Article. Contradictory results in pollen viability determination of Valeriana scandens L.

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1 Article Contradictory results in pollen viability determination of Valeriana scandens L. Erica Duarte-Silva 1, Lia R. Rodrigues 2, Jorge E.A. Mariath 1 Received April 15, 2011 Accepted May 20, Geneconserve 40: , Laboratório de Anatomia Vegetal, Instituto de Biociências, UFRGS, Porto Alegre, RS BRAZIL ( jorge.mariath@ufrgs.br) 2 FEPAGRO- Fundação Estadual de Pesquisa Agropecuária do Rio Grande do Sul, Porto Alegre, RS BRAZIL Abstract Methods for estimating pollen viability may offer contradictory inferences. Based on this, we investigated the pollen viability from the perfect flowers of Valeriana scandens based on three methods: (1) Alexander stain, (2) FDA fluorescein diacetate and (3) in vitro pollen germination. As results, V. scandens pollen viability was 95% with Alexander, 47% with FDA reaction and 30% with pollen germination. Analysis of variance indicated that the three techniques offer distinct inferences and it was not possible to estimate pollen germination performing Alexander or FDA reaction. A complementary test with propionic-carmine was performed and showed 98% of pollen stainability. Considering that in vitro pollen germination is the quantitative method that presents the highest correlation with seed set, Alexander and carmine tests overestimated the pollen viability in V. scandens. We conclude that

2 stainability tests were inconsistent to measure pollen viability and estimate pollen germination. Keywords: Alexander, carmine, FDA, pollen germination, germination medium, ANOVA Introduction Valeriana L. (Caprifoliaceae s.l.) contains metabolites used to produce phytomedicines that prevent anxiety and insomnia, and improve the quality of sleep (von Poser et al. 2004; Xena de Enrech 1993). The biodiversity center of Valeriana is located in South America (Xena de Enrech 1993), and Brazilian species present a high potential for economic exploitation due to their pharmacological properties (von Poser et al. 2004). Basic knowledge about reproductive biology, such as pollen viability, is a prerequisite for the domestication of native plants. Studies of Valeriana pollen viability and in vitro pollen tube germination can contribute to basic and applied studies in plant breeding of this group. Valeriana scandens, as well as many Valeriana species, presents floral polymorphism bearing pistillate flowers with staminodia and perfect flowers with three stamens (Xena de Enrech 1993). Staminodia of pistillate flowers do not present pollen grains or pollen without cellular content otherwise perfect flowers present pollen grains with a vegetative and two sperm cells (Duarte-Silva et al. 2010a). Viability tests of Valeriana are restricted to the carmine test of V. scandens (Duarte-Silva et al. 2010b). Current pollen viability techniques offer different inferences of pollen functionality. There are several methods for evaluating pollen quality including: (1) stainability tests of pollen cells; (2) tests based on integrity of pollen cell membranes; (3) in vitro pollen germination; and (4) pollen germination on stigma (Dafni and Firmage 2000). The first three methods are quantitative and the last one is qualitative (Dafni and Firmage 2000). In stainability tests, Alexander stain (Alexander 1969) or

3 carmine stain (Radford et al. 1974) associate viability with the presence of pollen cellular content. In fluoresceine diacetate (FDA), pollen viability is based on the integrity of vegetative cell (Widholm 1972; Heslop-Harrison and Heslop-Harrison 1970). In vitro pollen germination is the quantitative viability test that presented the highest correlation with seed set (Dafni and Firmage 2000). Valeriana scandens L. (Caprifoliaceae sensu APG IIII 2009) bears three anthers per flower, and two different flower morphs: perfect and pistillate, the latter with sterile pollen (Duarte-Silva et al. 2010a). Pollen viability test performed with aceto-carmine stain showed that pistillate flower pollen grains were fully inviable while most part of perfect flower pollen grains presented cellular content (94,50%±3,23) (Duarte-Silva et al. 2010b). At this work, we investigated the pollen viability from perfect flowers of V. scandens based on three different methods: (1) Alexander stain (Alexander 1969), (2) FDA reaction (Heslop-Harrison and Heslop-Harrison 1970) and (3) in vitro pollen germination (Santos and Mariath 1997). A complementary pollen viability test was performed with carmine. This test was made because it has been used in recent studies, in both cytogenetical and reproductive biology analyses. Material and methods During October 2008, 10 inflorescences of Valeriana scandens were collected from 10 individuals in Rio Grande do Sul state, Brazil. Five inflorescences were collected in Reserva Morro Santana, Porto Alegre municipality (30 04 S, W), and five others were collected in Dom Pedro de Alcântara municipality (29 23 S, W). Voucher specimens were deposited in the ICN Herbarium, Brazil (ICN , , , ).

4 Inflorescences with buds, at pre-anthesis stage, were kept in MS culture medium (Murashige and Skoog 1962) at room temperature until the beginning of anthesis (7h30min) when flowers were collected. Each individual contributed with one flower, each flower bears three anthers, and each anther was assigned to one method, as follows: the first anther was squashed in a Alexander stain drop and observed under light microscope (Alexander 1969); the second was squashed in a FDA work solution (Heslop-Harrison and Heslop Harrison 1970) with 0.5 M sucrose, incubated in darkness for 35min at 22 C and observed under fluorescence microscopy ( nm). The remaining anther of each flower was excised and dissected in germination medium (2% colorless gelatin; 20% sucrose and 0.01% boric acid) (Santos and Mariath 1997), in a Klein dish, on moistened absorbent paper, inside a closed Petri dish. Culture mediums were previously tested with different sucrose concentrations. Samples were kept in the dark for 3 hours at 38 C and observed using differential interference contrast microscopy (DIC). Observations and photomicrographs were made with a Leica DMR-HC microscope and a DFC 500 digital camera, respectively. For statistical analysis a two-way analysis of variance (ANOVA) was performed (ten individuals and three methods) to analyze an index of pollen viability. This index was defined as percentage of viable pollen grains, based on average of 121 observations per glass slide. Normality and equal variance tests were performed to check the assumption of the analysis of variance (ANOVA). Data were also submitted to correlation test. Statistical tests were performed in numbers but presented in percentage. For complementary analysis with propionic-carmine stain, 10 flowers were collected from the 10 studied individuals. One anther from each flower was squashed in a drop of propionic-carmine drop (Radford et al. 1974), and an average of 100 pollen grains were counted per glass slide.

5 Results Valeriana scandens pollen viability was 95% with Alexander stain (n=1176; standard error=4.7%); 47% with FDA reaction (n=1446; se=20.8%) and 30% with in vitro pollen germination (n=999; se=12.6%) (Fig. and Table 1). Data presented normal distribution and equal variance. The three techniques offer distinct inferences and it was not possible estimate pollen germinability with the Alexander stain or the FDA reaction. Using ANOVA, a highly significant difference among all techniques was detected (F (technique)= ; df=2; 18; p<0.001) but not among individuals (F (individual)= 1.540; df= 9; 18; p=0.208), indicating low variability in pollen quality of the species (Table 2). The viability index varied from 47.2% to 72.7% in the ten flowers. In correlation tests, all correlation coefficients (r) were not significant. The higher r was , between the positive reaction to FDA and in vitro germination. The r between the positive reaction to Alexander and FDA was negative ( ), as well as between Alexander and in vitro germination ( ). The result of complementary pollen viability test with propionic-carmine was 98.2% (n=1000; se=1.92). Discussion Pollen viability tests with Alexander stain, FDA and in vitro germination have been performed with Sorghum halepense to evaluate the nuclear state and to develop a suitable medium for in vitro germination (Burke et al. 2007). Sorghum halepense showed similar results between Alexander and FDA tests (93.1 to 98.4% and 92.6 to 97.9% respectively) (Burke et al. 2007). On the other hand, Valeriana

6 scandens presented no correlation between the Alexander stain, FDA and in vitro germination tests. The three stainability tests that have been performed with V. scandens presented high pollen viability rates: 94.5% with aceto-carmine (Duarte-Silva et al. 2010b); 95% with Alexander stain and 98% with propionic-carmine stain (shown at this work). These data show that the majority of pollen grains from perfect flowers have cellular content. Of pollen with cellular content (almost 100%), only 47% had a positive reaction to the FDA and were, therefore, possibly alive (according to Widholm 1972 and Heslop-Harrison & Heslop-Harrison 1970). In many cases, FDA has been considered a well-established test capable of estimating pollen germinability (Dafni and Firmage 2000). In the case of V. scandens, FDA and germination reactions have been significantly different. Considering that in vitro pollen germination is the quantitative method with the higher seed set correlation (Dafni and Firmage 2000), stainability tests of acetocarmine (Duarte-Silva et al. 2010b); propionic-carmine and Alexander stain overestimated the pollen viability in the studied species. Presence of cellular content does not guarantee that pollen is metabolically active or capable of growing pollen tubes (Rodrigues et al. 2006). Several methods for determinate pollen viability normally stain either killed or aborted pollen (Rodriguez-Riano and Dafni (2000). Histochemical tests indicate pollen stainability rather than pollen viability (Slomka et al. 2010). Therefore, we conclude that stainability tests are inconsistent when measuring pollen viability and estimating pollen germinability in V. scandens. Acknowledgments This research is part of the first author s PhD thesis, undertaken at the Post Graduate Program of Botany at UFRGS, Brazil, and it was supported by Coordenadoria de

7 Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and research grant for the last author; Dr. Luis Rios de Moura Baptista, for enabling the fieldwork at Dom Pedro de Alcântara, RS, Brazil, and Dra. Sidia Callegari Jacques for the statistics critical review. References Alexander MP (1969). Differential staining of aborted and non-aborted pollen. Biotech. and Histochem. 62: APG III (2009). An update of the Angiosperm Phylogeny Group classification orders and families of flowering plants: APGIII. Bot. J. Linn. Soc. 161: Burke IC, Wilcut JW, Allen NS (2007). Viability and in vitro germination of Johnsongrass (Sorghum halepense) pollen. Weed. Technol. 21: Dafni A, Firmage D (2000). Pollen viability and longevity: pratical, ecological and evolutionary implications. Plant. Syst. Evol. 222: Duarte-Silva E, Vanzela AA, Mariath JEA (2010a). Developmental and cytogenetic analysis of pollen sterility in Valeriana scandens L. Sex. Plant. Reprod. 23: Duarte-Silva E, Vieira MFV, Bittencourt Jr. NS, Garcia FCP (2010b). Polimorfismo floral em Valeriana scandens L. Acta. bot. bras. 24(3): Heslop-Harrison J, Heslop-Harrison Y (1970). Evaluation of pollen viability by enzimatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Biotech. and Histochem. 45: Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol. Plant. 15 (3):

8 Radford AE, Dickinson WC, Massey JR, Bell CR (1974). Vascular plant systematics. New York, Harper and Row. Rodrigues LR, Forte BC, Bodanese-Zanettini MH (2006). Isolation and culture of soybean microspores and pollen grains. Braz. Arch. Biol. Techn. 49: Rodriguez-Riano T, Dafni A (2000). A new procedure to asses pollen viability. Sex. Plant. Reprod. 12: Santos RP, Mariath JEA (1997). A Single method for fixing, dehydrating and embedding pollen tubes cultivated in vitro for optical and transmission electron microscopy. Biotech. and Histochem. 72: Slomka A, Kawalec P, Kellner K, Jedrzejczyk-Korycínska M, Rostanski A, Kuta E. (2010). Was reduced pollen viability in Viola tricolor L. the result of heavy metal pollution or rather the tests applied? Acta Biol. Cracov. Bot. 52 (1): Von Poser GL, Rech SB, Silva AL, Sobral M (2004). Valerianas brasileiras: potencial terapêutico de espécies do sul do país. Ciência Hoje 32: Widholm JM (1972). The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Biotech. and Histochem. 47: Xena de Enrech, N (1993). Contribución al studio del género Valeriana en Venezuela: distribución geográfica, caracteres morfoanatomicos, cariologicos y palinologicos de interes taxonomico y evolutivo. Acta Bot. Venez. 16: Table 1 ANOVA of viable pollen grains percentage from V. scandens using three different techniques: Alexander stain, FDA reaction and in vitro pollen germination.

9 Means indicated by different letters differ significantly using the Tukey test at the 0.05 significance level. Technique Means Percentage Tukey test Alexander stain 95.1 A FDA reaction 46.9 B Pollen in vitro germination 30.1 C General mean 57.3 Coefficient of variation (%) 23.4 Table 2 Percentage of positive reaction to Alexander, FDA and in vitro germination of pollen from 10 individuals of V. scandens. Individuals Alexander stain FDA reaction In vitro pollen germination Standard error of the difference between means of reactions = 5.03 Standard error of the difference between means of individuals = Fig. 1-3 Pollen viability of Valeriana scandens based on three different methods. 1 Alexander stain. 2 FDA reaction (fluorescence microscopy). 3 In vitro pollen tube germination (differential interference contrast microscopy). Bars = 50µm