Ploidy analysis among Citrus mutants using leaf meristematic tissue

A promising method for preparing metaphase spread for counting the number of chromosomes from the emerging shoot tissue is described in this report. In the present study, we adopted enzymatic digestion of shoot tips to analyse the chromosome number. The chromosomes in metaphase stage of cell division are highly condensed and easy to count in routine cytological technique. Even the morphological features like position of centromere can be seen in metaphase. In prophase it may not be clear as the chromosomes are getting ready for cell division. In enzymatic digestion even the prophase chromosomes are visible, which can be counted. Hence enzymatic digestion technique is more efficient in citrus as compared to acid digestion method as the citrus crop is a perennial crop with small-sized chromosomes. Furthermore, the sample collection in the field was easy and actively growing vegetative flush was available throughout the year. This technique was attempted in the tissue culture lab of ICAR- CCRI in various in vito and in vivo ploidy induction experiments in Citrus sinensis Osbeck (Sweet orange cv. mosambi), C. reticulata Blanco (Nagpur mandarin) and C. jambhiri Lush (Rough lemon), for confirmation of diploidy (2n=2x=18), triploidy (2n=3x=27), tetraploid (2n=4x=36), hexaploid (2n=6x=54).