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D.S. Achor, H. Browning, and L.G. Albrigo

Young expanding leaves of `Ambersweet' [Citrus reticulata Blanco × C. paradisi Macf. × C. reticulata) × C. sinensis (L) Osb.] with feeding injury by third larval stage of citrus leafminer (Phyllocnistis citrella) were examined by light and electron microscopy for extent of injury and tissue recovery over time. Results confirmed that injury is confined to the epidermal layer, leaving a thin covering over the mine tunnel that consisted of the cuticle and outer cell wall. Wound recovery consisted of two possible responses: the production of callus tissue or the formation of wound periderm. The production of callus tissue developed within 3 days of injury when the uninjured palisade or spongy parenchyma below the injured epidermis produced callus tissue through periclinal or diagonal cell divisions. After 1 month, the entire epidermis was replaced by callus tissue. In the absence of secondary microbial invasion, this callus tissue developed a thick cuticle, followed by development of a covering of platelet wax after 4 months. Alternatively, wound periderm formed if the outer cuticular covering was torn before the cuticle had developed sufficiently to prevent the exposed cells from being desiccated or invaded by fungi, bacteria, or other insects. The wound periderm consisted of a lignified layer of collapsed callus cells, a suberized phellem layer, and a multilayered phelloderm-phellogen. Since there were always cellular collapse or fungi and bacteria associated with wound periderm formation, it was determined to be a secondary effect, not a direct effect of leafminer feeding.

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Yihui Cui, Peng Zhao, Hongqiang An, Nan Lv, Zifeng Zhang, Wei Pei, and Wanjun Wang

were examined by ESEM (Quanta 200; FEI, Eindhoven, The Netherlands) in their natural form without additional sample preparation. Results Cytological characteristics of embryonic callus cells. Histologically, embryonic calli were composed of two

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Takaya Moriguchi, Tetsushi Hidaka, Mitsuo Omura, Toshiaki Motomura, and Tomoya Akihama

Interspecific hybridizations by electrofusion of embryogenic callus cells from `Seminole' tangelo (Citrus reticulata Blanco × C. paradisi Macf.), `Hazzara (Abohar)', or `Ohta' ponkan (C. reticulata Blanco) and leaf cells from `Lisbon' lemon [C. limon (L.) Burm. f.] or rough lemon (C. jambhili Lush.), respectively, were performed. Electrofusion of `Seminole' tangelo and `Lisbon' lemon, `Hazzara (Abohar)' and rough lemon, and `Ohta' ponkan and rough lemon resulted in 33, 43, and 36 plants, respectively. Seven to 10 plants in each combination were selected randomly and used to investigate nuclear and cytoplasmic genomes. Regenerated plants derived from electrofusion of `Seminole' tangelo and `Lisbon' lemon, and `Hazzara (Abohar)' and rough lemon possessed the same restriction fragment pattern for nuclear rDNA as that of the mesophyll parents and the same mitochondrial DNA (mtDNA) restriction pattern as that of the respective suspension parents, indicating that they were cybrids. In contrast, all the plants resulting from a combination between `Ohta' ponkan and rough lemon were confirmed by nuclear rDNA and mtDNA analysis to be somatic hybrids. The analysis of chromosome number supported the results of Southern blot hybridization. The results suggest that specific cell lines, parental combinations, or both can increase the efficiency of inducing cybrids in Citrus by electrofusion.

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Mustapha Benmousea and Yves Desjardine

We have developed tissue culture and protoplasts isolation protocols for Asparagus densiflorus in order to use this genetic material in the breeding of Asparagus officinalis. For tissue-culture of A. densiflorus, the conditions which optimize the induction and the production of callus are a full MS medium with 1 mg/L of both pCPA and BAP and 0.5 mg/L of thiamine. HCL in the dark. on this medium, we obtained a friable white callus. Indirect organogenesis was obtained if pCPA was omitted from the medium. Replacement of the plant growth regulators by 2,4-D and Kinetin produced a hard and compact callus which did not differentiate. Protoplast have been isolated from 10 days old friable callus. cell wall was digested with 0.3% macerase, 1% cellulase and 0.8% rhozyme for a period of 16h at a temperature of 27°C in a CPW medium. Protoplast yield was 2 ×106 protoplasts/g callus. osmolarity of the digestion solution was 0.8 M provided with a mixture of glucose (0.6 M) and mannitol (0.2 M). cells were then plated at a density of 1 × 105 cells per ml. Microcolonies formed on a 1/2 MS medium with 0,5 mg/L NAA and ZEA and 1 g/L glutamine in the dark.

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J. Michele Myers and Philipp W. Simon

We evaluated the efficiency of transformation in garlic for promoter activity, osmoticum effect and shaker speed using particle bombardment as the method of gene delivery. Callus was produced from root segments on a modified B-5 medium for four garlic clones. Suspension cultures were then established on a modified B-5 medium + 2,4-D using 6-month-old callus. Cells were collected by vacuum filtration and the Bio-Rad PDS-1000/He system was used to deliver genes. The activities of CaMV 35S, maize Adh1, and rice Act promoters were evaluated for transient expression using the β-glucuronidase (GUS) reporter gene. Osmotic conditioning of cells was performed by adding both mannitol and sorbitol to the medium. Osmoticum effect was evaluated for enhancement of transformation efficiency using GUS. The effect of shaker speed (120, 180 and 240 rpm) on cell type was evaluated for transformation efficiency using GUS. CaMV 35S promoter activity was much higher for garlic than either the maize Adh1 or rice Act promoters. Osmoticum did not enhance promoter activity, but differences in response to osmoticum among garlic clones were observed. Shaker speed did affect cell type, and transformation efficiency was greatly increased at higher shaker speeds. Confirmation of stable transformation and regeneration are in progress.

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Simon A. Mng'omba, Elsa S. du Toit, Festus K. Akinnifesi, and Helena M. Venter

, presence of phenols, and callus proliferation at the union. U, unknown stock. Fig. 5 shows a common trend for callus proliferation at the unions observed using a light microscope. Callus cells were prolific below the union, where a good union was

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Fan Cao, Xinwang Wang, Zhuangzhuang Liu, Yongrong Li, and Fangren Peng

D ). Based on a series of slice observations, it was found that there was no latent root primordium in pecan cutting stems. The root primordia of pecan hardwood cuttings are the differentiation of the callus cells. Therefore, promoting callus cell

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Napaporn Sae-Lee, Orapin Kerdchoechuen, Natta Laohakunjit, Benjawan Thumthanaruk, Dipayan Sarkar, and Kalidas Shetty

incubated at 25 ± 1 °C with 108 µmol·m −2 ·s −1 light for 16 and 8 h dark photoperiod with the 40 W cool-white fluorescent tubes (Phillips, Kolkata, India). Every 14 d, induced callus was subcultured for producing of friable cultured callus cells

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Haiyan Zhang

.R. Durzan, D. 2000 A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues J. Expt. Bot. 51 1027 1036 Pitman, M.G. Läuchli, A. 2002 Global impact of salinity and agricultural ecosystems, p. 3–20. In: Läuchli, A

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Ling Yu, Hongwei Chen, Peipei Hong, Hongli Wang, and Kefeng Liu

. splendens has been reported. Unfortunately, the regeneration cycle is comparatively long. Moreover, under in vitro conditions, because of long-term exposure to PGRs and chemicals, the callus cells are very sensitive ( Maruthi Rao et al., 2012 ), and they