Cytogenetics of two Indian varieties of Momordica charantia L. (bittergourd)☆
Introduction
Momordica charantia L.is the largest cultivated species of Momordica out of the 60 existing species (Schaefer and Renner, 2011) world-wide. It is a monoecious annual climber belonging to the Momordiceae tribe of the Cucurbitaceae family (Schaefer and Renner, 2011) characterized by lobed leaves and jagged fruits with crocodile skin-like sculptured seeds (Behera et al., 2011). The species has been mostly valued for its anti-diabetic effect among several other medicinal applications (Grover and Yadav, 2004) since ages. Due to ‘bitter’ taste of edible parts of the plant (especially the fruits) justifying the name ‘Bitter gourd’, M. charantia was thought to have hypoglycaemic effect and consumed commonly in most Indian households as an effective alternative-drug for diabetic patients (Bharathi and John, 2013). The anti-diabetic feature of the plant has been successfully examined in clinical trials (Miura et al., 2001; Chen et al., 2003; Lo et al., 2017; Thent et al., 2017) as well.
It is well known that a huge genetic diversity occurs in M. charantia which has been reported within domesticated and wild populations of this species (Behera et al., 2008). India is one centre of diversity of M. charantia owing to huge domestication and agronomical practices. Two prominent varieties of M. charantia namely M. charantia var. charantia (long fruited variety) and M. charantia var. muricata (small fruited variety) were reported to occur in India in taxonomy (Chakravarthy, 1990; Nayar et al., 2006; Joseph and Antony, 2008; Bharathi and John, 2013). Molecular marker based assessment of the accessions (Dey et al., 2006; Behera et al., 2008) also pointed to the existence of considerable genetic diversity in M. charantia all together.
The Momordica species are not easily amenable to cytogenetic analysis due to small size of chromosomes, dense cytoplasmic background and inefficient stainability (Trivedi and Roy, 1972). These are probably the reasons for overall dearth in karyomorphological information on M. charantia till date. Though there are some earlier reports on chromosome counts and conventional karyotypes for M. charantia (Bhaduri and Bose, 1947; Trivedi and Roy, 1972; Lombello and Pinto-Maglio, 2007; Zaman and Alam, 2009; Bharathi et al., 2011; Waminal and Kim, 2012; Kausar et al., 2014; Kido et al., 2016), detailed investigation involving Indian populations of the intraspecific varieties are negligible (Beevy and Bai, 2013). In Cucurbitaceae species, enzymatic maceration and air drying method, EMA (Fukui, 1996) followed by Giemsa staining has been applied previously for improved karyomorphological analysis by resolving structural details of chromosomes and overcoming the technical limitations of conventional aceto-orcein schedules (Bhowmick et al., 2012; Bhowmick and Jha, 2015). EMA method based chromosomal preparations enable easy application of fluorochrome banding technique as a suitable upgradation in karyotyping in the era of molecular cytogenetics. The use of base specific fluorochromes like the guanine-cytosine (GC)-specific chromomycin A3 (CMA) and adenine-thymine (AT)-specific 4′,6-diamidino-2-phenylindole (DAPI) facilitates distinction of morphologically alike chromosomes which in a broader sense contribute to karyotype refinements and characterization of individual genotypes or varieties in particular. In the present study, we report EMA based karyotyping and meiotic analysis for the first time in two varieties of Indian bittergourd, MCC and MCM collected from different regions of India. We addressed detailed investigation of the two varieties of M. charantia involving the karyomorphometric parameters, fluorochrome banding patterns accompanied by flow cytometric nuclear genome size estimation.
Section snippets
Plant materials
For the present study, seeds of 14 cultivated populations of M. charantia var. charantia (long-fruited variety of M. charantia, MCC) were collected from different parts of India viz. West Bengal, Odisha, Maharashtra, Karnataka and Kerala; seedling and fruits of a wild population of MCC were also collected from Kerala and seeds of 5 accessions of MCC were kindly provided by National Bureau of Plant Genetic Resources (NBPGR), Thrissur, Kerala. On the other hand, seeds of 6 cultivated populations
Karyomorphological analysis based on EMA-Giemsa staining
Standardization of pretreatment and EMA-Giemsa method for the populations of M. charantia var. charantia and M. charantia var. muricata enabled visualization of well-scattered metaphase plates with much prominent constrictions against a clear background as compared to conventional aceto-orcein squash technique reported previously. Increase in metaphase frequency was obtained mainly with cold water (4 °C) treatment as compared to hydroxyquinoline or pDB. The use of cold water (4 °C) for varied
Discussions and conclusions
In our present study, we revisited the chromosomes of M. charantia for explicit karyotype analysis with standardization of technical protocols for pretreatment, cell wall hydrolysis and chromosome staining using advanced cytogenetic tools. Application of HQ which was found suitable for other Cucurbits (Bhowmick and Jha, 2015) did not facilitate sufficient chromosome scattering and proper chromosome condensation in M.charantia varieties. EMA method was instrumental in bringing a clear background
Conflict of interest
Authors declare that they have no conflict of interest.
Acknowledgements
IG is grateful to Department of Biotechnology, Govt. of India for financial support in form of Senior Research Fellowship. SJ is thankful to the National Academy of Sciences (NASI, Allahabad, India), for award of Platinum Jubilee Senior Scientist Fellowship and providing the financial support to continue the research. BKB & IG express sincere gratitude to Prof. Timir Baran Jha for his help and guidance in fluorescence banding technique. Special thanks are due to Dr.B.Ghosh RKMVC Rahara, Prof.
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This paper is dedicated to the memory of our respected teacher & renowned Plant Cytogeneticist Professor Arun Kumar Sharma, who passed away on 6th July 2017.