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Ramana M. Gosukonda, C.S. Prakash, and Ananta Porobo Dessai

Studies were conducted to improve adventitious shoot regeneration in sweetpotato [Ipomoea batatas (L.) Lam.], specifically to extend the protocol to many genotypes and to elicit production of multiple shoots per explant. The use of a two-stage procedure where excised petioles were incubated on Murashige and Skoog (MS) (1962) medium with 2,4-D (0.2 mg·liter–1) for 3 days and transferred to a second medium containing MS salts with thidiazuron and 2iP (0.05 mg·liter–1 each) resulted in shoot regeneration from eight of 13 genotypes tested, including elite sweetpotato cultivars such as `Jewel' and `Rojoblanco'. PI 318846-3 was the most regenerable genotype, with up to 77% of explants producing one to three shoots per explant. The orientation of the petiole on the nutrient medium was critical; those placed vertically inverted developed multiple shoots. Wounding explants through epidermal peeling with normal horizontal orientation of the explants during incubation also resulted in multiple shoot production (about three shoots per explant). Interference with auxin transport due to explant inversion or wounding may have stimulated increased shoot induction. Chemical names used: 2,4 dichlorophenoxyacetic acid (2,4-D); N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron); N 6-(2-isopentenyl) adenine (2iP).

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David C. Zlesak*, Corinne M. Radatz, and Neil O. Anderson

Haploid (2x) roses derived from modern tetraploid breeding lines would allow for crosses to diploid species at the diploid level. In addition, inheritance studies are easier at the diploid level, using diploids derived from tetraploids possessing economically important traits. Haploidization of 4x roses through anther culture has not been successful due to challenges in callus induction and shoot regeneration. This study investigates rose anther responses to recently reported methods that optimize in vitro adventitious shoot regeneration in rose leaves. Anthers of three cultivars (Akito, Grand Gala, and Orlando) were put in a two-step callus induction (CI) and shoot regeneration procedure with varying CI factors. Experiment one (E1) compared continuous light/dark and silver nitrate (0,30,60 mg·L-1) and experiment two (E2) used the optimal E1 treatment comparing two and four weeks on CI media. Twenty-five anthers per treatment per cultivar were used in E1 and n = 100 for E2. Although no adventitious shoots were generated, callus formed on anther tissue and frequency of formation was variable across treatments. Continuous light resulted in 100% lethality. Darkness and silver nitrate (30 or 60 μm) favored callus generation and significant differences for callus generation were found among cultivars. Darkness and 30 μm silver nitrate were used in E2. Two and four weeks on initiation media were not significantly different for generation of anther-derived callus. Identification of factors which optimize callus formation on rose anthers is a positive step toward reliably generating rose haploids.

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Ming-yin Li

In Pelargonium, the plastid mutation in three independent cell layers L1, L2, and L3, can produce plastid chimeras with visible shoot colour difference such as GWG (green-white-green) and GGW (green-green-white). Chimera can be used to trace the relationship between the cell layers of different genotypes during shoot development and the effect of the mutated genes on shoot development. In this study, we have obtained different adventitious shoots with GGG, GWG, GGW, and WWW combinations of cell layers through tissue culture of petioles and internodes from GGW and GWG chimeras of Pelargonium zonale `Mrs Pollock'. Much higher percentage (14.9%) of chimeral adventitious shoots was obtained from GGW tissues than from GWG tissues (4.2%). Of the 10.8% chimeral adventitious shoots regenerated in this experiment, 8.6% are different from the original type of explants. This result indicated that cells at least in both L2 and L3 of the explants were involved in the regeneration of the adventitious shoots. The number of shoot types regenerated is likely dependent on the number and the type of cells that were in direct contact with the culture medium. It is suggested that the mixed cells can be used to produce the chimera by tissue culture. Three possible ways to form the chimeras in vitro culture were discussed. Chemical names used: TDZ =1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (Thidiazuron); IAA = Indole-3-acetic acid; PVP = polyvinylpyrrolidone.

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Mark H. Brand

To introduce desirable trait genes into Kalmia latifolia, efficient adventitious shoot regeneration methods are needed. Silver Dollar (S$) callus induction and growth in the dark was compared on Woody Plant (WP) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (1, 5, 10, 20 μM) or naphthaleneacetic acid (NAA) (1, 10, 20, 40 μM) with and without 5 μM isopentenyladenine (2iP). Both 2,4-D and NAA produced >450 mg of callus from leaf explants in 8 weeks. The addition of 2iP tripled growth for 2,4-D and doubled growth for NAA. Greatest callus growth was obtained on 20-40 μM NAA or 5-20 μM 2,4-D. Shoot regeneration on callus was achieved on WP medium containing 30 μM 2iP or 1 μM thidiazuron (TDZ), but a combination of the two was best, with 68% of dark-grown calli regenerating shoots in 4 weeks. 26% more dark-grown calli regenerated shoots than light-grown calli. The type of auxin (2,4-D or NAA) used to grow the calli did not affect shoot regeneration. For direct shoot regeneration, S$ leaf explants were tested on WP medium containing 5, 15, 30, 45 and 60 μM 2iP. The addition of 1 μM indole-3-butyric acid (IBA) doubled the percentage of leaves that regenerated shoots. 2iP concentrations between 15 and 45 μM supported excellent shoot regeneration, but optimal regeneration (95% of explants, 5.1 shoots/leaf) occurred on 30 μM 2iP+1 μM IBA. Leaf explants of six cultivars were grown on optimal medium with shoot regeneration ranging from 17% to 93% of leaves and 1.8 to 8.2 shoots per leaf, depending on the cultivar.

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M.W. George and R.R. Tripepi

Plant preservative mixture (PPM) is a new broad-spectrum biocide that may be useful for plant tissue culture. The objective of this study was to determine if PPM interfered with adventitious shoot regeneration on leaf explants from several plant species. Leaf explants from Dendranthema grandiflora `Iridon', Betula pendula, Rhododendron catawbiense var. album and R.c. `America' were made from the top two apical leaves on the microshoots. In the first experiment, 0, 0.5, 1, 2, or 4 mL·L-1 PPM were added to species-appropriate regeneration media. In the second experiment, only mum leaf explants were placed on regeneration media containing 0, 0.1, 0.2, 0.3, or 0.4 mL·L-1 PPM. The percentage of explants forming shoots and the number of shoots per regenerating explant were recorded after 4, 6, and 10 weeks, for mum, birch, and rhododendron leaves, respectively. The percentages of shoot regeneration from birch and rhododendron leaf explants were unaffected by up to 4 mL·L-1 PPM, and the number of shoots formed per R.c. album explant were also unaffected by the tested concentrations of PPM. In contrast, the numbers of shoots formed on birch and `America' explants were reduced 48% and 25%, respectively, when 4 mL·L-1 PPM was used in the media. The percentages of shoot regeneration and number of shoots per explant were drastically reduced on mum explants when only 0.5 mL·L-1 PPM was used in the medium. In fact, 0.3 mL·L-1 PPM or higher reduced shoot formation by more than 5-fold. This study demonstrates that the effects of PPM on shoot regeneration from leaf explants are species specific.

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Areej A. Alosaimi, Robert R. Tripepi, and Stephen L. Love

reproduced by tissue culture. Most of these species have been micropropagated via adventitious shoot regeneration from excised seedling tissues (roots, stems, leaves, etc.), with the exception of L. repens and F. magellanica that can be reproduced by

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Qingrong Sun, Meijuan Sun, Hongyan Sun, Richard L. Bell, Linguang Li, Wei Zhang, and Jihan Tao

shoot formation from pear (Pyrus communis) leaf explants in vitro Plant Cell Tiss. Org. Cult. 27 315 319 Ancherani, M. Rosati, P. Predieri, S. 1990 Adventitious shoot regeneration formation from in vitro leaves of MM.106 apple clonal rootstock Acta

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

in apple is the development of an efficient regeneration system that would increase adventitious shoot regeneration, because the regeneration ability of transformed tissues is much lower than that of non-transformed ones ( Pawlicki-Jullian et al

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I-Ling Lai, Chih-Wan Lin, Tsai-Yu Chen, and Wei-Hsin Hu

vitro hybrid regeneration techniques should be investigated and the corresponding control mechanism determined before this hybrid is promoted for commercial use. Furthermore, light quality influences adventitious shoot regeneration ( Burritt and Leung

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Dongliang Qiu, Xiangying Wei, Shufang Fan, Dawei Jian, and Jianjun Chen

that are equal to 1 cm. IBA = indole-3-butyric acid; WPM = woody plant medium; ZT = zeatin. Table 1. The number of adventitious shoots regenerated from leaf, stem, and callus explants of ‘Sunshine Blue’ cultured on woody plant medium supplemented with