LaCl3 Treatment Improves Agrobacterium-mediated Immature Embryo Genetic Transformation Frequency of Maize

Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive calluses, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40% to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Ploidy Mosaics - Does Endopolyploidy in Explants Affects the Cytogenetic Stability of Orchids Regenerated From PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry (FC) was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, protocorms bases and protocorms apexes, which were subsequently used as explants for PLB induction and plant regeneration. The ploidy screening showed contrasting ploidy patterns in the samples. Endopolyploidy was detected in all tissues, with C-values ranging from 1C to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apexes showed the lowest proportion. Flower parts presented high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate was observed at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants.

LaCl3 Treatment Improves the Agrobacterium-mediated Immature Embryo Genetic Transformation Efficiency in Maize

Agrobacterium mediated genetic transformation of immature embryo plays an important auxiliary role in the study of gene function and molecular breeding in maize. However, the relatively low genetic transformation efficiency is still the bottleneck of the application of this method, especially in commercial scale production application. In this study, we found that pretreatment of immature embryos with LaCl 3 , a Ca 2+ channel blocker, could improve the infection efficiency of Agrobacterium tumefaciens , increase the proportion of resistant calluses, obtain more positive regenerated plantlets, and finally improve the transformation efficiency in maize. This optimization provides a new direction for improving the efficiency of plant genetic transformation mediated by Agrobacterium tumefaciens .

Cytokinin Mediated Increased In Vitro Production of Secondary Metabolites with Special Reference to Solasodine in Solanum erianthum

Steroid alkaloid solasodine is a nitrogen analogue of diosgenin and has great importance in the production of steroidal medicines. Solanum erianthum D. Don (Solanaceae) is a good source of solasodine. The aim of this study was to evaluate the effect of different cytokinins on the production of secondary metabolites, especially solasodine in the in vitro culture of S. erianthum. For solasodine estimation, field-grown plant parts and in vitro tissues were extracted thrice and subjected to high-performance liquid Chromatography. Quantitative analysis of different secondary metabolites showed that the amount was higher in the in vitro regenerated plantlets compared to callus and field-grown plants. The present study critically evaluates the effect of the type of cytokinin used in the culture medium on solasodine accumulation in regenerated plants. The highest solasodine content (46.78±3.23 mg g-1) was recorded in leaf extracts of the in vitro grown plantlets in the presence of 6-γ,γ-dimethylallylamino purine in the culture medium and the content was 3.8-fold higher compared to the mother plant.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The cultivated diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function compared to the allotetraploid cotton species through genetic transformation, the reliable and efficient method for high frequency somatic embryogenesis and plant regeneration in G. arboreum is urgent need to be established. Callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB (MS salts and B5 vitamins) medium with 0.09 µM 2,4-D and 2.32 µM KT. The embryogenic callus was induced on MS5 medium from the suspended cultures of several cycles of alternate liquid-solid culture, which was critical step for somatic embryogenesis. The liquid medium of MS4 was supplemented with 0.1g/L NaCl, 0.1g/L KCl and 0.1g/L CuSO4. The solid medium of MS5 for embryogenic callus effective induction was supplemented with 37.59 mM KNO3 + 62.47 µM NH4NO3 and 2.46 µM IBA + 0.93 µM KT or 0.045 µM 2,4-D + 2.46 µM IBA + 0.465 µM KT. During callus growing on different media, callus was effectively selected for subculture or treatment according to cell morphology to induce embryogenic callus and somatic embryos. Somatic embryo maturation and germination were better on MS5 medium with maltose or glucose + maltose than the single glucose. The regenerated plantlets with well-developed roots were directly transferred to soil or grafted onto the germinated cotton plantlets. The feasible process of plant regeneration via somatic embryogenesis in diploid cultivated species was established and needed to be improved and optimized for the gene functional analysis and gene editing in the diploid cotton species.

Effect of water stress induced by polyethylene glycol 6000 on the somatic embryos conversion into whole plantlets in cocoa (Theobroma cacao L.)

Rainfall scarcity due to climate change is a major constraint that limits cocoa productivity in Côte d'Ivoire. This work aims to regenerate cocoa plants tolerant to water stress using in vitro methods. Staminode and petal explants of the genotypes C1, C9, C14, C15, C16, C18 and C20 were used to produce somatic embryos through two methods. Firstly, somatic embryos were induced under stressfull conditions on media containing different concentrations of polyethylene glycol (PEG) 6000 (0; 25; 50; 75; 100 and 125 g/l) and secondly; under non-stressed conditions. Somatic embryos were placed on a conversion medium in the same stress condition. The number of regenerants decreased with the increase in the concentration of PEG with all genotypes. Only genotypes C1 and C15 regenerated plantlets under water stress conditions. The sensitive genotypes C9, C14, C16, C18 and C20 have not developed plantlets on media containing PEG. The plantlets produced under water deficit conditions exhibited a reduction in stem length and leaves number and an increase in length or offset of the high number of roots. The survival rate of regenerants during acclimatization was higher on the sandsubstrate. The selected genotypes could be used in an improvement program of cocoa production.Keywords: Climate change; plant regeneration; genotype; tolerance; drought; in vitro

Genotype-dependent mass somatic embryogenesis: a chance to recover extinct populations of Pulsatilla vulgaris Mill.

The paper presents a technique for micropropagation of endangered in Europe and extinct in Poland Pulsatilla vulgaris for ex situ conservation of the genetic resources. Genotype-dependent induction of somatic embryogenesis and rooting was revealed in series of two experiments (I and II) performed under the same experimental conditions. Shoot tips of seedlings were the best explants in both experiments and Murashige and Skoog (MS) medium supplemented with 0.25 or 0.5 mg L−1 BAP was suitable for induction of somatic embryos (SE) and adventitious shoots. Mass SE was obtained in experiment I after explants transfer on ½ MS (2% sucrose) + 0.45 mg L−1 B1 and extending culture to 2–3 months without passages. Rooting of adventitious shoots was a critical point. Out of seven rooting media used in experiment I, only two, ½ MS hormone free (2% sucrose) + 0.45 mg L−1 B1 or MS + 5 mg L−1 NAA + 3.76 mg L−1 B2 resulted in altogether 36.4% rooted shoots. In experiment II, somatic embryogenesis, rooting and acclimatization of adventitious shoots failed. Regenerated plantlets and seedlings converted from SE from experiment I were acclimatized to ex vitro conditions. Both genome size, determined by flow cytometry, and genetic diversity analyzed by ISSR markers, confirmed the compatibility of regenerants from experiment I with P. vulgaris initial seedlings and commercial cultivar. Regenerants obtained in experiment II differed genetically from the regenerants of experiment I and cultivar. Propagated in vitro tissues/organs (SE, adventitious shoots) of P. vulgaris could be a source of material for cryopreservation, artificial seed production and/or for acclimatization of regenerated plantlets and could be used for restoration of the extinct populations. Key Message The micropropagation technique via organogenesis and somatic embryogenesis of endangered in Europe pasqueflower was developed as a tool for species recovery. The critical point is that somatic embryogenesis is genotype-dependent, which affects the repeatability of the experiments and also imposes applying molecular techniques to confirm the genetic fidelity of the regenerants with the initial material and other genotypes.

Original Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana spp.)

The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians ( Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5%-79.2% frequency of responding flower buds and 0.01-1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata . Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata . In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were haploid (32.5%) and diploid (46.2%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding.