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The highly conserved plant microRNA156, miR156, affects various aspects of plant development and stress response by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Our understanding of the role of miR156 and its mode of action in alfalfa’s (Medicago sativa L.) response to drought and flooding is still elusive, and thus this study was aimed at filling this gap in knowledge. Physiological parameters, metabolite and transcriptional analyses showed an interplay between miR156/SPL13 and WD40-1/DFR to mitigate drought stress. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune the DIHYDROFLAVONOL-4-REDUCTASE (DFR) level for enhanced anthocyanin biosynthesis. Moreover, RNAseq-derived weighted gene co-expression network analysis (WGCNA) of SPL13RNAi alfalfa plants showed tissue-and genotype-specific drought responses. Accordingly, transcripts mediating stress-mitigating metabolites, such as anthocyanin, were increased in stem tissues of drought-stressed plants, while those involved in photosynthesis were maintained in leaves. Moreover, drought-stressed roots showed elevated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. The role of miR156 in flooding tolerance was also investigated using flooding-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars, along with miR156OE and SPL13RNAi plants. Additionally, to examine the role of ABA and SnRK1 in regulating miR156 expression, ABA insensitive (abi1-2, abi5-8) Arabidopsis thaliana mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) SnRK1 were used. Investigation of physiological parameters, hormone profiling, and global transcriptomics showed a positive role for miR156 in flooding tolerance, and a comparison of Arabidopsis mutants and transgenic lines showed that miR156 expression was affected by SnRK1 to enhance anthocyanin and ABA metabolites. Transcriptomics analysis also revealed nine new alfalfa SPLs, three of which responded to flooding (SPL7a, SPL8, and SPL13a) along with the previously identified SPL4, SPL9, and SPL13. Characterization of the newly identified SPLs, along with understanding the mode of action of miR156 in alfalfa’s response to drought and flooding, will provide useful tools in marker-assisted breeding of alfalfa and resource to scientific knowledge.

Plant tissue culture is an inevitable technique to overcome healthy and limited planting materials problems using suitable energy sources. Different carbohydrates have diverse effect on in vitro growing plantlets in terms of growth performance, acclimatization and cost used for micro-propagation. Hence, this paper reports the effects of sucrose, fructose, glucose, table sugar and starch on pineapple in vitro mass propagation and acclimatization as well as the analysis of energy source required cost per a medium. A complete randomized design was used to compare analytic grade sucrose with other four energy sources at 2 and 3 % (w/v). The results revealed that the energy sources with varied concentration strongly influenced the in vitro growth and subsequent acclimatization of pineapple plantlets. Analytic grade sucrose and table sugar at 3 % performed well for in vitro survival rate (100%), shoot amplification (15.3-16.5 shoots), rooting ability (2.5cm long and 12 roots) and acclimatization (95.4-97%). However, fructose and glucose required high importation cost (229.1% and 121.9% over analytic grade sucrose, respectively), and have low growth and acclimatization performance next to starch and energy free medium. Thus, table sugar has found to be a suitable alternative energy source for pineapple mass propagation, which saved about 95-97% cost from that of laboratory grade sucrose.

Pineapple (Ananas comosus L.) is one of the most potential fruit crop growing in Ethiopia due to suitable agro-ecology and economic importance. However, it is difficult to meet the demand for planting materials using the conventional propagation techniques due to production inefficiency and disease transmission. The experiment is laid in Completely Randomized Design with three treatments of vessels used along its media type, Temporary Immersion Bioreactor, TIB (RITA®, Vitropic, France) , Glass jam jar and plastic jars , replicated six times on MS medium. A highly significant difference (p

Pineapple (Ananas comosuss, var. Smooth cayenne), which is a popular tropical fruit, is propagated vegetatively. Conventional propagation alone does not provide clean and adequate planting material demanded in Ethiopia. Recently, in vitro multiplication has become a promising technique for large-scale production. However, the acclimatization to the external environment procedure impedes the efficiency, which needs carefully optimized acclimatization techniques. We report optimized acclimatization procedures following first- and second-stage hardening methods for in vitro pineapple plantlets. Primarily, Jiffy-7 peat pellet allowed growing plants vigorously and provided above 8% survival rate over soil mix. Nevertheless, in Ethiopia, soil mix is cheaper and locally accessible. The primarily acclimatized plantlets are needed to be hardened further for better establishment and survival in the field. Black polybag and polysleeve pots filled with soil mix were evaluated in the greenhouse. A significant difference was obtained between pots for number of roots and substrate weight. Polybags had higher root number than polysleeves and saved about 27% of substrates per plant, which is a reduction of 25% of total transportation cost. Hence, the soil mix and polybags were found to be preferable over substrates and pots, for subsequent in vitro pineapple acclimatization.

Different mechanisms are tried so far to reduce the production cost of plant tissue culture through increasing multiplication rate and/or substituting expensive components for wider usage of the technology. Subsequently, replacing the most common gelling agent, agar, took an attention since higher proportion of media cost comes from it. Up to now different alternatives were tried though they have drawbacks due to their inherent chemical characteristics. Therefore the search for new alternatives like Enset ventricosum is important. Enset flour, ‘Bulla’, has been tried in this study for the first time to substitute agar and showed no significant difference for shoots number, root number, shoot height, leaf number and an associated fresh weight of the plantlets besides good gelling ability than Agar. Subsequently, ‘Bulla’ at 80g L-1 gelled well and gave 11.8 shoots with 0.95g and 13.33 roots having 1.37 cm length. This rate can also save up to 76 % cost of gelling though significant difference was found for root length of 3.23 cm with Agar, 8 g L-1. This indicates further study on biochemical and/ or hormonal activity and across crop genotypes to use ‘Bulla’ as a cheap alternative commercial gelling agent.

Grape-berries are exposed to a plethora of abiotic and biotic stimuli during their development. The developmental and temporal regulation of grape berry polyphenol metabolism in response to various cues was investigated using LC-QTOF-MS based metabolite profiling. High light (2500μmolm(-2)s(-1)), high temperature (40°C), jasmonic acid (200μM), menadione (120μM) and abscisic acid (3.026mM) treatments were applied to detached berries. Greater magnitudes of metabolite fluctuations characterize the pre-veraison berries than the veraison stage in response to the treatments. Furthermore, a tighter co-response of metabolic processes was shown at veraison, likely supporting the resilience to change in response to stress. High temperature and ABA treatments led to greater magnitudes of change during the course of the experiment. The present study demonstrates the occurrence of differential patterns of metabolic responses specific to individual cues and berry developmental stage, which in the field are commonly associated and thus hardly discernable.

Ginger ( Zingiber officinale Rosc.) is the second most widely cultivated spice in Ethiopia, next to chilies. Recently, there has been huge demand for clean planting material of improved ginger cultivars, though it is difficult to meet the demand of planting materials using the conventional propagation techniques due to production inefficiency and disease transmission. Therefore, the present study was carried out with the objective of assessing the potential of axillary buds and shoot tips as explant sources and determination of suitable growth regulators for in vitro propagation of two ginger cultivars. Murashige and Skoog (MS) medium with four levels of benzyl adenine (BA) and kinetin was used for shoot multiplication in combination with two explant sources. A highly significant difference (p