Plant regeneration from protoplast of Brazilian citrus cultivars

Abstract

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Citrus asymmetric somatic hybrids produced via fusion of gamma-irradiated and iodoacetamide-treated protoplasts

“…In AFLP performed to identify polymorphisms among donor and receptor genomes ('Ruby Red', 'Itaboraí', 'Succari', and 'Murcott'), very similar AFLP patterns were observed in 'Ruby Red' and the sweet oranges, which was explainable by their genetic proximity (Gloria et al, 2000).…”

“…Somatic hybridization has contributed tremendously to citrus improvement and many citrus somatic hybrids have been reported to be in use in various breeding programs (Gloria et al, 2000;Calixto, 2004). Even though symmetric somatic hybrids have great potential for rootstock improvement and as tetraploid breeding parents in interploid crosses, they may not have direct application as scion cultivars as they may present complex genetic constitution.…”

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

“…Genetic manipulation of citrus in Brazil has focused on the production of somatic hybrids through protoplast fusion (Glória et al, 2000a(Glória et al, , 2000bMendes et al, 2001).…”

Plantlets Regeneration via Somatic Embryogenesis from the Nucellus Tissues of Kinnow Mandarin (<i>Citrus reticulata</i> L.)

“…Our findings regarding to embryogenic callus induction for Citrus reticulata are in strongly agreement with Khan et al [28] who also reported similar results. Due to presence of auxins, Gloria et al [29] observed good embryoid formation in citrus cultivars and good response for somatic embryo induction when MT medium was supplemented with 500 mg/l malt extract. Embryogenic calluses were then cultured in different concentrations of BAP for somatic embryo induction.…”