Benzylaminopurine

The pear calli were induced from the flesh of young ‘Clapp's Favorite’ (P. communis) fruit on the NN69 (NITSCH and NITSCH 1969) solid medium with addition of sucrose (30 g/L), 6‐benzylaminopurine (0.5 mg/L) and 2,4‐dichlorophenoxyacetic acid (1.0 mg/L). The first‐generation calli were subcultured several times, and the rapidly growing soft calli were screened and maintained under dark condition on the MS (Murashige and Skoog) solid medium supplemented with sucrose (30 g/L), 6‐benzylaminopurine (0.5 mg/L) and 2,4‐dichlorophenoxyacetic acid (1.0 mg/L). The transformation of pear calli was performed as follows: pear calli were soaked in A. tumefaciens strain EHA105 (0.5 OD₆₀₀) containing either the pCambia1301‐PpBBX16 vector or the empty pCambia1301 vector for 10 min. After 3 days coculture, the calli were then screened on MS solid medium mentioned above by 10 mg/L hygromycin B under continuous dark conditions at 24 °C. For the light treatment, the newly subcultured empty or PpBBX16‐containing calli were moved to the light conditions (16‐h light/8‐h dark) for 2 day and then used for observation.

Transcriptome analyses were performed using RNA-Seq data generated by the PGSC described previously [3] (link). In this data set, transcriptome sequences were generated from 32 DM libraries using RNA-Seq with the Illumina Genome Analyzer II platform (Tables 1 and 2). The 32 DM libraries represent a wide range of developmental tissues/organs as well as abiotic and biotic stress treatments and are described in detail in reference [3] (link) (see Supplementary Material and Table S4). The developmental tissues represent vegetative (leaves, petioles, stolons, tubers sampled twice) and reproductive organs (Floral: carpels, petals, sepals, stamens, whole flowers; Fruit: mesocarp/endocarp, whole immature berries, whole mature berries) from greenhouse-grown plants. Shoots and roots from in vitro-grown plants were also included in the developmental series. Callus (10–11 week old) derived from leaves and stems were used to assess transcription in an undifferentiated tissue. The biotic stress conditions (pooled samples at 24 hr, 36 hr, 72 hr) were induced with Phytophthora infestans inoculum (Pi isolate US8: Pi02-007) and two chemical inducers, acibenzolar-S-methyl (BTH, 100 µg/ml) and DL-β-amino-n-butyric acid (BABA, 2 mg/ml) using detached leaves. Wounded leaves, primary and secondary, were included to mimic herbivory. The abiotic stress conditions (24 hr treatment of in vitro grown whole plants) include heat (35°C), salt (150 mM NaCl) and mannitol (260 µM) treatment. Abscisic acid (ABA, 50 µM), indole-3-acetic acid (IAA, 10 µM), giberellic acid (GA3, 50 µM), and 6 benzylaminopurine (BAP, 10 µM) were used to induce hormone stress responses. Expression levels as previously described in [3] (link) were determined by mapping the RNA-Seq reads to the DM potato reference genome using Tophat [23] (link) and expression levels were determined using Cufflinks [19] . Only representative transcripts, which were chosen by selecting the longest Coding Sequence (CDS) from each gene, were used for the analyses [3] (link). RNA-Seq reads are available in the NCBI Sequence Read Archive under study number SRA029323.

The preparation of embryogenic suspension cells and RNA extractions for the suppression subtractive hybridization (SSH) library constructions from the 30-day proliferation cycle and after 16 days of liquid pretreatment to initiate somatic embryogenesis was performed and described previously [17 (link)]. In addition, a portion of pretreated embryogenic suspension cells initiated to undergo somatic embryogenesis was plated on solid agar plates containing the basal medium with or without 6-benzylaminopurine (synthetic cytokinin) for further somatic embryo development and collected after 7 days for RNA extractions and SSH library constructions. The material collected for the shoot apex, female and male inflorescences, and zygotic embryos for the unnormalized library constructions was described previously [16 (link)]. The material for the normal and abnormal male inflorescences SSH libraries was described and performed previously [15 (link)]. The zygotic embryos (3-5.5 months of development) were isolated from tenera palm seeds collected from trees (Deli x La Mé origin) cultivated at CRAPP Pobé Station, Benin.

After data from the carbohydrate study were analyzed, all culture lines were transferred to version 5 medium (Figure 2) for two months. Afterward, shoot tips from all 115 available culture lines, including the 11 core and 16 additional lines used previously, were placed on version 5 medium with 0, 0.33, 0.66, or 1 mg L⁻¹ BAP and 0 or 0.5 mg L⁻¹ gibberellic acid (GA₃) (Phytotech). Four shoot tips from the eleven core and sixteen additional culture lines were placed on each of the eight treatments (864 shoots). The remaining 88 lines were divided in the following manner: culture lines with enough shoots contributed one shoot per treatment, and lines that had less than eight available shoots had their shoots randomly assigned to treatments without repeat. Necrosis, total shoots with lengths greater than 0.25 cm, and shoots greater than 2 cm were counted after 80 days of culture.