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  • Author or Editor: M. A. L. Smith 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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The bright red pigmentation in edible, anthocyanin-rich fruits and vegetables is a definite bonus in terms of market appeal. As a result, breeders have worked consistently to intensify anthocyanin levels or alter composition in crops. The positive links between consumption of crops and food products containing natural anthocyanin pigments, and reduced incidence of cardiovascular and other chronic diseases, have been established anecdotally and more recently validated in research trials including those from our laboratory group. The protective events, most attributed to the potent antioxidant properties of anthocyanin pigments and associated phytochemicals, place anthocyanin-rich crops in the category of “Functional Foods,” yielding health protection unrelated to nutritional value. In vitro bioactivity assays have identified components from these crops capable of blocking the initiation stages of carcinogenesis, while a completely separate class of phytochemicals and sets of assays establish efficacy against the promotion stages of tumorigenesis. Animal models for carcinogen-induced damage to mammary gland and skin DNA subsequently demonstrate the in vivo potency of the same target compounds. Similarly, to establish cardioprotective properties, demonstrations of ability to inhibit platelet aggregation, relax vascular muscle tissue, and reduce total serum cholesterol are demonstrated in a series of in vitro assays, and via animal models and human studies. While activity-directed fractionations seek to identify specific responsible compounds, it is increasingly evident that bioactivity is drastically attenuated once specific compounds are isolated, and the synergistic interaction of associated phytochemicals in horticultural crops is prerequisite to realizing health benefits. These complications have slowed the establishment of effective minimum “dosages,” but all the more strongly promote consumption of the crops.

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The structure and quality of the adventitious root system formed on a microcutting stem is crucial to the successful acclimation, survival, and ultimate performance of micropropagated plants. Despite increasing evidence that the rooting method impacts on the character of the framework root system, very little research has elucidated the consequences of standard rooting methods on plant quality and performance. Root initiation on microcuttings is handled by a wide range of strategies in commercial practice. In comparison to in vitro roots, ex vitro roots have smaller root diameters, larger vascular diameters, greater length, more branch root development, and root hairs. One important microenvironmental parameter - light - was investigated to determine its contribution to root character. Typically, in vitro root systems are exposed to light throughout the root initiation period. Parallel treatments were established of in vitro light and dark rooting and ex vitro rooting of four woody species. Regardless of light exposure, the overall diameter of adventitious roots was larger for in vitro treatments than the ex vitro treatment. Vascular development was significantly more advanced ex vitro. These results suggest that light is not a major influence contributing to the differences between ex vitro and in vitro root character.

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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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Abstract

University horticulture students often do not experience the real challenges of the industry until after graduation due to the constraints of classroom schedules and the traditional lecture/laboratory format (3, 10). Practical management of a nursery inventory is a classic example. Complete inventory records are essential for making key management and planning decisions in any business, but the peculiar traits of a woody plant materials inventory demand exceptional ingenuity on the part of nurserymen. Nursery stock may remain in inventory for 7-10 years (from propagation to sale), and is continually changing (in terms of size and caliper) during this period. Individual plants are moved through multiple production stages with characteristic production losses at each stage, a complication that requires constant updates in inventory records. The long lag time between propagation and finished production also makes the target market and number of plants ultimately available for sale very difficult to predict accurately. Each unique requirement compounds the difficulty of maintaining a useful nursery stock inventory record.

Open Access

Effects of three variables (vessel type, closure, and explant orientation) on microcutting quality were investigated using five woody species [low shadblow, Amefanchier spicata (Lam.) C. Koch (Syn. A. humilus Wieg.); red maple, Acer rubrun L. `Red Sunset'; border forsythia, Forsythia ×intermedia Zab. `Sunrise'; apple, Malus ×domestica Borkh. `McIntosh'; river birch, Betula nigra L.]. Uniform shoot explants were oriented vertically or horizontally in three vessel types (60-ml glass culture tubes, 200-ml glass baby food jars, and 350-ml polypropylene GA7 vessels) with and without a Parafilm seal. Visual density per explant obtained by image analysis was increased in larger vessel types, and significantly more shoots were produced from horizontally placed explants. Closure treatments influenced microshoot quality, but trends were species specific. Overall, horizontal explant orientation in larger vessels wthout parafilm maximized shoot response for most of the species studied. In vitro rooting of microcuttings was significantly enhanced in larger vessels.

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Ohelo (V. pahalae Skottsb.) and bilberry (V. myrtillus L.) shoots were regenerated via direct organogenesis from whole leaves and leaf sections and also from hypocotyl explants of bilberry. Explants preincubated for 1 to 2 weeks in darkness yielded ≈75% regeneration frequencies and the highest number of regenerating shoots/explant on TDZ-supplemented media (0.9 to 2.7 μm). When 2iP or zeatin were substituted as the cytokinin source, frequencies of regeneration and shoot productivity were significantly lower. Explants held under constant illumination (no dark pretreatment) had significantly lower regeneration frequencies in all tested cytokinin-supplemented media. 2,4-D stimulated callus formation, but did not support regeneration from vegetative explants. Cells from callus and suspension cultures did not exhibit regeneration in any of the media that supported organogenesis from leaves. Regenerants were successfully micropropagated, although callus formation caused by zeatin and high 2iP levels interfered with shoot proliferation. Zeatin induced hyperhydricity in shoots from both species, but more severely in ohelo. Ex vitro rooting after treatment with 4.9 μm IBA or 5.4 μm NAA was 95% and 60% successful for bilberry and ohelo, respectively, and plants were readily acclimatized after an interval in a fog chamber. Bilberry microshoots also rooted in vitro in the absence of growth regulator treatment. Chemical names used: 1H-indole-3-butanoic acid (IBA); N-(3-methyl-2-butenyl)-1-H-purine-6-amine (2iP); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA); thidiazuron=1-phenyl-3-(1,2,3-thiadiazio-5-yl)urea (TDZ); 2,4-dichlorophenoxyacetic acid (2,4-D); 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

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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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