Chromosome preparation optimization and karyotype analysis of Primulina

In order to optimize the chromosome preparation of Primulina (Gesneriaceae), evaluate the effects of various factors on chromosome preparation, and perform karyotype analysis of three Primulina species, providing technical support for chromosome research and hybrid breeding in this genus.

Three Primulina species were used as experimental materials to investigate the effects of different sampling sites, plant maturity, pretreatment methods and durations, dissociation time, and preparation techniques on chromosome preparation. The chromosome preparation protocol was optimized, and karyotype analysis was conducted based on the refined method.

1) The third leaf from each plant was used for leaf cutting hydroponics, with optimal chromosome preparation achieved when the new root tips reached 0.5-1.0 cm in length, allowing clear observation of cell division stages. Pretreatment with 0.02% colchicine for 3 hours yielded the best results, producing well-dispersed and appropriately concentrated chromosomes. For acid hydrolysis, the optimal dissociation occurred with 1 mol/L HCl + 45% acetic acid at 60 ℃ for 4 minutes. The most effective enzyme digestion formula was a mixture of 2.5% cellulase and pectinase, with a 5 minute dissociation time. Both methods produced clear, well-dispersed chromosomes. 2) All three Primulina species are diploid, with a chromosome number of 2n=36 and a basic number of X=18. The karyotype formula of Primulina eburnea is 2n=2x=36=28m+6sm+2st, with a satellite on chromosome 1. The relative chromosome length ranges from 1.12% to 4.27%. The chromosome composition is 8L+5M2+19M1+4S, and the karyotype asymmetry coefficient is 58.70%, classified as type “2B”. The karyotype formula of Primulina pungentisepala is 2n=2x=36=30m+6sm, with a satellite on chromosome 11. The relative chromosome length ranges from 1.89% to 4.22%. The chromosome composition is 3L+11M2+19M1+3S, and the karyotype asymmetry coefficient is 57.10%, also classified as type “2B”. The karyotype of Primulina ‘Spring of Beilin’ has a chromosome number of 2n=36, with a basic number of X=18, and the karyotype formula is 2n=2x=36=32m+4sm. The relative chromosome length ranges from 1.94% to 3.98%. The chromosome composition is 3L+14M2+17M1+2S, with a karyotype asymmetry coefficient of 56.60%, also classified as type “2B”.

The chromosome preparation protocol for the genus Primulina was successfully optimized using three Primulina species. The study concludes that these species are diploid, possess small chromosomes, and have karyotypes classified as relatively symmetric.