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Methods for production of Citrus triploids were investigated. Ten diploid, strictly zygotic clones were used as seed parents. Two tetraploid somatic hybrids [`Key' lime (Citrus aurantifolia) with `Valencia' sweet orange (C. sinensis), and `Hamlin' sweet orange with `Flying Dragon' trifoliate orange (Poncirus trifoliata)] and two autotetraploids were used as the male parents. Embryos were excised from underdeveloped seeds 3-6 months after pollination and cultured. Murashige and Tucker's medium was supplemented with either malt extract (500 mg·L-1) and adenine sulfate (25 mg·L-1) (EMA), with gibberellic acid (1 mg·L-1) (EG1), or with malt extract (1500 mg·L-1) (MTG). Embryo proliferation was observed from 11.5% of all cultured embryos excised 3-4 months after pollination, but varied among crosses. Ploidy level was determined by flow cytometry. Most recovered plants were triploid, but tetraploids and diploids were also identified at frequencies that varied among crosses. The greatest frequency of 3× plant recovery per embryo rescued resulted when embryos were harvested 3 months post-pollination and cultured on EG1 medium.
Protoplasm culture following the chemical fusion of `Valencia' sweet orange [Citrus sinensis (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon [Citrus limon (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 Citrus. 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 Citrus clones amenable to somatic hybridization and genetic transformation experiments.
Specific primers were designed for 61 cloned RAPD fragments and from 10 Citrus EST sequences for the production of SCAR, CAPS, and STS markers for a Citrus grandis `DPI 6-4' × Poncirus trifoliata `Rubideaux' F1 pseudo-testcross population. Fifteen SCAR, three CAPS, and one EST/STS markers were developed. An additional 17 SCAR and CAPS primer pairs developed at the Citrus Research and Education Center for a Citrus grandis `Thong Dee' × (Citrus grandis `Thong Dee' × Poncirus trifoliata `Pomeroy') BC1 population were screened in the pseudo-testcross population. A total of 27 markers were identified and scored in the pseudo-testcross population in which 24 were mapped; 13 in the Citrus parental linkage map on seven linkage groups and 11 in the Poncirus parental map on five linkage groups. In the BC1 population, 20 of 27 markers tested were found to be polymorphic and 13 mapped to seven of nine linkage groups. Of these, 11 were mapped in both populations and could be used for aligning presumed homologous regions on the three linkage maps.
Production of tetraploid somatic hybrids that combine complementary diploid rootstock germplasm via protoplast fusion has become a practical strategy for citrus rootstock improvement, with the overall objective of packaging necessary disease and pest resistance into horticulturally desirable, widely adapted rootstocks. Citrus somatic hybridization techniques have been advanced to the point where numerous somatic hybrid rootstocks can now be produced and propagated for evaluation on a timely basis. Herein we report the production of 11 new somatic hybrid rootstock candidates from 12 different parents, including Milam lemon hybrid (Citrus jambhiri Lush.), Cleopatra mandarin (C. reticulata Blanco), sour orange (C. aurantium L.), `Succari' sweet orange [C. sinensis (L.) Osbeck], `Redblush' grapefruit (C. paradisi Macf.), `Nova' tangelo [C. reticulata × (C. paradisi × C. reticulata)], `Kinkoji' (C. obovoidea Hort. Ex Takahashi), Swingle citrumelo [C. paradisi × Poncirus trifoliata (L.) Raf.], Carrizo citrange (C. sinensis × P. trifoliata), rough lemon 8166 (C. jambhiri), and Palestine sweet lime (C. limettoides Tan.). All hybrids were confirmed by cytological and VNTR-PCR analyses, and have been propagated, budded with a commercial scion, and field-planted for performance evaluation.
Allotetraploid somatic hybrid plants of `Hamlin' sweet orange (Citrus sinensis L. Osbeck) + `Femminello' lemon (C. limon L. Burm. f.), and Milam lemon (purported hybrid of C. jambhiri Lush) + `Femminello' lemon were regenerated via somatic embryogenesis following protoplast fusion. `Hamlin' and Milam protoplasts were isolated from undeveloped ovule-derived embryogenic callus cultures and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Femminello' lemon. Somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose isomerase, phosphoglucose mutase, and 6-phosphogluconate dehydrogenase leaf isozymes. The somatic hybrids will be used in interploid crosses with lemon in an effort to generate seedless triploid lemon types with improved tolerance to mal secco disease.
Seedlessness is an important breeding objective of most citrus scion improvement programs, but production of quality seedless triploid citrus via interploid crosses has historically been limited by the low quality of available tetraploid parents. Production of tetraploid hybrid parents from elite diploid scion cultivars via protoplast fusion is now a practical strategy, and numerous hybrids can be produced on a timely basis from a wide range of parents. Such hybrids can be used as pollen parents in interploid crosses to generate improved seedless triploid fresh fruit cultivars. Herein we report the production of 15 such hybrids from 17 different parents, including sweet orange [Citrus sinensis (L.) Osbeck], mandarin/tangerine (C. reticulata Blanco), grapefruit (C. paradisi Macf.), pummelo [C. grandis (L.) Osbeck], tangor (C. reticulata × C. sinensis), and tangelo (C. reticulata × C. paradisi) germplasm. All hybrids were confirmed by cytological and RAPD analyses, and have been budded to selected rootstocks to expedite flowering.