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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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Paula M. Pijut

Butternut (Juglans cinerea L.), a native hardwood to the northeastern United States, is a valuable species for its wood and edible nuts. Butternut is becoming endangered in its native range as a result of a virulent fungal (perennial canker) pathogen, Sirococcus clavigignenti - juglandacearum. Micropropagation techniques are being developed to clone disease-resistant specimens. Axillary buds, obtained from 2-3-month old seedlings, were induced to break buds in vitro and form a single shoot when cultured on Murashige and Skoog (MS) medium supplemented with 200 mg/l casein hydrolysate, 3% sucrose, and 2 mg/l 6-benzylaminopurine. Roots were initiated on microshoots when cultured on half-strength MS medium containing 100 mg/l casein hydrolysate, 1.5% sucrose, and 0.5 mg/l indole-3-butyric acid for seven days in the dark. Adventitious roots elongated when shoots were placed in the light on the same medium, but with 2% sucrose, and no growth regulators. Rooted plantlets were successfully acclimated ex vitro. These results provide a basis for the development of techniques to micropropagate selected, mature, disease-resistant butternut germ plasm.

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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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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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Yves Desjardins and André Gosselin

Strawberry plantlets (Fragaria X ananassa Duch. cv. Kent) were submitted to a factorial arrangement of 2 photosynthetic photon fluxes (PPF) (80 and 150 μmol·m-2·s-1, PAR) and 2 CO2 concentrations (330 and 3000 ppm) during the in vitro rooting stage. Leaves were tagged and placed in a growth chamber tor acclimatization. Photosynthetic capability of leaves from different origins was determined by measuring the initial and total activity of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco), but the contribution of Phosphoenolpyruvate carboxylase (PEPCase) to fixation was also examined High CO2 concentration and PPF significantly increased fresh weight and surface area in vitro and after 4 weeks ex vitro. Improved growth was not the result of increased autotrophy in vitro since initial rubisco activity was 10 times lower than that of de novo formed leaves and declined under high CO2 and PPF. Carbon dioxide concentration and PPF had no effect on total activity of rubisco. Low activation state and total activity of rubisco in in vitro leaves is the cause of poor photosynthetic activity in vitro Persistent in vitro leaves after 4 weeks of acclimatization did not have higher total activity of rubisco, but the activation state was 4 times larger than the corresponding activity in vitro which might thus provide for non-negligible contribution to photosynthetic carbon assimilation. The possible inhibition of photosynthesis by the presence of sugar in the medium is discussed.

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Nancy Phiman and Michael E. Kane

Beach stabilization by replanting dune species such as Uniola paniculata L. (Sea Oats), is an accepted practice to control erosion in the southeastern United States. Increased restrictions on collection of sea oat seed and plant material for propagation is of increasing concern. Development of micropropagation protocols for establishment and production of sea oats from donor plants of known phenotype would be useful for selecting and producing plants with commercially valuable characteristics. Terminal and lateral shoot tips (3 mm wide and 4 mm high) from containerized plants were surface sterilized and established on Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine solidified with 0.8% TC® Agar. Terminal tiller shoot tips were more responsive than lateral shoot tips. Four monthly subcultures were. required for stabilized shoot multiplication from culture lines established from terminal tiller shoot tips. Shoot organogenesis frequently occurred from the cut leaf surfaces of subcultured shoot clusters. Microcuttings were established ex vitro in plug cells containing sand or vermiculite.

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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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J.C. Vlahos, M. Dragassaki, and I. Assargiotaki

Achimenes is a summer-flowering pot plant commonly propagated by shoot tip cuttings taken from rhizomes released from dormancy. Micropropagation was used in this study in order to establish a protocol for producing plants in winter when Achimenes are not usually available. Leaf segments, taken in August 1993, from hybrids `Flamenco', `Rosenelfe', `Bella', and `Sandra' grown in a greenhouse, were cultured on a modified Murashige and Skoog (MS) medium supplemented with 0.1 mg·liter–1 BA and 0.5 mg·liter–1; shoots proliferated without callus formation. Leaf explants taken from the proliferated shoots were placed on MS medium with 0.5 mg·liter–1 BA and 0.1 mg·liter–1 NAA for 8 weeks for further shoots proliferation. `Bella' showed vigorous growth and produced the most shoots (82) with no rhizomes, whereas `Flamenco' had the least shoots (28) along with rhizomes. Shoot tips were then transferred on MS medium supplemented with 0.5 mg·liter–1 NAA for 6 weeks where more vigorous shoots developed along with roots. Microcuttings were directly stuck ex vitro under moisture and rooted well in 4 weeks before planting in individual culture and flowered normally. These results provide the basis for a successful production of Achimenes hybrids for growth and flowering in winter months provided optimal temperature and irradiance levels are given.

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Kullanart Obsuwan, Wayne B. Borth, John Hu, and Adelheid R. Kuehnle

A Cymbidium mosaic virus movement protein gene with a site-specific mutation (mut11) under control of a ubiquitin promoter was inserted using biolistics into two Dendrobium varieties with the intention of creating CymMV-resistant orchids. Presence of the transgene in regenerated plants of D. × Jaquelyn Thomas `Uniwai Mist' and D. x Jaq–Hawaii `Uniwai Pearl' was confirmed by PCR using genomic DNA, and mut11-positive plants were potted ex vitro. Forty-two transgenic plants and four non-transgenic control plants at the 4- to 6-leaf stage were inoculated with a 1:1000 dilution of CymMV obtained from infected orchids. Plants were analyzed for systemic infection using tissue blot immunoassay (TBIA). Seventeen plants from at least six independent transformations remained virus-free, whereas all control plants were infected with CymMV within 1 month. Further analysis by RT-PCR showed that the mut-11 mRNA was detectable in only two of these 17 plants. All plants were challenged again with a second CymMV inoculation as above, followed by TBIA analysis after 1 month. Thirteen of 17 plants remained free from virus. A third challenge of these plants with CymMV as above was followed by TBIA analysis at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 12 months after challenge. Results at 2 weeks post-inoculation showed that all six controls and four individual transgenic plants, including the RT-PCR-positive plants, became systemically infected. Nine transgenic plants from both varieties remained free from CymMV 12 months after the third challenge. Lack of detectable mut11 mRNA in these resistant lines suggests that a post-transcriptional gene silencing (PTGS) mechanism may be conferring resistance to CymMV.

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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].