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Nevena Mitić, Mariana Stanišić, Jelena Milojević, Ljiljana Tubić, Tatjana Ćosić, Radomirka Nikolić, Slavica Ninković, and Rade Miletić

An efficient in vitro shoot regeneration method from leaf explants of apple cultivars Golden Delicious and Melrose by optimization of regeneration medium, explant type and orientation, dark pre-treatment, and gelling agent is presented. Murashige and Skoog’s regeneration medium containing 22 μM thidiazuron (TDZ) and 1.5 μM indole-3-butyric acid (IBA) (M2 medium) was superior for regeneration as well as for subsequent shoot multiplication in both cultivars, providing regeneration frequency of 95% or higher in the best combination with other factors. Pre-incubation in the dark proved to be an essential factor for regeneration. The use of agar as a gelling agent provides satisfactory regeneration frequency compared with media gelled with PhytagelTM. Leaf explants of cv. Melrose with adaxial surface in contact with M2 medium and those of cv. Golden Delicious orientated contrary regenerated the highest mean number of shoots per explant. Under optimal conditions, a maximal index of shoot-forming capacity of 11.44 and 6.30 for ‘Melrose’ and ‘Golden Delicious’, respectively, was achieved. Regenerated shoots were successfully rooted and acclimated ex vitro.

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Kelly M. Oates, Thomas G. Ranney, and Darren H. Touchell

Rudbeckia spp. are adaptable and valuable ornamental wildflowers. Development of new varieties of Rudbeckia spp., with improved commercial characteristics, would be highly desirable. Interspecific hybridization and induced polyploidy may be avenues for improvement within the genus. The objective of this study was to evaluate fertility, morphology, phenology of flowering, and perennialness (overwintering survival) for lines of diploid and induced allotetraploids of R. subtomentosa × hirta and diploid and autotetraploids of R. subtomentosa ‘Henry Eilers’. Polyploid lines were developed and propagated in vitro and then grown ex vitro in a randomized complete block design with 12 replications. Compared with their diploid counterparts, autotetraploid lines of R. subtomentosa ‘Henry Eilers’ had similar internode lengths, plant heights, number of stems, flowering times (date at first anthesis), and fall and spring survival (100%); reduced number of inflorescences and male and female fertility; and increased inflorescence diameters. Compared with their diploid counterparts, allotetraploids of R. subtomentosa × hirta had similar internode lengths, reduced number of inflorescences, delayed flowering times, and increased pollen staining. Allotetraploids had limited male and female fertility compared with no detectable fertility in their diploid counterparts. Plant height and number of stems either decreased or showed no change with induced allotetraploidy. Spring survival of diploid hybrid genotypes ranged from 0% to 82% and was not improved in the allotetraploid hybrids. For a given genotype, some polyploidy lines varied significantly in certain morphological traits (e.g., plant height) indicating somaclonal variation may have developed in vitro or there were variable genomic or epigenetic changes associated with induced polyploidy.

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Darren Touchell, Zenaida Viloria, and Thomas Ranney

Weigela Thunb. consists of 12 species distributed throughout Northeast Asia. Diervilla Mill. is a closely related genus containing three species endemic to North America. Taxa from both of these genera are important nursery crops. Hybrids between these genera could potentially combine the excellent cold hardiness and adaptability of Diervilla with diverse forms, foliage colors, and flower colors found in Weigela. Prior attempts to create intergeneric hybrids between these genera were unsuccessful and resulted in embryo abortion before seeds matured. To overcome this barrier, ovule culture and micropropagation procedures were used to develop intergeneric hybrids. Cleaved amplified polymorphic sequences (CAPS) analysis was used to verify hybrids. Intergeneric crosses, D. lonicera × W. middendorfiana, D. sessilifolia × W. florida (two clones), and D. lonicera × W. florida were attempted. Crosses of D. lonicera × W. middendorfiana did not produce viable hybrids. From the remaining three crosses, a total of 544 plants were obtained from 1278 ovules. About 85% of the 544 plants appeared very chlorotic or had low vigor, and senesced when transferred to multiplication medium. Only 80 of the 544 plants were successfully maintained in tissue culture, of which 10 have been successfully transferred ex vitro. CAPS analysis indicated that a majority of these plants were hybrids. Further studies are focused on improving tissue culture procedures and other methods to develop tetraploids to increase plantlet vigor and fertility.

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Wenhao Dai, Cielo Castillo, and Victoria Magnusson

In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.

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Margarita Fraga, Mertxe Alonso, Philippe Ellul, and Marisé Borja

Meristem culture and/or thermotherapy were used to eliminate viruses from ornamental Dianthus gratianopolitanus Vill. (`Spotti' and `Frosty Fire') mother plants. Shoot tip, leaf, node, and ovary explants collected from greenhouse-maintained, virus-free plants were cultured in vitro for shoot initiation on Murashige and Skoog (MS) medium containing BAP, kinetin, or 2-iP with or without IAA or NAA. Culture of shoot tips in MS with 0.57 μm IAA and node explants in MS with 2.46 μm 2-iP is recommended for `Spotti' cultivar. In `Frosty Fire', optimum number of axillary shoots was obtained from shoot tip and node explants in MS without plant regulators. Leaves and ovaries were not adequate explants for D. gratianopolitanus micropropagation because none or only a low percentage of explants regenerated shoots. High levels of cytokinins increased the number of shoots per explant but also increased the production of aberrant phenotypes and induced hyperhydricity. Adventitious shoots rooted in vitro with auxins, but maximum rooting was 97% ex vitro without auxins. This study demonstrated that D. gratianopolitanus can be successfully micropropagated. Chemical names used: 6-benzyladenine (BAP); kinetin (KIN); 6-(γ,γ-dimethylallylamino)-purine (2iP); indole-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); gibberellic acid (GA3).

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Kris Pruski, Tina Lewis, and Mohyuddin Mirza

Chokecherries and pincherries are commonly used in landscaping. Some of the selections such as `Garrington', `Mary Liss' and `Jumping Pound' have large fruits of good quality suitable for food processing. The species are also very well adapted to severe winter conditions of the Prairie Provinces. In our studies, in vitro propagation of the selections was undertaken. The best results with initiation of cultures were observed when dormant buds were used as explants on MS medium with 30 g/L sucrose, 0.1 mg/L NAA and 1.0 mg/L BAP (4 wks, 24/22°C day/night, 16 hrs photoperiod 3000 lux). Optimal proliferation in both species was on MS medium with 1-2 mg/L BAP, 80 mg/L AdSO4 and 170 mg/L NaH2PO4. Rosettes produced were placed on medium without hormones prior to rooting. Rooting was performed ex vitro in root-trainers (soilless mix) on the greenhouse bench under mist. Basal dip in commercial rooting powder Stimroot 1 (0.1% IBA) was equally effective to spray application (2 mg/L IAA with 0.5 mg/L NAA). Average of 77% rooting with `Garrington, 72% and 81% rooting with `Jumping Pound' and `Mary Liss' was observed respectively.

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Surawit Wannakrairoj

Patumma, a native of Thailand, is a new ornamental crop. Its increased demand for export make the species vulnerable to extinction. Cloning methodology is thus needed for the production of both existing and newly developed clone. Young inflorescent segment and lateral bud from rhizome can be used as explants. For decontamination of the lateral bud, the success was depended both on pre-treatment and disintestation procedure. The bud from dried rhizome was better than one from fresh rhizome. Prior to disinfestation with sodium hypochlorite, pre-treatment of rhizome in 52°C water for 5 minutes could replaced the use of antibiotic. Plantlet were placed on modified MS media with 0, 6.67, 13.32, 19.98 and 26.64 μmol/l benzyladenine (BA) or 0.19, 0.56, 1.67 and 5 μmol/l kinetin. The best multiplication rate of 4.83 fold was obtained when longitudinally-divided rhizome was on the medium with 13.32 μmol/l BA. The result also showed that wild-collected and selected clones responded to the media similarly. When the MS media modified with 13.32, 15.54, 17.76 and 19.98 μmol/l BA in combination with 15, 30 and 45 g/l sucrose were tested, the multiplication rate of non-divided plantlets were all the same. Acclimatization by open the culture vessel for 3 day improved plantlet survival rate ex vitro.

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Michael Marcotrigiano and Susan P. McGlew

A two-stage micropropagation system was devised for cranberries (Vaccinium macrocarpon Ait.). Shoot-tip explants taken from four cultivars of greenhouse-grown plants were placed on media composed of Anderson's major salts, Murashige and Skoog's (MS) minor salts and organics, plus various concentrations of 2iP, IBA, and GA3. In other experiments, explant source, salt formulations for media, and rooting treatments were studied. Optimal multiplication and shoot quality occurred when nodal explants taken from greenhouse-grown or micropropagated plants were placed on medium containing 150 μm 2iP, 1.0 μm IBA, and no GA3. Histological examination revealed that the initial response of nodes to culture is axillary bud proliferation, but adventitious shoot formation occurred after 4 to 6 weeks. Cultures that contained only axillary shoots were not evident unless low levels of 2iP were used, at which point only axillary buds present on the explants were released. Proliferated shoots could be rooted ex vitro without auxin treatment. Optimal rooting occurred under high-light conditions. Plants were transplanted to the field for comparison to conventionally propagated material. Chemical names used: gibberellic acid (GA3), N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP), 1H-indole-3-butanoic acid (IBA).

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Luping Qu, James Polashock, and Nicholi Vorsa

A very efficient adventitious regeneration (shoot organogenesis) system for cranberry (Vaccinium macrocarpon Ait.) leaves was developed. A basal medium consisting of Anderson's rhododendron salts and Murashige and Skoog's (MS) organics, supplemented with 10.0 μm thidiazuron (TDZ) and 5.0 μm 2ip, was effective for adventitious regeneration from leaves for the five cranberry cultivars tested: `Early Black', `Pilgrim', `Stevens', `Ben Lear', and `No. 35'. Parameters examined included: 1) varying combinations of three plant growth regulators (TDZ, 2ip, and NAA); 2) explant orientation (adaxial vs. abaxial side in contact with the medium); and 3) leaf position relative to the apical meristem from the source plant. Cultivars varied in regeneration frequency, but cultivar × growth regulator interaction was nonsignificant. With optimal treatment conditions, regeneration occurred on more than 95% of the explants, with `Early Black' and `Pilgrim' producing as many as 100 shoot meristems per explant. At all concentrations tested, NAA (as low as 0.1 μm) increased callus formation and significantly reduced regeneration. Emerging adventitious shoots were always observed on the adaxial side of the leaves regardless of explant orientation on the medium. Regeneration was much greater when the abaxial side was in contact with the medium, and was not related to leaf position on the source plants. Elongation of adventitious shoots began ≈2 weeks after transfer to the basal medium without growth regulators. Cuttings of elongated shoots rooted 100% both in vitro in the basal medium and ex vitro in shredded sphagnum moss. The high regeneration efficiency achieved by using this system will be very useful in the application of techniques, such as Agrobacterium- and particle bombardment-mediated transformation. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ); N6-(γ-γ-dimethyallylamino) purine (2ip); α-naphthaleneacetic acid (NAA).

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Joseph Thomas, D. Sreedhar, S. Murali, S. Jose, K.Gopal Krishnan, P.K. Salama, V. Sayee, B. Bagyalakshmi, M. Narendran, and T.S. Lokeswari

Many researchers regard somatic embryogenesis as a system of choice for in vitro propagation of superior varieties of crops such as coffee, mango, datepalm, and rose. While there are advantages, commercialization has not been possible so far in coffee, mango, and rose. The work highlights some reasons for this and feasible alternatives. We have established somatic embryogenesis in four elite Indian arabica coffee genotypes. Plantlets (3500) of all the four varieties are now being field-evaluated. The cost of producing these propagules is 15 times the seedling cost at present. A major constraint is the long time (6 months) needed to reach the five-leaf stage in vitro prior to release for acclimatization. This period can be reduced to 2 months using exvitro development after the two leaf stage. There are many reports of somatic embryogenesis in mango. Results on establishing free-living plantlets have not been encouraging.We found a number of abnormalities in the shape of the somatic embryos in cv. Rumani. However, except for the “rod”-shaped ones (that lacked cotyledonary expansion), all embryos germinated satisfactorily (75% rooting).We have encouraging results in reducing the time required to generate suitable plantlets for field acclimatization and in standardizing the procedures for grafting. Our laboratory has developed methods for ex vitro germination of mature embryos in datepalm,which yield more numbers of free-living plantlets (50%–60%) in only 3 months with an average of four leaves per plant. This compared favorably with in vitro germination that takes 6 months and produces plantlets with one or two leaves only. A novel protocol for obtaining somatic embryogenesis in rose from petal derived calli was developed by us (Murali et al., 1996). The number of embryos induced was too low for commercial application. [Murali et al., 1996. Euphytica 91:271–275].