Development of flow cytometry protocol and genome size estimation in heeng (Ferula assa-foetida L.)

Ferula assa-foetida L., also known as heeng, is a herbaceous perennial plant of the Apiaceae family. Heeng being an important spice and medicinal crop, has received relatively limited attention globally. Recent breeding efforts to improve its status are hampered due to limited genetic information available in this crop. Hence, the present study was conducted to standardize the flow cytometry (FCM) protocol for F. assa-foetida L. and estimate the genome size of various accessions introduced in India. Four common standard lysis buffers (Galbraith buffer, Woody plant buffer, Tris.MgCl₂ buffer, and Otto’s buffer) and their eight modifications were used to isolate the heeng plant nuclei. Leaf tissues and two internal standard crops, i.e., Pisum sativum cv. Ctirad and Solanum lycopersicum cv. Stupice were analyzed with four popular buffers and their modifications using BD Accuri c6 + flow cytometer. All four standard lysis buffers did not yield distinct peaks and responded differently for all the accessions. Among the four modifications of the Otto I buffer, the best results were observed in the OB4 modification. The genome size of F. assa-foetida L. plants observed in twenty-two accessions ranged from 4.10 pg to 4.98 pg with an average of 4.48 pg (2192.15 Mbp). These results indicated OB4 modification of Otto I Buffer as the most appropriate buffer for the flow cytometry of F. assa-foetida L. This buffer may be employed in future cytogenetic and breeding investigations to decipher the relationship between chromosome number, ploidy, and genome size to FCM sample preparation in heeng plants.