Protuberances are organized distinct regions of long-term callus: histological and transcriptomic analyses in kiwifruit

Key message Macroscopic, ultrastructural, and molecular features—like a ball shape, the presence of starch granules, and the up-regulation of genes involved in carbohydrate metabolism and secondary metabolite biosynthesis—distinguish PT regions within a callus. Abstract The modification of the mass of pluripotent cells into de novo shoot bud regeneration is highly relevant to developmental biology and for agriculture and biotechnology. This study deals with protuberances (PT), structures that appear during the organogenic long-term culturing of callus (OC) in kiwifruit. These ball-shaped regions of callus might be considered the first morphological sign of the subsequent shoot bud development. Sections of PT show the regular arrangement of some cells, especially on the surface, in contrast to the regions of OC beyond the PT. The cells of OC possess chloroplasts; however, starch granules were observed only in PTs’ plastids. Transcriptomic data revealed unique gene expression for each kind of sample: OC, PT, and PT with visible shoot buds (PT–SH). Higher expression of the gene involved in lipid (glycerol-3-phosphate acyltransferase 5 [GPAT5]), carbohydrate (granule-bound starch synthase 1 [GBSS1]), and secondary metabolite (beta-glucosidase 45 [BGL45]) pathways were detected in PT and could be proposed as the markers of these structures. The up-regulation of the regulatory associated protein of TOR (RAPTOR1) was found in PT–SH. The highest expression of the actinidain gene in leaves from two-year-old regenerated plants suggests that the synthesis of this protein takes place in fully developed organs. The findings indicate that PT and PT–SH are specific structures within OC but have more features in common with callus tissue than with organs.

Morpho-histological study on shoot bud regeneration in cotyledon cultures of pepper (Capsicum annuum)

Adventitious organogenetic structures were produced in vitro using cotyledon explants excised from 14 day-old pepper seedlings. The best response was observed on MS medium containing 5.7 μM indole-3-acetic acid and 8.8 μM 6-benzylaminopurine. However, when transferred onto elongation medium (MS + 2.8 μM gibberellic acid), these structures frequently developed into leaf-like features rather than into normal shoots. Interestingly, the histological study conducted on the cut end of the cotyledonary petiole revealed a direct induction of numerous teratological protuberances that arise around the cut end of the explant. On the contrary, typical organized bud meristems were rarely observed. Thus, the low number of plants obtained after transfer onto elongation medium seems to be a consequence of this teratological developmental process, frequently associated with fasciated and degenerative meristems, rather than a defect in shoot development from available meristems.

REGENERATION OF TREMA ORIENTALIS (BLUME) LINN.: EFFECT OF GROWTH REGULATORS, CULTURE CONDITIONS, AND AGE AND SOURCE OF EXPLANTS

Optimal conditions for high frequency shoot bud regeneration from leaf callus of Trema orientalis (Blume) Linn. were studied. The regeneration rate was controlled by the growth regulators, the age and the source of the explants, and the illumination conditions. Irrespective of illumination conditions, shoot bud regeneration was achieved only in media containing benzyladenine (BA) + α-naphthaleneacetic acid (NAA) combinations, with the best results being obtained in the presence of 2.5 mg/1 BA and 0.25–0.5 mg/1 NAA. The morphogenic response was less frequent in the calluses derived from leaf explants of the mature trees compared to those of the in vitro-grown seedlings. The rate of shoot bud regeneration was more pronounced in the cultures maintained for 4 weeks in the light (16-h photoperiod) than the cultures incubated in the dark. Regenerated shoots were rooted on the medium containing 1/2 strength basal Murashige and Skoog (MS) salts supplemented with 0.01 mg/1 NAA or indole-3-butyric acid (IBA). The rooted plantlets were established in the greenhouse.