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.
Karim H. Al-Juboory, J. Al-Naimi, L.K. Al-Amiry, R. Shibli, and R.M. Skirvin
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).
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.
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.
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).
Michael Marcotrigiano and Susan P. McGlew
In an effort to accelerate breeding programs and to study somaclonal variation, a micropropagation system was devised for cranberries (Vaccinium macrocarpon). Using a factorial design, explants taken from greenhouse grown plants were placed on Anderson's medium containing different concentrations of 2ip' GA3, and IBA, with 4 cultivars tested over 3 subcultures. In other experiments, explant source, macro and micro salt formulations, and rooting treatments, were studied. Optimal multiplication and shoot quality occurred when single node explants taken from greenhouse grown plants were placed on Anderson's media containing 150 uM 2iP, 1.0 uM IBA and no GA3. Histological examinations indicate that initial response is axillary bud proliferation but upon subculture adventitious shoot formation may be possible. Proliferated shoots could be rooted ex vitro in plug trays under plastic tents and without hormone treatments. Optimal rooting occurred under high light conditions in a 1:1 (v:v) peat:sand mix. Plants were easily transplanted into the field in spring and will be evaluated by comparison to conventionally propagated material.
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.
Darren Touchell, Zenaida Viloria, and Thomas Ranney
Weigela Thunb. consists of 12 species distributed throughout Northeast Asia. Diervilla Mill. is a closely related genus containing three species endemic to North America. Taxa from both of these genera are important nursery crops. Hybrids between these genera could potentially combine the excellent cold hardiness and adaptability of Diervilla with diverse forms, foliage colors, and flower colors found in Weigela. Prior attempts to create intergeneric hybrids between these genera were unsuccessful and resulted in embryo abortion before seeds matured. To overcome this barrier, ovule culture and micropropagation procedures were used to develop intergeneric hybrids. Cleaved amplified polymorphic sequences (CAPS) analysis was used to verify hybrids. Intergeneric crosses, D. lonicera × W. middendorfiana, D. sessilifolia × W. florida (two clones), and D. lonicera × W. florida were attempted. Crosses of D. lonicera × W. middendorfiana did not produce viable hybrids. From the remaining three crosses, a total of 544 plants were obtained from 1278 ovules. About 85% of the 544 plants appeared very chlorotic or had low vigor, and senesced when transferred to multiplication medium. Only 80 of the 544 plants were successfully maintained in tissue culture, of which 10 have been successfully transferred ex vitro. CAPS analysis indicated that a majority of these plants were hybrids. Further studies are focused on improving tissue culture procedures and other methods to develop tetraploids to increase plantlet vigor and fertility.
Kris Pruski, Tina Lewis, and Mohyuddin Mirza
Chokecherries and pincherries are commonly used in landscaping. Some of the selections such as `Garrington', `Mary Liss' and `Jumping Pound' have large fruits of good quality suitable for food processing. The species are also very well adapted to severe winter conditions of the Prairie Provinces. In our studies, in vitro propagation of the selections was undertaken. The best results with initiation of cultures were observed when dormant buds were used as explants on MS medium with 30 g/L sucrose, 0.1 mg/L NAA and 1.0 mg/L BAP (4 wks, 24/22°C day/night, 16 hrs photoperiod 3000 lux). Optimal proliferation in both species was on MS medium with 1-2 mg/L BAP, 80 mg/L AdSO4 and 170 mg/L NaH2PO4. Rosettes produced were placed on medium without hormones prior to rooting. Rooting was performed ex vitro in root-trainers (soilless mix) on the greenhouse bench under mist. Basal dip in commercial rooting powder Stimroot 1 (0.1% IBA) was equally effective to spray application (2 mg/L IAA with 0.5 mg/L NAA). Average of 77% rooting with `Garrington, 72% and 81% rooting with `Jumping Pound' and `Mary Liss' was observed respectively.
Margarita Fraga, Mertxe Alonso, Philippe Ellul, and Marisé Borja
Meristem culture and/or thermotherapy were used to eliminate viruses from ornamental Dianthus gratianopolitanus Vill. (`Spotti' and `Frosty Fire') mother plants. Shoot tip, leaf, node, and ovary explants collected from greenhouse-maintained, virus-free plants were cultured in vitro for shoot initiation on Murashige and Skoog (MS) medium containing BAP, kinetin, or 2-iP with or without IAA or NAA. Culture of shoot tips in MS with 0.57 μm IAA and node explants in MS with 2.46 μm 2-iP is recommended for `Spotti' cultivar. In `Frosty Fire', optimum number of axillary shoots was obtained from shoot tip and node explants in MS without plant regulators. Leaves and ovaries were not adequate explants for D. gratianopolitanus micropropagation because none or only a low percentage of explants regenerated shoots. High levels of cytokinins increased the number of shoots per explant but also increased the production of aberrant phenotypes and induced hyperhydricity. Adventitious shoots rooted in vitro with auxins, but maximum rooting was 97% ex vitro without auxins. This study demonstrated that D. gratianopolitanus can be successfully micropropagated. Chemical names used: 6-benzyladenine (BAP); kinetin (KIN); 6-(γ,γ-dimethylallylamino)-purine (2iP); indole-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); gibberellic acid (GA3).