الفهرس 
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Abstract
This study was carried out through four successive years (2010-2014) in the Citrus orchard and the plant tissue culture laboratory of Breeding Research Department, HRI, ARC, Giza, Egypt. The main targets of this investigation were; i) production of the tetraploids from doubling of the diploid nucellar embryos with colchicine treatment, ii) induction of homozygous plants by in vitro anther culture and iii) induction of different ploidy levels by culture of small, large and aborted seeds.
For tetraploids production, the detached nucellar embryos of Balady mandarin were either treated with different colchicine concentrations and durations or directly cultured for three weeks on germination medium. For colchicine concentrations, the control treatment (without colchicine) showed the highest significant mean percentage of survival and germination (96.8%) and (99.0%), respectively. For colchicine durations, the highest significant mean percentage of survival and germination was (85.0%) and (91.4%), respectively for 24 h. For three weeks culture, the control treatment showed the highest survival percentages (93.3% and 84.4%) for (control and 0.01% colchicine), respectively. The highest significant percentage of germinated embryos was (100%) for control.
For haploids induction, the anthers containing microspores at uninucleate stage of three mandarin, two Clementine and five orange cultivars were excised and cultured on N6 medium supplemented with N&N vitamins plus different additives. The addition of Nitsch vitamins, 40 gl-1 sucrose, 2 mgl-1 2,4-D, 0.5 mgl-1 BA, 1 mgl-1 TDZ, 200 mgl-1 ascorbic acid, 500 mgl-1 casein hydrolysate, 200 mgl-1 glutamine and 2 mgl-1 AgNO3 to N6 medium achieved the highest percentage of embryogenic callus of anthers (20.18%) of all genotypes under investigation. Anthers of Sanguinelli Blood orange and Olinda Valencia orange showed the highest mean percentage of embryogenic calli (24.95 and 24.13%, respectively). The somatic embryogenesis percentage reached 85.30 and 84.40% for Cutter Valencia orange and Sanguinelli blood orange, respectively after five month from anther culture. The highest mean number of embryos from embryogenic calli was 14.80 embryos per callus mass for Cutter Valencia orange on MT medium supplemented with 50 gl-1 sucrose and 500 mgl-1 malt extract. The highest germination percentage of embryos was 55.00 % in Sunburst mandarin on MS medium supplemented with 30 gl-1 sucrose and 1 mgl-1 GA3. The highest germination percentage of embryos was 55.00% in Sunburst mandarin on MS medium supplemented with 30 gl-1 sucrose and 1 mgl-1 GA3.
To induce different ploidy levels, the large, normal, small and aborted seeds from open pollination of two Clementine and three mandarin cultivars were cultured in vitro on MS supplemented with either casein hydrolysate or malt extract. For the normal seeds, the highest significant mean percentage of germination was obtained by Balady mandarin (93.50%). For the small, large and aborted seeds, Kara mandarin showed the highest significant mean percentage of germination (70.56%), (54.89%) and (50.91%), respectively. For all seed types, MS medium supplemented with casein hydrolysate recorded higher significant value than MS supplemented with malt extract.
Chromosome number counting application showed that out of 80 examined plantlets derived from colchicine-treated embryos, only three (3.75%) were tetraploids. Only four (4%) haploids from 100 examined plantlets derived from anther culture that were regenerated from anther culture. Out of 176 plantlets obtained from selected seeds, two (1.14%) were haploids and two (1.14%) were triploids.
For obtaining homozygous plants via doubling of haploid plantlets, the axillary buds of these plantlets were treated with 0.05% colchicine for 10 days. For haploids obtained from anther culture, five of the colchicine-treated axillary buds produced shoots in Balady mandarin, one in Kara mandarin. For haploids obtained from selected seeds, one of the colchicine-treated axillary buds produced shoots in Balady mandarin. Ploidy level analysis by chromosome counting of new shoots arising showed that, only four of resulting shoots of Balady mandarin and one of Kara mandarin of plantlets resulted from anther culture were diploid.
Key words: Anther culture - chromosome counting - Citrus - Clementine - Colchicine - In vitro - Mandarin - MS - MT - N6 - Orange - Ploidy