Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivative

Somatic embryos were regenerated in vitro from calamondin style-stigma explants cultured in the presence of N6-benzylaminopurine (BAP) cytokinin and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), N-phenyl-N¢-benzothiazol-6-ylurea (PBU) and N,N¢-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at micromolar level (12 lM) were able to induce a percentage of responsive explants significantly higher from that obtained with BAP and hormone-free (HF) conditions. In order to verify the genetic stability of the regenerants, 27 plants coming from different embryogenic events were randomly selected from each different culture condition and evaluated for somaclonal variations using inter-simple sequence repeat and randomamplified polymorphic DNA analyses. We observed that 2,3-MDPU and PBU gave 3.7% of somaclonal mutants, whereas 4-CPPU gave 7.4% of mutants. No somaclonal variability was observed when plantlets were regenerated in BAP or HF medium. Although diphenylurea derivatives show a higher embryogenic potential as compared to BAP, they induce higher levels of somaclonal variability. This finding should be taken in consideration when new protocols for clonal propagation are being developed.