Ploidy investigation of bananas (Musa spp.) by flow cytometry

Abstract: 【Objective】Banana (Musa spp.), origined from the southeast Asia, is the forth food crop in theworld, which are important staple food and income-generating crops, especially to the people in the tropi⁃cal and subtropical regions Therefore, global banana production plays an important role in dealing withthe problems derived from the global warming and the population expansion of the world. However, thereare some problems exsiting in banana genetic improvement due to uncertainty of ploidy of varieties orbreeds. The accurate determination of banana ploidy level is important for proper genetic manipulationand is a prerequisite for interspecific hybridization. So far, various methods, including conventional breed⁃ing, genetic transformation, morphological classification, cytological study, biochemical means and molec⁃ular markers, have been used to genetically manipulate bananas so as to improve its production. Chromo⁃some counting is tedious and laborious to determine the ploidy of bananas. Flow cytometry(FCM) was em⁃ployed to detect the ploidy of bananas in this study. 11 banana varieties, named Musa AAA Cavendish‘Honghe Ai’, M. AA acuminate‘Lingshui YeshengJiao’, M. balbisiana‘Shixing BB’,‘Red banana’,M. ABB Cavendish‘Dongguan Zhongba Dajiao’,‘Pisang Ceylan’(AAB),‘FHIA-18’(AAAB),‘Cachaco’(ABB),‘FHIA-17’(AAAA),‘FHIA-03’(AABB) and‘TMB×5259-1’(ABBB) were chosen as materials.【Methods】Approximately 0.5 cm2 of fresh leaves were chopped with a sharp razor blade in a plastic Petridish containing 2 mL HRA buffer for each sample. The suspension of nuclei was filtered through a 30 μmnylon mesh to remove large cellular materials. After that, the nuclear DNA of the sample was stained byadding 1 600 μL of 0.02 g·L^-1 propidium iodide. The sample was mixed gently and incubated briefly onice before mixing again. A known diploid wild banana germplasm resource, M. AA acuminate‘LingshuiYeshengJiao’, was used to test the standardisation of flow cytometry by using a Partec CyFlow® SpaceFlow Cytometer with a 405 nm laser. At least 5 000-10 000 nuclei were analyzed for each sample. Threeleaf samples were tested from each of four plants of each accession.【Results】The FCM was successfullyused to analyze the ploidy of 11 banana germplasm resources in present study,. The ploidy of 11 bananaclones were divided into four groups. The first group included M. AAA Cavendish‘Honghe Ai’and M.AA acuminate.‘Lingshui YeshengJiao’, identified as 2 N. The second group contained M. BB balbisiana‘Shixing BB’, M. ABB Cavendish‘Dongguan Zhongba Dajiao’,‘Pisang Ceylan’and‘FHIA-18’, identi⁃fied as 3 N. The third group consisted of‘Cachaco’‘FHIA-17’‘FHIA-03’and‘TMB×5259-1’, identi⁃fied as 4 N. The fourth group only included‘Red banana’, which showed two peak values in the analysisatlas, 52.79 and 103.24, with good repeatability. Our study had some different results from the prevous re⁃search. M. AAA Cavendish‘Honghe Ai’previously was classified as triploid, M. BB balbisiana.‘Shix⁃ing BB’as diploid,‘FHIA-18’as tetraploid, and‘Cachaco’as triploid, , they were found to be diploid,triploid, triploid and triploid, respectively ,in our study.【Conclusions】The ploidy of 11 banana varietieswere identified by means of the FCM method and were divide into four groups.‘Red banana’might be amixoploid derived from two diploid and tetraploid, and this needs further investigation to confirm.