Effect of activated charcoal and ascorbic acid on in vitro morphogenesis and o-dihydroxyphenols content in Paphiopedilum insigne

Phenolic compounds limit micropropagation of many orchids in vitro. The aim of the study was to estimate the effect of activated charcoal (AC);1, 2 or 4 g/L) or ascorbic acid (AA; 10, 20 or 30 mg/L) added to the half strength MS medium on the growth and o-dihydroxyphenols content in Paphiopedilum insigne in vitro. A positive effect of AC on the shoot and root formation has been found. The highest multiplication rate (5.6 shoots/explant) and rooting frequency were obtained on medium containing 2 g/L of AC. However, AC reduced the leaf number as compared to the control. The lowest content of o-dihydroxyphenols was marked in Paphiopedilum insigne leaves when the shoots were grown on medium with 10 mg/L AA, followed by AC at 1 or 2 g/L.

Self-pollination of the orchid Cycnoches haagii from Brazilian Cerrado results in albino phenotype seedlings

Cycnoches haagii Barb. Rodr. is an epiphytic orchid very targeted by collectors, but no reference was found in the literature about its reproductive biology. Thus, the purpose of this study was to obtain initial information regarding pollination types and its influence on seed viability of this native orchid of the Brazilian Cerrado, in order to enable future propagation and preservation programs. Pollination among flowers of the same plant (geitonogamy) or different plants (xenogamy) were carried out. Seeds extracted from the capsules were sown in B&G medium, with full and half strength. Seeds from geitonogamic resulted in 25% of albino protocorms and consequently in albino seedlings. This phenomenon did not occur in seedlings derived from xenogamic pollination. Pigment analysis showed that even the albino seedlings presented chlorophylls and carotenoids, however, in significantly minor concentrations, 16% and 37% respectively, in relation to green seedlings. Geitonogamic and xenogamic pollinations resulted in C. haagii viable seeds with high germination percentage (90%) under in vitro conditions. The germination of seeds from xenogamic pollination resulted in chlorophyll or normal seedlings only, and can be recommended at conservation programs. On the other hand, although geitonogamic pollination should be avoided at conservation programs of this orchid species as it leads to albino seedlings, it showed a very interesting system to obtain seedlings with this phenotype, an interesting plant material to future investigation.

In vitro Propagation of Banana (Musa paradisiaca L.) Plant Using Shoot Tip Explant

Banana is a fruit crop which has high demand in Ethiopia, but its production is constrained by lack of disease free planting material with conventional propagation methods. For shoot initiation, shoot tip explants were cultured on MS medium supplemented with 0.5, 1.0, 1.5 and 2.0 mg/L BAP. Similarly, MS medium supplemented with BAP at 1.0, 1.5, 2.0 mg/L in combination with IBA at 0.25 and 0.50 mg/L were used for shoot multiplication. Half- strength MS medium augmented with IBA at 1.0, 2.0, 3.0 and 4.0 mg/l were used for root induction. MS medium without PGRs were used as controls. Finally, hardening of the in vitro derived plantlets was carried out in green house both in the primary and secondary acclimatization stages. Results showed that the highest shoot initiation percent (93.40%), highest mean number of shoots per explant (4.67) and lesser day for shoot induction (11.00) were observed in explant cultured on MS + 1.0 mg/L BAP. With shoot multiplication, highest shooting percent (92.60%), maximum number of shoots (7.67) and highest shoot length (5.27 cm) were recorded on MS + 1.5 mg/L BAP + 0.5 mg/L IBA. The highest rooting percent (93.40%), maximum root number per shoot (7.67) and highest root length (11.00 cm) were found on a half strength MS medium + 2.0 mg/L IBA. The survival rate of plantlets were 96.00% in coco peat substrate in primary acclimatization and 97.92% in forest soil, sand and manure substrates mixed at 3:2:1 ratio in secondary acclimatization. Overall, the result showed that the PGRs type, concentrations and combinations used are effective for mass propagation of banana variety studied in this experiment.

Combination of yeast antagonists and Acibenzolar-S-Methyl reduced the severity of Fusarium head blight of wheat incited by Fusarium graminearum sensu stricto

The combination of yeast antagonists and Acibenzolar-S-Methyl (ASM) was tested against Fusarium graminearum on a spring wheat cultivar PAN3471. Two strains of Papiliotrema flavescens (Strains WL3 and WL6) and a strain of Pseudozyma sp. (MGO1) were combined with full strength ASM at anthesis, half strength ASM at anthesis and quarter strength ASM at late boot stages. The yeast and ASM treatments were applied prior to F. graminearum inoculation and disease progress was assessed over time. The combination of yeast and ASM treatments effectively reduced Fusarium Head Blight (FHB) severity and deoxynivalenol (DON) concentration compared to when the treatments were used alone. A positive correlation was observed between the Area Under Disease Progress Curve (AUDPC) and Percentage Seed Infection (PSI) (r = 0.44) whereas a negative correlation was observed between AUDPC and Hundred Seed Weight (HSW) (r = -0.77) and PSI and HSW (r = -0.44). The best combination treatment providing the highest reduction in final disease severity (41.83%), high HSW and moderate PSI was 0.075 g/l ASM at anthesis plus P. flavescens strain WL3. The highest DON reduction (19.35%) was by the treatment 0.075 g/l ASM at anthesis plus P. flavescens strain WL6. The best treatment was P. flavescens combined with 0.075 g/l ASM at anthesis. Although Pseudozyma sp. strain MGO1 did not provide the best FHB and DON reduction, its combination with ASM application improved disease control efficacy. To the best of our knowledge, this study presents the first report of the combination of P. flavescens and ASM in the management of FHB caused by F. graminearum in wheat plants.

Regeneration potential of different explants during micropropagation of neem tree (Azadirachta indica A. Juss.)

Azadirachta indica A. Juss., (Neem), a prodigious multipurpose tree, has immense potential to benefit mankind and to protect the environment. In order to investigate the effects of three different explants for its regeneration potential, de embryonated cotyledon, immature zygotic embryo and nodal segments from a 30 year old neem plus tree were used. Half strength MS medium with benzyl amino purine (3 mg/L) and naphthalene acetic acid (0.5 mg/L) and casein hydrolysate (1 g/L) was effective in shoot bud sprouting from both nodes and cotyledons. Half strength MS medium fortified with TDZ (0.2 mg/L) was effective for induction of somatic embryogenesis from zygotic embryos. Shoot buds initiated from the cotyledons produced a maximum number of shoots per explants (4.33) which on further sub culturing induced maximum multiple shoots (15) on half strength MS medium fortified with BAP (1.5 mg/L), NAA (0.5 mg/L) and CH (400 g /L) and the nodal explants induced only 4-5 axillary shoots on further sub culturing. Even though immature zygotic embryos produced more number of somatic embryos per explant (24.97) within a short time (30-45 days), the plantlet conversion was poor (25.52 %). In vitro rooting was observed in half strength MS medium supplemented with IBA (2 mg/L). The regeneration potential of de embryonated cotyledons through a simple regeneration system may be beneficial for efficient mass propagation of selected plus trees of neem.

In Vitro Propagation And Rejuvenation of Senescent Maternal Plant of Ardisia Crenata Var. Bicolor (Primulaceae)

Ardisia crenata var. bicolor is an ornamental shrub, owing to its declined wild population, recalcitrant seeds and few high-quality cuttings, the main objective of this study was to optimize an in vitro propagation protocol by using tip shoot and nodal segment as explants from senescent plant. Explants were sterilized and cultured on Muraghige and Skoog medium contained 1.0 mg·L-1 benzylaminopurine and 0.05 mg·L-1 1-naphthaleneacetic acid for shoot initiation. For shoot proliferation, explants were cultured on MS medium with 1.0 mg·L-1 BAP, 0.1 mg·L-1 NAA, and 0.5 mg·L-1 kinetin, and the proliferation coefficient were 3.1 and 2.5. Rooting was achieved by two explants in half-strength MS medium containing 0.5 mg·L-1 indole-3-butyric acid + 0.1 mg·L-1 or 0.2 mg·L-1 NAA, and 0.5 g·L-1 activated charcoal. The highest rooting rate were 72.7% and 65.1% with the highest mean number of roots (4.2 and 2.8, respectively). After acclimatization, 83.3% and 81.2% of plants were survived in the greenhouse. The plant can be rejuvenated via in vitro propagation and provide a reference for supplying the planting materials quickly with an uniform genotype.

Sand Particle Size and Phosphorus Amount Affect Rhizophagus irregularis Spore Production Using In Vitro Propagated Spore as a Starter Inoculum in Rhizosphere of Maize (Zea mays) Plantlets

Microbial inoculants, particularly arbuscular mycorrhizal (AM) fungi, have great potential for sustainable crop management. In this study, monoxenic culture of indigenous R. irregularis was developed and used as a tool to determine the minimum phosphorus (P) level for maximum spore production under the in vitro conditions. This type of starter AM fungal inoculum was then applied to an in vivo substrate-based mass-cultivation system. Spore production, colonization rate, and plant growth were examined in maize (Zea mays L.) plant inoculated with the monoxenic culture of R. irregularis in sand graded by particle size with varying P levels in nutrient treatments. In the in vitro culture, the growth medium supplemented with 20 µM P generated the maximum number of spores (400 spores/mL media) of R. irregularis. In the in vivo system, the highest sporulation (≈500 spores g−1 sand) occurred when we added a half-strength Hoagland solution (20 µM P) in the sand with particle size between 500 µm and 710 µm and omitted P after seven weeks. However, the highest colonization occurred when we added a half-strength Hoagland solution in the sand with particle sizes between 710 µm and 1000 µm and omitted P after seven weeks. This study suggests that substrate particle size and P reduction and regulation might have a strong influence on the maximization of sporulation and colonization of R. irregularis in sand substrate-based culture.

Continuous gastric saline perfusion elicits cardiovascular responses in freshwater rainbow trout (Oncorhynchus mykiss)

When in seawater, rainbow trout (Oncorhynchus mykiss) drink to avoid dehydration and display stroke volume (SV) mediated elevations in cardiac output (CO) and an increased proportion of CO is diverted to the gastrointestinal tract as compared to when in freshwater. These cardiovascular alterations are associated with distinct reductions in systemic and gastrointestinal vascular resistance (RSys and RGI, respectively). Although increased gastrointestinal blood flow (GBF) is likely essential for osmoregulation in seawater, the sensory functions and mechanisms driving the vascular resistance changes and other associated cardiovascular changes in euryhaline fishes remain poorly understood. Here, we examined whether internal gastrointestinal mechanisms responsive to osmotic changes mediate the cardiovascular changes typically observed in seawater, by comparing the cardiovascular responses of freshwater-acclimated rainbow trout receiving continuous (for 4 days) gastric perfusion with half-strength seawater (½ SW, ~ 17 ppt) to control fish (i.e., no perfusion). We show that perfusion with ½ SW causes significantly larger increases in CO, SV and GBF, as well as reductions in RSys and RGI, compared with the control, whilst there were no significant differences in blood composition between treatments. Taken together, our data suggest that increased gastrointestinal luminal osmolality is sensed directly in the gut, and at least partly, mediates cardiovascular responses previously observed in SW acclimated rainbow trout. Even though a potential role of mechano-receptor stimulation from gastrointestinal volume loading in eliciting these cardiovascular responses cannot be excluded, our study indicates the presence of internal gastrointestinal milieu-sensing mechanisms that affect cardiovascular responses when environmental salinity changes.

Non-symbiotic Seed Germination and In vitro Plant Development of Pholidota articulata

Pholidota articulata is an epiphytic orchid mostly used in ornamental cut/pot flower and in traditional medicine. As it has high ornamental and medicinal values, its population from natural habitats is decreasing, therefore, it is listed in the Appendix-II of Convention on International Trade in Endangered Species (CITES). The objective of the present study is to obtain the in vitro plants of P. articulata from seed culture to conserve its germplasm. The in vitro seed germination was carried out in different strengths of Murashige and Skoog (MS) and Knudson C (KnC) medium supplemented with various plant hormones. On the half-strength of MS medium, seeds were started to germinate after 4 weeks of primary culture and they were developed into protocorms with first leaf primordium earlier than on the other medium. Therefore, in vitro developed protocorms were sub-cultured on the half-strength of MS medium supplemented with different concentrations of 6-benzylaminopurine (BAP), gibberellic acid (GA3) and α-naphtalene acetic acid (NAA). They were successfully developed into shoots on the 1.5 mg/l BAP supplemented half-strength of MS medium. Later, they were inoculated on the half-strength of MS medium supplemented with different concentration of α-napthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) for the root formation, where IBA supplemented medium was found effective for the development of roots. Thus, this study provides a reliable protocol for non-symbiotic seed germination and plant production, and reveals that seed-derived protocorms are good explants for the in vitro mass propagation for conservation and sustainable utilization in horticulture.

In vitro propagation of an economically important medicinal plant Lawsonia inermis L. through nodal segments

The present investigation aimed to standardize efficient plant regeneration protocol through in vitro culture by using nodal segment for mass multiplication of Lawsonia inermis an economically important medicinal plant species. Mass multiplication of shoots induced on Murashige and Skoog (MS) medium supplemented with different growth regulators like auxins and cytokinins separately and in different combinations. The medium fortified with 6-Benzylaminopurine ( BAP) 1.0 mg/l + kinetin (KN) 1.5mg/l explained best compared to all other combinations. In vitro raised plantlets were excised and transferred in half strength MS medium supplemented with different growth regulators like Indole Butyric acid ( IBA) and naphthalene acetic acid (NAA ) (0.5-3.0 mg/l) in an experiment that gave rise to rooting. The half strength of MS medium additive with IBA in separate and in different combinations with NAA concentrations (0.5-3.0 mg/l) supported root development. The best response of rooting was obtained on half MS medium fortified with 1.0 mg/l IBA. The regenerated plantlets were successfully transplanted to pots. Regenerants were transferred to the field conditions and recorded the survival rate.. Among all the carbon sources and gelling agents used, sucrose (3%) in combination with 0.8 per cent agar-agar has proved significantly better. Multiple shoots formation with longer shoots were achieved on medium with 1.0mg/l BAP and 1.5mg/l Kn. Thus, it is possible to develop a large number of plants of L. inermis through shoot bud regeneration which can cater for the need of pharmaceutical as well as other industries.