Mark P. Bridgen

Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid Alstroemeria aurea × A. caryophyllaea, resulted in no seeds. Backcrosses of the hybrid with parent A. aurea did not produce any seeds. In an attempt to restore the hybrid fertility, an efficient in vitro procedure has been developed and applied effectively in the chromosome doubling of the diploid hybrid. Forty-one percent of the treated plants were proven to be truly tetraploid by chromosome counts and stomatal measurements after applying 0.2 to 0.6% colchicine for 6 to 24 hours. Over 87.5% of these colchicine-induced tetraploids were stable and retained their tetraploidy after one year of growth. The fertility of the hybrid was not restored although the pollen stainability was increased from 0 to 12% after chromosome doubling. Cytological studies on the pollen mother cells (PMCs) of the sterile diploid hybrids revealed abnormal meiotic behaviors. In addition, aneuploid chromosome numbers, ranging from 2n = 1 to 2n = 18, were observed in over 45% of the PMCs examined. PMCs of the colchicine-induced tetraploids showed that meiotic chromosome pairings were normal in most cases (1.59I + 15.07II + 0.05III + 0.03IV). These results indicate that the sterility of this hybrid is not only caused by parental chromosome differences, but other complex fertility/sterility-regulating mechanisms are involved too.

Effects of genotype, culture medium and embryo developmental stage on the in vitro responses from ovule cultures of interspecific hybrids of Alstroemeria

An ovule culture procedure to rescue hybrid embryos from incompatible interspecific crosses of Alstroemeria was developed. Genotype, the developmental stage of embryo and the culture medium were found to have essential roles in the success of ovule culture. Ovules from 10 interspecific crosses showed different in vitro responses in the percentages of embryo germination, callus production and/or shoot development from the germinated embryos. Responsive genotypes with greater than 15% embryo germination were identified. Ovules collected at 7 days after pollination resulted in 53.3% embryo germination after 10 weeks of culture on Murashige and Skoog medium supplemented with 146 mg/l glutamine, 30 g/l sucrose and pH 5.5. This was significantly higher than those from 4 and 10 DAP, which had a germination rate of 14.2 and 12.5%, respectively. Ovules cultured on MS basal medium had a significantly higher embryo germination rate of 33.3% than those cultured on B5 basal medium which averaged 10%. The addition of 2,4-dichlorophenoxyacetic acid and benzyladenine reduced the rate of embryo germination and shoot development.

Rhizome splitting : a new micropropagation technique to increase in vitro propagule yield in Alstroemeria

The growth of in vitro Alstroemeria hybrids was investigated through morphological studies. Hybrids maintained the same sympodial growth described in wild species. Rhizome tip internodes were extremely short, axillary buds were very close to each other and not lined up. Rhizome halves, that were produced by cutting the rhizome with a horizontal or vertical longitudinal cut, could regenerate a new rhizome. With this new technique, propagule production increased up to 194% and 213% in Alstroemeria ‘Sweet Laura’ and Alstroemeria ‘A30’, respectively, over a period of 12 weeks. In vitro plants produced with the split technique had greater fresh weight than the control. Plants produced with the split technique rooted and flowered regularly and were true-to-type.

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing μM 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.

Somaclonal variation as a tool to develop pest resistant plants of Torenia fournieri 'Compacta Blue'

Callus cultures of Torenia fournieri ‘Compacta Blue’ were initiated on a modified Murashige and Skoog salt medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid. Shoots were regenerated from these cultures using MS medium amended with 2.46 μM indolebutyric acid and 8.88 μM benzyladenine. These shoot cultures were subjected to two-spotted spidermite (Tetranychus urticae Koch.) and the greenhouse whitefly [Trialeurodes vaporariorum (Westwood)]. Pests were allowed to feed until such time that their populations started to decrease due to lack of food. The remaining live tissue of the Torenia was placed on MS medium amended with 2.28 μM zeatin to induce new adventitious shoots and plantlets. Newly regenerated plantlets were acclimated to greenhouse conditions and evaluated for resistance to the pest to which they were subjected in vitro. Highly significant differences in pest numbers were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls.

Micropropagation procedures for Leontochir ovallei

Leontochir ovallei Phil., an endangered Chilean species in the Alstroemericeae, was micropropagated on Murashige & Skoog medium supplemented with 4 μM benzyladenine, 1 μM indolebutyric acid and 146 mg l-1 glutamine. Over 88% of the shoots rooted in vitro when treated with 10 μM naphthaleneacetic acid and micropropagated plantlets were successfully transplanted into the greenhouse.

Micropropagation of Mussaenda Cultivars

The genus Mussaenda (family Rubiaceae) consists of ornamental shrubs or small trees grown in the subtropical and tropical regions of the world for the one to many colorful, large petaloid sepals of scarlet, pink, picotee, or white (Fig. 1). According to Rosario (1984), the development of the Mussaenda cultivars is the greatest contribution from the Philippines to the field of ornamentals. Current cultivars were developed through a breeding program at the University of the Philippines at Los Banos (Rosario 1987). The cultivars were named after important women in Filipino history (Rosario 1984).