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Michael E. Kane, Nancy L. Philman, and Matthew A. Jenks

Only a few plants are suitable for reliably demonstrating rapid direct and indirect shoot organogenesis in vitro. A laboratory exercise has been developed using internodes of Myriophyllum aquaticum, an amphibious water garden plant. Stock shoot cultures are established and maintained in vitro from nodal explants cultured on agar-solidified medium consisting of half-strength Murashige & Skoog salts (MS) and 30 g·liter-1 sucrose. Students use these cultures as the source of internode explants. Explants are cultured on agar-solidified full-strength MS with 30 g·liter-1 sucrose, 100 mg·liter-1 myo-inositol, and 0.4 mg·liter-1 thiamine·HCL and factorial combinations of 0 to 10 μM 2iP and 0 to 1.0 μM NAA. Adventitious shoot development occurs directly from the explant epidermis within 4 days and is promoted in media supplemented with 2iP alone. Cytokinin-supplemented media amended with NAA induce organogenetic callus formation, but reduce 2iP promotion of direct shoot organogenesis. After 4 weeks, shoot organogenesis on the various media is quantified and can be analyzed statistically. Chemical names used: N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP); α-naphthaleneacetic acid (NAA).

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Yiqin Ruan and Mark Brand

Combinations of me auxins 2.4-dichlorophenoxyacetic acid (2.4-D). indolebutyric acid (IBA). and naphthaleneacetic acid (NAA), with isopentenylademne (2-iP) were studied in Woody Plant (WP) medium for callus induction and shoot organogenesis from leaves of Rhododendron `Besse Howells' (BH) and `Catawbiense Album' (CA). IBA was more effective than NAA and 2,4-D at inducing shoot organogenesis when combined with 2-iP. Addition of 1 uM IBA and 15 or 30 uM 2-iP to WP medium resulted in the highest percentage of explants producing shoots (90% in BH, 100% in CA), and the greatest number of shoots per explant (18.4 in CA, 10.1 in BH) after 12 weeks of culture. Shoot organogenesis also occurred using 1 uM NAA and 2-iP combinations, but me number of shoots produced was much less than for IBA treatments. 2,4-D and NAA were more productive than IBA for callus induction. Media containing l-10 uM 2,4-D plus 5 uM 2-iP. or 10 uM NAA plus 15 uM 2-iP. were me best for callus production. In studies using thidiazuron instead of 2-iP as the cytokinin, leafy buds and very short shoots developed.

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Rida A. Shibli and M.A.L. Smith

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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Wenhao Dai, Victoria Magnusson, and Chris Johnson

Chokecherry (Prunus virginiana L.) was transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pBI121 carrying the neomycin phosphotransferase gene (nptII) and β-glucuronidase (GUS) gene (uidA). Plants were regenerated from the Agrobacterium-infected leaf tissues through organogenesis on woody plant medium (WPM) supplemented with MS (Murashige and Skoog) vitamins, 10 μm 6-benzyladenine (BA), and 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 15 mg·L−1kanamycin (CCK15). Transformation was verified with polymerase chain reaction (PCR) and Southern blot analysis. Four of 150 (2.67%) initial explants produced GUS- and PCR-positive shoots. Southern blot analysis confirmed that the transgenes were integrated into the chokecherry genome. Transgenic in vitro shoots were rooted in half-strength MS medium containing 10 μm naphthalene acetic acid. Rooted plants were transferred to potting mix and grown in the greenhouse. This research shows a potential for future improvement of chokecherry and other Prunus species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime, carbenicillin, kanamycin.

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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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Wenhao Dai, Yuanjie Su, Hongxia Wang, and Ceilo Castillo

Two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were transformed using Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBI121 carrying the neomycin phosphotransferase gene and β-glucuronidase gene (uidA). Transgenic plants were recovered from the Agrobacterium-infected leaf tissues through organogenesis in the selection medium (woody plant medium containing 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 40 mg·L−1 kanamycin). The rate of shoot regeneration from transformed leaf tissues increased from 5.7% to 32% through extending cocultivation time from 3 to 9 days. Integration of marker genes was verified with polymerase chain reaction (PCR) and Southern blot analysis. Southern blot analysis confirmed that one to three copies of transgenes were integrated into the buddleia genome. This transformation system could be used for improvement of buddleia or other related species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime (Cef), carbenicillin (Carb), kanamycin (Km).

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Enaksha R. Wickremesinhe and Richard N. Arteca

Fast growing callus was derived from Cephalotaxus harringtonia stem explants placed on MS basal salt medium with B-5 vitamins, 3% sucrose, 1 μM kinetin and 4.5 μM 2,4-D. Callus placed on basal medium with 10 μM kinetin and 0.45 μM 2,4-D turned green and organogenesis was observed upon subculture onto basal medium without hormones. Shoots were excised and placed on 1/2 strength MS salts and 10% sucrose for further shoot development. During the process of organogenesis, we also observed the differentiation of roots. Rapidly growing root cultures were established by culturing them under a 24 hour light regime of 35 μM/m2/s. Two grams of root tip explants cultured on B-5 medium with 2% sucrose were capable of producing an average of 24 grams of roots within 11 days. A 20-fold increase in fresh weight was achieved within 3 weeks when 15 grams of these roots were cultured in a 3 liter air-sparged bioreactor. C. harringtonia contains a number of alkaloids that exhibit cytotoxicity and are being evaluated as chemotherapeutic agents. We are currently in the process of establishing growth characteristics for these roots and assaying roots for the presence of these alkaloids. All cultures were grown under a 12 hour light regime unless otherwise stated.

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Xiaoling Cao, Freddi A. Hammerschlag, and Larry Douglass

As part of a program to improve highbush blueberry (Vaccinium corymbosum L.) cultivars via tissue culture and genetic engineering, studies were conducted to determine optimum conditions for organogenesis from leaf explants of the previously recalcitrant cv. Bluecrop. The effects of a pretreatment, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% explants regenerated shoots with a mean of 11 shoots per leaf explant after 62 days when explants of 2-week-old shoot cultures were incubated on the following regime: pretreatment medium #1 containing 5 μm TDZ and 2.6 μm NAA for 4 days, pretreatment medium #2 containing 7 μm zeatin riboside and 2.6 μm NAA for 3 days, regeneration medium containing 1 μm TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatment prior to incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on regeneration medium containing 1 μm TDZ was about three times that on 0.5 μm TDZ or 20 μm zeatin riboside, and nine times that on 5 μm TDZ. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(β-D-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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K.A. Malik, Christena Visser, and praveen K. saxena

In vitro regeneration by shoot organogenesis and-or somatic embryogenesis is accomplished by culturing the explants on a nutrient medium supplemented with phytohormones. Auxins in general, and 2,4-D in particular, have been shown to induce somatic embryogenesis whereas shoot regeneration is stimulated by cytokinins. In studying the morphoregulatory role of thidiazuron (TDZ) - a substituted urea with cytokinin-like activity - we found that it induces a high frequency of both organogenesis and somatic embryogenesis depending upon the plant species. For instance, whole seedlings of peanut developed somatic embryos and those of bean and pea produced shoots in response to culture on TDZ (1-40 μM)-supplemented media. In cultured explants of geranium, the use of TDZ (0.2-1 μM) effectively replaced the requirement of 2,4-D or BAP and IAA for obtaining somatic embryos. The frequency of regeneration was two to ten times higher than that achieved with auxin-cytokinin combinations. While no direct evidence is currently available to establish a relationship between TDZ and endogenous phytohormones, our results suggest that it may act by establishing endogenously the auxin:cytokinin ratio permissive of induction and expression of morphogenically competent cells.

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Hyo-Geur. Park, Kyung-Young Choi, and Do-Hyun Lee

Hot pepper is the most important vegetable in Korea in terms of both acreage and product value. Morphogenic response in tissue culture of hot pepper varied greatly with explant source and growth regulators. Somatic enbryogenesis, which we believe had not been reported yet, was only induced from 5-month-old callus derived from anther culture. Embryogenic callus was solely observed in the MS medium supplemented with 2 mg/l of NAA and 2 mg/l of BA. Better somatic embryogenesis and plant regeneration was observed on hormone free medium. Adventitious organogenesis was well induced from both hypocotyl and cotyledon. Polarity of explant for shoot regeneration was observed. Success rate of regeneration has been continuously improved mainly due to combinations of growth regulators. The best combination so far was 2 mg/l of zeatin and 1 mg/l of IAA, resulting in 92% of regeneration from cotyledon explant.