Optimization of chromosome preparation methods and karyotype analysis of 13 species of Camellia sinensis plants

This study aims to explore the effects of different treatment conditions on chromosome root tip squashing, optimize the chromosome preparation method, conduct karyotype analysis of 13 Camellia species, and to provide a theoretical basis for the research on Camellia plants.

The effects of different pretreatment and dissociation conditions on chromosome slide preparation were compared to select a universally applicable method for Camellia species. Karyotype analysis of 13 Camellia species was conducted using the optimized method, yielding karyotype diagrams, parameters, and evolutionary trends for each species.

The optimized pressing method suitable for Camellia is to treat the root tips with saturated 1,4-dichlorobenzene for 6 h, then place in a 1 mol/L hydrochloric acid water bath at 60 ℃ for 15 minutes and stain with a modified carbol-fuchsin solution for 15 minutes for the conventional pressing operation. All 13 Camellia species exhibit a chromosome base number of x=15, the results of the karyotype analysis show that the karyotypes include diploid, tetraploid, hexaploid, and octoploid. Among diploids, the karyotype formula for C. semiserrata is 2n=2x=30=24m+6sm, and for the C. chekiangoleosa, it is 2n=2x=30=22m+8sm; for the tetraploid C. meiocarpa, the karyotype formula is 2n=4x=60=40m+20sm; among the hexaploid species, the karyotype formula for C. oleifera is 2n=6x=90=42m+44sm+4st, and for C. drupifera, it is 2n=6x=90=60m+26sm+4st; additionally, the karyotype formula for the octaploid C. gauchowensis is 2n=8x=120=94m+26sm. The range of asymmetry indexes for 13 Camellia species is 47.90%–64.63%, the relative length of chromosomes ranges from 2.78 to 13.59, the arm ratios range from 1.00 to 4.22, and the Lt/St ranges from 1.62 to 4.89. According to the Stebbins' karyotype classification system, the C. sinensis, C. chekiangoleosa, C. transarisanensis, and C. meiocarpa are of type 2A, C. boreali-yunnanica is type 1A, and C. osmantha is type 2C, while the remaining eight species are type 2B.

This study explored chromosome staining techniques widely applicable to Camellia, determined the chromosome numbers and ploidy levels of 13 Camellia species, and reported the karyotypes of C. transarisanensis, C. cordifolia, and C. osmantha for the first time. It provides an important reference for chromosome preparation and karyotype analysis of Camellia sinensis plants.