Abstract:

【Objective】The Camellia oleifera Abel. has a wide distribution and a long cultivation history, but its ploidy has always been controversial, and there is a lack of research on ploidy and karyotype. The chromosome is the genetic basis of interspecies variation; the chromosomal ploidy and karyotype characteristics of the main varieties of C. oleifera are defined, providing a basis for the production and cultivation of new varieties. The chromosome mounting technique of C. oleifera is no longer suitable for current experimental requirements. This study aims to optimize the chromosome mounting technique of C. oleifera, compare and analyze the chromosome numbers of cuttings and seedlings, and the karyotype of some main varieties of C. oleifera.【Method】In this study, the root tips of cutting stecklings and seedlings of three main cultivars (‘Huashuo’, ‘Huajin’ and ‘Huaxin’) were used as test materials. Firstly, the chromosome mounting technique was optimized by modifying key steps of the wall degradation hypotonic method, including pre-treatment, enzymatic hydrolysis and posterior hypotonicity. Secondly, to identify the material for a karyotype analysis, the modified wall degradation hypotonic method was used to unlock the chromosome number of the three main cultivars of C. oleifera. Finally, the karyotype of the three cultivars of C. oleifera was unlocked, and the karyotype formula and pivotal karyotype parameters were clarified.【Result】The following three points can be summarized from this study: ① The optimized chromosome mounting technique, which included pre-treatment of the tender root tip with a solution of 0.002 mol/L 8-hydroxyquinoline for 5-6 hours, hypotonicity for 60 min in 0.075 mol/L KCl solution, enzymolyzation with a mixture of 1.75% cellulase and 1.75% pectinase for 120 min at 20 ℃ in the dark, posterior hypotonicity in distilled water, and finally fixing with fresh Carnoy solution for more than 30 min, was found to be the best for chromosome preparation. The results showed that the modified wall degradation hypotonic method is the most suitable for preparation of C. oleifera chromosomes. ② The chromosome number of cuttings of three main cultivars was hexaploidy (2n=6x=90), and the chromosome number of seedlings of the main cultivars was 90, 87, 85, 75 and so on. As there is a tendency for chromosomal deletion in some seedlings, the stem tip or root tip of cutting stecklings should be used for a karyotype analysis of C. oleifera. ③ The cells at the mitotic metaphase stage of the root tip of cutting were used for a karyotype analysis; ‘Huashuo’, ‘Huaxin’ karyotypes are 2A, and ‘Huajin’ karyotypes are 2B. The karyotype formulas are 2n=90=63m(3SAT)+27sm (‘Huashuo’), 2n=90=58m(SAT)+32sm (‘Huaxin’) and 2n=90=64m+26sm (SAT) (‘Huajin’), and the karyotype asymmetry coefficient is 61.73%, 61.13% and 61.44%, respectively. The relative length of the chromosomes ranged from 4.81% to 8.64%, and the arm ratio ranged from 1.07 to 2.96.【Conclusion】There are many varieties of C. oleifera, and it is crucial to explore their genetic diversity, genetic relationships and the evolutionary trends among populations. Moreover, chromosome studies of C. oleifera can reflect the regular karyotype pattern of different cultivars. Through a comparative analysis of chromosome numbers of cuttings and seedlings of ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ by optimizing the chromosome mounting technique, it was found that the offspring of C. oleifera undergo polyploidy germplasm, reflecting the importance of choosing suitable test materials when studying C. oleifera karyotypes. The karyotype analysis of the three main cultivars further reflects the large karyotype difference between C. oleifera cultivars.

Key words: Camellia oleifera Abel., chromosome mounting technique, chromosome, ploidy, karyotype