Variability in somatic embryo-forming capacity of spinach

High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80–100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20–60% of regenerating explants. The explants from the majority of seedlings (52–100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10–20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1–10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.

Application of Artificial Neural Network for Modeling and Studying In Vitro Genotype-Independent Shoot Regeneration in Wheat

Optimizing in vitro shoot regeneration conditions in wheat is one of the important steps in successful micropropagation and gene transformation. Various factors such as genotypes, explants, and phytohormones affect in vitro regeneration of wheat, hindering the ability to tailor genotype-independent protocols. Novel computational approaches such as artificial neural networks (ANNs) can facilitate modeling and predicting outcomes of tissue culture experiments and thereby reduce large experimental treatments and combinations. In this study, generalized regression neural network (GRNN) were used to model and forecast in vitro shoot regeneration outcomes of wheat on the basis of 10 factors including genotypes, explants, and different concentrations of 6-benzylaminopurine (BAP), kinetin (Kin), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), zeatin, and CuSO4. In addition, GRNN was linked to a genetic algorithm (GA) to identify an optimized solution for maximum shoot regeneration. Results indicated that GRNN could accurately predict the shoot regeneration frequency in the validation set with a coefficient determination of 0.78. Sensitivity analysis demonstrated that shoot regeneration frequency was more sensitive to variables in the order of 2,4-D > explant > genotype < zeatin < NAA. Results of this study suggest that GRNN-GA can be used as a tool, besides experimental approaches, to develop and optimize in vitro genotype-independent regeneration protocols.

An efficient protocol for regenerating shoots from paper mulberry (Broussonetia papyrifera) leaf explants

Paper mulberry (Broussonetia papyrifera) is a tree species that has many economic, ecological, and social uses. This study developed an efficient protocol for regenerating shoots from leaf explants using Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs), which play vital roles in shoot regeneration. The best result, 86.67% induction frequency and 4.35 shoots per explant, was obtained in the MS medium containing 2.0 mg/L N6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid. The effects of explant age, orientation, and genotype were also investigated. Explants from young leaves had a greater regeneration frequency than those from old leaves, and the results were better when the distal end of the leaf explant contacted the medium versus the proximal end. Approximately 70.96% of the shoots rooted well in the MS medium containing 0.4 mg/L α-naphthalene acetic acid (NAA). Although some genotypes achieved poorer results, the regeneration protocol is still applicable for mass multiplication and genetic transformation.

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.

Effect of the plant growth stimulant zeatin on regeneration capacity of some Physalis species in vitro culture

The aim of the study was to find an efficient culture medium for regeneration of Physalis species in vitro to provide their further propagation ex vitro and obtain fructiferous plants from the regenerants. Physalis peruviana L., P. ixocarpa Broth. (cv. Likhtaryk), and P. pubescens L. (cv. Zarynka) were taken as plant material for the research. Plant introduction into culture and regenerant production were carried out in vitro; the rooting of mature plants and obtaining plants with ripe fruits took place in a greenhouse and in open ground (ex vitro). To obtain regenerants, we used Murashige and Skoog (MC30) medium supplemented with the growth stimulant zeatin (Zea) at a concentration of 0.5–3 mg/l. The growth stimulant 6-benzylaminopurine (BAP) was used to elongate the regenerant stalks, and the growth stimulator α-naphthylacetic acid (NAA) was used to initiate root formation. Plant regeneration frequency and the number of regenerants per explant served as indicators of the efficiency of various zeatin concentrations on the physalis regenerative capacity. The most effective media for the shoot regeneration from cotyledonous leaf explants were MC30 + 1 mg/l Zea and MC30 + 2 mg/l Zea. Regeneration frequency on these media was 46.15 % and 53.84 % for P. ixocarpa (cv. Likhtaryk), 38.46 % and 45 % for P. peruviana, and 27 % and 34 % for P. pubescens (cv. Zarynka) respectively. The emerged regenerants were separated from explants and transferred to MC30 medium supplemented with 1 mg/l of BAP + 0.1 mg/l of NAA for stalk growth and rooting. After a month of cultivation, juvenile plants were obtained. They were transferred to a greenhouse for adaptation, and later to open ground at the experimental plot. Three months after the regenerant emergence, we obtained fertile plants, which bloomed and bore fruit. The regenerants for domestic varieties of P. ixocarpa (cv. Likhtaryk) and P. pubescens (cv. Zarynka) were obtained for the first time. We established a direct relationship between the concentration of zeatin and both the frequency of plant regeneration and the number of regenerants per explant.

Efficient Shoot Regeneration via Indirect Organaogenesis of a Highly Recalcitrant Species of Cissampelos pareira (L.)

An efficient protocol was standardized for successful regeneration of Cissampelos pareira (L.) through indirect organogenesis. Nodal explants were cultured on MS fortified with 0.5 ± 1.0 mg/l BAP, Kn either single or in combination with NAA 0.5 mg/l. The combinations induced profuse, compact, light green to greenish coloured calli. Some differences in the morphology of callus such as change in the colour and texture was also observed with increasing the concentration of BAP 0.5 ± 2.0 mg/l + NAA 0.5 mg/l. Maximum callus induction was observed on 1.0 mg/l BAP and 0.5 mg/l NAA showed greenish, friable and granular lush colour. The calli were subcultured on fresh MS that contained BAP and Kn single or in combination with NAA (BAP 0.5 ± 2.0 mg/l, Kn 0.5 ± 2.0 mg/l, NAA 0.5 mg/l). The maximum regeneration frequency of shoot organogenesis was recorded on BAP (2.0 mg/l) + NAA (0.5 mg/l). Healthy microshoots were separated and transferred to the rooting medium. Here, MS augmented with IBA 1.0 mg/l showed maximum rooting. Well rooted plantlets were transferred to the field and maximum survival frequency was recorded when BAP (1.0 mg/l) + NAA (0.5 mg/l) for callus induction, for shooting BAP (2.0 mg/l) + NAA (0.5 mg/l) and for rooting IBA (1.0 mg/l) was used. The regenerated whole plants were subjected for hardening where the maximum survival frequency was found to be 80%. This reproducible protocol can be used for regeneration and genetic transformation studies.

Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica

In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in transformation experiments. In this study, we evaluated the morphogenesis potential of four commercial cultivars (Faisal canola, Punjab canola, Aari canola, Nifa Gold) and one model, Westar, from four different explants namely cotyledons, hypocotyls, petioles and roots on three different Brassica regeneration protocols, BRP-I, -II and -III. The regeneration efficiency was observed in the range of 6–73%, 4–79.3%, 0–50.6%, and 0–42.6% from cotyledons, petioles, hypocotyls and roots, respectively, whereas, the regeneration response in terms of average shoots per explant was found to be 0.76–10.9, 0.2–3.2, 0–3.4 and 0–2.7 from these explants. Of the commercial varieties tested, almost all varieties showed poorer regeneration than Westar except Aari canola. In comparison to Westar, its regeneration frequency from cotyledons was up to 7.5-fold higher on BRP-I, while it produced up to 21.9-fold more shoots per explant. Our data show that the explant has strong influence on the regeneration response, ranging from 24% to 92%. While the growth of commercial cultivars was least affected by the regeneration conditions provided, the effect on Westar was twice that of the commercial cultivars. After determining the optimal explant type and regeneration conditions, we also determined the minimum kanamycin concentration levels required to selectively inhibit the growth of untransformed cells for these cultivars. Regenerated shoots of Aari canola could be successfully grown to maturity within 16–18 weeks, with no altered phenotype noted and normal seed yields obtained. Therefore, the commercial variety, Aari canola, could be a good candidate for future genetic transformation studies.

Development of In vitro Tetraploid Plants of Hevea brasiliensis

Increase in global consumption of natural rubber necessitates crop improvement of Hevea aimed at increased productivity. As conventional breeding of Hevea is very elaborate and time consuming. Hence in the present study development of tetraploids through chromosome doubling of diploid callus obtained from cultured immature inflorescence of Hevea using colchicines were attempted. Chromosome doubling of the diploid callus occurred when treated with 1.25 µM colchicine for 3 days. In higher concentrations as well as at longer exposure periods, the callus texture and viability were affected. 48 % embryo induction and a maturation frequency of 45 % were obtained. Embryo germination and plant regeneration with a germination frequency (30 %) and a regeneration frequency (20 %) were obtained. Cytological and flow cytometric analyses confirmed the tetraploid nature of the colchicines treated callus. In vitro tetraploid plant developed through these in vitro techniques can be further used in Hevea brasiliensis breeding.

Experimental Study on the Influence of an Extension Tube on the Evolution Process and Characteristic Parameters of a Gliding Arc

The influence of the discharge cone size on the characteristic parameters of a rotating gliding arc, e.g., arc length, arc rotation frequency, and regeneration frequency was investigated when the grounding electrode was equipped with an extension tube. In addition, the effect of air flow rate (100~400 standard liter per minute, SLPM) on the characteristic parameters was also studied (with an extension tube, using a No. 5 discharge cone). The results showed that the disturbance of the outlet flow was intense without an extension tube, which led to the occurrence of the short-circuit regeneration of the gliding arc when the discharge gap was small, and the short-circuit regeneration of the gliding arc could be effectively avoided by adding an extension tube at the grounding end. The existence of the extension tube could significantly increase the arc length by up to 140 mm (250 SLPM, No. 3 discharge cone). The arc lengthening was mainly caused by the unilateral extension of the high voltage side, and the arc shape changed from a “C” to an “L” shape. This also reduced the regeneration frequency to one-quarter of the arc regeneration frequency without extending the tube. Without lengthening the tube, the arc could not achieve complete circumferential rotation. The arc distribution in the annular area between the anode and the cathode was discrete, and the arc distribution was extremely uneven. The existence of the extension tube made the arc rotate uniformly, and the maximum rotation frequency was 228 Hz. The influence of the air flow rate on arc length was mainly reflected in the small flow rate range. When the air flow rate was less than 200 SLPM, the arc length increased first, and then decreased with the increase of the air flow rate, reaching the maximum arc length of 143.6 mm at 150 SLPM. The arc frequency was positively correlated with the air flow rate, but the rotation frequency increased faster than the regeneration frequency: when the flow rate was 150 SLPM, the gliding arc rotated one circle in a tensile fracture period; when the flow rate reached 400 SLPM, the gliding arc rotated two circles in a tensile fracture period.