Cytogenetic, chromosome count optimization and automation of Neolamarckia cadamba (Rubiaceae) root tips derived from in vitro mutagenesis


  • Wei-Seng HO Universiti Malaysia Sarawak, Forest Genomics and Informatics Laboratory (fGiLab), Faculty of Resource Science and Technology, 94300 Kota Samarahan, Sarawak (MY)
  • Wee-Hiang ENG Universiti Malaysia Sarawak, Forest Genomics and Informatics Laboratory (fGiLab), Faculty of Resource Science and Technology, 94300 Kota Samarahan, Sarawak (MY)
  • Kwong-Hung LING Sarawak Timber Association, Wisma STA, 26, Jalan Datuk Abang Abdul Rahim, 93450 Kuching, Sarawak (MY)



ImageJ, mitotic chromosomes, polyploid, Rubiaceae, single and double staining


Chromosome count is the only direct way to determine the number of chromosomes of a species. This study is often considered trivial that seldom described and discussed in detail. Therefore, it is inevitable that the chromosome count protocol should be revised and revisited before it becomes obliterated. In the present study, we encountered challenges in obtaining a clear micrograph for the chromosome count of active mitotic cells of Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) root tips. Several obstacles were determined through micrograph observation, such as existing unwanted particles in cells, poor chromosome staining and chromosome clumping. To overcome these, root tip types, staining methodologies, squashing methods were among the factors assessed to obtain clear micrographs. The chromosome counts of N. cadamba under optimized procedure showed 2n = 44 chromosomes. We also apply digital technology in chromosome counts, such as online databases and graphic software that are open source and freely accessible to the public. Only basic laboratory equipment and chemicals were used throughout the study, thus making this study economical and applicable in a basic laboratory. The availability of online digital software and databases provide open-source platforms that will ease the efforts in chromosome count.


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Barbat C, Rodino S, Petrache P, Butu M, Butnariu M (2013). Microencapsulation of the allelochemical compounds and study of their release from different products. Digest Journal of Nanomaterials and Biostructures 8(3):945-953.

Bedi YS, Bir SS, Gill BS (1981). Cytopalynology of woody taxa of family Rubiaceae from North and Central India. Proceeding Indian Natural Science Academy B47(6):708-715.

Bedini G, Peruzzi L (2015). A comparison of plant chromosome number variation among Corsica, Sardinia and Sicily, the three largest Mediterranean islands. Caryologia 68(4):289-293.

Bosser J (1984). Bulletin du Muséum National d'Histoire Naturelle Section B, Adansonia, botanique, phytochimie. Muséum, Paris.

Bostan C, Butnariu M, Butu M, Ortan A, Butu A, Rodino S, Parvu C (2013). Allelopathic effect of Festuca rubra on perennial grasses. Romanian Biotechnological Letters 18(2):8190-8196.

Candan F (2013). Some observations on plant karyology and investigation methods. Current Protocol Biology Research 217-55.

Corréa AM, Forni-Martins ER (2004). Chromosomal studies of species of Rubiaceae (A. L. de Jussieu) from the Brazilian cerrado. Caryologia 57(3):250-258.

de Alencar, LD, Azevedo P, Latado RR (2020). Mothers’ command: phenotypes changes resulting from reciprocal interploidy crosses. Euphytica 216:21.

Eng WH, Ho WS, Ling KH (2021). Effects of colchicine treatment on morphological variations of Neolamarckia cadamba. International Journal of Agricultural Technology 17(1):47-66.

Gujendran V, Rodriguez JJ (2004). Chromosome counting via digital image analysis. International Conference on Image Processing 2929-2932.

Ho WS, Pang SL, Julaihi A (2014). Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser). Physiology and Molecular Biology – Plants 20(3):393-397.

Huang H (2016). Systematics and genetic variation of Actinidia. Academic Press, United States.

Ianculov I, Palicica R, Butnariu M, Dumbrava D, Gergen I (2005). Achieving the crystalline state of chlorophyll of the Fir–tree (Abies alba) and the pine (Pinus ylvestris). Revista de Chimie 56(4):441-443.

Jiang Y, Liu S, Hu J, He G, Liu Y, Chen X, … Gao S (2020). Polyploidization of Plumbago auriculata Lam. in vitro and its characterization including cold tolerance. Plant Cell, Tissue and Organ Culture 140:315-325.

Khoshoo TN, Sc FA, Bhatia SK (1963). Cytology of some Rubiaceae of the North-western Himalayas. Proceedings of the Indian Academy of Sciences 58(B):36-44.

Kiehn M (1986). Karyosystematic studies on Rubiaceae: Chromosome counts from Sri Lanka. Plant Systematics and Evolution 154:213-23.

Kiehn M (1995). Chromosome survey of the Rubiaceae. Annals of the Missouri Botanical Garden 82(3):398-408.

Kiehn M (2010). Chromosomes of neotropical Rubiaceae. I: Rubioideae. Annals of the Missouri Botanical Garden 97(1):91-105.

Kiehn M, Lorence DH (1996). Chromosome counts on Angiosperms cultivated at the National Tropical Botanical Garden, Kauai, Hawaii. Pacific Science 50(3):317-323.

Kim JS, Seo MS, Moon MS, Won SY, Kwon SJ (2019). Insight on doubled haploid production with an amphidiploid species ‘Dolsangat’ in Brassica juncea. Korean Journal Breeding Science 51(4):341-350.

Lavania UC (2020). Plant speciation and polyploidy: in habitat divergence and environmental perspective. Nucleus 63:1-5.

Lee YS (1979). Remarks on chromosome number in Rubiaceae. Korean Journal of Plant Taxon 9(1):57-66.

Maluszynska J (2003). Cytogenetics test for ploidy level analyse-chromosome counting. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (Eds). Doubled haploid production in crop plants. Springer, Netherland pp 391-395.

Martin C, Viruel MA, Lora J, Hormaza JI (2019). Polyploidy in fruit tree crops of the Genus Annona (Annonaceae). Frontiers in Plant Science 10:99.

Mathew PM, Philip O (1986). The distribution and systematic significance of pollen nuclear number in the Rubiaceae. Cytologia 51:117-124.

Matsushima R (2015). Morphological variations of starch grains. In: Nakamura Y (Ed). Starch. Springer, Japan pp 425-441.

Mehra PN, Bawa KS (1969). Chromosomal evolution in tropical hardwoods. Evolution 23(3):466-481.

Mehrvarz SS, Ghadim SM (2015). Chromosome counts of some species of the genus Galium (Rubiaceae) from Iran. Feddes Repertorium 126:31-36.

Midin MR, Nordin MS, Madon M, Saleh MN, Goh H, Noor NM (2018). Determination of the chromosome number and genome size of Garcinia mangostana L. via cytogenetics, flow cytometry and k-mer analyses. Caryologia 1017:1-10.

Ochatt SJ, Patat-Ochatt EM, Moessner A (2011). Ploidy level determination within the context of in vitro breeding. Plant Cell, Tissue and Organ Culture 104:329-341.

Pang SL, Ho WS, Mat-Isa MN, Julaihi A (2015). Gene discovery in the developing xylem tissue of a tropical timber tree species: Neolamarckia cadamba (Roxb.) Bosser (Kelampayan). Tree Genetics & Genomes 11:47.

Pfister B, Zeeman SC (2016). Formation of starch in plant cells. Cellular and Molecular Life Sciences 73:2781-807.

Puangsomlee P, Puff C (2001). Chromosome numbers of Thai Rubiaceae. Nordic Journal of Botany 21(2):165-176.

Rice A, Šmarda P, Novosolov M, Drori M, Glick L, Sabath N, Meiri S, Belmaker J, Mayrose I (2019). The global biogeography of polyploid plants. Nature Ecology and Evolution 3:265-273.

Rivero R, Emily B, Sessa EB, Zenil-Ferguson R (2019). EyeChrom and CCDBcurator: Visualizing chromosome count data from plants. Application in Plant Sciences 7(1):e1207.

Selvaraj R (1987). Karyomorphological studies in South Indian Rubiaceae. Cytologia 52:343-56.

Sharma A, Sen S (2019). Chromosome botany. CRC Press, Boca Raton.

Sharma AK, Sharma A (1980). Chromosome techniques theory and practice. Butterworth & Co. Ltd., United Kingdom.

Singh RJ (2016). Plant cytogenetics. CRC Press, USA.

Singh RJ (2017). Practical manual on plant cytogenetics. CRC Press, Boca Raton.

Tchin BL, Ho WS, Pang SL (2018a). Isolation and characterisation of cinnamate 4-hydroxylase (C4H) gene controlling the early stage of phenylpropanoid biosynthetic pathway in developing xylem tissues of kelampayan (Neolamarckia cadamba, Rubiaceae). Asian Journal of Agriculture and Biology 6(2):278-286.

Tchin BL, Ho WS, Pang SL (2018b). Isolation and in silico characterization of full-length cinnamyl alcohol dehydrogenase gene involved in lignin biosynthesis in Neolamarckia cadamba. Journal of Applied Biology and Biotechnology 6(2):1-5.

Windham MD, Pryer KM, Poindexter DB, Li F, Rothfels CJ, Beck JB (2020). A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited. Applications in Plant Sciences 8(4):e11342.




How to Cite

HO, W.-S., ENG, W.-H., & LING, K.-H. (2021). Cytogenetic, chromosome count optimization and automation of Neolamarckia cadamba (Rubiaceae) root tips derived from in vitro mutagenesis. Notulae Scientia Biologicae, 13(3), 10995.



Research articles
DOI: 10.15835/nsb13310995