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  • Author or Editor: Paul E. Read x
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Abstract

This symposium is sponsored jointly by the Tissue Culture and Propagation Working Groups, with cosponsorship by ASHS Working Groups on Viticulture and Small Fruits, Citrus, and Fruit Breeding. The interest of these groups indicates the significance of this subject to so many areas of horticulture. Many professionals in both commercial and public sectors devote their efforts to, and earn their livelihood from, a dependency on clonal propagation. Most fruit crops, many nursery species and types, a significant number of floricultural crops, and several vegetables are propagated clonally (6, 7). Recently, many of these clones have been propagated commercially through various tissue-culture methods (7, 14).

Open Access

The temperate native terrestrial orchids are endangered species. Their propagation from seeds poses specific problems. It is well known that orchid seeds are devoid of endosperm and in nature they need microscopic fungi in a symbiotic relationship for germination. We developed a successful asymbiotic in vitro culture method for germinating seeds of several temperate orchid species and for maintaining the cultures of young plantlets. The medium used for both germination and seedling culture was a modified FAST medium. Seeds were surface-disinfested for 10 minutes in a 10% calcium hypochlorite solution. After sowing, the cultures were kept under dark condition at 10–12°C for 4 weeks. After that the cultures remained in the dark, but the temperature was raised to 25–26°C until germination occurred. Thereafter cultures required alternating seasonal temperatures: 25–26°C from the beginning of April to the end of September and 17–19°C from October to March. For the development of the young plantlets natural dispersed light and prevailing day-length was favorable. After 2 years of aseptic culture they were suitable for transfer ex vitro. Different stages of seed germination and plant development were observed using a scanning electron microscope and will be included in this presentation. Further observation of the effects of different environmental factors is currently under investigation.

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A certain period of cold is needed to break bud dormancy for almost all woody species. A pre-forcing bleach soak has been demonstrated to at least partially replace this requirement (Yang and Read, 1989). Therefore, new softwood growth can be produced in the off-season. Such supple softwood growth is excellent material to be used either as explants for in vitro culture, or as cuttings for macropropagation of woody species. Further studies on pre-forcing bleach soaks were conducted to investigate optimum concentration and duration of soak, and to find the most suitable depth of bleach solution soak, in order to maximize the breaking of bud dormancy. Optimum bud break was obtained by soaking the basal 1/3 of dormant stems in 10% bleach solution for 10 minutes prior to forcing. Soaking dormant woody stems in alcohol solutions prior to placing stems in the forcing solution was also studied. The alcohol soak had negative effects on bud break of spirea, although it showed positive effects for lilac and privet.

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BA, IBA and GA3 were incorporated into softwood tissues to be cultured in vitro or rooted as cuttings by adding the plant growth regulators (PGR) at various concentrations to a forcing solution containing 200 mg/l 8-hydroxyquinoline citrate and 2% sucrose. BA and GA3 helped break bud dormancy in autumn-collected stems and increased percent bud-break. IBA inhibited bud break and shoot elongation. Rooting of forced softwood cuttings was enhanced by IBA in the forcing solution, while GA3 inhibited the rooting of plant species tested. When dormant stems were forced with periodic additions of BA (10 mg/l) in the forcing solution, in vitro shoot proliferation was enhanced. However, inclusion of GA3 in the forcing solution reduced shoot proliferation. A pre-forcing NaOCl soak and a pre-forcing treatment with wetting agents accelerated bud break, size and number of shoots available for both micro- and macro-propagation of the woody plant species tested. The forcing solution protocol described is an effective PGR delivery system and it can be used by the propagator to extend the season for obtaining softwood growth suitable for use as in vitro explants or softwood cuttings.

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A forcing solution containing 200 mg 8-hydroxyquinoline citrate per liter and 2% sucrose has enhanced availability of cutting materials by forcing dormant woody stems in the off-season. Anxins, such as IBA, included in the forcing solution promoted subsequent rooting by increasing root number per cutting and root length for privet. Inclusion of IBA in the forcing solution following the initial use of GA3 in the forcing solution counteracted the undesirable effects of GA3 on rooting and stimulated rooting after taking advantage of the favorable effects of GA3 on bud break and shoot elongation. However, the ability of IBA to counteract the negative effects of GA3 on rooting was dependent on the length of GA3 treatment. The modification of forcing solution system by sequentially including GA3 and then replacing GA3 with IBA expedited propagation of privet. Production of candidate cuttings or explants was stimulated by including GA3 in the forcing solution, and rooting of the cuttings was promoted by subsequent auxin or cytokinin inclusions in the forcing solution to replace GA3 This modified forcing solution system also proved to be a successful and efficient model for propagation of other difficult to propagate woody species.

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Microshoots of four hazelnut genotypes grown in vitro on Nas and Read medium (NRM) containing various combinations of CuSO4 • 5H2O and myo-inositol were successfully rooted and acclimatized ex vitro without any need of in vitro hardening treatments. Dipping of shoot bases in 1000 ppm indole-3-butyric acid (IBA) solution for 5 or 10 seconds followed by placement of shoots in plant growth regulator free NRM gave rise to formation of roots as early as 8 days. Shoots treated for 5 and 10 seconds rooted similarly, and depending on genotype, 88% to 98% rooting was observed within 15 days after treatment with IBA. Ex vitro survival of shoots three months after in vitro-root induction was 73% when shoots were treated with IBA for 5 seconds and 66% when shoots were treated for 10 seconds. The highest ex vitro survival rate (97%) 3 months after root induction was observed when shoots were treated with IBA solution for 10 seconds, and then cultured directly in peat pellets. Shoots developed good roots, and grew up to 70 cm in height 3 months after root induction. The potential use of rooting and acclimatization protocol for commercial micropropagation of hazelnut is presented.

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In an effort to increase somaclonal variation in blueberry, a protocol was developed to regenerate viable shoots from internode segments. The explant consisted of the last-formed, fully developed internode taken from 3 different genotypes of Vaccinium grown in vitro. Explants were cultured 6 weeks on Zimmerman's Z-2 medium supplemented with 2iP, zeatin, thidiazuron, kinetin, or BA at concentrations of 5, 25, 50, and 100 uM. Explant response to the treatments varied and included: no response, callus growth only, callus growth and subsequent shoot formation originating from the callus mass, and adventitious shoot formation directly from the internode segment without an intervening callus. Greatest shoot regeneration (20-25 shoots/explant) was obtained on medium supplemented with zeatin at 5, 25, and 50 uM, however treatment response was not consistant across all genotypes. Regenerated shoots could be readily sub-cultured, rooted in soil mix and will be evaluated for somaclonal variation.

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A forcing solution containing 200 mg 8-hydroxyquinoline citrate per liter and 2% sucrose has been demonstrated to extend the season for obtaining softwood growth suitable for use as explants in micropropagation (Yang & Read 1989). Forcing dormant woody stems in the off-season in this fashion also enhances the macropropagation of woody plant species by providing softwood outgrowth that can be rooted as softwood cuttings. GA 3 , IBA, IAA and NAA were incorporated into softwood growth which was later used as cuttings for rooting by adding plant growth regulators at various concentrations to the forcing solution. GA 3 incorporated into the forcing solution hastened bud break, increased shoot elongation, but inhibited rooting of softwood cuttings taken from stems forced in this manner. IBA, IAA and NAA in the forcing solution exhibited typical auxin effects on rooting of cuttings by increasing root number per cutting and root elongation. In order to expedite macropropagation of woody plants, GA 3 and IBA were added SEQUENTIALLY to the forcing solution. Addition of IBA to fresh forcing solution following initial use of GA 3 in the forcing solution counteracted the negative effects of GA 3 and stimulated rooting. This protocol is proposed as a method to assist propagation in rooting difficult species by softwood cuttings in the off-season.

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Axillary buds of `Valiant' grapevine (Vitis spp.) grown in vitro were transferred onto Murashige and Skoog (MS) medium supplemented with different cytokinin and auxin combinations and concentrations. It was found that culture medium caused statistically important differences in number of nodes, number of fully expanded leaves, number of multiple shoots, number of roots, and length of shoots. MS medium supplemented with 1.0 mg BA/liter in combination with 0.01 mg NAA/L was found to be the best medium for shoot growth and callus production. MS medium supplemented with the combination of 0.5 mg BA/L and 0.01 mg NAA/L was the best medium for explant rooting. The medium containing BA and NAA encouraged better shoot growth than those containing BA alone. When the concentration of BA in the medium was increased, multiple shoot proliferation and teratological structures of explants increased, but the number of small leaves and length of internode decreased. Axillary bud culture led to better shoot growth than was found for shoot apex culture. The presence of leaves positively affected shoot growth from axillary buds. Also placing the axillary buds horizontally onto the medium gave better shoot proliferation and growth than placing them vertically.

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