Grabowska-Joachimiak, Aleksandra

Joachimiak, Andrzej

Acta Biologica Cracoviensia s Botanica

English

Jagiellonian Univeristy Repository

ACTA BIOLOGICA CRACOVIENSIA Series Botánica 42/1: 103-107, 2000Stomatal Cell Length and Ploidy Level in Four Taxa Belonging to the Phleum Sect. PhleumAndrzej Joachimiak12* and Aleksandra Grabowska-Joachimiak2‘Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, ul. Grodzka 52, 31—044 Cracow, Poland“Cytogenetics Group, Department of Plant Breeding and Seed Science, Agricultural University, ul. Łobzowska 24, 31—140 Cracow, PolandReceived December 13, 1999; revision accepted March 28, 2000Stomatal cell length was examined in four closely related taxa of sect. Phleum in the genus Phleum (P. nodosum, P. pratense and in two cytotypes ofP. commutatum}. It was found that the polyploid taxa (P. pratense, 2n - 6x = 42 and P. commutatum, 2n = 4x = 28) have longer stomatal cells than their diploid relatives (P. nodosum, 2n = 2x = 14 andP. commutatum, 2n = 2x = 14). In these two pairs of taxa, stomatal cell length can be a rapid and useful indirect ploidylevel indicator and can assist in their identification. Material taken from live and dried specimens of a given taxondid not differ in stomatal cell length, meaning that different cytotypes can be identified from herbarium material aswell.Key words: Phleum, grasses, stomata, ploidy level. INTRODUCTIONRoot tip cell chromosome counting is time-consum­ing and not suited for routine screening of ploidy level. For this purpose, the stomatal cell length can be a rapid and reliable indirect ploidy indicator. The length of stomatal guard cells is correlated with the ploidy level in a range of natural and commercial cytotypes/varieties of different plant species (Speck­man et al., 1965; Przywara et al., 1988; Cohen and Yao, 1996; Mishra, 1997). Grasses are a particularly promising group of plants in which stomatal features (stomatal fre­quency, stomatal guard cell length and stomatal index) generally can be used to determine of ploidy level. These plants often show significant intraspe­cific and intrageneric chromosome number vari­ability, and rapid identification of their different cytotypes is of great importance in taxonomy, pre­liminary selection and breeding. For this reason the*e-mail: joachim@grodzki. phils. uj. edu. plcorrelations between stomatal features and ploidy level in grasses have been examined many times (Speckman et al., 1965; Miskin and Rasmusson, 1970; Heichel, 1971; Teare et al., 1971; Liang et al., 1975; Sapra et al., 1975; Tan and Dunn, 1973, 1975; Lea et al., 1977a, b; Santen and Casler, 1986; Borrino and Powell, 1988). In the majority of results publish­ed so far, guard cell length was measured manually using an ocular micrometer on a microscope, and the number of stomata measured per plant was small (6-25). To rationalize this kind of study, measure­ments should be automated at least in part, to make it easier to obtain and compare a larger number of measurements. Here we attempted to determine stomatal guard cell length in four taxa from the section Phleum in the genus Phleum. The examined taxa (P. nodosum, 2n = 2x = 14, P. pratense, 2n = 6x = 42, and P. commutatum, 2n = 2x = 14, 2n = 4x = 28) representthree different ploidy levels and are closely relatedPL 1SSN OCX) 1-5296 © Polish Academy of Sciences, Cracow 2000104 Joachimiak and Grabowska-JoachimiakTABLE 1. Provenance and chromosome number of the Phleum samples studiedTaxon Locality Chromosome Number ofnumber (2n) plantsPhleum commutatum Gaud. 11] Austria, Alps - Nationalpark Hohe Tauern 14 {a} 5(2] Sweden, Are vicinity 14 (a) 5[3] Poland, Tatra Mts. 14 <b} 5141 Sweden, Are vicinity 28 (a) 5|5] Scotland, Aonach Beag 24 (a) 5Phleum nodosum L. |6] Poland, Cracow vicinity 14 {b} 5|7] Sweden, Stockholm vicinity 14 {c} 5Phleum pratense L. [8] Poland, Cracow vicinity 42 {b} 5[9] Sweden, Stockholm vicinity 42 (c) 5Chromosome numbers established in: {a} - Joachimiak and Kula (1996); (b) - Joachimiak and Kula (1993); (c) - this study. TABLE 2. Stomatai guard cell length in representatives of four Phleum taxa from different populationsP. contr2nNonutatum= 2xACLP. comr2nNonutatum= 4xACLP. no2nNodosum= 2xACLPpr 2nNoátense= 6xACLLU 24. 5±2. 4 15] 39. 8±3. 5 [6] 29. 0±3. 7 [8| 35. 9±3. 9(*) 29. 2±2. 9 (*) 36. 0±3. 7121 99 5+9 Я 141 9Q 9+9 Q (71 99 1+9 9 Г91 97 1+9 fi[3] 25. 3±2. 4Mean: 24. 1±2. 4 Mean: 34. 5±3. 2 Mean: 25. 5±2. 9 Mean: 31. 5±3. 4No - population number (see Tab. 1); ACL - average stomatai guard cell length in pm; ± - standard deviation; (*)- herbarium material. (Joachimiak and Kula, 1997). There is a lack of good morphological features to enable differentiation of P. nodosum {-P. bertolonii DC) from P. pratense and between the diploid and tetrapioid forms of P. com­mutatum, frequently occurring side by side in Eu­rope. In particular, morphological identification of different cytotypes within the P. pratense group (P. pratense and P. nodosum) is of great importance for taxonomy and for practical purposes. P. pratense is largely used as a pasture species and P. nodosum is used as a turf grass, and both species are used in various breeding programs in Europe and other parts of the world. However, no morphological characters previously studied (plant height, flag leaf length and width, inflorescence diameter, glume length, awn length, cilia length and num­ber) are reliable in distinguishing different ploidy levels (for review: Cenci et al., 1984). So far, rep­resentatives of these taxa can be reliably identi­fied only on the basis of differences in chromosome number (Nordenskiold, 1945; Joachimiak and Kula, 1993, 1996) or cytometric DNA estimations. The amounts of 2C DNA in different races of P. pratense and P. nodosum are well correlated with their ploidy level (Alan Steward, personal communi­cation). We also checked whether stomatai length meas­urements can be facilitated by automated computer procedures and whether stomatai cell length can be used to identify herbarium specimens. MATERIALS AND METHODSThe research material consisted of fragments of upper epidermis from fully developed leaves. For stomatai measurements the third leaf below the panicle was sampled from each analyzed plant. The majority of plants had been collected from various natural populations in Europe and transferred to an experimental field. Two years later, leaves were cut from them for further analysis. In the case of P. pratense andP. nodosum, fragments of epidermis from herbarium specimens were examined also. All the Phleum specimens were cytologically examined and identified (Tab. 1). Each piece of upper epidermis was removed with forceps from the central part of the leaf andStomatai cell length and ploidy level in Phleum 105Fig. 1. Phleum stomatai apparatus and measurement of its length, (a) Vertical view, (b) Lateral view, scl - stomatai guard cell length. placed in commercial chlorine bleach for 30 min, then rinsed with distilled water and stored in 70% ethanol. Leaves from herbarium specimens were soaked in distilled water for 24 h before the epider­mis fragments were removed. The material was stained with a 1% solution of safranin in alcohol for 5 min, rinsed in 70% alcohol and distilled water, counterstained with an acidic solution of alcyan blue for 10 min, and mounted in a drop of water on a microscope slide. The preparations were analyzed under an Eclipse E800 microscope (Nikon Corp., Japan) equipped with a Panasonic video camera. Image grabbing and stomatai guard cell length measurements were done with Lucia G software (Laboratory Imaging Ltd. ). During the measure­ments, special attention was paid to measure the total length of the guard cells, together with those parts which can be poorly visible in preparations because of slipping under epidermal cells (Fig. 1). In each specimen the lengths of at least 200 guard cells were measured. By using a number of leaves for each examined collection, at least 1000 measurements were obtained. RESULTS AND DISCUSSIONEach of the examined taxa showed a strictly defined range of stomatai guard cell length variability (Tab. 2). The ranges overlap partially, thus stomataiTABLE 3. Phleum pratense I nodosum (6x/2x) andP. commu- tatum (2x/4x). Results of two-tailed Student’s test for mean stomatai cell lengths. Compared means are statistically sig­nificant if P<0. 0001. AU - Austria, PL - Poland, SW - Sweden; ** significant, (-) not significantP. pratense /P. nodosum P. nodosum P. pratenseSW(6x/2x) PL SWP. pratense PL ** ** **SW (-) **P. nodosum SW **P. commutatum 4x 2x 2x(2x/4x) SW AU PL2x SW ** (-) (-)2x PL ** (-)2x AU **length alone cannot be an absolute criterion for taxonomic identification of particular specimens within the section Phleum. In spite of this, measure­ment of stomatai length can be very useful for proper taxonomic identification of morphologically indis­tinguishable specimens belonging to closely related pairs of taxa (P. nodosum IP. pratense or diploid/te- traploid P. commutatum), because both polyploid forms (P. pratense, 2n = 6x = 42 andP. commutatum, 2n = 4x - 28) have definitely longer stomatai cells than their diploid relatives (P. nodosum, 2n = 2x = 14 and P. commutatum, 2n = 2x = 14) (Tabs. 2, 3). The stomata of P. nodosum and P. pratense originating from Sweden are generally smaller than those of plants from other areas (Tab. 2, and unpubl. results). The observed differences between Polish and Swedish representatives of these taxa are statistically significant (Tab. 3). Intraspecific differences in guard cell length were previously observed among various cultivars of Triticum aestivum (Teare et al., 1971) and Bromus inermis (Tan and Dunn, 1975). Another question is the assessment of stomatai cell length in P. commutatum from Scotland. Speci­mens of this taxon were obtained from seeds col­lected from a natural population on Aonach Beag, received from the Royal Botanic Gardens, Kew Seed Bank, England. In previous research (Joachimiak and Kula, 1996) it was stated that plants grown from these seeds were hypotetraploid with somatic chro­mosome number 2n - 24. The mean length of their stomata was about 10 pm more (mean - 39. 8 pm) than in Swedish representatives of this tetrapioid taxon (mean - 29. 2 pm). Such a difference can sug­gest the possible influence of aneuploid changes on the stomatai cell length, but may well be that the106 Joachimiak and Grabowska-JoachimiakFig. 2. Stomatai length and distribution in leaf epidermis of Polish material [6, 8|. (a) P. nodosum, (b, c) P. pratense. a, b - from living specimens, c - from herbarium specimens. Id - origin of Phleum samples (see Table 1). Bar = 50 pm. P. commutatum from that area is of different origin and is a separate taxon within the P. alpinum group (in which the diploid and tetrapioid P. commutatum as well as P. alpinum subsp. rhaeticum are in­cluded). It is even more probable because previous authors have suggested different (auto- or allopoly­ploid) origins of the tetrapioid races of P. commuta­tum from different areas (Gregor and Sansome, 1930; Miintzing, 1935; Nordenskiold, 1945; for re­view: Joachimiak and Kula, 1993, 1996). The adopted procedure yielded epidermis frag­ments and stained stomata from both live and dried specimens. Neither stomata morphology nor stoma­ta staining ability underwent significant changes due to storage in a herbarium. The mean stomatai guard cell length and the range of their variability were very similar between the two types of speci­mens (Tab. 2; Fig. 2b, c). This way of identifying cytotypes in herbarium material provides an extra tool in taxonomic research. These results indicate that stomata length measurements can be useful not only for diploid and polyploid conspecifics (P. commutatum) but also for species with different ploidy level if they are very closely related to each other (Fig. 2a, b). This was the case of P. nodosum and P. pratense, which are considered two separate species, how­ever closely related. Previous studies found at least four genomes of P. nodosum in the karyotype of the hexapioid P. pratense (Joachimiak and Kula, 1993; Cai and Bullen, 1994). Measurements of stomatai guard cell length are especially useful for identifying different cytotypes of plants growing in mixed populations or in geographically near localities (Tabs. 2, 3). ACKNOWLEDGEMENTSThe authors wish to thank Joanna Klos (Agricultu­ral University, Cracow) for technical assistance, and Dr. Alan Steward (Pyne Gould Guiness Ltd, Christ­church, New Zealand) for information about his results on DNA estimations in Phleum. 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Stomatal cell length and ploidy level in four taxa belonging to the Phleum sect. Phleum.

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Stomatal cell length and ploidy level in four taxa belonging to the Phleum sect. Phleum.

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