Genetic analysis and population structure of wild and cultivated wishbone flower (Torenia fournieri Lind.) lines related to specific floral color

Background The wishbone flower or Torenia fournieri Lind., an annual from tropical Indochina and southern China, is a popular ornamental plant, and many interspecific (T. fournieri × T. concolor) hybrid lines have been bred for the international market. The cultivated lines show a pattern of genetic similarity that correlates with floral color which informs on future breeding strategies. This study aimed to perform genetic analysis and population structure of cultivated hybrid lines comparing with closely related T. concolor wild populations. Methods We applied the retrotransposon based iPBS marker system for genotyping of a total of 136 accessions from 17 lines/populations of Torenia. These included 15 cultivated lines of three series: Duchess (A, B, C); Kauai (D, E, F, G, H, I, J); Little Kiss (K, L, M, N, P) and two wild T. concolor populations (Q and R). PCR products from each individual were applied to estimate the genetic diversity and differentiation between lines/populations. Results Genotyping results showed a pattern of genetic variation differentiating the 17 lines/populations characterized by their specific floral colors. The final PCoA analysis, phylogenetic tree construction, and Bayesian population structural bar plot all showed a clear subdivision of lines/populations analysed. The 15 cultivated hybrid lines and the wild population Q that collected from a small area showed the lowest genetic variability while the other wild population R which sampled from a larger area had the highest genetic variability. Discussion The extremely low genetic variability of 15 cultivated lines indicated that individual line has similar reduction in diversity/heterozygosity from a bottleneck event, and each retained a similar (but different from each other) content of the wild genetic diversity. The genetic variance for the two wild T. concolor populations could be due to our varied sampling methods. The two wild populations (Q, R) and the cultivated hybrid lines (I, K, M, N, P) are genetically more closely related, but strong positive correlations presented in cultivated lines A, C, E, M, and N. These results could be used to guide future Torenia breeding. Conclusions The genetic variation and population structure found in our study showed that cultivated hybrid lines had similar reduction in diversity/heterozygosity from a bottleneck event and each line retained a similar (but different from each other) content of the wild genetic diversity, especially when strong phenotypic selection of floral color overlaps. Generally, environmental factors could induce transposon activation and generate genetic variability which enabled the acceleration of the evolutionary process of wild Torenia species. Our study revealed that wild Torenia populations sampled from broad geographic region represent stronger species strength with outstanding genetic diversity, but selective breeding targeting a specific floral color decreased such genetic variability.

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration occurs mediated by auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia) without intervening callus mass formation in culture with cytokinin, yet its molecular mechanisms remain unaddressed. Here we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding, and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

First Report of Binucleate Rhizoctonia AG-L Causing Root and Stem Rot of Wishbone Flower (Torenia fournieri) in Taiwan

Wishbone flower (Torenia fournieri L.) is a common ornamental plant for flower bed in Taiwan. In August 2018, root and stem rot of wishbone flower occurred on the flower bed in the campus of National Chung Hsing University, Taichung city, with 100% incidence. Symptoms were dark brown discoloration of basal stems and brown necrosis of roots. Lesions from base of stems were excised into 5 mm long fragments, which were then surface sterilized in 1% sodium hydrochloride for 1 min, rinsed in sterile distilled water, dried on filter paper and thereafter placed onto 2% water agar. After 24 h, hyphae characteristic of Rhizoctonia (Sneh et al. 1991) appeared and dominated in every isolation. Hyphal tips were transferred to potato dextrose agar (PDA). After 5 days of incubation at 28°C, characteristic brown colonies of Rhizoctonia (Sneh et al. 1991) were observed. Hyphal width was 4.29±0.52 μm. No sclerotia were visibly present after 21 days of growth on PDA at 28°C. Hyphae were stained by 0.3% safranin-O and 1% KOH. There were two nuclei in each hyphal compartment, suggesting a binucleate Rhizoctonia fungus. ITS sequence has been used as the best tool to identify specific anastomosis group (AG) of Rhizoctonia as shown by Sharon et al. (2006, 2008). ITS sequence was amplified using the primers Bd1a and ITS4 (Goka et al. 2009; White et al. 1990). Blast search analysis of this acquired sequence (acc. no. LC498494) revealed the highest similarity (98.75 to 99.83%) with the reference sequences (acc. nos. AB286934, AB286933, and AB196653) of binucleate Rhizoctonia AG-L, namely Ceratobasidium sp. AG-L. Pathogenicity test was carried out using seedlings of 4-week-old wishbone flower each grown in a pot of 6.35 cm diameter. To prepare the inoculum, a PDA agar block (6 mm in diameter) excised from the growing front of 5-day-old colony was transferred into a flask with 200 ml of potato dextrose broth (PDB) incubated in a shaker at 26°C and 120 rpm for 6 days. The PDB broth was then blended into slurry. Ten pots each with a seedling were inoculated by pouring 50 ml of slurry onto the potting medium. Five pots were served as the controls by pouring PDB only. Pots were maintained in a greenhouse (26 to 33°C). Three days after inoculation, all inoculated plants exhibited symptom of root and stem rot. The same fungus was reisolated and confirmed by sequencing rDNA-ITS. This is the first report of root and stem rot of wishbone flower caused by binucleate Rhizoctonia AG-L in Taiwan and in the world. Although this is the second cases, since Wang and Hsieh (1993), for binucleate Rhizoctonia AG-L to be pathogenic, this study shows that this fungus has the potential to cause damages and is worth of further investigations.

Nuclear DNA replicates during zygote development in Arabidopsis and Torenia fournieri

The progression of the cell cycle is continuous in most cells, but gametes (sperm and egg cells) exhibit an arrest of the cell cycle to await fertilization to form a zygote, which then continues through the subsequent phases to complete cell division. The phase in which gametes of flowering plants arrest has been a matter of debate, since different phases have been reported for the gametes of different species. In this study, we reassessed the phase of cell-cycle arrest in the gametes of two species, Arabidopsis (Arabidopsis thaliana) and Torenia fournieri. We first showed that 4’, 6-diamidino-2-phenylindole staining was not feasible to detect changes in gametic nuclear DNA in T. fournieri. Next, using 5-ethynyl-2’-deoxyuridine (EdU) staining that detects DNA replication by labeling the EdU absorbed by deoxyribonucleic acid, we found that the replication of nuclear DNA did not occur during gamete development but during zygote development, revealing that the gametes of these species have a haploid nuclear DNA content before fertilization. We thus propose that gametes in the G1 phase participate in the fertilization event in Arabidopsis and T. fournieri.

The endogenous responses during the flowering stage of Torenia fournieri L. under LED light

The quality of light has played an important role not only in the vegetative growth, but also in the reproductive stage of the plant. Normally, the endogenous transformation has hardly been observed under the general condition. This study analyzed the endogenous changes, which were particularly influenced by the spectrum of light emitting diode (LED) with induction flowering time from 30th to 40th day. In the 40th day of the flowering process, when the average number of flower buds get the highest, endogenous carbohydrate content was decreased. However, in this phase, plants need more energy; therefore, the net photosynthetic rate tends to increase in order to supply energy for flowering. Throughout 40 days of culture, the net photosynthetic rate had an increase in the concentration of CO2. In particular, it reached the peak when being induced under the light combination ratio 7:3 of red and blue light (0.222 µmol mol-1 h-1). The sugar content also followed the same trajectory; however, it dropped at the end of the period. Among all the experiments, the ratio of red and blue light 5:5 resulted in the highest content of endogenous carbohydrate source (722.30 µg g-1). Moreover, the morphological anatomy of shoot apical meristem in flowering stage was also studied. The floral transition at meristem and floral architecture is as similar as that of Arabidopsis. A typical flower of T. fournieri also consists of a sequence: sepals – petals – stamens – carpels. The SAM is organized into three different zones such as the central zone (CZ), the peripheral zone (PZ) surrounding the CZ and the rib zone (RZ) underneath the CZ.