Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Relationship between the oil and sugar content in olive oil fruits from Moraiolo and Leccino cultivars during ripening

In our previous work (Cherubini et al., 2009), sugar concentration was proposed as an accurate, reproducible index for technological ripening of olive oil fruits. During 2009 crop season, sugar and oil content of olive oil fruits from Moraiolo and Leccino cultivars were measured during ripening. The sugar content of olive oil fruits was determined both by titration and by using a portable refractometer.     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Production protocol development for greenhouse cut Linaria, Lupinus, and Papaver flowers

Linaria maroccana Hook. f. Ann., ‘Lace Violet’, Lupinus hartwegii ssp. cruikshankii Lindl. ‘Sunrise’ and Papaver nudicaule L. ‘Meadow Pastels’ seeds were directly sown into 105 cell plug trays and received either ambient light or supplemental high intensity discharge (HID) lighting. For each species, a 2 × 3 × 3 factorial was used with two light intensities during propagation, three transplant stages, and three night temperatures. Seedlings were transplanted at the appearance of 2–3, 5–6, or 8–9 true leaves. Transplanted Linaria and Papaver seedlings were placed at 5/11, 10/16, or 15/21 ± 1 °C night/day temperatures and Lupinus seedlings were placed at 15/24, 18/25, or 20/26 ± 2 °C night/day temperatures. For this study, the optimum production temperature for Linaria was 10/16 °C as the cut stems produced at 15/21 °C were unmarketable and production time was excessively long at 5/11 °C. At 10/16 °C, Linaria seedlings should be transplanted at the 2–3 leaf stage to maximize stem number, stem length and profitability. For Lupinus the optimum temperature was 15/24 °C due to long stems and high profitability per plant. Lupinus seedlings should be transplanted at the 2–3 leaf stage when grown at 15/24 °C to obtain the longest and thickest stems; however, $/m² week was higher for plants transplanted at the 8–9 leaf stage due to less time in finishing production space. For Papaver, the 15/21 °C temperature was optimal as that temperature produced the longest stems in the shortest duration, resulting in the highest $/m² week. At 15/21 °C Papaver plants should be transplanted at the 2–3 leaf stage. Supplemental HID lighting had no effect on any of the species.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.     

Confirmation of Clematis hybrids using molecular markers

The hybrid origin of progeny from crosses of Clematis tubulosa and Clematis brevicaudata was investigated using randomly amplified polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) from sequence analysis of chloroplast rbcL, accD genes, and the C. brevicaudatamatK gene. Plants collected from three and four populations of C. brevicaudata (C. brev) and C. tubulosa (C. tubu), respectively, from Mt. Songshan, Beijing, China were used as parents for hybridization. Morphological characters of pollen, seeds, and leaves were recorded in 2007. DNA from leaf samples of both parents and of C. brev × C. tubu and C. tubu × C. brev were extracted, and used for RAPD and SNPs from sequence data. A dendrogram was constructed by the branching neighbor-joining (IB-NJ) method. Proportionate population scores were generated by the admixture model using the STRUCTURE software. Based on morphological characters, C. brevicaudata was quite uniform. However, variations were detected in C. tubulosa. Hybrids of C. brev × C. tubu and C. tubu × C. brev showed intermediate morphological characters of the parents. Accessions of C. tubu × C. brev were clustered into 2 groups, with the majority of hybrids belonging to group IV b in the RAPD dendrogram, suggesting that this resulted from variations within C. tubulosa. In general, the hybrid origin of all progeny characterized by morphological characters was supported by the RAPD and SNPs data. These results indicate that RAPD results supported by SNPs data will be useful tool to verify hybrids.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.     

6-Benzylamino purine stimulates in vitro shoot organogenesis in Eucalyptus erythronema, E. stricklandii and their interspecific hybrids

In vitro bud and shoot organogenesis was investigated for the ornamental plants Eucalyptus erythronema var. erythronema, E. stricklandii and their interspecific hybrids cv. ‘Urrbrae Gem’ and ‘Hybrid 2.5’ by using 0.0, 0.1, 0.25, 0.5 or 1.0 μM BAP on apex and leaf explants. Callus developed on all explants and increased with all concentrations of BAP without significant differences between BAP concentrations. Buds formed on apex and leaf explants of E. erythronema and E. cv. ‘Urrbrae Gem’ especially with 1.0 μM BAP, but these buds rarely developed into shoots. Bud clusters formed on E. erythronema and E. cv. ‘Urrbrae Gem’ apex and leaf explants whereas E. stricklandii and ‘Hybrid 2.5’ produced fewer, individual buds on the explant. Shoots regenerated from apex explants of all genotypes with all levels of BAP, whereas few shoots of any genotype regenerated from leaf explants regardless of the number of buds formed. Shoots from apex explants could be multiplied successfully. Light microscopy showed meristems developed within the callus, and at the callus and bud surfaces. However, few shoots developed considering the level of bud and meristem formation. This report is the first for successful shoot organogenesis and multiplication in an ornamental eucalypt.     

Genetic diversity of Rehmannia glutinosa cultivars based on sequence-related amplified polymorphism markers

SRAP analytic system was used to assess genetic diversity of Rehmnnia glutinosa. Twenty-three Rehmnnia glutinosa cultivars were screened with 288 primer combinations, of which 13 produced stable and reproducible amplification patterns in three repetitive experiments. Among a total of 338 amplified fragments, 306 (90.5%) were polymorphic, with an average of 23.5 fragments for each primer combination. The percentage of polymorphic bands for each primer combination varied from 58.3 to 100%. The cultivars had a similarity ranging from 0.335 to 0.713 with a mean of 0.518. Shannon's diversity index and expected heterozygosity were 0.3217 and 0.2008, respectively. Based on the cluster, which were conducted on the similarity matrix of SRAP marker data, the cultivars were divided into four groups at the 20 rescaled distance cluster combine. The results demonstrated that SRAP is a stable marker technique for the assessment of genetic diversity of Rehmannia glutinosa cultivars, and that the level of genetic diversity among them from different production areas was relatively high.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.     

Compositional quality of virgin olive oils from cultivars introduced in Tunisian arid zones in comparison to Chemlali cultivars

This work is an evaluation of the quality of three introduced European olive cultivars Arbequina, Coratina, and Koroneiki grown under a biological agricultural system in the arid zone of Sfax in Tunisia compared to Chemlali cultivars. Various olive parameters were analyzed, such as ripening index, pulp/stone (P/S) ratio and oil content. We have considered the regulated physicochemical analytical parameters, fatty acids and minor compounds for olive oil. The most of the quality indices and fatty acid composition showed significant variations among olive cultivars. The Koroneiki, Coratina and Chemlali Zarzis cultivars had highest values of oleic acid (62.7%) (76.8%, 75.8% and 73.9%, respectively), Arbequina, had lowest value of oleic acid and similar to Chemlali Sfax. Coratina was noteworthy for its higher content of phenolic compounds (287.8 mg kg⁻¹), oil content (42.4%) and P/S ratio (4.7%). Although their low phenol contents, autochthonous cultivars presented high contents of α-tocopherol (577.8 and 434.6 mg kg⁻¹ for Chemlali Boughrara and Chemlali Zarzis, respectively) except for Chemlali Sfax. In conclusion, European cultivars seem to have adapted to the area studied according to their oil biochemical composition while the increase observed in their α-tocopherol levels. The cultivar Arbequina showed a similar composition of Chemlali Sfax.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental

Lilium Oriental is of great commercial value, but large losses in production can result from its susceptibility to diseases caused by Fusarium ssp. Here we report a mutant of Lilium Oriental resistant to Fusarium, Cai-74, which was generated from crossing tetraploid (from ‘Star fighter’) and diploid (‘Cor. Amore’ × ‘Acapulco’) plants. Hybrid progeny were screened via inoculating Fusarium oxysporum into tissue cultured plantlets in a greenhouse. Although Cai-74 had a saponin content of 3.81 mg g⁻¹, which is much higher than its parents, the highly Fusarium-resistant wild species Lilium dauricum had the highest content of 4.11 mg g⁻¹ among the tested genotypes. Cai-74 had a 3A karyotype rather than 3B found in normal plants of Lilium Oriental. Taken together, our results suggest that Cai-74 bears a chromosomal variation in its structure, and that the saponin content in the lily scales correlate with the Fusarium resistance of Lilium Oriental. Cai-74 could be used as a gene resource for breeding Fusarium-resistant cultivars of Lilium Oriental.     

Breaking the intergeneric hybridization barrier in Carica papaya and Vasconcellea cauliflora

The present investigation was undertaken to develop PRSV (Papaya ringspot virus) resistant hybrids through intergeneric hybridization. Intergeneric hybridization was done involving nine Carica papaya cultivars as female and Vasconcellea cauliflora as male. To break the intergeneric hybridization barrier, various nutrient combinations were used. Among the combinations used, sucrose 5%, sucrose 5% + boron 0.5% and sucrose 5% + CaCl₂ 0.5% improved the fruit set and seed set percentage. A total number of 1197 flowers were pollinated and 308 fruits were obtained. On extraction, 721 seeds were obtained from CO 7, Pusa Nanha and CP 50. Out of 721 F₀ seeds (crossed seeds) sown, 419 seeds germinated and artificial screening for PRSV was carried out 27 days after sap inoculation. Out of 29 F₁ hybrid plants from CO 7 x V. cauliflora cross, only six plants namely CO 7V1 to CO 7V6 were found free from PRSV symptoms. Similarly, out of 55 F₁ hybrids from cross involving Pusa Nanha x V. cauliflora only 23 plants namely PNV1 to PNV23 were found free from the symptoms and 70 plants namely CPV1 to CPV70 out of 335 plants of CP50 x V. cauliflora cross were found free from PRSV symptoms. Among the crosses, Pusa Nanha x V. cauliflora had higher yield under PRSV infected conditions, however, total soluble solids and total sugars were found lesser than the CO 7 x V. cauliflora cross. The hybridity of the progenies were confirmed by using ISSR (Inter Simple Sequence Repeats) primers by the amplification of DNA from progenies and their parents. ISSR primers UBC 856, UBC807 and ISSR primer combinations UBC 856-817, UBC 810-817, UBC 861-817, UBC 856-810, UBC 861-810 and UBC 856-817 clearly amplified specific bands of the male parent, which were present in F₁ progenies, but it was absent in female parents.     

Taxonomic revision of Dendrobium moniliforme complex (Orchidaceae)

Taxonomic revision of Dendrobium moniliforme complex is presented. D. moniliforme complex is characterized by the even slim stems, bracts with brownish zone, semi-spherical anther cap and the hairy disc of lip. Dendrobium tosaense, Dendrobium officinale and Dendrobium guangxiense were excluded by having membranous bracts lacking brownish zone, anther cap conical and bifid. Two species are recognized in this complex, i.e., D. moniliforme and Dendrobium wilsonii. D. wilsonii differs from D. moniliforme by having elliptic leaves about 1.3–2 cm wide, dorsal sepal 3.0–4.0 cm long, 0.6–0.9 cm wide, petals elliptic to oblong, 3.0–4.0 cm long, 1.0–1.5 cm wide, lip elliptic to ovate–lanceolate, 2.6–3 cm long, 1.2–1.5 cm wide.     

Relationship between inbreeding coefficients and plant height of 1-year-old seedlings in crosses among Japanese pear (Pyrus pyrifolia Nakai) cultivars/selections

Plant height, a vigor trait, in 1-year-old seedlings made from Japanese pear (Pyrus pyrifolia) cultivars/selections was measured using 994 individuals from 29 families. The family mean of plant height was negatively correlated (r = −0.72**) to the inbreeding coefficients (F). The regression of the family mean (Fm) on the F value (Fm = 130 − 104F) showed that inbreeding depressions were 8%, 20%, and 40% for F = 0.1, 0.25, and 0.5, respectively. According to the regression, the family mean at F = 0 was estimated at 130 cm. These results showed that the vigor was greatly influenced by inbreeding in Japanese pear. Within-family variances, the genetic segregation of offspring in a family, differed according to family. The proportions of offspring with plant height above 130 cm (estimated Fm for F = 0) were extremely low, i.e., 0–17% for 0.5 ≤ F < 0.60 and 0–8% for F = 0.75.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.