Clonal micropropagation studies with silver maple (Acer saccharinum L.) included experiments with various shoot. explant types, cytokinins, and stock plant maturation levels. These trials led to successful explant establishment, axillary shoot proliferation, rooting of microshoots, and establishment of plantlets in the greenhouse. Overall, the best cytokinin tested was the phenylurea derivative TDZ. Shoot proliferation on juvenile explants was poor with kinetin, 2iP, and BA. Only zeatin at 10 μm was comparable to TDZ. TDZ at 10 nm was optimal for both juvenile and adult nodal explants. Juvenile explants that were held in vitro for 4 months commonly had at least 60 axillary shoots that could be subculture or excised for rooting. Microshoots rooted within 2 weeks. Following rooting, silver maple plantlets could be transplanted into a growing medium and placed directly onto a greenhouse bench. Studies were also conducted on rooting stem cuttings (macropropagation). Single nodes from juvenile plants rooted under intermittent mist, regardless of auxin application; however, shoot-tip cuttings from adult trees rooted best when auxin in ethanol solution was applied. Chemical names used: N- phenyl- N' -1,2,3 -thiadiazol-5-ylurea (thidiazuron, TDZ), N- (2-furanylmethyl)-1H-purin-6-amine (kinetin), isopentenyladenine (2iP), benzyladenine (BA), (E)-2-methyl-4-(1H-purin-6-ylamino)-2-buten-1-ol (zeatin).
John E. Preece, Carl A. Huetteman, W. Clark Ashby, and Paul L. Roth
Michael E. Kane, Edward F. Gilman, Matthew A. Jenks, and Thomas J. Sheehan
Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).
M.L. Arrebola, O. Socorro, A. Barceló-Muñoz, E. Simón-Pérez, and Fernando Pliego-Alfaro
A micropropagation procedure for juvenile and adult savory (Satureja obovata Lag.) explants is described. Pretreatment of the nutlets with gibberellic acid (0.57 mm) did not improve in vitro germination. Optimum shoot proliferation of juvenile and adult material was obtained on medium containing 2.22 μm N6-benzyladenine. Rooting and acclimatization of juvenile shoots were accomplished in vivo, while adult shoots were rooted in vitro after 3 days of exposure to 4.92 μm indole-3-butyric acid followed by subsequent transfer to auxin-free medium. More than 95% survival of adult rooted plants was obtained during the acclimatization phase. Chemical names used: gibberellic acid (GA3); N6-benzyladenine (BA); indole-3-butyric acid (IBA); isopentenyladenine (2iP).
Leopold M. Nyochembeng and Stephen Garton
Studies were conducted to determine the response of cocoyam shoot tips, petioles, cotyledons and hypocotyls in various media for callus formation, adventitious shoot development and somatic embryogenesis. In all experiments, B5 basal medium or low N B5 were supplemented with various growth regulators.
High frequency adventitious shoot proliferation was obtained using cotyledons and hypocotyls in medium supplemented with 1 mg/l IBA and 0.5 mg/l TDZ. Embryogenic callus was obtained using shoot tips in media containing 1 mg/l Dicamba, hile somatic embryos were observed in media containing 0.3 mg/l 2, 4 - D and 1 mg/l Kinetin, using hypocotyl and petiole explants. The impact of these results on micropropagation of cocoyam is discussed.
Chi Won Lee, Chun-Ho Pak, and Harrison G. Hughes
Stevia (Stevia rebaudiana Bert.) leaves produce stevioside and rebaudioside that can be used as a natural source of low-calorie sweetener which is heat-stable. Because of low fertility, this plant is often vegetatively propagated for field production. This study was conducted to optimize tissue culture procedures for propagating selected clones and explore the feasibility of producing the sweetener compounds by callus cultures. Shoot proliferation was best in Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthaleneacetic acid (NAA) Plus 10 mg/l kinetin. Kinetin as a cytokinin source was better than benzyladenine (BA) especially when NAA was present. Callus production fronm leaf disc cultures was most prolific when a combination of 0.1 mg/l NAA and 3 mg/l BA was used in MS medium. The relative sweetener contents of callus cultures are currently being-analyzed.
Guochen Yang and Marihelen Kamp-Glass
Exochorda racemosa is an ornamental shrub with white flowers that is spiraea-like, deciduous, and hardy. The buds resemble pearls. Normally it is propagated by seeds, layers, and cuttings of softwood. However, it is a slow process that takes a few years to produce a reasonable size plant for the demanding market. Our objective was to establish a successful in vitro culture and to rapidly multiply this ornamental species. Softwood explant materials were collected from a local nursery and were disinfested with 15% bleach solution and rinsed three times with sterile distilled and deionized water. In vitro cultures were established and maintained in woody plant medium (WPM) supplemented with BA at 0.1 mg·L-1, 3% sucrose, and 0.7% agar with the pH adjusted to 5.8. Then shoots were transferred to the multiplication medium containing BA, CPPU, or thidiazuron (TDZ) at various concentrations. Preliminary results show that explants cultured on medium containing TDZ produced the best shoot proliferation.
Richard K. Kiyomoto and Mark H. Brand
Experiments were conducted on tissue proliferation (TP) development and in vitro and ex vitro growth of tissues from plants with (TP+) and without TP (TP-). In 1993 the increase in TP in one-, two-, and three-yr-old `Holden' and `Besse Howells' was 3%, 52%. and 32% and 10%, 26% and 21%, respectively. No differential mortality was observed. Shoot tip cultures initated from TP+ and TP- `Montego' showed 10-12 mo were required for miniaturiziation and multiplication in TP- shoot tips and 4 mo in TP+ shoot tips. TP- cultures require 10 uM 2-iP for normal shoot proliferation; whereas TP+ cultures had to be transferred to hormone-free medium after 6 mo to maintain normal shoot morphology. Cutting propagation from TP- and TP+ plants older than 5 yr, showed persistence of morphological aberrations associated with TP+ plants.
The morphological development of lingonberry (Vaccinium vitis-idaea L.) plants propagated either by conventional softwood cuttings or by in vitro shoot proliferation from nodal explants or by shoot regeneration from excised leaves of micropropagated shoots, was studied in cultivars `Regal', `Splendor', and `Erntedank'. Significant differences were observed between the treatments. In vitro-derived plants produced more shoots branches and rhizomes in contrast to conventional cuttings which rarely produced rhizomes. Plants propagated from cuttings had a lower number but vigorous shoots and thicker rhizomes than in vitro-derived plants. Source propagule had significant effect on multiplication rate. Another experiment evaluated the effect of indole-3-butyric acid (IBA) application to softwood cuttings on subsequent rooting, shoot development, and rhizome production. Treating cuttings with IBA did not significantly improve rhizome formation and elongation. In vitro culture on nutrient medium apparently induces the juvenile branching characteristics that favored rhizome production. The advantage of rhizome production of in vitro-derived plants over stem cuttings varied among genotypes.
O. Pérez-Tornero, F. Ortín-Párraga, J. Egea, and L. Burgos
Apricot (Prunus armeniaca L. cv.'Helena') shoots grown on a proliferation medium containing 3% sucrose, 0.4 mg·L–1 benzyladenine (BA), and 0.04 mg·L–1 indolebutyric acid (IBA) and solidified with 0.6% agar were stored at three different temperatures in the dark for up to 24 weeks. All shoots remained viable for 24 weeks when stored at 3 °C, while at 14 °C the percentage of survival decreased quickly after 12 weeks of storage. At 7 °C, percentage of survival started to decline after 18 weeks of storage. Shoots stored at 3 °C had the highest regeneration rates and shoot lengths following transfer to standard proliferation conditions. This temperature also had a beneficial effect on shoot proliferation during the first 12 to 18 weeks of the experiment.
Yrina P. Ferreras and Harry Jan Swartz
Shoot proliferation of Acer ginnala Maxim. (Amur maple) from nodal-segments was obtained on Murashige and Skoog salts and vitamins supplemented with 25 nM thidiazuron and 3% sucrose. Higher concentrations of cytokinin resulted in callus formation at the base of explants. Explant orientation had a significant effect on shoot elongation. Explant elongation and proliferation were correlated. Plants inverted in the medium elongated and proliferated readily. Branching was obtained primarily from axillary buds several nodes basal to the apex. Gelling agent type did not affect proliferation. Vitrification was significantly affected by type of gelling agent, gelling agent concentration and thidiazuron concentration. In vitro shoots rooted readily even in medium containing adenine. Greater than 95% of the in vitro plants established in the greenhouse.