Assessment Various Concentrations of ZnO-Nanoparticles on Micropropagation for Chenopodium quinoa Willd. Plant

Assessment influences various concentrations of ZnO-Nanoparticles (ZnO-NPs) on Chenopodium quinoa Willd. Micro-propagation by cotyledonary nodes explants was achieved. We used different concentrations of ZnO-NPs (0.2, 2, 10, 20 mg/l) and used the medium free from ZnO-NPs as control. The results indicated that the presence ZnO-NPs in medium was good effect on germination rate of quinoa seeds at 2 mg/l concentration, and we noticed a density in roots hair and number or length roots of seedling. In addition, the highest responding of explant to ZnO-NPs 93.33, 80.8% were in MS that supplemented 0.0, 2.0 mg/l respectively. Maximum numbers of roots 4.0 were also observed in MS containing 0.0, 2.0 mg/l. Although, there was positive clearly effect on number leaves of shoots, but there was a sudden decline (from 8.43 to 1.0) occurred by increasing ZnO-NPs concentrations from 2.0 to 20.0 mg/L. However, the ZnO-NPs do not effect on length of shoots, where the lengthiest shoot occurred in MS without ZnO-NPs. Regarding rooting shoots, there was significant effect of ZnO-NPs on both percentage of root and root number, where percentage of root reached to 100% in 10.0 mg/l concentration and roots number was 4.80 roots in the same concentration.

In vitro: Influence of Various Concentrations of Plant Growth Regulators (BAP & NAA) and Sucrose on Regeneration of Chenopodium quinoa Willd. Plant

In vitro: regeneration of Chenopodium quinoa Willd. was achieved from cotyledonary nodes explants. In this study, used 6-Benzylaminopurine (BAP) and α-Naphthalene Acetic Acid (NAA) of plant growth regulators with different concentrations individually as well as in combination and used different concentrations of sugar (sucrose) with different concentrations. For was rooting, used half strength (½MS), full-strength MS and ½ MS supplemented with 0.2 mg/l of NAA. The results mentioned, explant responding (%) to multiplication was about 73% for all BAP treatments compared with control and average numbers of shoot increased with increased BAP concentration except 5 mg/l of BAP. The highest explant responding (%) was in media supplemented BAP without NAA compared other treatments noted that the media with combination of BAP and NAA gives formation of callus in bases of the plantlets. Also, the result inducted the combinations between (BAP–NAA) was highly significantly (P≤ 0.001) and less effective on number of shoots where the highest number of shoot was 3.40 in media with 3 mg/l BAP compared other treatments. The highest of explant responding 93.33% was in media supplement with 10 g/l sucrose and (10 g/l sucrose + 3 mg/l BAP), but sucrose level for good greening and developed shoots (4 shoots) was in medium supplement with 10 g/l sucrose. The shoots rooted well on half-strength MS medium with 60% percentage of root. The rooted shoots were acclimatized and transferred to green house to follow their development.

In Vitro Propagation via Organogenesis and Formation of Globular Bodies of Salvia Plebeia: A Valuable Medicinal Plant

Background: As a highly valued medicinal plant, Salvia plebeia R. Brown belongs to the Lamiaceae family that has been subjected to over exploitation in its natural habitat for phytochemical and pharmacological studies. Alternative collection methods need to be developed for the large-scale propagation of Salvia plebeian. Results: Here, efficient and simple, direct organogenesis (from shoot tips and cotyledonary nodes explants) and Globular bodies (GBs) induction (from hypocotyl explants) systems were developed for the in vitro propagation of Salvia plebeia. The highest and number of regenerated shoots (7.0±0.82) per shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 0.1 mg L-1 indole-3-acetic acid (IAA) and 1.0 mg L-1 6-benzyladenine (6-BA), the proliferation of shoots and shoots rooted were carried out on the same medium treatments almost synchronously. Similarly, MS medium supplemented with 0.1 mg L-1 IAA and 1.0 mg L-1 thidiazuron (TDZ) yielded the maximum number of shoots (37.5±1.34) with 100% shoot sprouting frequency. Simultaneously, a protocol was developed for GBs induction from hypocotyl explants, and it produced 17.4 GBs per explant with 82.7% response on MS medium supplemented with TDZ (1.0 mg L-1) and IAA (0.1 mg L-1), and produced GBs that were morphologically similar to globular embryos and successfully germinated on hormone-free MS medium. The acclimatized plantlets with well-developed root systems were successfully shifted to the natural soils with a 100% survival rate. Conclusions: Taken together, this protocol can be efficiently used for mass propagation, germplasm preservation and likely also for gene transfer of Salvia plebeia.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.

In vitro culture as a source of biodiversity for soybean breeding

The paper shows the effectiveness of methods of somaclonal variability in combination with multiple individual selection for creating new soybean varieties characterized by early ripeness and increased seed productivity for sub-arid regions of Russia and Kazakhstan with cold continental climate. Soybean breeding material was created using biotechnology methods combined with multiple individual selection. Higher regeneration potential of soybean tissues of SibNIIK 315 variety compared to Omskaya-4 variety was revealed. The ability to regenerate is associated with the adaptability of SibNIIK 315 variety, which is confi rmed by the wider area of its cultivation in various ecological and geographical conditions in fi ve regions of Russia and Kazakhstan. Tissues of apexes and cotyledonary nodes of R21 somaclonal line differed from the original SibNIIK 315 variety by the increased activity of callus formation, regeneration and growth of shoots. Tissues of regenerated plants passed repeatedly through the regeneration cycle according to the protocol of recurrent regeneration. Due to this, high morphogenic activity of R21 tissues can be the result of autoselection in vitro. The analysis of phenotypic variations in early generations of soybean somaclones showed an asymmetric distribution of deviations by the main breeding and marker traits: duration of vegetation, plant height and seed productivity, as well as dependence of distribution on weather conditions. In a dry year most somaclonal lines had a higher seed productivity, whereas in favourable conditions of a humid summer only every third line was superior to the original variety. This indicates increased levels of ontogenetic adaptation of somaclones in extreme drought conditions.

Developing a rapid and highly efficient cowpea regeneration and transformation system using embryonic axis explants

SummaryCowpea is one of the most important legume crops planted worldwide, especially in Sub-Saharan Africa and Asia. Despite decades of effort, genetic engineering of cowpea is still challenging due to inefficient in vitro shoot regeneration, Agrobacterium-mediated T-DNA delivery and transgenic selection. Here, we report a rapid and highly efficient cowpea transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem by cutting through the middle of the epicotyl stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. Furthermore, the application of a ternary transformation vector system using an optimized pVIR accessory plasmid provided high levels of Agrobacterium-mediated gene delivery. The utilization of spectinomycin as the selection agent enabled more efficient transgenic selection and plant recovery. Transgenic cowpea shoots developed exclusively from the cotyledonary nodes at high frequencies of 4.5 to 37% across a wide range of cowpea genotypes. We believe that the transformation principles established in this study could also be applied to other legumes to increase transformation efficiencies.

Establishment of an efficient alfalfa regeneration system via organogenesis and the co-expression of Arabidopsis SOS genes improves salt tolerance in transgenic alfalfa (Medicago sativa L.)

The Salt Overly Sensitive (SOS) signal transduction pathway is pivotal in Na+ efflux and facilitates ion transport and homeostasis for improved salt tolerance in plants. Ten alfalfa varieties were used as experimental materials and two alfalfa regeneration systems were established and optimized. Cotyledons and hypocotyls were initially used as explants to induce embryogenic callus via the indirect production of somatic embryos to establish a callus acceptor system. Cotyledonary nodes were used as explants to induce adventitious bud formation via direct organogenesis, thereby establishing an in vitro regeneration system that could be used for the genetic transformation. Agrobacterium-mediated transformation of the cotyledonary nodes of the alfalfa ‘Golden Empress b’ was used to generate 25 independent sources of transformed plants exhibiting herbicide tolerance. Four of the positive transgenic plants were randomly selected for southern blot analysis, and three hybridization signals with one or two copies were detected. Reverse transcription polymerase chain reaction showed that the Bialaphos resistance (Bar) and SOS1 genes were expressed in transgenic plants and that multiple exogenous salt-tolerant genes were integrated into the transgenic plant genome and expressed at the transcriptional level. The overexpression of Arabidopsis SOS genes in alfalfa conferred a high degree of salinity tolerance, enhanced plant growth, lowered the accumulation of Na+, increased the accumulation of K+ in the leaves, and altered physiological and biochemical parameters in response to salt stress.

Micropropagation of Chinquapin (Castanea henryi) Using Axillary Shoots and Cotyledonary Nodes

Castanea henryi is an important woody grain tree species native to China. The objective of the current study was to find the suitable plant growth regulators (PGRs) and the optimal concentrations for direct organogenesis by using axillary shoots and cotyledonary nodes. Seeds were collected from the field, sterilized, and germinated in vitro. Axillary shoots and cotyledonary nodes of 3-week-old seedlings were used as explants. To find the suitable PGR for adventitious shoot induction, 0.5 mg·L–1 6-benzylaminopurine (6-BA), 0.1 mg·L–1 indole-3-acetic acid (IAA), 0.1 mg·L–1 2,4-dichlorophenoxyacetic acid (2,4-D), or 0.1 mg·L–1 1-naphthaleneacetic acid (NAA) was supplemented to Murashige and Skoog (MS) medium containing 0.65% agar and 3% sucrose. A high induction percentage of adventitious shoots (85.67%) was obtained from cotyledonary nodes supplemented with 0.1 mg·L–1 2,4-D. The type of explant influenced shoot proliferation rates and quality. Apical explants produced more and longer shoots than nodal segments. For shoot multiplication, 1 mg·L–1 6-BA + 0.05 mg·L–1 indole-3-butyric acid (IBA) supplemented with MS medium produced 12.33 and 6.25 shoots per explant, respectively, from apical and nodal explants. For shoot elongation and strengthening, 2 mg·L–1 6-BA + 0.05 mg·L–1 IBA supplemented with MS medium was the best combination, producing shoots with a mean length of 3.50 cm, a diameter of 0.46 cm, and about eight leaves per shoot. The greatest rooting of 76.70% and 11.33 roots per shoot was achieved when cultured in MS medium supplemented with 3.5% perlite + 1.5 mg·L–1 IBA. For acclimatization of the rooted plantlets in the greenhouse, a survival rate of 80% was achieved. This protocol—from multiplication to acclimation—is helpful to realize mass propagation of high-quality trees of chinquapin for increasing production and nut quality.

Micropropagation of White Palash tree (Butea monosperma (Lam.) Taub. Var. lutea (Witt.)).

<p class="Default"><span>An efficient and reproducible protocol is established for rapid <em>in vitro</em> multiplication of an endangered, valuable medicinal plant, <em>Butea monosperma </em>(Lam.) Taub. Var. <em>lutea</em>, through cotyledonary nodes of mature seeds. Among various cytokinins tested, high frequency of direct shoot regeneration was induced on Murashige and skoog (MS) medium supplemented with BAP, which found to be more effective and showed optimal response at 2 mg/L with a maximum number of </span><span>8.35±0.32 multiple shoots per explant. Proliferation of shoots was established by repeated subculturing on to same regeneration medium with 2-3 weeks of time interval. Rooting of regenerated shoots was achieved after 3 weeks of culture on MS medium containing 1 mg/L IBA. <em>In vitro</em> raised plantlets were transferred to pots containing sterilized soil and vermiculate mixture in 1:1 ratio and then shifted to greenhouse. Well established plantlets exhibited 75% survival rate.</span></p>