Karyotyping of Spondias L. (Anacardiaceae) using fluorescent microscope

doi:10.15406/apar.2017.07.00280

Advances in

eISSN: 2373-6402

Plants & Agriculture Research

Research Article Volume 7 Issue 6

Karyotyping of Spondias L. (Anacardiaceae) using fluorescent microscope

Mohannad G Al Saghir

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Department of Environmental and Plant Biology, Ohio University, Zanesville, USA

Correspondence: Mohannad G. Al-Saghir, Department of Environmental and Plant Biology, Ohio University, Zanesville, Ohio, USA

Received: October 23, 2017 | Published: December 8, 2017

Citation: Al-Saghir MG. Karyotyping of Spondias L. (Anacardiaceae) using fluorescent microscope. Adv Plants Agric Res. 2017;7(6):441-442. DOI: 10.15406/apar.2017.07.00280

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Abstract

The genus Spondias L. belongs to the Anacardiaceae family and includes 18species. Cytogenetic studies addressing the genus Spondias are rather few. Previous studies did not include all Spondias species and the chromosome number is questionable due to the fact that poor chromosome counting protocols were used. In this paper, the karyotypes of five Spondias species were studied using fluorescent microscope to provide in-depth insights to understanding the cytogenetics and phylogeny of the genus Spondias. The results show that all studied Spondias species have the same chromosome number which is 2n=32 based on all previous morphological and molecular studies, which clearly suggest a very close genetic relationship among Spondias species, and the chromosome numbers reported by various cytogenetic studies.

Keywords: spondias, cytogenetics, chromosome numbers, fluorescent microscopy

Introduction

The genus Spondias L. belongs to the Anacardiaceae family and includes 18species with at least nine Neotropical species.¹ Spondias is native to tropical America and Asia, and Madagascar. Spondias has been used as far as 6500 B.C., in the Tehuacán Valley of Mexico.² S. mombin L. (Cajá), S. tuberosa Arruda Câmara (Umbu) and S. purpurea (Serigüela) are the most economically important species in the genus for agro-industrial applications. Chromosomal data are a valuable resource for cytogeneticists and breeders as they provide better insights into taxonomic and phylogenetic relationships among the species.^3,4 Cytogenetic studies addressing the genus Spondias are rather limited. Previous studies showed that studied Spondias species were diploids with somatic chromosome number of 2n=32. The only cytogenetic data available are the chromosome numbers (2n=32) of three species: S. pinnata^5–7 S. mombin,⁸ and S. tuberose.⁹ Recently, De Souza Alemdia et al.,¹⁰ studied the karyotype differentiation among five Spondias species and the putative hybrid Umbu-cajá. All the studied species and hybrid presented the same chromosome number (2n=32) and morphology. Previous studies did not include all Spondias species and chromosome numbers of the different Spondias species are questionable because inefficient chromosome counting protocols were employed.¹¹ These protocols are hampered by the small sized chromosomes of Spondias species and a few cell divisions were visible in a single root tip.¹¹ The aim of this study was to develop more effective method to resolve the chromosome numbers in Spondias species using fluorescent microscope and provide more insight into understanding the cytogenetic and phylogeny of the genus Spondias.

Materials and method

Plant materials

Leaves from the herbarium specimens of six Spondias specieswere used. The species studied are listed in (Table 1). The specimens were obtained from University of Florida Herbarium (FLAS), Gainesville, Florida. For this study, the Sigma Plant Protoplast Digest/Wash Solution protocol (Sigma, St. Louis, MO, USA) was modified as the following:¹² 1 g of dried leaf tissue was collected from each specimen and cut into 1mm sections using a sharp blade. The sections were placed in 50 mL conical vials, each filled with 20 mL of Plant Protoplast Digest/Wash Solution (Sigma, St. Louis, MO, USA).After being mixed via inversion for 5 min, the Digest/Wash Solution was removed leaving only the leaf tissue. Ten mL of the digestion enzyme solution was added and mixed via inversion for 2 min. The mixture was gently agitated on the platform shaker for one hour. After 1 h, 50 µL of each mixture was diluted into 4 micro centrifuge tubes each containing 450 µL of Digest/Wash Solution.

Species	Chromosome Count (2n)	Plant ID
Spondias mombin L.	32	148031
Spondias x robe Vrbar	32	179743
Spondias dulcisForst. F.	32	188070
Spondias purpurea L.	32	80867
Spondias radlkoferi J.D. Sm.	32	140725
Spondias laevis Griseb.	32	78925

Table 1 List of Spondias species used; Their chromosome number and plant identification numbers

The mixtures were spun at 100 x g-forces for 5 min; the supernatant was removed leaving the pellets intact. Twenty mL of Digest/Wash Solution was added to the pellets and was mixed. The mixture was spun at 100x g-forces for 5 min. The supernatant was removed and 10 µL of fixative was added to each pellet; the pellets were re-suspended via inversion and chilled on ice for one hour. Then, the mixtures were dropped onto room-temperature slides. The slides were stained with 4'-6-Diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO, USA) and placed under fluorescent microscope (Leica, Wetzlar, Germany).

Results and discussion

The results show that Spondias species are diploid and they have the same chromosome number of 2n=32 (Table 1 & Figure 1). This study is the first to report the chromosome numbers using fluorescent microscope and the first to report the chromosome number for all studied species except S. mombin. The method was developed initially for Pistacia L. species because root-tips from the field are not possible to obtain and cuttings do not make roots easily. Roots from seedlings are rather small. Moreover, the Pistacia species havevery small chromosomes.¹² This study shows that the same method can be used successfully to study the chromosome number among Spondias species. Somatic chromosome number of 2n= 32 for S. mombin has earlier been reported.^8,10 These findings were supported by the current study (Figure 1). Chromosome number of S. purpurea was reported as 2n=32.¹⁰ The current study is in agreement with these findings. Moreover, De Souza Alemdia et al.¹⁰ found that all the analyzed Spondias species exhibited similar karyotypes and small chromosomes. This study provides valuable chromosomal data for potential use by the cytogeneticists and plant breeders. This study also provides additional insight into understanding the taxonomic and phylogenetic relationships among Spondias species.

Figure 1 A typical cell with chromosomes (2n=32) of Spondias mombin L.

Acknowledgements

The author is grateful to Ohio University Zanesville for funding this project. The author is very thankful for University of Florida Herbarium and Manager of collections Kent D. Perkins for loaning the specimens. The author would like to thank Dr. Philip Cantino, Director of the Bartley Herbarium at Ohio University for his help in sending this loan.