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Marijana Jakše, Pablo Hirschegger, Borut Bohanec, and Michael J. Havey

Although haploid induction has been used in onions (Allium cepa L.) for over 20 years, several obstacles limit its use in plant breeding programs. To address these limitations, we evaluated the responsiveness of doubled haploid (DH) lines and their selfed progenies, an alternative protocol for chromosome doubling using somatic regeneration of haploid lines, and pollen viability of DH lines. Twenty-one DH lines were self pollinated and tested for haploid induction in the second generation. Among the DH lines, 18 lines showed an average of 20% decrease in gynogenic responsiveness compared with the original lines, while three lines registered an average increase of 5.7%. Using a two-step induction/regeneration procedure, 8,589 somatic regenerants were obtained from 16,170 flower buds from haploid plants, and shoot culture was established. A more laborious procedure using extraction of ovaries in the regeneration stage was found equal to flower bud culture. Chromosome doubling via somatic regeneration was found to be 83% and 100% efficient when the source material was haploid or mixoploid, respectively. Based on the results achieved in this and previous studies, an alternative protocol for chromosome doubling of gynogenic haploids is proposed.

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Mark Bridgen

The potential value of somaclonal variation for economically important plants is well-documented. The process of somaclonal variation can arise from a controlled or a random source of variation. Variability can be obtained by applying cellular pressures and selection. Valuable resistance to diseases and nematodes has already been accomplished with somaclonal variation; now, plant tolerance to pests has been realized. Tetranychus urticae, the two-spotted spidermite, and Trialeurodes vaporariorum, the greenhouse whitefly, were disinfected and introduced to aseptic shoot cultures of Torenia fournieri. These pests were allowed to feed until such time that their populations decreased due to the absence of food. The plant cells that remained after feeding were induced to form adventitious shoots and plantlets. These regenerated plantlets were acclimated to greenhouse conditions and evaluated for tolerance to the pest to which they were subjected in vitro. Highly significant differences were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed-sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls. The potential for somaclonal variation to be used as a method to develop insect resistant plants will be discussed.

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Ryutaro Tao, Abhaya M. Dandekar, Sandra L. Uratsu, Patrick V. Vail, and J. Steven Tebbets

Japanese persimmon (Diospyros kaki L. `Jiro') was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA101, carrying the binary plasmid vector, pDU92.710. The T-DNA region of pDU92.710 contained the kanamycin resistance gene (nptII), the β-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of Bacillus thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with Agrobacterium and cultured on a callus-induction medium containing kanamycin and cefotaxime. Among 720 infected leaf discs, 17 putative transformed callus lines showing kanamycin resistance were obtained after 8 weeks of culture. When these were cultured on a regeneration medium containing kanamycin, 15 formed adventitious buds. Of the 15 shoot lines, 11 grew well on a shoot-proliferation medium containing kanamycin, while 4 lines did not grow well. Of the 11 shoot lines, 10 showed GUS activities by fluorometric assay and were subjected to polymerase chain reaction (PCR) and Southern analyses. Except for two lines, all results were consistent with a stable integration of T-DNA into the persimmon genome. The production of CryIA(c) protein in transformed shoot lines was confirmed with Western analysis using anti-CryIA(c) serum. Insect bioassays were conducted with 10 lines showing GUS activity. Many of these lines showed high significant mortality of the test insects, Plodia interpunctella Hüber and Monema flavescens Walker, when compared to nontransformed controls.

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Xiaoling Cao and F.A. Hammerschlag

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of in vitro-propagated shoot cultures. The effect of either thidiazuron at 1 or 5 μM, or zeatin riboside at 20 μM, and two lit levels (18 ± 5 or 55 ± 5 μmol·m-2·s-1) on shoot organogenesis were investigated. With the exception of `Bluecrop', which did not regenerate shoots, maximum shoot regeneration of 13, 12.7, 12.6 and 4.6 shoots per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occured on regeneration medium with zeatin riboside and under a light intensity of 55 μmol·m-2·s-1. Whereas `Duke' regenerated equally well on regeneration medium with either zeatin riboside or 5 μM thidiazuron, regeneration frequencies for `Georgiagem' and `Sierra' were significantly higher on zeatin riboside. A light intensity of 55 μmol·m-2·s-1 significantly increased regeneration of cultivars Duke, Jersey, and Sierra on zeatin riboside, but inhibited regeneration of Duke on 5 μM thidazuron.

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Yan Ma, David H. Byrne, and Jing Chen

A high priority in rose (Rosa spp.) breeding research is the transfer of disease resistance, especially to black spot (Diplocarpon rosae Lib.), from wild diploid Rosa species to modern rose cultivars. To this end, amphidiploids (2n = 4x = 28) were induced with colchicine from five interspecific diploid (2n = 2x = 14) hybrids involving the black spot resistant diploid species R. wichuraiana Crép, R. roxburghii Thratt., R. banksiae Ait., R. rugosa rubra Hort., and R. setigera Michaux. Two application procedures (agitation of excised nodes in colchicine solution or tissue culture of shoots on medium with colchicine), five colchicine concentrations (0.0, 1.25, 2.50, 3.76, and 5.01 mmol), and five durations (2, 3, 5, 8, and 10 d) were used. After colchicine treatment, the materials were cultured in vitro and the surviving explants were examined for the “gigas” characteristics typical of doubled diploids. Chromosome counts of morphologically suspect genotypes confirmed 15 amphidiploids among 1109 plants that survived colchicine treatment. Although the effect of colchicine treatment varied some among interspecific hybrids, 2.50 mmol for 48 h of node agitation or 1.25 mmol for at least 5 d of shoot culture were optimal.

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Xiaoline Yu and Barbara M. Reed

Adventitious shoots were regenerated from stem segments or leaf discs of hazelnut (Corylus species) in vitro shoot cultures. Five to 10% of stem segments of `Nonpareil' regenerated adventitious shoots on modified MS medium and NCGR-COR medium supplemented with 200 mg·1-1 glutamine and combination of 1 or 5 μM thidiazuron (TDZ) and 0.1 μM naphthaleneacetic acid (NAA). Callus derived from stem segments of `Nonpareil', `Tonda Gentile Romana', and `Willamette' and leaf discs of `Dundee' cultured on medium with TDZ and NAA also produced shoots (buds) after transfer to NCGR-COR medium or modified MS medium with benzyladenine (BA) and indole-3-butyric acid (IBA). Adventitious roots were produced from leaf discs and stem segments on medium with NAA alone or with high levels of IBA or NAA combined with low levels of BA. Regenerated shoots of `Nonpareil' and `Willamette' were multiplied, rooted, and acclimatized in the greenhouse. This provides a starting point for improving the plant regeneration frequency to a level useful for genetic manipulation.

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M. Cristina Pedroso, M. Margarida Oliveira, and M. Salomé S. Pais

Nodal segments and shoot tips of axenic shoot cultures of `Hayward' kiwifruit were inoculated on modified Murashige and Skoog (MS) medium supplemented with zeatin at 1 mg·liter-1 and IAA at 0.05 mg·liter-1 (H1) or on MS medium without growth regulators (H2). Inocula cultured on H2 medium all developed into normal plantlets, while those cultured on H1 medium developed into shoots, 18% of them abnormal. Rooting of H1 shoots was induced by a 24-h immersion in a solution of IRA at 20 mg·liter-1. H2 plantlets were directly transferred to soil. Statistical treatment of the results revealed no significant differences, in terms of plant development, between the two micropropagation methods used. However, the presence of a functional root system on 5-week-old H2 plantlets resulted in 100% plant survival, but only 70% of in vivo-rooted shoots from H1 survived. Nevertheless, H1 still allowed for an important reduction of costs and manipulation. Chemical names used: indole3-acetic acid (IAA).

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Fabiola Domínguez, Xavier Lozoya, and James Simon

An efficient whole plant regeneration method from callus cultures of Piper auritum was achieved through organogenesis derived from leaf tissue. Proliferating callus and shoot cultures derived from leaf tissue explants placed on Murashige and Skoog (MS) medium supplemented with 2.0 mg·L–1 2, 4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg·L–1 kinetin. Optimum combination of hormones (mg·L–1) for shoot induction was 0.5 2,4-D: 1.5 mg·L–1 kinetin (by volume), that resulted in a high rooting rate (49.6 shoots per explant). All of the plants elongated when using a medium consisting of 0.1 mg·L–1 2,4-D plus 1 mg·L–1 kinetin. Elongated shoots were successfully rooted (100%) on half-strength MS medium supplemented with 2.0 mg·L–1 indole-3-acetic acid. All plantlets survived to the growing conditions of a greenhouse. This study demonstrates that leaf tissue of P. auritum is competent for adventitious shoot regeneration and establishes an efficient and useful protocol for the multiplication and conservation of P. autirum for further investigation of its medicinally active constituents.

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Michael E. Kane, Edward F. Gilman, Matthew A. Jenks, and Thomas J. Sheehan

Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).

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W. David Lane, Basdeo Bhagwat, Susan Wahlgren, and John D. Armstrong

A protocol for micrografting shoot tips harvested from in vitro shoot cultures directly to transplanted rootstock plants in the greenhouse was developed. Shoot tips of the apple (Malus domestica) cultivars Golden Delicious, Granny Smith and Fuji clone, Nagafu12, were harvested, stored in a water bath then prepared for grafting by cutting the stem immediately below the tip into a wedge shape leaving the tip approximately 3 mm (0.12 inch) long. The rootstock cultivar, Malling 9 (M.9) (M. domestica), was prepared by cutting into a young fast growing side branch to expose the cambium, creating a pocket into which the shoot tip was inserted. The cut section of the tip was oriented so as to contact the exposed rootstock cambium and was held in place by wrapping with a strip of pliable plastic film. Two weeks later the wrapping was loosened and the grafted branch cut back. Side branches of the rootstock were not removed until later in order to support rootstock growth. The scion shoots developed into nursery whips suitable for transplanting to a screen house or field after 2 months. The protocol proved to be a simple efficient way to rapidly grow nursery trees from tissue culture clones developed in genetic modification experiments and was used to propagate several hundred plants. Grafting success was often 100% but was reduced if quality of shoot tips was poor due to injury indicated by brown tip color. The protocol eliminates the steps of rooting, acclimatizing and growing shoots into plants to serve as a scion wood source.