Plant Regeneration of `Valencia' Sweet Orange, `Femminello' Lemon, and the Interspecific Somatic Hybrid following Protoplasm Fusion

Abstract

<jats:p>Protoplasm culture following the chemical fusion of `Valencia' sweet orange <jats:italic>[Citrus sinensis</jats:italic> (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon <jats:italic>[Citrus limon</jats:italic> (L.) Burro. f.] leaf protoplasts resulted in the regeneration of an interspecific allotetraploid somatic hybrid plant, two autotetraploid lemon plants, and diploid plants from both parents. The regeneration of plants from lemon leaf protoplasts is an example of protoplast-to-plant regeneration from non-nucellus-derived tissue for <jats:italic>Citrus</jats:italic>. Regenerated plants were classified according to leaf morphology, chromosome number, and analyses of phosphohexose isomerase (PHI), peroxidase (PER), and 6-phosphoglucose dehydrogenase (PGD) zymograms. The somatic hybrid plant was vigorous, with leaves morphologically intermediate to the parents. The tetraploid lemon plants were similar to diploids, although less vigorous and with thicker leaves. The tetraploid lemon and somatic hybrid plants, if fertile, could be used in interploid sexual crosses to breed triploid seedless lemon cultivars with tolerance of mal secco disease from sweet orange. Further investigation of plant regeneration from leaf protoplasts could increase the number of totipotent <jats:italic>Citrus</jats:italic> clones amenable to somatic hybridization and genetic transformation experiments.</jats:p>

Assessment of the origin of new citrus tetraploid hybrids (2n = 4x) by means of SSR markers and PCR based dosage effects

“…Even though in nature there are few tetraploid citrus genotypes, we widely use this approach with the various tetraploid somatic hybrids produced by somatic hybridization in our institute (Scarano et al , 2003Tusa et al 1990Tusa et al , 1992Tusa et al , 1996Tusa et al , 2002. Unfortunately, in Citrus, conventional breeding methods are limited by their complex reproductive biology that often prevents sexual hybridization and the production of segregating zygotic populations for selection (Grosser et al 2000;Swingle and Reece 1967).…”

Culturing embryogenic protoplasts of ?Hamlin? sweet orange in calcium alginate beads

“…Large starch granules have been found in embryogenic Citrus protoplasts by other researchers (Kobayashi et al 1985). Tusa et al (1990) purified protoplasts by flotation in a 25% sucrose solution, and Vardi et al (1982) used sucrose or Ficoll. …”

Growth of embryogenic sweet orange callus on media varying in the ratio of nitrate to ammonium nitrogen

“…been successfully used to generate somatic hybrids Tusa et al 1990;Ohgawara et al 1991), cybrids (Vardi et al 1987), and transgenic plants (Vardi et al 1990;Hidaka et al 1990;Moore et al 1992). Physiological studies and genetic manipulations require cultured cells or tissues that are healthy and rapidly growing.…”