From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (Coffea arabica L. × Coffea canephora Pierre ex A. Froehner)

Abstract

Since 1966, chromosome doubling has been performed mainly in vitro, associating anti-tubulin treatment and different plant tissues showing proliferative cells. Despite the achieved improvements, some bottlenecks have been pointed out, such as the low rate of polyploids and high rate of mixoploid plantlets. To overcome these hurdles, some approaches have indicated that indirect somatic embryogenesis (ISE) constitutes an alternative trigger for chromosome doubling, especially for homoploid and anorthoploid germplasms. In this way, a guideline has been developed for hexaploidization of the Coffea line “Híbrido de Timor” (HT) ‘CIFC 4106’ (anorthoploid, 3x = 33 chromosomes, 1C = 2.10 pg, Coffea canephora × Coffea arabica) from friable embryogenic calli (FEC) treated with colchicine. From this, a relatively high percentage (49.3%) of HT hexaploids (6x = 66 chromosomes, 2C = 4.20 pg) was obtained, without recovery of mixoploids. Besides confirmation of endomitosis induction through the obtained hexaploids, SSR markers revealed that the FEC/colchicine strategy also resulted in loss of allelic diversity in 39 regenerated HT plantlets, demonstrating its genotoxic effect. Considering these results, the present procedure resolved the main bottlenecks for chromosome doubling, which have been reported since the discovery and isolation of the anti-tubulin colchicine in 1930. Hexaploid HT plantlets have enriched Coffea germplasm banks as a new genetic resource since the resolution of their karyotype and DNA sequence. Just as the true allotetraploid C. arabica and the allotriploid HT ‘CIFC 4106’, the hexaploid HT is relevant to investigate the genomic and phenotypic changes arising from polyploidization events.