Achimenes is a summer-flowering pot plant commonly propagated by shoot tip cuttings taken from rhizomes released from dormancy. Micropropagation was used in this study in order to establish a protocol for producing plants in winter when Achimenes are not usually available. Leaf segments, taken in August 1993, from hybrids `Flamenco', `Rosenelfe', `Bella', and `Sandra' grown in a greenhouse, were cultured on a modified Murashige and Skoog (MS) medium supplemented with 0.1 mg·liter–1 BA and 0.5 mg·liter–1; shoots proliferated without callus formation. Leaf explants taken from the proliferated shoots were placed on MS medium with 0.5 mg·liter–1 BA and 0.1 mg·liter–1 NAA for 8 weeks for further shoots proliferation. `Bella' showed vigorous growth and produced the most shoots (82) with no rhizomes, whereas `Flamenco' had the least shoots (28) along with rhizomes. Shoot tips were then transferred on MS medium supplemented with 0.5 mg·liter–1 NAA for 6 weeks where more vigorous shoots developed along with roots. Microcuttings were directly stuck ex vitro under moisture and rooted well in 4 weeks before planting in individual culture and flowered normally. These results provide the basis for a successful production of Achimenes hybrids for growth and flowering in winter months provided optimal temperature and irradiance levels are given.
J.C. Vlahos, M. Dragassaki, and I. Assargiotaki
Erika Szendrák, Paul E. Read, Eszter R. Eszéki, Elizabeth Jámbor-Benczúi, and Aniko Csillag
Cultures of several orchid species [Barlia robertiana (Loisel.), Dactylorhiza fúchsii Soó, D. incarnata (L.) Soó, D. maculata (L.) Soó, D. majalis (Rchb.), D. saccifera (Brong) Soó, D. sambucina (L.) Soó, Gymnadenia conopsea (L.) R.Br., Himantoglossurn hircinum (L.) Spreng., Ophris sphegodes Mill., Orchis coriophora ssp. fragrans L., Orchis laxiflora ssp. palustris Lam., Orchis mascula L., Orchis morio L., Platanthera bifolia (L.) Rich., Spiranthes aestivalis (Poir.) Rich.] were initiated with fresh ripe seeds from desiccated fruit and 4-month-old in vitro seedlings. The medium used for both germination and seedling culture was a modified FAST medium. Samples for the scanning electron microscope (SEM) surveys were taken from the in vitro cultures and some plant materials were collected from their native habit. Samples were observed with a Tesla BS 300 SEM. Seeds ranged from 300 to 450 μm in length and were flask-shaped. The first germination step is opening of the seedcoat, when the first few white cells will be visible. After a few weeks, the apical meristem appears. The young protocorm is covered with numerous translucent rhizoids. In the last stage of germination, the first root and the first true leaf start to develop. After 2 years, they are suitable for transfer ex vitro. Structure of the mature organs and tissues can be examined at this stage.
Paula M. Pijut
Butternut (Juglans cinerea L.), a native hardwood to the northeastern United States, is a valuable species for its wood and edible nuts. Butternut is becoming endangered in its native range as a result of a virulent fungal (perennial canker) pathogen, Sirococcus clavigignenti - juglandacearum. Micropropagation techniques are being developed to clone disease-resistant specimens. Axillary buds, obtained from 2-3-month old seedlings, were induced to break buds in vitro and form a single shoot when cultured on Murashige and Skoog (MS) medium supplemented with 200 mg/l casein hydrolysate, 3% sucrose, and 2 mg/l 6-benzylaminopurine. Roots were initiated on microshoots when cultured on half-strength MS medium containing 100 mg/l casein hydrolysate, 1.5% sucrose, and 0.5 mg/l indole-3-butyric acid for seven days in the dark. Adventitious roots elongated when shoots were placed in the light on the same medium, but with 2% sucrose, and no growth regulators. Rooted plantlets were successfully acclimated ex vitro. These results provide a basis for the development of techniques to micropropagate selected, mature, disease-resistant butternut germ plasm.
Karim H. Al-Juboory, J. Al-Naimi, L.K. Al-Amiry, R. Shibli, and R.M. Skirvin
Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.
Erika Szendrák and Paul E. Read
The temperate native terrestrial orchids are endangered species. Their propagation from seeds poses specific problems. It is well known that orchid seeds are devoid of endosperm and in nature they need microscopic fungi in a symbiotic relationship for germination. We developed a successful asymbiotic in vitro culture method for germinating seeds of several temperate orchid species and for maintaining the cultures of young plantlets. The medium used for both germination and seedling culture was a modified FAST medium. Seeds were surface-disinfested for 10 minutes in a 10% calcium hypochlorite solution. After sowing, the cultures were kept under dark condition at 10–12°C for 4 weeks. After that the cultures remained in the dark, but the temperature was raised to 25–26°C until germination occurred. Thereafter cultures required alternating seasonal temperatures: 25–26°C from the beginning of April to the end of September and 17–19°C from October to March. For the development of the young plantlets natural dispersed light and prevailing day-length was favorable. After 2 years of aseptic culture they were suitable for transfer ex vitro. Different stages of seed germination and plant development were observed using a scanning electron microscope and will be included in this presentation. Further observation of the effects of different environmental factors is currently under investigation.
Nancy Phiman and Michael E. Kane
Beach stabilization by replanting dune species such as Uniola paniculata L. (Sea Oats), is an accepted practice to control erosion in the southeastern United States. Increased restrictions on collection of sea oat seed and plant material for propagation is of increasing concern. Development of micropropagation protocols for establishment and production of sea oats from donor plants of known phenotype would be useful for selecting and producing plants with commercially valuable characteristics. Terminal and lateral shoot tips (3 mm wide and 4 mm high) from containerized plants were surface sterilized and established on Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine solidified with 0.8% TC® Agar. Terminal tiller shoot tips were more responsive than lateral shoot tips. Four monthly subcultures were. required for stabilized shoot multiplication from culture lines established from terminal tiller shoot tips. Shoot organogenesis frequently occurred from the cut leaf surfaces of subcultured shoot clusters. Microcuttings were established ex vitro in plug cells containing sand or vermiculite.
Yong Cheong Koh and Fred T. Davies Jr.
The leaves of vegetative stolons of greenhouse grown Cryptanthus `Marian Oppenheimer' (wide leaf clone) were cultured in modified MS media to induce adventitious shoot formation via callus formation. The best callus induction medium was basal MS medium with 10 μM NAA, IBA and BA. Pure green (843), maroon (3), striped (2) and albino plantlets were obtained. Most of the albino plantlets were stunted, tightly clumped together and impossible to score. The medium which produced the highest average number of non-albino plantlets was basal MS medium with 0.3 μM NAA, IBA and BA All non-albino plantlets were rooted in MS medium with 5.4 μM NAA and transplanted ex vitro with a survival rate of 96.7%. The maroon plantlets became green two weeks after transplanting. Histological studies revealed that C. `Marian Oppenheimer' (wide leaf clone) has two tunicas (L1 and L2) and a corpus (L3). Callus on the leaf explant arose mainly from the L2 and L3. Apparently C. `Marian Oppenheimer' (wide leaf clone) is a GWG periclinal chimera.
Jeffrey P. Schnurr and Zongming Cheng
A selection of Betula platyphylla, from an open pollinated population, was made for upright growth habit, cold hardiness, and a dark green canopy. A micropropagation system was developed to overcome the difficulty with conventional propagation techniques. Shoot-tip cultures were best established in 3/4 strength MS medium supplemented with 0.1 μM thiadiazuron. After 5 weeks in culture, shoots were transferred to woody plant medium (WPM) with 4.4 μM BA. The highest proliferation rate occurred at 24 C on WPM, solidified with agar, and supplemented with 2.2 μM BA. Shoots rooted in vitro and ex vitro and have been established in the field. A regeneration system has also been developed using leaves from aseptic cultures. The optimum conditions for shoot regeneration include a 2-week dark treatment before exposure to a 16-h day/8-h night cycle. Large, healthy leaf explants cultured on WPM with 20 μM BA regenerated shoots at the highest frequency. Regenerated shoots, when transferred to the micropropagation system, proliferate successfully. Currently, a transformation system for this selection is being developed.
Jung Eek Son, Yil Jang, and Jung Hyuk Seo
Supporting materials for rooting have a considerable influence on the growth and quality of in vitro plantlets. Various supporting materials (rockwool, perlite, vermiculite, and polyurethane) and nutrient supply cycles (12, 24, 36, and 48 hours) were examined to find the optimum conditions for photoautotrophic micropropagation of potato plantlets in the nutrient-circulated micropropagation (NCM) system. In the NCM system, nutrient solution was circulated between the culture vessel and the nutrient reservoir. A plug cell tray with 70 plantlets was placed inside. The number of air exchanges was 10 hours under forced ventilation. Nodal leafy cuttings of plantlets were cultured at CO2 concentrations (mol·mol-1)/PPF s (mol·m-2·s-1) of 350/80, 700/120, and 1500/250 on day 5-11, 12-18, and 19-28, respectively, for all treatments. All growth factors of in vitro plantlets grown for 28 days using rockwool, perlite and vermiculite were greater than those grown using polyurethane. Dry weight of plantlets grown using rockwool was eight times greater than those grown using polyurethane. The same results were obtained in the growth and survival percentages 14 days after transplanting to ex vitro conditions. Optimum nutrient supply cycles were 12, 24, and 48 hours when perlite, rockwool, and vermiculite were used as supporting materials, respectively. It was considered that the range of optimum nutrient supply cycle was affected by water retention characteristics of supporting materials. This study proved that the supporting material and the nutrient supply cycle were very important environmental factors in photoautotrophic mass propagation.
Margarita Fraga, Mertxe Alonso, and Marisé Borja
Meristem culture and/or thermotherapy were used for virus elimination from ornamental Phlox paniculata L. (`Blue Boy', `Orange perfection' and `Starfire') mother plants. Shoot tip, leaf, node and flower ovary explants collected from greenhouse-maintained virus free plants were cultured in vitro for shoot initiation. Adventitious shoot initiation was observed on Murashige and Skoog (MS) medium containing the cytokinin BA with or without the auxin NAA. The addition of 0.4 mg·L-1 thiamine, 0.4 mg·L-1 folic acid, and 40 mg·L-1 adenine sulfate to the MS medium did not improve the regeneration rate. Multiplication and rooting were genotype dependent. Blue Boy and Orange Perfection cultivars regenerated the maximum number of shoots from leaf explants. `Blue Boy' leaf explants from in vitro plants had a lower regeneration rate than explants from greenhouse plants. Cultivar `Starfire' had the highest shoot formation with open flower ovary explants and failed to regenerate from leaf explants. In vitro rooting of adventitious shoots in the presence of auxins (IAA, NAA, or IBA) with or without BA was less effective than ex vitro rooting. Chemical names used: 6-benzyladenine (BA); indole-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA).