Methods of transformation

Most of the reports on pepper regeneration deal with two main types, bell and chile. Shoot regeneration is generally dependented on organogenesis from cotyledons and hypocotyls, but the regeneration capacity is highly dependent upon thecultivar,^{46, 47} the developmental stage⁴⁹ and thelocation⁵⁰ of the plant tissue. Zhu et al.⁵¹ found that leaves of a Chinese sweet pepper variety were by far the best material for successful regeneration, transformation with young leaves giving the highest rates. However, Arroyo and Revilla⁵² had previously described an efficient regeneration procedure for some commercially important spanish cultivars using hypocotyls and especially cotyledons with high regeneration rates. Fari et al.⁵³ by screening a seed collection of chile peppers for in vitro regeneration selected plants that, after selfing, generated an inbred line n°40017-13 with a high capacity for regeneration. Regeneration from protoplasts has also been achieved⁵⁴ in one cultivar of C. annuum ‘Dulce Italiano’, while three other cultivars (Americano, Florida Gynat and Nigrum) and a wild species C. chinense produced meristem-like structures but no shoots. Regeneration of chile peppers has also been achieved by somatic embryogenesis. ^{55, 56}

Concerning bell peppers, an in vitro regeneration protocol including transformation via A. tumefaciens harbouring a GUS reporter gene has been defined by Liu et al.⁵⁷ using six cultivarsand one wild accession. Regeneration of whole transgenic plants proved unsuccessful. A critical step appears to be the elongation and rooting of the shoots during kanamycin selection.⁵⁸ The virulence of Agrobacterium strains towards peppers seems to be variable and dependent on the cultivar,^{51, 57} using leaves as starting material and a specific protocol, specially for bud elongation, have succeeded in generating a Chinese sweet pepper variety harbouring a CMV coat protein.

Concerning chile peppers, Manoharan et al.⁵⁹ established a protocol for regeneration-transformation of a hot chile pepper variety from India using a hypervirulent A. tumefaciens strain (EHA 105) and involving the use of thidiazuron as a cytokinin as suggested by Szàsz et al.58 for bell peppers. An effective protocol has also been established for a Korean variety of hot pepper that uses a complex cocktail of plant growth regulators and a pre-culture of cotyledons or hypocotyls in the presence of the ethylene inhibitor AgNO₃.⁶⁰

Despite the elevated number of protocols published so far, their efficiency is still very low and peppers can still be considered as recalcitrant to genetic transformation. It is obvious that a good combination of an efficient protocol for regeneration-transformation, a virulent. Atumefaciens strain, and highly responsive cultivars is the key for the generation of transgenic peppers.