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Chionanthus retusus is a small deciduous tree that is widely used in landscaping due to its beautiful white spring flowers and ornamental value. Conventional propagation through seeds requires one to two years of breaking dormancy. The objective of this study was to determine the conditions of in vitro germination in C. retusus. In vitro embryo culture was carried out to investigate the effects of six factors: basal media (McCown Woody Plant Medium (WPM) and Murashige and Skoog (MS)); plant growth regulators (different combinations and concentrations of naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BA), and gibberellic acid (GA₃)); embryo age (collected weekly beginning 36 days after fruit setting); low temperature pretreatment (storing 4°C for 1, 2, 3, and 4 weeks); coconut additives (100, 200, and 300 ml·L-1); and genotype (grouping plants depending on their flowering nature). The basal medium used in this study was WPM with 2 mg·L-1-1 GA₃, 20 g·L-1 sucrose, and 6 g·L-1 Agar. WPM medium mixed with GA₃, resulted in higher germination rate as compared to when using a combination of auxin and cytokinin. GA₃ at 2 mg·L-1 was the most effective of all combinations and concentrations of PGRs. WPM medium with 2 mg·L-1 GA₃ resulted in better and faster germination (75.93%). Embryos collected at 57 days after fruit setting had the highest percent of germinated seeds (87.04%) while low-temperature pretreatment of fruits at 4°C for two weeks produced the highest germination (95.37%). These results of this study could be an open ground for development of an efficient protocol for commercial production of the ornamental tree.

We established an in vitro system to investigate transcription levels of the RsMYB1 gene expressed in T2 20-day-old transgenic Petunia plants (three independent lines: PhRs1, PhRs2, and PhRs3), and the association between those transcription levels and anthocyanin production at various pH values (3.0 to 8.0) for a period of 10 days. All the lines treated with pH 5.0-7.0 exhibited increased anthocyanin content and delays in growth compared to the wild-type (WT) seedlings. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that the enhancement of anthocyanin production in the transgenic lines was due to the upregulation of RsMYB1 transcription at various pH values. The results suggest that pH value can control expression of RsMYB1 which is associated with anthocyanin production.

Anthocyanin accumulation and plant growth were examined in petunia (NT and transgenic plants) by determining the effects of different sources of light and varying light/dark cycles. Red light significantly enhanced anthocyanin content of B-peru+mPAP1; however, it had a negative effect on anthocyanin production in RsMYB1 plants. In general, white light was found to be reasonable for anthocyanin accumulation in all plants. In case of light/dark cycles, application of seven days of light:14 days of dark significantly enhanced anthocyanin content. We found that anthocyanin content detected in transgenic plants expressing anthocyanin regulatory transcription factor genes (B-peru+mPAP1 or RsMYB1) was higher than that in NT plants in all treatments. Plant growth was also influenced by the different light sources and dark/light cycles. Taken together, our results suggest that light source and light/dark cycle play an important role in anthocyanin production and plant growth. The choice of the optimal conditions is also important for anthocyanin production and plant growth depending on NT or transgenic plants carrying anthocyanin regulatory transcription factors.