Maximizing Hybrid Seedlings Recovery and Early Identification of Highly Polyembryonic Acid Lime (Citrus aurantifolia Swing.) × Lemon (Citrus limon Burm.) Hybrids using SSR Markers

Nucellar embryony is the major obstacle in getting hybrid seedlings in interspecific crosses in citrus. Hence, the present study was conducted to standardize embryo age, culture media for maximizing germination, and subsequent identification of simple sequence repeat (SSR) markers to differentiate the hybrids. A factorial experiment was conducted with three embryo ages – 80–90, 110–120, and 130–140 days after pollination (DAP). The germination of the rescued immature embryos of acid lime × lemon crosses was initiated on three different growing media. The fruits harvested at 130–140 and 80–90 DAP had a higher number of functional seeds (90%). Older embryos (130–140 DAP) and middle-aged embryos (110–120 DAP) germinated in 100% on Murashige and Skoog (MS) medium supplemented with 500 mg·l−1 malt extract, 1.5 mg·l−1 gibberellic acid (GA3), and 0.02 mg·l−1 naphthalene acetic acid (NAA). MS medium supplemented with 500 mg·l−1 malt extract proved better for the germination of embryos taken at 80–90 DAP. Plantlet survival was the highest in younger embryos (80–90 DAP) cultured on MS basal medium (84.21%) and the lowest in older embryos cultured on MS medium supplemented with 500 mg·l−1 malt extract plus 1.5 mg·l−1 GA3 and 0.02 mg·l−1 NAA. The seedlings obtained from the culture 80–90 DAP had the highest root length (4.9 mm) and shoot length (5.3 mm) at 60 days after inoculation (DAI) on the above medium. SSR marker analysis revealed that CCSM-4 and CAC-33 markers expressed polymorphism between female and male parents, proving their ability to identify the hybrids of ‘Kagzi’ acid lime × ‘Konkan Seedless’ lemon.

Nucellar and integumentary embryony in angiosperms

The form of adventive embryony is determined by the type of ovule. Nucellar embryony is possible in crassinnucellate ovules, and integumentary embryony in tenuinucellate ovules.

Anther, ovule, seed, and nucellar embryo development in Citrus sinensis cv. Valencia

'Valencia' orange, a commercially important cultivar of Citrus, forms polyembryonic seeds by an apomictic process called nucellar embryony in which many embryos initiate directly from nucellar cells surrounding the sexual embryo sac. We observed anther, ovule, seed, and fruit development in relation to nucellar embryo development in seeds and unfertilized ovules of 'Valencia'. Pollination and fertilization are required to set fruit in 'Valencia', and low seed set was found to be related to defects in both male and female gametogenesis. Nucellar embryo initial cells were evident histologically in ovules of flowers just prior to anthesis. However, in vitro culture of ovules from flowers at different prepollination stages showed that embryos could develop from ovules cultured as early as the binucleate stage of megagametogenesis in which nucellar initial cells were absent histologically. During fruit development, the timing and sequence of the early events of nucellar embryo formation were synchronous in seeds and unfertilized ovules, indicating a co-ordinated control of embryo development in spatially and developmentally distinct structures. In both developing seeds and unfertilized ovules, embryo initial cells first formed thick walls, which isolated them from surrounding maternal tissue. In later stages, the cell walls thinned in some initial cells and embryogenesis became asynchronous. Cleavage of embryogénie cells coincided with degenerative processes linked to embryo sac expansion in seeds and to a previously unreported, localized degeneration in the central portion of the nucellus in unfertilized ovules. Some initial cells never divided. Nucellar embryo development was restricted to the central portion of unfertilized ovules and to the micropylar region of seeds. Only fertilized ovules had the capacity to form mature polyembryonic seeds. In unfertilized ovules a specialized vascular structure formed linking developing embryos to the chalazal vasculature of the ovule. Embryo development arrested at the globular stage in unfertilized ovules and the integuments differentiated to form a seed coat. The timing of reproductive events described was linked to floral and fruit morphological characteristics to facilitate molecular characterization of nucellar embryogenesis and seed formation in this cultivar. Key words: Citrus, nucellar embryony, seed, ovule, apomixis.

Contemporary Approaches to Improving Citrus Cultivars

Traditional genetic manipulation methods have proven ineffective or irrelevant for many citrus breeding objectives. Alternative approaches to Citrus genetic improvement are now available as a result of technological developments in genetics and tissue culture. For example, mapping DNA marker polymorphisms should lead to identifying markers closely linked to important loci, thereby facilitating early selection and minimizing costs associated with plant size and juvenility. Genetic transformation methods allow trait-specific modification of commercial cultivars. By selecting beneficial variants from sectored fruit chimeras and the recovering plants via somatic embryogenesis, the problems of nucellar embryony and the hybrid nature of commercial cultivar groups can be avoided. Induced mutagenesis from mature vegetative buds may overcome these problems, as well as juvenility. Ploidy level manipulation in vitro can increase the number and diversity of tetraploid breeding parents, leading to the development of seedless Citrus triploids and mitigating sterility, incompatibility, and nucellar embryony.

CONTEMPORARY APPROACHES TO CITRUS CULTIVAR IMPROVEMENT

Traditional methods of genetic manipulation have proven ineffective or irrelevant for many citrus breeding objectives. Alternative approaches to genetic improvement of citrus are now available as a result of technological developments in genetics and tissue culture. Mapping DNA markers on the Citrus genome should lead to identification of markers closely linked to important loci, thereby facilitating early selection and minimizing costs associated with plant size and juvenility. Genetic transformation methods provide opportunities for trait-specific modification of commercial cultivars. The selection of beneficial variants from sectored fruit chimeras, and the recovery of plants via somatic embryogenesis, can overcome the problems of nucellar embryony and the hybrid nature of commercial cultivar groups. Induced mutagenesis, using mature vegetative buds, may overcome size and juvenility, as well as nucellar embryony and hybridity. Ploidy level manipulation in vitro provides methods to overcome sterility, incompatibility, and nucellar embryony, and it can increase the number and diversity of tetraploid breeding parents available for development of seedless citrus triploids.

Virus indexing in the New South Wales citrus improvement scheme

A virus indexing program was initiated in the 1950s to test local citrus clones with commercial potential, for the presence of citrus exocortis viroid and psorosis. As indexing techniques improved, clones have also been tested for the presence of citrus cachexia viroid and other viroids, citrus infectious variegation virus, citrus tatter leaf virus, and strain severity of citrus tristeza virus. Of 123 candidate clones tested, only 2 were infected with psorosis virus. Crinkly leaf (infectious variegation virus) was widespread in lemons but has been eliminated by nucellar embryony. The widespread incidence of citrus exocortis and other lower molecular weight viroids has been reduced by screening mother trees on Poncirus trifoliata stocks, biological indexing, and, more recently, by sequential polyacrylamide gel electrophoresis of RNA extracts. Cachexia is rarely found in Australian citrus clones.