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Nancy A. Reichert, Nathan R. Oakley and Brian S. Baldwin

An adventitious regeneration protocol developed for Hibiscus cannabinus L. (kenaf) was attempted on various ornamental hibiscus species. Hibiscus syriacus (Althea, Rose of Sharon) has been successfully regenerated using the kenaf protocol. Leaf tissue from two cultivars (`Double Pink' and `Diana'—a triploid) was placed on kenaf regeneration media. Adventitious shoots emerged from both cultivars within 8 to 10 weeks. Shoots were then excised and placed on a medium for rooting. Additional hibiscus species have been evaluated for regeneration ability. Previous studies with kenaf determined the adventitious regeneration protocol could induce mutations (somaclonal variation) in the regenerants. Variations in kenaf stem color and flower shape were noted. Since many ornamental hibiscus are asexually propagated, once a desired mutant is identified, it could be maintained and propagated without loss of the unique trait(s).

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Freddi A. Hammerschlag, Ghazala Hashmi, Robin Huettel, Dennis Werner and David Ritchie

One approach for obtaining useful genetic variation is to select for somaclonal variants generated by tissue culture techniques. Increased levels of resistance to bacterial leaf spot (Xanthomonas campestris pv. pruni) have been observed in toxin-selected and unselected peach regenerants in vitro, in the greenhouse and under field conditions. Peach regenerants have also demonstrated increased levels of bacterial canker (Pseudomonas syringae pv. syringae) and root-knot nematode (Meloidogyne incognita) resistance. Random amplified polymorphic DNA (RAPD) primers have been used to study genetic variation at the DNA level among the somaclonal variants. Sixty RAPD primers (10-mers) were screened and 10 proved useful as markers to detect polymorphisms, thus establishing a genetic basis for somaclonal variation. These studies demonstrate the feasibility of using tissue culture techniques to generate fruit trees with increased levels of disease resistance.

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Björn A. Gustavsson and Vidmantas Stanys

Field performance in lingonberry (Vaccinium vitis-idaea L. cv. Sanna) was compared in 1995–97 for plants produced by tissue culture (TC) vs. stem cuttings (SC). Pot plants of about the same size were transplanted from the nursery to an infertile, sandy moraine soil. Survival was 97% for the TC plants but only 83% for the SC plants. Fruit yield was significantly greater for TC plants than for SC plants in both the second (+79%) and third (+190%) years, but mean fruit weight was not influenced by propagation method. Rhizome production and total plant weight were also greater for the TC plants. Although micropropagation may give rise to somaclonal variation, no obviously variant plants were apparent in the field.

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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.

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Francisco A. Ferreira, Dae-Geun Oh and Edward C. Tigchelaar

Six F1's involving 6 multiple genetic marker stocks and a common inbred parent (PU812) were cultured to study the genotypic effect on regeneration ability and frequency of somaclonal variation in R0 for the known heterozygous marker genes. Leaf discs 7 mm in diameter were excised from young fully expanded leaves of 6-7 week old plants, and cultured on MS medium supplemented with cytokinins (Kinetin, Benzyladenine) and IAA. With few exceptions, the parents and F1's responded similarly to different hormone combinations. The beat hormone combinations for shoot formation were 4 mg/l Kinetin + 0.5 mg/l IAA and 2.3 mg/l BA + 0-0.18 mg/l IAA.

Only 2 of the 6 multiple marker stocks and the common inbred parent (PU812) were successfully regenerated. Four of the six hybrids between PU812 and the multiple markers were readily regenerated, whereas 2 hybrids failed to regenerate with several different hormonal combinations. No mutations have been observed for the known heterozygous markers in 76 R0 tissue culture regenerants.

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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.

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Gerson R. de L. Fortes and Silvio L. Teixeira

The aim of this work was to study different apple of somatic material as callus and adventitious shoots are concerned, for further utilization in the research of somaclonal variation. The somatic materials were: leaf discs, cotyledons and hypocotyls of Gala apple seedlings, cultivated in a MS medium added by B5 vitamins in addition to (in mg/l): BAP (1,0), NAA (0,5) mio-inositol (100,0) sucrose (30,0 g/l) and solidified in agar (6,0 g/l). The several times of explant exposition to the dark affected the final callus weight. Callus weight derived from leaf discs were higher than those for cotyledons and hypocotyls. Explants exposed directly under light or up to two weeks in the dark showed less percentage of regenerative callus as compared to those of three weeks in the dark. The leaf explants presented the highest percentage of regenerative callus. The least response was obtained for those derived from hypocotyls. The highest number of adventitious shoots was obtained keeping the explants three weeks in the dark as compared to directed light exposition.

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Wenhao Dai, Victoria Magnusson and Andrea Swanberg

Many woody plants, including some birch species, can be cloned using such in vitro techniques as pre-existing meristem culture, organogenesis, and embryogenesis. However, clonal fidelity of in vitro-derived plants is always a big concern because somaclonal variations may be induced during the entire in vitro process. To address this issue, we used random amplified polymorphic DNA (RAPD) markers to determine the genetic stability of in vitro-propagated plants of Betula platyphylla `Fargo'. Forty-two greenhouse-grown birch plants derived from a 10-year shoot tip culture (shoot-derived) and 42 in vitro plants regenerated from leaf tissues (regenerated) were randomly selected and evaluated for their genetic fidelity by RAPD. To date, 20 primers (C1-C20, Operon Technologies) were screened for all 84 plants. Only strong bands that are conservative were scored. Each primer generated a unique set of amplification products. Most of scoreable bands are ranged from 350 to 1800 bp. A total of 3696 fragments were amplified from 42 shoot-derived plants by all 20 primers with an average of 4.4 bands per primer, in which 6 primers produced polymorphic bands, indicating some genetic variations within shoot-derived plants. Nineteen out of 20 primers yielded 2772 clear and reproducible bands (an average of 3.47 per primer) from 42 regenerated plants with no significant variations being detected. Our preliminary results showed that in vitro regenerated plants are genetically uniform. However, a long-term tissue culture might result in a few genetic variations of birch species.

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Stan C. Hokanson, Kelvin G. Grant, Elizabeth L. Ogden and Lisa J. Rowland

Commercial strawberry plantings in the mid-Atlantic region are often quickly infected with one or more aphid-transmitted viruses, resulting in the loss of plant vigor, stunting, lowered yields, etc. To produce virus-free plant material for the strawberry industry and for cultivar development programs, heat therapy and/or meristem tip culture protocols are generally employed. One of the problems associated with meristem culturing is the potential for somaclonal mutations to occur in the meristem or surrounding proliferating tissue. As a result, distinct “bud lines” displaying functionally insignificant to distressingly high levels of phenotypic variation can arise from individual meristems. It would be desirable to differentiate these off-types by genetic fingerprinting to maintain trueness-to-type. Randomly amplified polymorphic DNA (RAPD) markers were evaluated for the potential to differentiate six pairs of strawberry bud lines that exhibit slight to fairly extreme levels of phenotypic variation. Reproducible RAPD marker profiles were generated using 10 primers in amplification reactions with genomic DNA obtained from multiple extractions. While five of the bud line pairs remained indistinguishable, three primers distinguished two variants of the Mohawk cultivar that are now in existence in the strawberry industry. Results suggest that typical somaclonal variation produced in the meristem culture process is of a magnitude that is not readily detectable with the RAPD protocol. The two Mohawk lines were probably produced by a higher magnitude mutation event than generally occurs or a cultivar mix-up.

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Nilvane T.G. Müller, Gerson R. de L. Fortes, Gioconda C. Nascimento and Julio Daniels

Garlic (Allium sativum L.) belongs to the Alliaceae family and originated from Asia and Mediterranean countries. Their bulblets are rich in starch and aromatic substances. The rate of garlic propagation in field conditions takes several years for the production of a certain number of seed bulbs for the release of a new variety. The use of tissue culture techniques is a useful tool for overcoming this problem. The aim of this work was to increase the mean number of shoots derived from the meristem isolation and to verify the percentage of callus formation and to analyze vigor of the material. The initial meristems were inoculated in a salt and vitamin B5 media except for the iron element, which was provided by MS medium added to in mg·L-1: myo-inositol (100.0), nicotinic acid (1.0), piridoxine (1.0), thiamine (10.0), sucrose (20.0 g·L-1), agar (6.0 g·L-1). BAP and TDZ were added at: 0.0; 1.0; 1.5; 2.0; and 2.5 μM This material remained in a growth room for a 16-h photoperiod, radiation of 20 μMol·m-2·s-1 and 25 °C for 40 days. Although `Sao Marcos' produced more vigorous shoots, no significant difference was found for the mean number of shoots. `Sao Valentim' cultivar shows more callus at the shoot base, making this cultivar more prone to somaclonal variation On the other hand, BAP estimulates the appearance of callus, but it has been shown that this is cultivar-dependent.