endangered tree species. Adventitious shoot regeneration and rooting is a prerequisite for use in genetic modification technology. Adventitious shoot regeneration has been reported for common or European ash ( F . excelsior ) ( Hammatt, 1996 ; Mockeliunaite
Rochelle R. Beasley and Paula M. Pijut
Maria Cantor*, Rodica Pop, and Ioana Pop
The Streptocarpus is propagated ease vegetatively from leaf cuttings all year round, but is grown on a very limited scale commercially in Romania. Successful protocol for direct shoot regeneration from in vitro Cape primrose (Streptocarpus × hybridus Voss.) leaf explants has been developed. The ease of tissue culture propagation can promote Streptocarpus production and facilitated the rapid introduction of this new species. Adventitious shoot regeneration was inducted in vitro on MS basal medium, using different concentration of NAA (1, 1.5, 2 mg·L-1) and cyokinin TDZ (0.1, 0.5, 1 mg·L-1). High frequency regeneration was obtained from leaves when cultured in the media supplemented with 1 mg·L-1 NAA plus 0.5 mg·L-1 TDZ and the percent of regeneration resulted is between 70% to 100%. Complete plantlets were acclimatized and successfully transplanted to glasshouse conditions. The total duration of the cycle from leaf explants through complete plantlets was 10 weeks.
Karen E. Hokanson and Margaret R. Pooler
Callus formation and adventitious shoot regeneration in vitro from mature stored seed were evaluated in eight ornamental cherry (Prunus) taxa: P. campanulata Maxim., P. maackii Rupr., P. sargentii Rehd., P. serrula Franch., P. serrulata Lindl., P. subhirtella Miq., P. virginiana L., and P. yedoensis Matsum. Several portions of the embryo (cotyledons and hypocotyl sections) and nine combinations of growth regulators (BA, 2,4-D, IBA, NAA, and TDZ) were compared. Effects of embryo portions and growth regulator treatments were generally small within taxa, but shoot formation differed among taxa. About 20% to 50% of the embryos from P. virginiana and P. serrula and ≈5% to 30% of those from P. maackii produced shoots. The other taxa generally did not produce shoots. Regeneration from mature stored seed in the responsive taxa represents a potential system for genetic transformation. Chemical names used: 6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); thidiazuron (TDZ).
Michael E. Compton
Organic competence of different explant sizes and locations on watermelon seedlings was determined by calculating the percentage of cotyledon explants that produced adventitious shoots. About 52% (214/412) of explants prepared from the proximal region of cotyledons formed shoots, whereas only ≈6% (24/411) of distal explants did so. Shoot formation was limited to the proximal end of basal explants but was not restricted to any specific region on distal ones. The percentage of explants that produced harvestable shoots was greater from basal halves than basal quarters in `Sweet Gem', `Crimson Sweet', and `Minilee', but explant size did not affect adventitious shoot regeneration of `Yellow Doll', resulting in significant interaction between cultivar and explant size. This study indicates that cultivars that respond poorly to in vitro procedures may have fewer cells competent for shoot regeneration, requiring special care during explant preparation.
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.
Orlando McMeans and Robert M. Skirvin
To isolate unique fruit colors and look for somaclonal variation among regenerants, a regeneration protocol was established for various cultivars of striped apples (`Mailing 26', `Mutsu', `Regal Gala', `Summerland Red', and `Fuji'). Leaves were harvested from in m-grown plants, transferred to regeneration media [MS + NAA (5.4 μM) + TDZ (3 μM)], stored in the dark for 3 to 4 weeks, then moved to controlled light conditions, where adventitious shoot regeneration was observed. Developing shoots were transferred to proliferation medium and screened for their red or green phenotype by placing them on MS media containing various concentrations of sucrose (30, 45, 60, 75 and 90 g\L) and BA (0, 2.5, 5, 7.5, 10, 12.5 and 15 μM). Some of the regenerated apple shoots exhibited red color soon after being taken from the dark treatment. Others were less distinct, with colors ranging from dull green to a green-pink mixture. The red and green shoots are now being rooted and will be transferred to the field, where they will be grown to maturity.
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.
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.
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).
Agustin Huerta and Ramon Dolcet-Sanjuan
Adventitious shoots and viable plants were regenerated from bell pepper (Capsicum annuum L.) cultivars and dihaploid lines (DHLs) obtained from F1 hybrids via androgenesis (Dolcet-Sanjuan et al., in press). Hypocotil and cotyledon sections from in vitro-germinated seeds were used as explants. A modified MS medium (Murashige and Skoog, 1962) supplemented with IAA (0 to 3.2 μM) and BAP (0 to 100 μM) was used in a 3-week-long shoot primordia induction phase. Shoot elongation was best performed in the same basal medium, but supplemented with silver thiosulfate and GA3. Shoots were regenerated from eight selected DHLs (`C213', `C215', `C218', `C2123', `C2125', `C3111', `C3113', and `P493') and two cultivars (`Padrón' and `Yolo Wonder'). The percentage of cotyledon sections with shoot primordia after the induction phase was not genotype-dependent and always higher than with hypocotil sections (93.4% and 17.9%, respectively). The number of shoot primordia per responsive cotyledon section was also higher than with hypocotil sections (3.3 and 1.7, respectively). The genotype had a significant effect on the number of shoots regenerated per responsive cotyledon (1.1 to 5.5) or hypocotil (0.5 to 3.5) section. All adventitiously regenerated plants were fertile. This adventitious shoot regeneration protocol is being used to obtain transgenic plants from sweet bell pepper genotypes.