months after their estab-ishment in soil) might have reduced the number of mixoploidsetected.

In our study, traits such as the stomatal length and width, theollen grain diameter and the spikelet length were evaluated toetermine whether they could be used to identify or provide sup-orting evidence for putative polyploids in a preliminary scanning.olyploids had significantly greater values for stomatal length andollen grain diameter than controls. Determination of ploidy byxamination of stomatal size and pollen grain diameter or spikelet

ength are relatively laborious methods, mainly because of the highumber of required replications. However, examination of differ-nt parts of plants can provide reassurance of their stability andack of mixoploidy. The utility of stomatal size in distinguishinglants with different ploidies has been demonstrated in other plant

ig. 4. Configuration of the objects characterised by the stomatal length and width,he pollen grain diameter and the spikelet length, jointly in the space of the firstwo canonical variates V1 and V2, with the shortest dendrite connecting the nearestbjects.

−2.17 2.22 −0.67

rified by the F-test.

types (Borrino and Powell, 1988; Campos et al., 2009; Pozzobon andValls, 2000; Speckmann et al., 1965). Even in fossil plants, stom-atal size has been used as evidence for polyploidy in the majority(70%) of angiosperms (Masterson, 1994). In autotetraploid Sorghumplants, measurement of pollen grain sizes was helpful in discov-ering ploidy instability (Tsvetova and Elkonin, 2002). In itchgrass(Rottboellia cochinchinensis), the shape of the glume tip on the pedi-cellate spikelet was found to be a reliable marker distinguishingdiploids from polyploids (Millhollon and Burner, 1993).

Increases in ploidy can affect very specific traits, including flow-ers with increased diameter in pomegranates (Shao et al., 2003),thicker and rounder flowers in Spathiphyllum wallisii (Eeckhaut etal., 2004), thicker peduncles and larger peltate glandular trichomesin Lavandula angustifolia (Urwin et al., 2007) and a 300% increasein the volatile oil content in Acorus calamus (Dhawan and Lavania,1996).

Since Miscanthus plants are important in biomass production,we conducted a preliminary investigation of the impact of poly-ploidy on plant height, tillering, tuft diameter, stem diameter andtuft weight (dry matter) under field conditions. Comparison of thepolyploids obtained and the corresponding controls showed thatthe polyploidisation effects varied by plant genotype. Significantdifferences were found between the analysed induced polyploidsand corresponding controls in Ms10, Ms11, Ms16, and MG1, wherestem diameter was greater for tetraploid and hexaploid plants.Additionally, the tetraploids of Ms11 and Ms16 had significantlygreater dry matter weight of tuft, and in polyploids of Ms16, theplants were also significantly higher. Potentially, induced tetraploidplants in Ms11 and Ms16 genotypes might have enhanced biomassyield, particularly due to the greater tuft weight (dry matter), butalso because of other traits such as greater stem diameter and, incase of Ms16, taller stems. In contrast, comparisons of hexaploidsand control Ms19 plants showed significant greater values for alltraits in triploids. In Scutellaria baicalensis, comparison of diploidswith 20 induced tetraploid lines showed that the tetraploid lineshad both taller (1 line) and lower (19 lines) baicalin contents (Gaoet al., 2002).

In conclusion, doubling chromosome number was most effec-tively achieved using 313 �M colchicine for 18 h for all genotypestested, and either 6 or 18 h treatments with 313 �M colchicinewere optimal for shoot survival. Stomatal length and pollen graindiameter were identified as useful parameters for distinguishingboth diploid and tetraploid M. sinensis plants as well as triploidand hexaploid M. sinensis and M. x giganteus plants. According topreliminary characterisation of polyploids, the effect of enhancingploidy on agronomic traits depended on genotype. In four out offive genotypes tested, the stem diameter significantly increased incolchicine-induced polyploids compared with corresponding con-

trols. Tetraploid forms of Ms11 and Ms16 were found to havesignificantly greater dry matter weight of tuft, significantly greaterstem diameter and, in the case of Ms16, even significantly tallerplants when compared with controls. Therefore, they may be valu-able materials in breeding efforts to create new, high biomass forms

9 rops a

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6 K. Głowacka et al. / Industrial C

ithin the genus Miscanthus. Further studies will be needed for aull evaluation of the effects of induced polyploidisation on biomassield in biofuel crops.

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