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  • Author or Editor: Ahmed Obeidy x
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Endophytic fungi associated with mature pecan nuts (cultivars `Smart', `Desirable' and `Owens') prevented successful, contaminant-free. in vitro culture of embryo explants, even after rigorous surface disinfestation of the nuts and careful aseptic shelling. Disinfestation with sodium hypochlorite after removing the shells was also unsuccessful, because even dilute concentrations which were ineffective against the fungal contaminants destroyed embryo viability. An alternative disinfestation technique is required to establish in vitro cultures from mature pecan explants. Explanting media with low water availability were developed to capitalize on the relatively stringent requirement of most fungal contaminants for free water. The explanting media were supplemented with 0.9-1.5% agar. Other media components were selectively included or omitted to test their influence on water activity and fungal growth. After four weeks of culture, the incidence of contamination was reduced to 30% or less on modified explanting media, compared to 100% loss to contamination on control medium (0.5% agar). The brief initial culture period effectively eliminated fungal contamination from cultures by plasmolyzing fungal hyphae or denaturing fungal protein, without effecting pecan embryo viability. Cultures remained contaminant-free after transfer to control medium. Axillary and adventitious bud development from disinfested embryo axes was subsequently induced in a medium with 18 μM BAP and 5 μM IBA, and regeneration from cotyledon explants was accomplished in a medium with 50 μM NAA. This disinfestation technique allows mature pecan embryo explants to be used in biotechnology.

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Endophytic fungi associated with mature pecan nuts (cultivars `Smart', `Desirable' and `Owens') prevented successful, contaminant-free. in vitro culture of embryo explants, even after rigorous surface disinfestation of the nuts and careful aseptic shelling. Disinfestation with sodium hypochlorite after removing the shells was also unsuccessful, because even dilute concentrations which were ineffective against the fungal contaminants destroyed embryo viability. An alternative disinfestation technique is required to establish in vitro cultures from mature pecan explants. Explanting media with low water availability were developed to capitalize on the relatively stringent requirement of most fungal contaminants for free water. The explanting media were supplemented with 0.9-1.5% agar. Other media components were selectively included or omitted to test their influence on water activity and fungal growth. After four weeks of culture, the incidence of contamination was reduced to 30% or less on modified explanting media, compared to 100% loss to contamination on control medium (0.5% agar). The brief initial culture period effectively eliminated fungal contamination from cultures by plasmolyzing fungal hyphae or denaturing fungal protein, without effecting pecan embryo viability. Cultures remained contaminant-free after transfer to control medium. Axillary and adventitious bud development from disinfested embryo axes was subsequently induced in a medium with 18 μM BAP and 5 μM IBA, and regeneration from cotyledon explants was accomplished in a medium with 50 μM NAA. This disinfestation technique allows mature pecan embryo explants to be used in biotechnology.

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The regenerative capacity of mature pecan [Carya illinoinensis (Wangenh.) K. Koch] embryonic tissues was demonstrated after pretreating mature nuts to eliminate associated endogenous contaminants. Cultured cotyledon segments were induced to form adventitious roots in a medium with 50 μm NAA. A regeneration medium with 20 μm BA and 5 μm IBA stimulated prolific axillary shoot production from the embryonic axis without causing cotyledon abscission. Cotyledon retention was essential for shoot initiation and long-term development. Eighty-five percent of the shoots emerging from embryonic axes formed at the cotyledonary nodes. Thirty percent of the microshoots rooted on an auxin-free medium after preculture in a medium with 20 μm IBA. TDZ (25 μm) stimulated callus production from the cotyledonary nodes and radicles. Adventitious buds emerged on the callus surface and internally in callus. Chemical names used: a -naphthaleneacetic acid (NAA); 6-benzylaminopurine (BA); indole-3-butyric acid (IBA); N-phenyl-N'-1,2,3-thidiazol-5-ylurea (TDZ).

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Micrografting is an effective technique for elimination of viruses, early diagnosis of grafting incompatibilities, rejuvenation of mature tissue, and bypassing the juvenile phase in fruit trees. Current micrografting procedures are difficult, impractical, expensive, and generally result in an inefficient rate of successful graft production. In order to alleviate some of these limitations, a unique apparatus was designed to splice the in vitro-derived scion and rootstock together during the micrografting process. The dual-layer device was constructed with a pliant outer layer to facilitate manipulation during the grafting of micro-scale plants, and a delicate, absorbent inner layer to cushion the plant tissue and retain hormones and other compounds. These chemicals are slowly released at the grafting zone to alleviate oxidation and enhance callus formation at the cut surface of scion and rootstock. After healing, it is easy to remove the grafting apparatus from the grafted plant without damaging the tissues. This apparatus may be used to unite a scion and a rootstock with different stem diameters. Shoot-tip cultures of `McIntosh' and `M-7' apple and `North Star' sweet cherry, and in vitro seedlings of lemon, orange and grapefruit were used as a source of in vitro scions and rootstocks. Successful graft unions were developed, and the grafted plants were transplanted into the greenhouse environment Micrografted plants were sectioned to determine the anatomical characteristics of the graft union.

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Micrografting is au effective technique for elimination of viruses, early diagnosis of grafting incompatibilities, rejuvenation of mature tissue, and bypassing the juvenile phase in fruit trees. Current micrografting procedures are difficult, impractical, expensive, and generally result in an inefficient rate of successful graft production. To alleviate some of these limitations, a unique apparatus was designed to splice the in vitro-derived scion and rootstock together during the micrografting process. The dual-layer device was constructed with an outer layer of aluminum foil, with flexibility to facilitate manipulation during the grafting of micro-scale plants. A delicate, absorbent inner layer of paper toweling cushions the plant tissue. It also may be treated with hormones and other compounds. After healing, it is easy to remove the grafting apparatus from the grafted plant without damaging the tissues. This apparatus may be used to unite a scion and a rootstock with different stem diameters. Shoot-tip cultures of `McIntosh' and M.7 apple and `North Star' sour cherry, and in vitro seedlings of lemon, orange, and grapefruit were used as a source of in vitro scions and rootstocks. Successful graft unions were developed, and the grafted plants were transplanted into the greenhouse environment.

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