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Optimization of somatic embryogenesis and selection regimes for particle bombardment of friable embryogenic callus and somatic cotyledons of cassava (Manihot esculenta Crantz)
Abstract
Although a number of transformation systems and selection regimes have been developed for cassava (Manihot esculenta Crantz), they have only been applied in a limited number of genotypes. This limitation of the applicability of the systems is due largely to variation in morphological responses of different genotypes to the regeneration and transformation procedures employed, which underscores
the need to study all possible parameters for any given cultivar. Using two cultivars (“Rosinha” and “Bujá Preta”) we made an attempt to improve the frequency of somatic embryogenesis (SE), establish
friable embryogenic callus (FEC) lines from developing somatic embryos and evaluate the effect of different concentrations of the antibiotics kanamycin, on secondary somatic embryogenesis (SSE) and
of paromomycin, on FEC in proliferation and histodifferentiation media. Further, we report on transient expression of the visual marker gene uidA following particle bombardment of FEC from ‘Bujá Preta’ and
of cut pieces of green somatic cotyledons from ‘Rosinha’, using the plasmid pBI426. Higher number of embryos, which emerged as early as 8 days after culture on MS medium (CIM), was obtained when
abaxial part of the explant was in direct contact with the medium as against the adaxial side, and this did not depend on the explant size (0.5 cm² or 2.5 cm²). Highly friable and embryogenic callus was obtained on GD medium supplemented with 2% (w/v) sucrose and 12 mg/L picloram. While paromomycin, at concentration of 60 mg/L arrested the proliferation and histodifferentiation of FEC, kanamycin in CIM containing explants undergoing SSE led to a decrease in their embryogenic potential resulting in tissue death at 50 mg/L. The highest transient expression of uidA gene in FEC was observed combining
plasmolysis, M5 particle and a helium pressure of 1,200 psi, while in somatic cotyledons, the highest expression was observed when M5 particle was used along with the helium gas pressure of 900 psi.
the need to study all possible parameters for any given cultivar. Using two cultivars (“Rosinha” and “Bujá Preta”) we made an attempt to improve the frequency of somatic embryogenesis (SE), establish
friable embryogenic callus (FEC) lines from developing somatic embryos and evaluate the effect of different concentrations of the antibiotics kanamycin, on secondary somatic embryogenesis (SSE) and
of paromomycin, on FEC in proliferation and histodifferentiation media. Further, we report on transient expression of the visual marker gene uidA following particle bombardment of FEC from ‘Bujá Preta’ and
of cut pieces of green somatic cotyledons from ‘Rosinha’, using the plasmid pBI426. Higher number of embryos, which emerged as early as 8 days after culture on MS medium (CIM), was obtained when
abaxial part of the explant was in direct contact with the medium as against the adaxial side, and this did not depend on the explant size (0.5 cm² or 2.5 cm²). Highly friable and embryogenic callus was obtained on GD medium supplemented with 2% (w/v) sucrose and 12 mg/L picloram. While paromomycin, at concentration of 60 mg/L arrested the proliferation and histodifferentiation of FEC, kanamycin in CIM containing explants undergoing SSE led to a decrease in their embryogenic potential resulting in tissue death at 50 mg/L. The highest transient expression of uidA gene in FEC was observed combining
plasmolysis, M5 particle and a helium pressure of 1,200 psi, while in somatic cotyledons, the highest expression was observed when M5 particle was used along with the helium gas pressure of 900 psi.
