Evaluation of pollen and productive parameters, their interrelationship and clustering of eight Corchorus spp. (Tiliaceae) Aninda Mandal, Animesh K. Datta & Arnab Bhattacharya The Nucleus An International Journal of Cytology and Allied Topics ISSN 0029-568X Volume 54 Number 3 Nucleus (2011) 54:147-152 DOI 10.1007/s13237-011-0044-y 1 23 Your article is protected by copyright and all rights are held exclusively by Archana Sharma Foundation of Calcutta. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Nucleus (December 2011) 54(3):147–152 DOI 10.1007/s13237-011-0044-y ORIGINAL ARTICLE Evaluation of pollen and productive parameters, their interrelationship and clustering of eight Corchorus spp. (Tiliaceae) Aninda Mandal & Animesh K. Datta & Arnab Bhattacharya Received: 5 July 2011 / Published online: 27 December 2011 # Archana Sharma Foundation of Calcutta 2011 Abstract Pollen characteristics namely, pollen fertility (2% acetocarmine), viability (stain tests: Aniline blue, Lugol’s iodine, x-gal and Amido black), pollen production/flower (direct count using improved Neubauer Hemocytometer), pollen nuclei composition (following DAPI staining - 4′,6diamidino-2-phenylindole) and pollen size along with parameters like capsule/plant, capsule length and seed set/ capsule were studied in two cultivated (Corchorus capsularis L. and C. olitorius L.) and six wild (C. aestuans L., C. fascicularis Lamk., C. pseudocapsularis L., C. pseudoolitorius I. and Z., C. tridens L. and C. trilocularis L.) species of Corchorus under the Experimental field conditions of Kalyani University (West Bengal plains; latitude 22°50′–24°11′ N, longitude 88°09′–88°48′ E, elevation 48 ft above sea level, sandy loamy soil, organic carbon 0.76%, soil pH 6.85). Results obtained were statistically analyzed and the information accumulated may provide necessary impetus for formulating efficient breeding strategies and crop improvement. Further, clustering of the species on the basis of the attributes analyzed by UPGMA will be helpful for designing crossing experiments. Keywords Corchorus . Pollen parameters . Stain tests . DAPI . Hemocytometer . UPGMA . Efficient breeding . Genetic diversity A. Mandal : A. K. Datta (*) : A. Bhattacharya Department of Botany, Cytogenetics and Plant Breeding Section, University of Kalyani, Kalyani 741235, West Bengal, India e-mail: dattaanimesh@gmail.com Introduction Corchorus capsularis L. (white jute) and C. olitorius L. (tossa jute) belonging to the family Tiliaceae (2n014) are the important fibre yielding plants (phloem fibre) of commerce (India contributing about 40% of the world production [16]) distributed throughout the warmer regions of the world [19, 25]. Apart from the cultivated members, 8 wild species, namely C. aestuans L., C. depressus (L.) Stocks, C. fascicularis Lamk., C. pseudoolitorius I. and Z., C. tridens L. and C. trilocularis L. C. urticaefolius W. and A. and C. velutinus Her. are also reported from India [21]. The wild germplasms though poor fibre yielder could serve as the important genetic resources for abiotic stress tolerance (C. trilocularis—tolerant genotype to water inundation), disease resistance (C. pseudoolitorius and C. pseudocapsularis— resistant to fungal diseases) and fine fibre (C. aestuans, C. tridens and C. trilocularis) traits [22]. To utilize genetic resources in efficient breeding programs it is important to understand the interrelationships between/among wild and cultivated species. Bots and Mariani [5] were of opinion that hybrid formation rate include parameters like related species, pollen viability, suitable pollination vector, efficiency of seed set, seed viability, hybrid fitness and fecundity.Of these factors, knowledge of pollen viability which is a prerequisite for hybrid production is rather meagre and received relatively little attention [7, 8, 12, 26]. Pollen viability is often equated to the quantity of the pollen produced by a flower, an important component of fitness [18]. Pollen viability can be studied through different techniques [14, 30] but assessment of it following staining technique is advantageous as it is faster, easier and reliable [4, 6], apart from aiming to determine pollen enzymatic activity and membrane integrity [20]. Keijzer et al. [17] developed a Author's personal copy 148 Nucleus (December 2011) 54(3):147–152 method for tracing pollen nuclei using the fluorescent vital deoxyribonucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI), which enable to trace 3 nucleated condition in pollen grains [3, 11]. In the present investigation some pollen characteristics namely, pollen fertility and viability, pollen production/flower, pollen nuclei composition and pollen size were assessed along with parameters like capsule/plant, capsule length and seed set/capsule in 2 cultivated (C. capsularis and C. olitorius) and 6 wild species (C. aestuans, C. fascicularis, C. pseudocapsularis, C. pseudoolitorius, C. tridens and C. trilocularis) of Corchorus under the uniform Experimental field conditions. Clustering of the species by UPGMA was also performed to determine interrelationship among the studied parameters and species which may provide necessary information for designing efficient breeding strategies. Materials and methods The study was conducted with 2 cultivated (C. capsularis— JRC 321, C. olitorius—JRO 524) and 6 wild (C. aestuans— WCIJ 088, C. fascicularis—WCIJ 150, C. pseudocapsularis— CIM 036, C. pseudoolitorius—OIN 507, C. tridens—WCIJ 149 and C. trilocularis—KBA 222) species of jute. Mother seed stock of the studied species were obtained from Central Research Institute for Jute and Allied Fibres (CRIJAF), Nilganj, West Bengal, India. The seeds were sown in the Experimental field plots of Department of Botany, University of Kalyani (West Bengal plains, Nadia; latitude 22°50′–24°11′ N, longitude 88°09′–88°48′ E, elevation 48 ft above sea level, sandy loamy soil, organic carbon 0.76%, soil pH 6.85) during the months of March to August (2009 and 2010). The first formed flowers were assessed at maturity (yellow anthers before anthesis) for pollen fertility and viability, pollen count and pollen nuclei parameters in 5 randomly selected plants of each species for 2 consecutive years (data were pooled over the plants, species and each year). Agro meteorological data (temperature 31.6°C to 39.7°C max., 24.0°C to 26.8°C min., relative humidity 92.2–99.2% max., 56.8–84.7% min., average rainfall 2.08 mm to 18.18 mm) were noted in the months of May to August, during first flowering flash of the studied species (data was obtained from Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal). Pollen fertility and viability For pollen fertility analysis, pollen grains from matured flowers were stained in 2% acetocarmine, and fully stained pollen grains (Fig. 1a) were considered as fertile [24]. Pollen grain viability (Lugol’s iodine—detects the presence of starch, viable pollen turns black, Fig. 1b; Aniline blue in lacto phenol—detects the presence of callose on pollen wall, Fig. 1 Pollen photomicrographs showing fertility, viability and nuclei composition in Corchorus spp. (a). Fertile (uniformly stained) and sterile pollen grains (weak acetocarmine staining); (b-e). Viable (b stains black, Lugol’s iodine; c stains blue, Aniline blue; d stains green, x-gal; e stains black; Amido black) and non-viable pollen grains; (f-j). DAPI stained pollen grains (f 1v+2 g; g 0v+2 g; h 1v+1 g; i 1v+0 g; j 1v+3 g; v - vegetative nuclei, g - generative nuclei) Scale bar 0 20 μm Author's personal copy Nucleus (December 2011) 54(3):147–152 149 viable pollen turns blue, Fig. 1c – [2]; x-gal - 5-bromo-4chloro-3-indolyl-β-D-galactopyranoside - detects the presence of β-galactosidase, viable pollen turns green, Fig. 1d – [1]; Amido black - detects the presence of protein in pollen wall, viable pollen turns black, Fig. 1e – [27]) was analyzed following the application of staining technique. Further, pollen size in μm (polar diameter was considered after acetolysis—[9]); capsule length (cm), capsule number/plant, seed set/capsule and seed size (mm2) were also studied in the species. Quantification of pollen grains Direct count of pollen grains were made from pollen suspension (10 anthers squashed in 50 μl of distilled water for each species) using a Hemocytometer (improved Neubauer), counted (3 replications/species, 10 observations/replication) using a light microscope (Olympus CH 20i; 10x×40x) and calculated from the formula c 0 n×104/ml (c 0 cell concentration in cells/ml, n 0 average number of cells/mm2) as per Mather and Roberts [23]. DAPI staining DAPI (4′, 6-diamidino-2-phenylindole, a fluorescent vital stain binding DNA—[31]; and RNA—[13]) staining of pollen grains were performed as per the methodology described by Johnson and McCormick [15] to assess pollen nuclei composition (3 nucleate stage; v - vegetative nuclei, g - generative nuclei) per pollen grain (Figs. 1f-j). Observations were recorded under fluorescence microscope (Carl Zeiss Fig. 2 Dendogram showing clustering of 8 Corchorus spp. following UPGMA Axio fluor 900EX, Carl Zeiss MagAnalytic 10.1; DAPI excitation range 350–360 nm, emission maximum 460 nm). Statistical analysis χ2 test of heterogeneity was performed at 7 DF (degree of freedom) for different parameters namely, pollen fertility, pollen viability (pooled over the stain tests for a species), pollen grains/flower, anther/flower, pollen area, pollen nuclei composition, capsule length, capsule/plant and seed set/fruit among the species to assess significant variations, if any, as well as in each species for different stain tests. Further, Pearson’s correlation coefficient analysis (DF07) between the traits were also conducted to study interrelationship considering each of the species as independent variables. UPGMA analysis Eighty one discrete parameters were considered from the studied attributes. Their presence (1) and absence (0) in 8 jute species were scored and entered in a binary matrix. Based on the analysis, proximity matrix was generated for all possible pairs from Squared Euclidean Distance and used to construct a dendogram (Fig. 2) by the Unweighted Pair Group Method with Arithmetic mean (UPGMA) using the software IBM SPSS statistics (V. 19, 2010). Results and discussion Different parameters studied in Corchorus spp. are presented in Table 1. Heterogeneity (p<0.001) was noted for Dendogram using Single Linkage Rescaled Distance Cluster Combine 0 C. capsularis C. olitorius C. aestuans C. tridens C. pseudocapsularis C. pseudoolitorius C. trilocularis C. fascicularis 5 10 15 20 25 150 Table 1 Pollen, capsule and seed parameters in Corchorus spp. Attributes C. capsularis Pollen 84.29 fertility (%) Pollen viability (%) C. olitorius C. aestuans C. fascicularis C. pseudocapsularis C. pseudoolitorius C. tridens C. trilocularis 81.18 60.28 56.67 62.03 63.92 70.19 37.67 56.28 61.25 43.25 50.23 46.34 64.12 64.98 59.83 Aniline blue 65.90 69.76 52.54 48.67 52.24 49.59 52.35 28.82 x-gal 62.73 56.24 50.92 51.15 44.19 60.26 61.76 35.64 Amido black 62.77 59.64 35.63 40.53 58.71 50.41 70.53 41.61 30±0.01 15±0.01 10±0.01 19±0.01 16±0.01 15±0.01 29±0.01 12210 3600 4940 5282 8352 5295 10150 56.74±0.32 49.64±0.52 42.50±0.37 49.30±0.22 40.46±0.15 52.36±0.33 46.24±0.22 Anther 10±0.01 number/flower Pollen quantity/ 3590 flower Pollen size (μm) 41.48±0.16 Author's personal copy Lugol’s iodine Pollen nuclei composition (%) 70.29 73.90 90.27 14.81 39.32 40.68 7.90 38.76 0v + 2 g 0.00 0.77 0.24 3.40 22.17 24.19 10.57 15.12 1v + 1 g 7.28 14.10 2.61 62.75 21.92 17.17 73.26 24.28 1v + 0 g 6.21 6.72 4.86 8.70 15.27 13.82 6.64 20.87 v-dispersed 11.69 3.30 1.42 0.24 2.12 2.93 1.12 0.75 1v + (2-4)g 4.53 1.21 0.59 9.52 1.20 0.21 0.00 0.21 Capsules/plant 41.40±3.20 16.80±0.30 274.20±15.60 302.40±8.40 96.50±6.90 263.80±5.20 50.30±4.20 98.80±4.60 Capsule length (cm) Seed-set/fruit 0.90±0.10 8.20±1.20 1.90±0.10 2.70±0.20 0.60±0.40 7.00±0.20 2.20±0.10 7.60±0.10 76.40±0.90 174.12±3.80 77.44±0.60 92.60±2.40 35.40±2.60 93.02±1.60 110.20±1.40 129.20±1.60 Seed size (mm) 2.83 ±0.06×1.93 ±0.07 2.02 ±0.05×1.31 ±0.04 1.23 ±0.05×0.83 ±0.03 1.26 ±0.05×0.95 ±0.03 1.18±0.05×0.95±0.03 1.16 ±0.06×0.92 ±0.03 0.92 ±0.06×0.64 ±0.03 1.12±0.06×0.74±0.03 v vegetative nuclei; g generative nuclei; p value <0.001 for χ2 test (DF07) of heterogeneity (for all parameters among the species). Nucleus (December 2011) 54(3):147–152 1v + 2 g Author's personal copy Nucleus (December 2011) 54(3):147–152 151 each parameter thereby suggesting a wide range of interspecific variations. Pollen fertility was higher in cultivated species (81.2–84.3%) than the wild members (37.7– 70.2%). Stain tests (data of stain tests pooled over each species: C. capsularis—61.9%, C. olitorius—61.7%, C. aestuans—45.6%, C. fascicularis—47.7%, C. pseudocapsularis—50.4%, C.pseudoolitorius—56.1%, C. tridens—62.4% and C. trilocularis—41.5%) assessed over the species was random (χ200.79–3.9, DF03, p>0.05) as evidenced from χ2 test of heterogeneity, excepting for C. trilocularis (χ20 12.81, DF03, p<0.01) . Such uniformity is rather unique and reflects possibly coherent relationship of the biochemical constituents. However, Bolat and Pirlak [4] reported that pollen viability differed with respect to stain tests in some stone fruits. Pollen viability was studied in the species at the onset of anthesis; although first flowering flash varied among them. Environmental factors [5, 10] were reported to influence pollen viability under field conditions. Nuclei composition in pollen grains (Figs. 1f-j) analyzed following DAPI staining and upon considering 1v+2 g (Fig. 1f), 0v+2 g (vegetative nuclei already degenerated; Fig. 1g) and 1v + 1 g (2nd division yet not completed; Fig. 1h) as near normal associations, was found to range between 77.6% (C. capsularis) and 91.8% (C. tridens) among the species. Nuclei frequency in pollen grains was significant and negatively correlated with Lugol’s iodine (r0−0.84, p<0.01), Aniline blue (r0−0.88, p<0.01) and Amido black (r0−0.66, p<0.05). Results of DAPI stained pollen grains indicated that probably optimum balance of the metabolites is essential for pollen development. Pollen viability and pollen nuclei composition are significant components for pollen germination. Bolat and Pirlak [4], Davarynejad et al. [6], Ferri et al. [10], Lyra et al. [20] amongst others were of opinion that pollen germination in in vitro is an important criteria for reproductive biology in different plant species. Sedgley and Griffin [28] suggested that pollen germination is controlled by the vegetative nucleus and may occur even in the absence of a viable generative nucleus. However, such condition may not be appropriate for proper fertilization and subsequently seed setting. Results of correlation analysis between attributes of Corchorus spp. are presented in Table 2. It is evident from correlation studies that anther number/flower, pollen quantity/flower and capsule length are important criteria for seed setting. Anther number/flower is positively and significantly correlated with pollen quantity/flower and capsule length; while, pollen quantity and capsule length are also interrelated between themselves. Therefore, these traits seem to be important components for reproductive outcome. Pollen size seems not to be interrelated with any of the studied parameters; although, Sharma et al. [29] reported positive and significant association between pollen size and viability in Saccharum officinarum and S. spontaneum clones. Present investigation also revealed that pollen fertility and pollen viability were positively and significantly associated between themselves, thereby indicating validity of acetocarmine staining technique for estimation of pollen fertility. Clustering of the species following UPGMA (Fig. 2) using the studied attributes revealed a close proximity among C. capsularis, C. olitorius and C. aestuans and when the 3 species were considered together they showed positive and significant correlation (DF02) between different traits namely, pollen viability with fertility (r00.955, p<0.05), capsule/plant (r0−0.992, p<0.01) and seed/fruit (r00.960, p<0.05); pollen quantity/flower with seed/fruit (r00.953, p<0.05) and seed/fruit with capsule/plant (r00.961, p< 0.05). This result is nearly in accordance to the result obtained when each of the species was considered as independent variable (Table 2). Further, C. pseudocapsularis, C. pseudoolitorius, C. fascicularis and C. trilocularis also seem to be interrelated among themselves. The present investigation underpinn that pollen parameters specifically the anther number/flower, pollen quantity/flower, capsule length and seed set/capsule are Table 2 Correlation analysis between attributes of Corchorus spp. Attributes Pollen fertility Pollen viability Anther number/flower Pollen quantity/flower Pollen size Pollen nuclei composition Capsule number/plant Capsule length Pollen fertility Pollen viability 0.82* Anther number/flower Pollen quantity/flower −0.24 −0.15 −0.12 0.04 0.88* *, ** and *** significant at 0.05, 0.01 and 0.001 probability level respectively Pollen Pollen nuclei Capsule size composition number/plant 0.21 0.31 0.58 0.36 0.22 −0.63 0.28 0.12 0.11 −0.43 −0.49 −0.45 0.45 −0.46 0.48 Capsule length Seed set/capsule −0.22 −0.55 0.75* 0.94* 0.20 −0.11 −0.07 0.09 −0.49 0.66* 0.80* 0.06 −0.14 −0.32 0.81* Author's personal copy 152 important components in jute species for effective hybridization but it would be rather logical to assess other related parameters along with the pollen attributes for efficient breeding. Acknowledgement The Research is grant aided by University Grants Commission (India). Nucleus (December 2011) 54(3):147–152 13. 14. 15. References 16. 1. Atiaksheva LF, Pilipenko OS, Poltorak OM. Mechanism of the thermoinactivation of the β-galactosidase from Escherichia coli. In: International Symposium, Biocatalysis-2000: undamentals & Applications, Lomonosov Moscow State University, Khimiya, Moscow, Russia. 2000;41(No.6. Supplement):95-97. www.chem.msu.su/ eng/journals/vmgu/00add/95.pdf. Accessed 30 November 2011. 2. Bengtsson, S. Evaluation of transgenic Campanula carpatica plants. In: Epsilon Undergraduate Theses Archive: vol. 6. Dept. of Agricultural Sciences, The Royal Veterinary and Agricultural University. 2006. http://ex-epsilon.slu.se:8080/archive/00001970/. Accessed 28 March 2011. 3. Bhattacharya A, Datta AK. A report on genetic male sterility in Uraria picta (Jacq.) DC. Cytologia. 2011;76:55–62. 4. Bolat I, Pirlak L. An investigation on pollen viability, germination and tube growth in some stone fruits. Turk J Agric For. 1999;23:383–8. 5. Bots M, Mariani C. Pollen viability in the field. In: COGEM report, Radboud Universiteit Nijmegen, Netherlands. 2005. http:// www.cogem.net/showdownload.cfm?objectId0FFFE6554-151764D9-CC081ED9E42EF28C&objectType0mark.apps.cogem. contentobjects. publication.download.pdf. Accessed 04 April 2011. 6. Davarynejad GH, Szabo Z, Nyeki J, Szabo T. Phenological stages, pollen production level, pollen viability and in vitro germination capability of some sour cherry cultivars. Asian J Plant Sci. 2008;7:672–6. 7. de Vries FT, van der Meijden R, Brandenburg WA. Botanical files: a study of the real chances for spontaneous gene flow from cultivated plants to the wild flora of the Netherlands. Gorteria Supplement. 1992;1:1–100. 8. Eijlander R, Stiekema WJ. Biological containment of potato (Solanum tuberosum): outcrossing to the related wild species black nightshade (Solanum nigrum) and bittersweet (Solanum dulcamara). Sex Plant Reprod. 1994;7:29–40. 9. Erdtman G. Pollen Morphology and Plant Taxonomy: Angiosperms (An Introduction to Palynology). The Chronica Botanica Co./Almqvist & Wiksell: 1952. 10. Ferri A, Giordani E, Padula G, Bellini E. Viability and in vitro germinability of pollen grains of olive cultivars and advanced selections obtained in Italy. Adv Hortic Sci. 2008;22:116–22. 11. Ghosh BK, Datta AK, Mandal A, Paul R. Cytological and palynological aspects and andrographolide content in Andrographis paniculata. J Trop Med Plants. 2011;12:71–6. 12. Groot MHM, van de Wiel CCM, van Tienderen PH, den Nijs HCM. Hybridisation and introgression between crops and wild relatives. In: Current knowledge and research priorities in lieu of impending introductions of GM crops: COGEM research report. University of Amsterdam & Plant Research International, 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Amsterdam & Wageningen. 2003. http://www.nwo.nl/files.nsf/ pages/NWOA_6KNGVQ/$file/03%20COGEM%20-% 20Uitrkuising%20-%20CGM2003-02.pdf. Accessed 05 April 2011. Hard T, Fan P, Kearns DR. A fluorescence study of the binding of Hoechst 33258 and DAPI to halogenated DNAs. Photochem Photobiol. 1990;51:77–86. Heslop-Harrison J, Heslop-Harrison Y, Shivanna KR. The evaluation of pollen quality, and a further appraisal of the fluorochromatic (FCR) test procedure. Theor Appl Genet. 1984;167:367–75. Johnson S, McCormick S. Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis thaliana gametophytic mutant. Plant Physiol. 2001;126:685–95. Karmakar PG, Hazra SK, Ramasubramanian T, Mandal RK, Sinha MK, Sen HS, editors. Jute and Allied Fibre Updates: Production, & Technology. CRIJAF, Brrackpore, India. 2008. Keijzer CJ, Reinders MC, Leferink-ten Klooster HB. A micromanipulation method for artificial fertilization in Torenia. In: Cresti M, Jori P, Pacini E, editors. Sexual Reproduction in Higher Plants. New York: Springer; 1988. p. 119–24. Kelly JK, Rasch A, Kalisz S. A method to estimate pollen viability from pollen size variation. Am J Bot. 2002;89:1021–3. Kundu BC. Origin of Jute. Indian J Genet Plant Breed. 1951;2:95– 9. Lyra DH, Sampaio LS, Pereira DA, Silva AP, Amaral CLF. Pollen viability and germination in Jatropha ribifolia and Jatropha mollissima (Euphorbiaceae): Species with potential for biofuel production. Afr J Biotechnol. 2011;10:368–74. Mahapatra AK, Saha A, Basak SL. Origin, taxonomy and distribution of Corchorus species in India. J Green. 1998;1:64–82. Mahapatra AK, Saha A. Genetic resources of jute and allied fibre crops. In: Karmakar PG, Hazra SK, Ramasubramanian T, Sinha MK, Sen HS, editors. Jute and allied fibres updates: production and technology. Brrackpore: CRIJAF; 2008. p. 18–37. Mather JP, Roberts, PE. Introduction to cell and tissue culture: Theory and Technique. 1st ed. Springer; 1998. Marks GE. An aceto-carmine glycerol jelly for use in pollenfertility counts. Biotechnic Histochem. 1954;29:277–8. Purseglove JW. Tropical Crops: Dicotyledons 2. Longmans: Green; 1968. Ramsay G, Thompson C, Squire G. Quantifying landscape-scale gene flow in oilseed rape. In: Final Report of DEFRA Project RG0216: An experimental and mathematical study of the local and regional scale movement of an oilseed rape transgene. Scottish Crop Research Institute, Dundee: DEFRA. 2003. http://www.scri.ac.uk/scri/file/EPI/ Agroecology/Landscape_scale_geneflow_in_oilseed_rape_rg0216. pdf. Accessed 30 June 2011. Regan SM, Moffatt BA. Cytochemical analysis of pollen development in wild-type Arabidopsis and a male-sterile mutant. Plant Cell. 1990;2:877–89. Sedgley M, Griffin AR. Sexual Reproduction of Tree Crops. Academic Press: 1989. Sharma MLP, Singh ML. Floral behaviour in Saccharum officinarum hybrids and S. spontaneum clones. Indian J Agr Sci. 1996;66:455–8. Stanley RG, Linskens HF. Pollen: biology, biochemistry, management. 1st ed. Springer; 1975. Willemse MTM, Keijzer CJ. Tracing pollen nuclei in the ovary and ovule of Gasteria verrucosa (Mill.) H. Duval after pollination with DAPI-stained pollen. Sex Plant Reprod. 1990;3:219–24.