Quantitative and Qualitative Analyses of Genus Pyrus L. ( Tree) in West Azerbaijan Province, Iran

Hedieh Karimi, Bahman Eslami, Abbas Ali Dehpouri & Ali Shokuhi Rad

Iranian Journal of Science and Technology, Transactions A: Science

ISSN 1028-6276 Volume 43 Number 3

Iran J Sci Technol Trans Sci (2019) 43:715-724 DOI 10.1007/s40995-017-0460-1

1 23 Your article is protected by copyright and all rights are held exclusively by Shiraz University. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.

1 23 Author's personal copy

Iran J Sci Technol Trans Sci (2019) 43:715–724

https://doi.org/10.1007/s40995-017-0460-1 (0123456789().,-volV)(0123456789().,-volV)

RESEARCH PAPER

Quantitative and Qualitative Analyses of Genus Pyrus L. (Pear Tree) in West Azerbaijan Province, Iran

1 1 2 3 Hedieh Karimi • Bahman Eslami • Abbas Ali Dehpouri • Ali Shokuhi Rad

Received: 16 October 2017 / Accepted: 6 December 2017 / Published online: 22 December 2017 Ó Shiraz University 2017

Abstract Pollen morphology of Pyrus L. spp. genus from West Azerbaijan Province of Iran using light microscope and scanning electron microscope was studied. The species under study included , , , and . The measured characteristics were longitudinal axis, transverse axis, length-to-width ratio, depth of groove or pore, shapes, and decorations of wall, and size and type of pollen grain (number of tracks). The images and details of the internal structure of all these four pollen species are provided comprehensively. The results showed that the depth of groove or pore was in the range of 1.0–3.12. The pollen species were small or medium-sized and the most common type among them was type III. The pollen grains of these species were symmetrical with three tracks and their wall decorations were striate and striate and micro-reticulate.

Keywords Pollen grain Á Morphology Á Pyrus genus Á family Á West Azerbaijan Province

1 Introduction Lorestan, and Kohgiluyeh and Boyer Ahmad Provinces are the main producers of this product (Khatamsaz 1992). This Pyrus L. (pear) genus belongs to the family of Rosaceae genus and its variants have a wide variety in germplasms and the subfamily of Maloideae. Pear trees are native to and there are about 21–26 primary species throughout the Europe and Western Asia. Pear is the third most important world. In general, it can be said that it is difficult to temperate region fruit species after apple (Wu¨nsch and mention a precise amount for the different varieties of pear Hormaza 2007). Size, taste, and appearance are the vari- throughout the world, because the are easily crossed eties in this fruit that are its outstanding properties that are with each other and the obtained crosses can be placed in less observed in the other fruit species (Abdollahi 2009). various taxonomic categories. On the other hand, all Pyrus Gilan, Mazandaran, Fars, Isfahan, Azerbaijan, Kurdistan, variants have self-incompatible gametophytes system. Thus, there is no inconsistent barrier for interbreeding in this genus (Abdollahi 2009). The first steps to identify the & Abbas Ali Dehpouri [email protected] genetic resources are evaluating the morphological char- acteristics of genetic resources and collecting desirable & Ali Shokuhi Rad [email protected] traits in a variety (Poralijan and Rad 2016; Mohseni and Rad 2017). This is an easy, cost-effective, and available Hedieh Karimi shimifi[email protected] method, and provides a general view on the germplasm to breeders (Li et al. 2009). What have encouraged botanists Bahman Eslami [email protected] to investigate pollen grains in the studies are some unique features of the pollen grain that make it 1 Department of Biology, Islamic Azad University, Ayatollah superior to other biomaterials including the following. Amoli Branch, Amol, Iran – The existence of a tight outer cover (exine) that makes 2 Department of Biology, Islamic Azad University, Qaemshahr Branch, Qaemshahr, Iran them survive better and longer than many other biomaterials. 3 Department of Chemical Engineering, Islamic Azad University, Qaemshahr Branch, Qaemshahr, Iran 123 Author's personal copy

716 Iran J Sci Technol Trans Sci (2019) 43:715–724

– Diversity in shape and decorations of these resistant on each tube. Cytolysis conventional method was per- covers that has a lot of taxonomic values. formed to wash pollen grains and permanent slides were – Pollen grains are generated in large numbers that solve prepared (Li et al. 2009). the problem of sample abundance. An ocular micrometer performed measurements. For – Useful data can be obtained only through studying a each species, the parameters of longitudinal axis, trans- very small sample. Such properties are rarely seen in verse axis, and length-to-width ratio were recorded. For other biologically tested species. Thus, palynology has any species, a healthy, mature, and completely dried bud had special position in the taxonomy (Ishimizu was isolated from a suitable sample. On the microscopic et al. 1999). Iran has been recognized as one of the slide, at first, the flags were separated from the other parts most important genetic resources of pear in the world of the flowers and then the pollen grains were removed with over ten species and proximity to pear centers from the anthers. The surface of the pollen grains was (Khatamsaz 1992). According to the World Grocer covered with special double-sided adhesives. To prevent Organization, the global pear production in 2011 was mistakes, on the other side of the samples, the number about 24 million tons and Iran had a share of 145 labels were affixed and a specific number was assigned to thousand tons (FAO 2014). West Azerbaijan Province each species. The samples were placed in Sputter JFC 1100 has been considered as important due to the high level for 30–45 min to create vacuum, keep the sample dry, of production and different varieties of the species avoid wrinkles of samples, and also to sprinkle gold dust resulting in differences in the morphology of the pollen with a thickness of 100–120 A˚ on the pollens. The sample of this genus. The presence of the majority of fruit trees pollen grains covered with gold dust were placed in the in the Iranian traditional gardens has caused a lot of scanning electron microscope, Scanning Micro Analyzer genetic variations, which may lead to specific com- JEOL JXA-840 model, and on the dusts were observed on mercial or breeding properties. Studies have been the monitor screen. Statistical calculations including anal- conducted on the genetic variation of this fruit (Elshihy ysis of variance, and mean comparisons of traits (Duncan et al. 2004; Karadeniz and Sen 1990; Davoodi 1998; test) were performed and the coefficients were calculated Katayama and Uematsu 2006; Khoshghalb 2001). using the SPSS software. However, there is still no accurate information on the condition of pollen grains and genetic studies in this field. Therefore, this study was conducted in West 3 Results and discussion Azerbaijan attempting to provide quantitative and qualitative data on the morphology of the pollen grains Based on Table 1 and LM and SEM images (see Figs. 1, of Pyrus L. spp. and their differences. 2), it can be concluded that pollen grains of four species including Pyrus amygdaliformis, Pyrus syriaca, and Pyrus salicifolia, and Pyrus communis in Pyrus genus have usu- 2 Materials and methods ally symmetrical radius and are iso-polar (homo polar), i.e., the features of the near and far poles are similar. They have three pores and are normally tricolporate. The pollen grains In this study, pollen grains of four genus including Pyrus diameter in the 20 samples and two studied species of syriaca, Pyrus amygdaliformis, Pyrus salicifolia, and Pyrus Pyrus amygdaliformis and Pyrus syriaca from Marmisho communis were collected from Marmisho, Sardasht, Pirgol, region range from 26.69–26.52 to 26.50–35.61 lm. This and Kahriz regions in West Azerbaijan Province from 2016 diameter for the 35 samples of the Pyrus salicifolia species to 2017. To study the pollen grains, at first, the anthers from Pirgol region is 26.76–40.21 lm. For the 30 samples were removed from the perfect flowers that had mature of the Pyrus communis species from Sardasth forests, it is and anthers. After preparing all the samples by 22.61–37.86 lmL, and for the 80 samples of the Pyrus Cytolysis procedure, the samples were carefully investi- communis species from Kahriz region, the diameter is gated in terms of their shapes and sizes and other param- 21.86–40.88 lm. The pollen wall decorations in most eters using light and electron microscopes. The model of samples have striate or long exines separated with grooves. the electron microscope was Scanning Micro Analyser In some samples such as Pyrus syriaca from Marmisho and JEOL JXA-840. From each species, then, a healthy flower some varieties from Kahriz region such as Abate fetal and bud was chosen. The flower bud was boiled for about Pachens Tricumph, the wall decorations were striate micro- 3 min in water, and then with the help of a dissection reticulate (see Fig. 1, nos. 14 and 15). Moreover, the wall needle, the anthers were removed and were separated from decorations were irregular and striate in Kaiser variety in the other parts of the flower. Then, breaking the anthers Kahriz (see Fig. 1, no 11). A series of gemmate redun- drew out the pollens. The species names labels were pasted dancies were also observed on the wall of Butirra variety 123 rnJSiTcnlTasSi(09 43:715–724 (2019) Sci Trans Technol Sci J Iran Table 1 Results of analysis of different systems: region, number of population, transverse axis, the longitudinal axis, length-to-width, depth of pore ratio, shapes of wall, decorations of wall, and type of pollen grain Species and variety Region Number of The longitudinal Transverse Length-to-width Depth of Size Shapes of Decorations of wall Type of pollen grain population axis or P (lm) axis or ratio P/E (lm) groove or pore wall (number of tracks) E (lm) (lm)

Pyrus syriaca Marmisho 8 26.69–26.52 23.90–24.19 1.10–1.14 1.49–1.78 Medium Irregular Striato- 3 shape microreticulate Pyrus Marmisho 12 26.50–35.61 25.33–32.04 1.04–1.11 1.67–1.84 Medium Irregular Striate 3 amygdaliformis shape Pyrus salicifolia Pirgol 35 2.76–40.21 18.89–28.02 1.21–1.91 1.15–1.68 Medium Irregular Striate 3 shape Pyrus communis Sardasht 30 22.61–37.86 17.26–24.65 1.30–1.53 1.97–2.36 Medium Irregular Striate 3 (1) shape Pyrus communis var., Kahriz 5 23.09–24.55 22.06–22.68 1.0–1.04 1.58–1.68 Small Irregular Striate 3 Author's Tashkandia shape Pyrus communis var., Kahriz 5 23.04–24.04 20.55–21.08 1.02–1.09 1.48–1.60 Small Irregular Striate 3 Dargazia shape Pyrus communis var., Kahriz 5 22.67–24.04 19.18–22.10 1.06–1.10 1.44–1.60 Small Irregular Striate 3 Sebria shape Pyrus communis var., Kahriz 5 22.67–24.01 20.53–21.43 1.04–1.06 1.50–1.63 Small Irregular Striate 3 personal sardroodia shape Pyrus communis var., Kahriz 5 23.67–24.06 21.68–22.17 1.02–1.08 1/68–1/50 Small Irregular Striate 3 Shah Miveha shape Pyrus communis var., Kahriz 5 21.33–21.89 19.87–20.67 1.01–1.04 1.00–1.50 Medium Irregular Striate 3 Natanzia shape copy Pyrus communis var., Kahriz 5 30.87–31.02 30.90–30.97 1.00–1.04 1.90–1.99 Medium Irregular Striate with 3 Kaisera shape Fossulate or Irregular shape Pyrus communis var., Kahriz 5 36.89–37.27 19.60–20.16 1.80–1.84 1.40–1.44 Medium Irregular Striato- 3 Conference shape microreticulate Pyrus communis var., Kahriz 5 35.67–36.44 28.78–29.08 1.20–1.25 2.0–2.08 Medium Irregular Striate 3 General Licera shape Pyrus communis var., Kahriz 5 33.88–34.10 26.89–27.0 1.22–1.26 3.00–3.12 Medium Irregular Striato- 3 Abate Fetala shape microreticulate Pyrus communis var., Kahriz 5 25.87–26.36 23.34–24.95 1.00–1.05 2.44–4.57 Medium Irregular Striato- 3 Pachens Tricumpha shape microreticulate Pyrus communis var., Kahriz 5 33.55–35.85 19.54–20.60 1.70–1.74 1.60–1.62 Medium Irregular Striato- 3 Narta shape microreticulate Pyrus communis var., Kahriz 5 31.70–31.73 27.48–27.52 1.10–1.15 1.60–1.63 Medium Irregular Striato- 3 a 123 Decantel shape microreticulate Pyrus communis var., Kahriz 5 25.37–25.40 23.90–23.88 1.00–1.05 1.38–1.40 Medium Irregular Striato- 3 William Rossea shape microreticulate 717 Author's personal copy

718 Iran J Sci Technol Trans Sci (2019) 43:715–724

(no. 18). The studied species were mostly small-to-med- ium-sized among which the smallest grain belonged to the varieties of Pyrus communis in Kahriz region including Tashkandi, Dargazi, Sibri, Sardroodi, and Shahmiveh with polar axis of 21.33–23.09 lm and equatorial axis of (number of tracks) 5 3 1.18 lm (nos. 5–9), and the biggest grain belonged to the variety of Butirra from the Pyrus communis species in Kahriz with polar axis of 40.88–41.82 lm and equatorial and Anjo (the dominant kinds axis of 39.66–40.62 lm (no. 18). Regarding the P/E ratio … in all of the studied species, the pollen grains were tinian)

ortant kinds of native are Shah Miveh, observed to have triangular to round shapes with raised Bartlet), gemmate microreticulate pores in three corners, which are thick in the middle of Decorations of wall Type of pollen grain Pentaorate with Striato- Lumen (see LM images in Fig. 2). There are two known classes in the pollen shape including the stretched shape with the P/E ratio of 1.02–1.00 in the P/E and a small

shape shape change with the P/E ratio of 1.74–1.70 (types III and V). wall The greatest wall thickness was observed in the equatorial axes and margins. The thickness of the exine wall was 1 lm in all species and all their shapes were irregular Size Shapes of except for Butirra variety from Kahriz that had spheroidal shape. However, all the pollen grains were homologous in other spices and genus, and there are often large discrep- ancies in the structure and decoration of the pollen grains. m)

l The research has shown that the pollen grains are geneti- Depth of groove or pore ( cally much better for hybridity and introducing different

m) kinds of trees and other . l ( On the other hand, geographical differences and species E /

P dispersal can make differences (Zamani and Attar 2010). The obtained results from the present study are consistent Length-to-width ratio with the previous ones (Zamani and Attar 2010). Introducing and classification of fruit trees are difficult and complex. The environmental and agricultural factors m)

l can affect phenotypic changes and that makes the intro- (

Transverse axis or E ducing of the plant unreliable. The characteristics of the classification existing in pollen grains include morphology, m)

l polarity, symmetry, shape, and their size. In some genuses, (

P there are not any differences, but in some ones, there are some differences (Li et al. 2002).

The longitudinal axis or The research on pollen grains of pear has shown that there are many differences among different kinds, and the existing differences may be the relation of type and development (Javady and Arzani 2001).

population The research results show that the differences on pollen grains can be used to classify and determination of its origin and evolutionary of different types, and epidemiol- ogy has allotted the specific place in studying plant clas- KahrizKahriz 5 5 40.88–41.82 21.89–22.42 39.66–40.62 1.00–1.02 19.99–20.06 1.09–1.11 2.40–2.41 1.00–1.04 Medium Spheroidal Medium Irregular sification (Lanza et al. 1996). This research confirms the results of Zhang et al. (1999). var., var., They reported that there is a difference between pear cul- tivars in terms of polar length (P) and equatorial width a (continued) a (E) and ratio P to E. According to their reports, the length of the polar axis Butirra William There are a great number of different kinds of pear existing in Iran. Some of these pears are native (internal) and some others are external. The most imp Pyrus communis Pyrus communis of native pears in Iran are Shah Miveh, Natanzi, Sardorudi, Sabri, and Dargazi, and immortal types are Williams, Dushes, Espaduna, Beiruti, and Palen Natanz, Se Fasle, Sabri, Ghossi, Shakari, Hosseini, Dom Kaj, Dargazi, Sardorud or Peighambari .Some external pesrs cultivated in Iran are Williamz ( Table 1 Species and variety Region Number of a varied from 37.5 to 50 lm and the tropical width varied 123 Author's personal copy

Iran J Sci Technol Trans Sci (2019) 43:715–724 719

Fig. 1 SEM micrographs from pollen grain of Pyrus L. spp

123 Author's personal copy

720 Iran J Sci Technol Trans Sci (2019) 43:715–724

Fig. 1 continued from 20.8 to 25 lm, and pollen grains were of the tricol- In parallel to the result of Zhou et al. (2000), research porate type. They also had lace-shaped decorations with a conducted by Eti (1996) on pear showed that the penetration in their surface, and all pollen was of the pollen shape varies from subspherodial to prolate, and prolate spheroid type. pollen is of the tricolporate type. They also concluded that differences in the level of approach can be used to classify

123 Author's personal copy

Iran J Sci Technol Trans Sci (2019) 43:715–724 721

Fig. 1 continued and determine the origin and evolution of different species many reports about the ability to use the results of these and their cultivars. Pollen surveys have been carried out by studies in breeding and ranking. scanning electron images in many plants, and there are

123 Author's personal copy

722 Iran J Sci Technol Trans Sci (2019) 43:715–724

Fig. 1 continued

4 Conclusion much better for hybridity and introducing different kinds of trees and other plants. It can be concluded that variations and differentiations are The pollen grains which are classified in most taxons are observed in various species and genus which is due to the so similar. By studying the characteristics of morphology hybridization among the individuals of a species and the and the characteristics like polar axis, and tropical width distribution among them. Therefore, the observed similar- and the proportion of polar axis and tropical width, one can ity of the studied pollen can be justified accordingly. The obtain the differences among different races or types. research has shown that the pollen grains are genetically

123 Author's personal copy

Iran J Sci Technol Trans Sci (2019) 43:715–724 723

Fig. 2 LM images and triangular grains (1–9) References Elshihy OM, Sharaf AN, Muzher BM (2004) Morphological, anatomical and biochemical characterization of Syrian pear (Pyrus syriaca Boiss) genotypes. Arab J Biotechnol Abdollahi H (2009) Pear tree, planting to harvest. Extension Service 7(2):209–218 of Agricultural Organization of Tehran Province, Tehran, p 10 Eti S (1996) Investigation on the fertilization biology of some foreign (in Persian) pear cultivars. Bache 25(1–2):11–19 Davoodi A (1998) Evaluation of resistance of some apple and pear FAO (2014) FAOSTAT, FAO Statistical Databases (Food and cultivars to fire blight. MSc. Thesis, College of Agriculture, Agriculture Organization of the United Nations). http://www. Tabriz University, Tabriz, p 200 (in Persian) fao.org/ag/agl/agll/spush/. Accessed 25 Jan 2014

123 Author's personal copy

724 Iran J Sci Technol Trans Sci (2019) 43:715–724

Ishimizu T, Inoue K, Shimonaka M, Saito T, Terai O, Norioka S Li P, Wang Y, Sun X, Han J (2009) Using microsatellite and (1999) PCR-based method for identifying the S-genotypes of morphological markers to asses the genetic diversity 12 falcata Japanese pear cultivars. Theor Appl Genet 91:691–698 (Medicago sativa spp. falcate) population from Eurasia. Afr J Javady T, Arzani K (2001) Pollen morphology of five Iranian olive Biotehnol 8(10):2102–2108 (Olea europaea L.) cultivars. J Agric Sci Technol 3:37–42 Mohseni S, Rad AS (2017) Determination of compositions of Thymus Karadeniz T, Sen SM (1990) Morphological and pomological pubescens; the comparison of different solvents towards extrac- properties of pears grown in Tirebolu and vicinity. J Fac Agric tion. Iran J Sci Technol A. https://doi.org/10.1007/s40995-017- (Hokkaido University) 1:152–165 0159-3 Katayama H, Uematsu CH (2006) Pear (Pyrus species) genetic Poralijan V, Rad AS (2016) Extraction of eugenol from carnation: a resources in Iwate, Japan. Genet Resour Crop Evol 53:483–498 quantitative and qualitative analysis by aqueous and ethanolic Khatamsaz M (1992) Rosacea family, flora of Iran, 1st edn. Iranian solvents. J Essent Oil Bear Plants 19(2016):1495–1502 Research Organization of Forests and Pastures, Tehran (in Wu¨nsch A, Hormaza JI (2007) Characterization of variability and Farsi) genetic similarity of European pear using microsatellite loci Khoshghalb H (2001) Study on early growth, performance and developed in apple. Sci Hortic 113:37–43 survival of Asian pear cultivars (Pyrus serotina Rehd) on Zamani A, Attar F (2010) Pyrus longipedicellata sp. nov. (Rosaceae) European Pear (Pyrus communis L.) seedling rootstock under from Central Alborz, Iran. Nord J Bot 28:484–486 environmental condition. MSc. Thesis, Tarbiat Modares Univer- Zhang Y, Li R, Zhang YY, Li RT (1999) Studies on the local pear sity, Tehran (in Farsi) resources of Hunan Province: I. SEM observation of pollen Lanza B, Marsilio V, Martinelli N (1996) Olive pollen ultrastructur- morphology of Baojin local pear. Acta Scientiarum Naturalium ization of exine pattern through image analysis-scanning elec- Universitatis Normalis Hunanensis 22:91–94 tron microscopy (IA-SEM). Sci Hortic 65:283–294 Zhou L, Wei Z, Wu Z (2000) Pollen morphology of Maloideae Li X, Yang J, Li XG, Yang J (2002) Application of numerical (Rosaceae) in China. Acta Botanica Yunnanica 22:47–52 taxonomy of pollen morphology on origination, evolution and classification of Pyrus L. in China. J Fruit Sci 19(3):145–148

123