A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant

Abstract

Background: Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. Results: In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. Conclusions: An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.

Development of a Direct in vitro Plant Regeneration Protocol From Cannabis sativa L. Seedling Explants: Developmental Morphology of Shoot Regeneration and Ploidy Level of Regenerated Plants

“…In this way, we tested the media used in a study regarding the regenerative capacity of cotyledons through addition of thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA) to the culture medium ( Chaohua et al, 2016 ), one work concerning in vitro plant regeneration from cotyledons and epicotyls by means of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) ( Movahedi et al, 2015 ), and another one from leaves and hypocotyls through BAP and 2,4-dichlorophenoxyacetic acid (2,4-D) ( Movahedi et al, 2016a ). Additionally, it was added to our schedule an effective and newly released protocol developed for eggplant in which the use of zeatin riboside (ZEA RIB ), provided good results not only in terms of shoot organogenesis, but also in polyploidization of regenerants ( García-Fortea et al, 2020 ).…”

“…The developmental morphology process of in vitro shoot organogenesis from cotyledons, hypocotyls and true leaves was followed and registered with images, and the duration of each of the developmental stages of organogenesis was also recorded. Additionally, due to the lack of studies concerning polysomaty in this species and its potential usefulness to obtain polyploid plants as it has occurred in other species like tomato ( Van den Bulk et al, 1990 ), cucumber ( Colijn-Hooymans et al, 1994 ), or melon ( Ren et al, 2013 ), and more recently in eggplant ( García-Fortea et al, 2020 ), authors not only studied the regenerative capacity of cotyledons, hypocotyls and true leaves, but also analyzed the ploidy level of these explants together with that of the in vitro regenerants.…”

A Deep Learning-Based System (Microscan) for the Identification of Pollen Development Stages and Its Application to Obtaining Doubled Haploid Lines in Eggplant

“…Two different androgenesis protocols were applied, E6 (based on [ 35 ]) and Cb (based on [ 12 ]). For each of the three genotypes tested, three experimental replicates were performed with 15 anthers per replicate.…”

“…Following the procedure described in the previous sections, the cell concentrates were prepared and subjected to the analysis of the prediction model. Two different androgenesis protocols were applied, E6 (based on [35]) and Cb (based on [12]). For each of the three genotypes tested, three experimental replicates were performed with 15 anthers per replicate.…”

“…Once the buds were sterilized, they were left to dry for a few seconds on sterile filter paper. The anthers were extracted from inside the buds, measured, selected, and finally cultivated with their concave side down in medium E6 (Table 1) [35]. The plates were left at 35 • C for 8 days in dark conditions.…”