`Marion' is the most important blackberry cultivar in the world, primarily due to its outstanding processing characteristics. Ideally, `Marion' could be enhanced via transformation while maintaining its fruit quality. As a successful in vitro regeneration is the prerequisite for genetic transformation, a series of experiments were conducted to optimize the conditions for in vitro regeneration for `Marion' blackberry. Parameters studied included types (three cytokinins: BA, kinetin, and zeatin; four auxins: IBA, IAA, NAA, and 2,4-D) and concentrations of plant growth regulators, explants (leaf and petiole), medium formulations (MS, WPM, and BMM), and the duration of TDZ pretreatment (3 to 6 weeks) of in vitro-grown stock plants. The highest shoot regeneration rate (65.7%) and highest number of shoots (5.1 shoots/explant) were obtained under the following conditions: stock plants were pretreated with TDZ (1 mm) for 3 weeks, followed by leaf explants dark pretreatment for 1 week on the regeneration medium (WPM with 5 mM BA and 0. 5 mm IBA). After dark treatment, regeneration plates were placed under 16-h photoperiod at light intensity of ≈50 μmol·m-2·s-1 at 23 + 2 °C for 4 weeks.
Rengong Meng, Tony H.H. Chen and Chad E. Finn
Jorge H. Siller-Cepeda, Leslie H. Fuchigami and Tony H.H. Chen
The effects of hydrogen cyanamide (H2CN2) on budbreak and phytotoxicity of l-year-old potted peach trees [Prunus persica (L.) Batsch. cv. Redhaven] over a wide range of concentrations at several stages of dormancy were studied. Endodormancy (180° GS; degree growth stage) began on 1 Oct. Maximum intensity of endodormancy (270° GS) was reached after the plants were exposed to 320 chill units on 1 Nov., and 50% of the buds were broken at 860 chill units on 1 Dec. Five concentrations of H2C N2 (0, 0.125, 0.25, 0.5, and 1.0 m) were applied on 1 and 15 Oct., 1 and 15 Nov., and 1 and 15 Dec. 1990. All concentrations promoted budbreak; however, percent budbreak and phytotoxicity depended on concentration and timing of application. The most effective concentration (greatest budbreak and lowest phytotoxicity) was 0.125 m H2CN2 on all treatment dates. Phytotoxicity was evident at all application dates but was greatest at the highest concentrations. Plants were most resistant to H2CN2 at maximum intensity of endodormancy. Hydrogen cyanamide-induced budbreak was highest during the later stages of endodormancy (295 to 315° GS). Treatments applied during the ecodormancy stage (340° GS) inhibited and delayed budbreak and damaged buds and stems. Chemical name used: hydrogen cyanamide (H2CN2, Dormex).
Jorge Siller-Cepeda, Leslie Fuchigami and Tony H. Chen
Glutathione content was determined in buds of peach (Prunus persica L.) trees during rest development and release. Reduced (GSH) and oxidized (GSSG) glutathione content changed with the accumulation of chill units (CU). GSH content decreased in the early phases of rest, and then increased at maximum rest. GSH content continued to increase and peaked on 1 Dec at 860 CU, and then dropped during the quiescent stage. It appears that the increase of GSH during chilling was closely associated with the breaking of rest. In contrast GSSG showed a continuous increase from Oct to Dec. Five concentrations of cyanamide were applied every 2 weeks from Oct to Dec. All cyanamide treatments depleted GSH within 12 hr followed by a large increase 24 hr after treatment. The changes in GSH content induced by cyanamide were inversely related to the concentration. The extent of GSH change was dependent on both the physiological status of the bud and the cyanamide concentration. At maximum rest, the plants were more resistant to cyanamide treatment and this coincided with the highest level of cyanamide-induced GSH.
Stephen P. Lee, Paul M. Chen, Tony H.H. Chen, Diane M. Varga and Eugene A. Mielke
A proportion of `d'Anjou' pear fruit (Pyrus communis L.) developed a disorder, “black speck” or “skin speckling”, after prolonged controlled atmosphere (CA) storage (1% O2, - 0.5 C). A comparative study of biochemical components revealed that there was no significant difference in succinic, citric, fumaric, and pyruvic acids between the speckled' and normal skin tissues. The content of malic acid in the affected tissue was almost three times lower than that in the normal tissue. The specific activity of NADP-malic enzyme (EC 22.214.171.124) in the affected tissue was also lower, but the total activities were similar. The affected tissue contained higher percentages of dry matter and soluble proteins than the normal tissue. Two-dimensional gel electrophoresis of proteins showed that two groups of novel polypeptides appeared only in the affected skin tissue. This study indicated that a certain proportion of `d'Anjou' pear fruit might have been exposed to unfavorable preharvest environmental stresses, and, therefore, could no longer tolerate the subsequent semi-anaerobic and chilling stresses during prolonged CA storage.
Steven P. Castagnoli, Leslie H. Fuchigami, Tony H. H. Chen and Liping Zhen
Studies were performed on the development of dormancy, cold hardiness, and desiccation tolerance, and the effect of manual defoliation timing on performance of `Fuji' and `Braeburn' apple nursery stock. Dormancy development, response to defoliation, and desiccation tolerance of apple differed from those reported for other temperate woody plant species. Dormancy development in `Fuji' was approximately two weeks ahead of `Braeburn', and was strongly regulated by temperature. Photoperiod had no influence on dormancy development of `Fuji'. Desiccation tolerance of both varieties was greatest just prior to the onset of dormancy and early dormancy. This pattern in the seasonal development of tolerance to desiccation is not typical of temperate woody plant species. Early defoliation was detrimental to performance of `Braeburn', but had little effect on `Fuji'. Early defoliation promoted earlier spring budbreak in `Fuji'. Development of freezing tolerance in both apple varieties was typical of other woody plants, and coincided with the onset of dormancy. Maximum hardiness was achieved after the requirements for dormancy were completely satisfied.
John M. Englert, Gary D. Coleman, Tony H.H. Chen and Leslie H. Fuchigami
A 32kDa bark storage protein (BSP) which accumulates in the fall and is degraded in the spring has been identified in Populus deltoides bark. The BSP gene has been shown to be regulated by short day (SD) photoperiod (8 h). The physiological condition of the plant and the environmental factors necessary for the degradation and retranslocation of BSP are of considerable interest for determining the role of this protein in the remobilization of nitrogen in trees.
Poplar plants were placed in a SD growth chamber for 4 or 7 weeks to induce growth cessation (bud set) or dormancy, respectively. BSP accumulated to high levels in bark tissues after 3 weeks SD and remained high through 7 weeks SD. Plants in which growth had stopped (4 weeks SD), or in which dormancy (7 weeks SD) was broken with hydrogen cyanamide (0.5 M) or chilling (4 weeks 0C) broke bud within 1 week of being placed into long day (LD) conditions. Dormant plants which were not chilled broke bud after 3 weeks LD. BSP levels decreased around the time of budbreak, suggesting that the degradation of BSP is dependent on the need for a nitrogen sink, ie. budbreak and new shoot growth.
John M. Englert, Keith Warren, Leslie H. Fuchigami and Tony H.H. Chen
Desiccation stress during the postharvest handling of bare-root deciduous trees can account for dieback and poor regrowth after transplanting. Desiccation tolerance of three bare-root deciduous hardwood species was determined at monthly harvest intervals from Sept. 1990 through Apr. 1991. Among the three species tested red oak (Quercus rubra L.) was most tolerant to desiccation, followed by Norway maple (Acer platanoides L.) and Washington hawthorn (Crataegus phaenopyrum Medic.). Maximum desiccation tolerance of all three species occurred during the January and February harvests. Of 20 film-forming compounds tested, the antidesiccant Moisturin was the most effective in reducing water loss from bare-root trees during desiccation stress and in improving survival and plant performance during re-establishment in the laboratory, greenhouse, and field. Moisturin-treated plants lost up to 80% less water than untreated plants. Washington hawthorn seedlings treated with Moisturin before severe desiccating conditions had the highest survival, lowest dieback/plant, and highest root growth ratings. The results indicate that Moisturin is an effective means of overcoming postharvest desiccation stress in desiccation sensitive plants, such as Washington hawthorn.
Zoran Jeknic, Stephen P. Lee, Joel Davis, Richard C. Ernst and Tony H.H. Chen
A protocol was developed for production of transgenic iris plants (Iris germanica L. `Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII-positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering.
Yuexin Wang, Zoran Jeknić, Richard C. Ernst and Tony H.H. Chen
To improve the efficiency of iris plant regeneration, we tested the influence of several combinations of Kin and NAA in culture media on the induction of morphogenesis and the subsequent development of plantlets. The highest rates of regeneration (67%) were consistently observed in induction media containing 0.5 μm NAA and either 2.5 or 12.5 μm Kin. Developing medium containing 1.25 μm BA was optimal for high regeneration rates and a high percentage of plantlets simultaneously developing shoots and roots. Rooted plantlets were easily acclimatized and transplanted to various soil mixtures, then grown in the greenhouse. Under optimal conditions as many as 8000 plantlets could be regenerated from 1 g of cells in ≈4 months. Chemical names used: kinetin (Kin); 1-naphthaleneacetic acid (NAA); N6-benzyladenine (BA).
Rengong Meng, Tony H.H. Chen, Chad E. Finn and Yonghai Li
Experiments focusing on plant growth regulators' concentrations and combinations, mineral salt formulations, and TDZ pretreatment formations were conducted to optimize in vitro shoot regeneration from leaf and petiole explants of `Marion' blackberry. Optimum shoot formation was obtained when stock plants were incubated in TDZ pretreatment medium for 3 weeks before culturing leaf explants on regeneration medium (Woody Plant Medium with 5 μm BA and 0.5 μm IBA) in darkness for 1 week before transfer to light photoperiod (16-hour photoperiod at photosynthetic photon flux of ≈50 μmol·m-2·s-1) at 23 °C ± 2 °C for 4 weeks. Under these conditions, ≈70% of leaf explants formed ≈40 shoots per petri dish that could be harvested and rooted to form plantlets. Chemical names used: N6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); gibberellic acid (GA3); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); N-phenyl-N'-1,2,3-thidiazol-5-ylurea [thidiazuron (TDZ)].