Direct Shoot Organogenesis Research Articles

Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

Allele-specific DNA methylation and gene expression during shoot organogenesis in tissue culture of hybrid poplar.

Plant tissue regeneration is critical for genetic transformation and genome editing techniques. During the regeneration process, changes in epigenetic modifications accompany the cell fate transition. However, how allele-specific DNA methylation in two haplotypes contributes to the transcriptional dynamics during regeneration remains elusive. Here we applied an inter-species hybrid poplar (Populus alba × P. glandulosa cv. 84K) as a system to characterize the DNA methylation landscape during de novo shoot organogenesis at allele level. Both direct and indirect shoot organogenesis showed a reduction in genome-wide DNA methylation. At gene level, non-expressed genes were hypermethylated in comparison with expressed genes. Among the genes exhibiting significant correlations between levels of DNA methylation and gene expression, the expression patterns of 75% of genes were negatively correlated with DNA methylation in the CG context, whereas the correlation patterns in the CHH context were the reverse. The allele-biased DNA methylation was consistent during shoot organogenesis, with fewer than one-thousandth of allele-specific methylation regions shifted. Analysis of allele-specific expression revealed that there were only 1909 genes showing phase-dependent allele-biased expression in the regeneration process, among which the allele pairs with greater differences in transcription factor binding sites at promoter regions exhibited greater differences in allele expression. Our results indicated a relatively independent transcriptional regulation in two subgenomes during shoot organogenesis, which was contributed by cis-acting genomic and epigenomic variations.

Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants

Key messageWe transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants.Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

Efficient plant regeneration through direct shoot organogenesis and two-step rooting in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver (E. ulmoides Oliver), a multipurpose woody plant, holds great economic significance due to its expansive medicinal, food and industrial applications. The rapid advancement of E. ulmoides in various fields has resulted in the inadequacy of existing breeding methods to meet its growth and annual production demands. Consequently, there is an urgent need for innovative propagation strategies. This study introduces an optimized micropropagation protocol for E. ulmoides, facilitating direct shoot organogenesis from nodal segments with axillary buds. We systematically examined the impact of basal medium composition, plant growth regulators, photosynthetic photon flux density, and sucrose concentration on bud sprouting. Employing cuttings with axillary buds as propagation material, we achieved a shortened cultivation period of merely 4 weeks for bud elongation and proliferation, marking a substantial enhancement in propagation efficiency. Notably, the Driver Kuniyuki Walnut medium, supplemented with 20.0 g L-1 sucrose and 2.0 mg L-1 trans-zeatin, induced shoots sprouting with a 100% success rate and an average length of 5.18 cm per nodal segment, equating to a great bud propagation rate of approximately 500%. Furthermore, a light source with an intensity of 80 μmol m-2 s-1 was shown the most economical choice. To address the primary challenge of inducing roots in regenerated plants, we employed a refined two-step rooting technique. This method yielded the optimal rooting frequency of 93.02%, producing an average of 5.90 adventitious roots per plantlet, each with an average length of 2.77 cm. The micropropagation program developed in this work will be the cornerstone for the preservation of the germplasm of E. ulmoides and its long-term use in medicinal and industrial applications.

Direct Shoot From Root and True-to-Type Micropropagation of Limonium "Misty Blue" in Partially Immersed Culture on an Aluminum Mesh Raft.

Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog's (1962) (MS) basal medium supplemented with 1.6μM NAA and 1.1μM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44μM BA and 1.07μM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4-5 shoots is formed from a single shoot explant within 4weeks and are rooted simultaneously within 6weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined.

Control of media browning during micropropagation and assessment of biochemical and clonal fidelity of in vitro-derived and mother plants in Thottea siliquosa (Lamk.) Ding Hou., an important ethnomedicinal shrub

BackgroundThottea siliquosa (Lamk.) Ding Hou., an important medicinal shrub, is widely used in both ayurvedic and indigenous systems of medicine. Root being the most useful part, the plant is constantly uprooted and thus puts pressure on the natural population. Until date, no micropropagation study is available in this plant. The objective of the study is to develop an efficient in vitro propagation protocol and assessment of clonal fidelity of T. siliquosa. ResultsMedia browning was a serious issue during micropropagation, and the addition of 40.0 mg/L ascorbic acid reduced the media browning. For direct shoot regeneration, the optimum response (92% frequency with 20.9 shoots per explant) was obtained when 7-day-old cotyledons were cultured on WPM supplemented with 1.0 mg/L thidiazuron and 0.25 mg/L α-naphthalene acetic acid. The cultures were transferred to WPM augmented with 0.4 mg/L thidiazuron for shoot elongation and growth. On this medium, 100% of cultures responded with a mean number of 27.6 shoots. For callus induction, MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L N6-benzylaminopurin was used. Shoot organogenesis was initiated on the same medium, and calli with minute shoots were transferred to MS medium fortified with 0.5 mg/L N6-benzylaminopurin and 0.25 mg/L α-naphthalene acetic acid for highest shoot regeneration (100% cultures responded with a mean number of 26.5 shoots per explant). Maximum rooting frequency (82%) and number (20.8) were obtained on half-strength MS medium with 1.0 mg/L indole-3-butyric acid. The rooted plants were acclimatized and transferred to the field. The HPTLC and SCoT analysis revealed the phytochemical and clonal similarity between the in vitro propagated plants and mother plant. ConclusionsIn this study, it is confirmed that cotyledon is an excellent explant for direct and indirect shoot organogenesis in T. siliquosa. For direct shoot induction WPM and indirect organogenesis, MS medium was found to give better response. The true-to-type nature of in vitro-derived plants were confirmed by phytochemical and SCoT analysis. The protocol described here could be used for the large-scale propagation of elite clones of T. siliquosa.

Assessment of the In-vitro Potential of Two Different Genotypes of Bacopa Monnieri L. Pennell

Jal Brahmi (Bacopa monnieri L.) is an important medicinal herb rendering mental health-promoting activities. In-vitro mass multiplication of elite genotypes of Jal Brahmi may bring off its high industrial demand. In the present study, the comparative in-vitro responses of two diverse genotypes (DBM-4 and DBM-12) were assessed. Direct shoot organogenesis was reported in both genotypes studied in basal MS medium media. However, both genotypes formed calli in the presence of 2, 4-D and IAA. Germplasm DBM-4 observed significantly higher antioxidant potential than that of DBM-12. Further, in both genotypes, methyl jasmonate elicitation significantly improved the antioxidant potential and total phenolic contents of the callus. Hence, DBM-4 has the potential for in-vitro mass multiplication and secondary metabolites production.

High Frequency Direct Organogenesis in Five Romanian Tomato (Lycopersicon esculentum Mill.) Cultivars

Tomato (Lycopersicon esculentum Mill.) as the most economically important vegetable crop worldwide has been investigated intensively for the development of new and improved varieties. Most of these technologies require efficient protocols for in vitro regeneration and propagation of plant material. In the present study, an efficient and reproducible in vitro regeneration system for five Romanian tomato genotypes (cvs. ‘Capriciu’, ‘Darsirius’, ‘Kristin’, ‘Pontica’ and ‘Siriana’) has been established. The tomato genotypes were selected based on their horticultural and economically valuable traits. To study the in vitro morphogenic response, various explants, such as cotyledons, cotyledonary nodes, hypocotyls, leaf explants, internodes, stem nodes and apical buds have been selected. The highest efficiency in terms of direct shoot organogenesis was obtained in cv. ‘Capriciu’ (98% for apical buds and 94% for stem nodes) on culture media with zeatin and indole-3-butyric acid. One advantage of this regeneration procedure is beside its feasibility in handling, the high percentage of regenerated shoots and their rooting. The present protocol contributes to the existing information regarding the response of tomato cultivars to in vitro culture conditions.

Meta-Topolin enhanced direct shoot organogenesis and regeneration from leaf explants of Coleus forskohlii (Willd.) Briq

A novel and effective direct shoot regeneration technique from stomatal complexes was developed for a highly sought after medicinal plant Coleus forskohlii. Meta-Topolin was more efficient than 6-benzylaminopurine (BAP) in promoting direct organogenesis from leaf surface. Highest direct organogenesis was achieved from in vitro leaf explants on Murashige and Skoog (MS) medium augmented with meta-Topolin at the concentration of 2.0 mg/L. The regenerated leaves were fixed at weekly intervals and sectioned longitudinally to evaluate the anatomical developments. The results of light microscopic analysis suggested that the in vitro developed stomatal complexes showed new altered cell divisions, after a week of incubation, and the rapid periclinal divisions resulted in the formation of a meristematic dome. Distinct regions of meristematic activity were visible in the second week characterized by promeristems with small and densely stained parenchymatous cells. Promeristems lead to the development of a distinct tunica layer, which grew into shoot-like protuberances converting into adventitious shoots after 4 weeks of incubation. A clear three phases of shoot organogenesis viz. induction (7 days), initiation and organization (14 days), and shoot formation (28 days onwards) were identified. The shoots were proliferated (21.8 shoots/ single leaf explant) and elongated (5.0 cm length) using half-MS medium fortified with 0.5 mg/L mT and indole-3 acetic acid (IAA) at 0.1 mg/L. Notable rhizogenesis of the elongated shoots was achieved when cultured on half-MS medium containing indole-3 butyric acid (IBA) at the concentration of 1.5 mg/L. About 92.0% of the plantlets survived upon field transfer. Start Codon Target (SCoT) markers were employed to examine the regenerated plants’ genetic uniformity with the mother plant, similar banding patterns confirming the genetic uniformity. Hence, the developed protocol of direct in vitro regeneration of C. forskohlii using mT can be used for the commercial production.

Potential of In Vitro Culture of Scutellaria baicalensis in the Formation of Genetic Variation Confirmed by ScoT Markers.

The in vitro culture technique can be used for micropropagation of medicinal plants as well as for creating genotypes with an improved profile of phytochemical compounds. For this purpose, somaclonal variability may be used for the induction of genetic diversity among regenerants. The paper presents a protocol for obtaining Scutellaria baicalensis regenerants by indirect organogenesis and the assessment of their genetic variability with the use of start codon-targeted markers. The most intense process of indirect shoot organogenesis was observed on Murashige and Skoog medium supplemented with kinetin and 6-Benzylaminopurine (0.5 mg × dm-3 each)-7.4 shoot per explant on average. The callogenesis process occurred on the medium supplemented with TDZ, while the medium supplemented with GA3 allowed for direct shoot organogenesis and was used for the micropropagation of regenerants. In the analysis of plantlets obtained by indirect organogenesis, 11 ScoT markers generated a total of 130 amplicons, 45 of which were polymorphic. This analysis showed genetic diversity of regenerants in relation to the donor plant as well as within them, with mean similarity among the analyzed genotypes at the level of 0.90. This study confirms that the use of in vitro cultures allows for the possibility to generate genetic variability in Scutellaria baicalensis, which can be effectively revealed with the use of the SCoT marker.

Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of Plectranthus amboinicus (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers

Plectranthus amboinicus (Lour.) Spreng is a medicinally important aromatic perennial herb used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache as well as a flavoring ingredient in traditional drinks, food, and meat stuffing. In this study, a high-performance in vitro propagation system of P. amboinicus through direct shoot organogenesis was developed using axillary node explants cultured on MS (Murashige and Skoog) medium augmented with 0.5, 2.5, 5.0, 7.5, and 10.0 µM of 6-benzyladenine (BA) or kinetin (Kin), alone or with 0.1, 0.5, 2.5, and 5.0 µM of indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). To optimize the regeneration potential of node explants, the effects of basal media strength and pH were also investigated. After 8 weeks of culture, explants cultured in full strength MS basal medium (pH 5.7) with 5.0 µM BA and 2.5 µM NAA exhibited the highest percentage (97.1%) of regeneration and the maximum number (19.3) of shoots per explant. Individual elongated shoots were rooted on half strength MS basal medium containing 0.25 µM indole 3-butyric acid (IBA) after 4 weeks of culture, producing 5.3 roots/shootlets with a root induction frequency of 93.7%. First time genetic stability of in vitro raised P. amboinicus plants was determined using SPAR markers, such as DAMD and ISSR, as well as flow cytometric tests, assuring the availability of authenticated raw materials for commercial production of the plant and its bioactive components.

In vitro Induction and Phenotypic Variations of Autotetraploid Garlic (Allium sativum L.) With Dwarfism.

Garlic (Allium sativum L.) is a compelling horticultural crop with high culinary and therapeutic values. Commercial garlic varieties are male-sterile and propagated asexually from individual cloves or bulbils. Consequently, its main breeding strategy has been confined to the time-consuming and inefficient selection approach from the existing germplasm. Polyploidy, meanwhile, plays a prominent role in conferring plants various changes in morphological, physiological, and ecological properties. Artificial polyploidy induction has gained pivotal attention to generate new genotype for further crop improvement as a mutational breeding method. In our study, efficient and reliable in vitro induction protocols of autotetraploid garlic were established by applying different antimitotic agents based on high-frequency direct shoot organogenesis initiated from inflorescence explant. The explants were cultured on solid medium containing various concentrations of colchicine or oryzalin for different duration days. Afterward, the ploidy levels of regenerated plantlets with stable and distinguished characters were confirmed by flow cytometry and chromosome counting. The colchicine concentration at 0.2% (w/v) combined with culture duration for 20 days was most efficient (the autotetraploid induction rate was 21.8%) compared to the induction rate of 4.3% using oryzalin at 60 μmol L–1 for 20 days. No polymorphic bands were detected by simple sequence repeat analysis between tetraploid and diploid plantlets. The tetraploids exhibited a stable and remarkable dwarfness effect rarely reported in artificial polyploidization among wide range of phenotypic variations. There are both morphological and cytological changes including extremely reduced plant height, thickening and broadening of leaves, disappearance of pseudostem, density reduction, and augmented width of stomatal. Furthermore, the level of phytohormones, including, indole propionic acid, gibberellin, brassinolide, zeatin, dihydrozeatin, and methyl jasmonate, was significantly lower in tetraploids than those in diploid controls, except indole acetic acid and abscisic acid, which could partly explain the dwarfness in hormonal regulation aspect. Moreover, as the typical secondary metabolites of garlic, organosulfur compounds including allicin, diallyl disulfide, and diallyl trisulfide accumulated a higher content significantly in tetraploids. The obtained dwarf genotype of autotetraploid garlic could bring new perspectives for the artificial polyploids breeding and be implemented as a new germplasm to facilitate investigation into whole-genome doubling consequences.

Rapid and Efficient Regeneration of Populus ussuriensis Kom. from Root Explants through Direct De Novo Shoot Organogenesis

Populus ussuriensis is an important tree species with high economic and ecologic values. However, traditional sexual propagation is time-consuming and inefficient, challenging afforestation and wood production using P. ussuriensis, and requires a rapid and efficient regeneration system. The present study established a rapid, efficient, and stable shoot regeneration method from root explants in P. ussuriensis using several plant growth regulators. Most shoot buds (15.2 per explant) were induced at high efficiency under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 4.54 μM TDZ within two weeks. The shoot buds were further multiplicated and elongated under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 57.74 μM GA3 for four weeks. The average number and efficiency of elongation of multiplication and elongation for induced shoot buds were 75.2 and 78%, respectively. All the shoots were rooted within a week and none of them showed abnormality in rooting. The time spent for the entire regeneration of this direct shoot organogenesis was seven weeks, much shorter than conventional indirect organogenesis with the callus induction phase, and no abnormal growth was observed. This novel regeneration system will not only promote the massive propagation, but also accelerate the genetic engineering studies for trait improvement of P. ussuriensis species.

Efficient plant regeneration system for New Guinea Impatiens (Impatiens hawkeri W. Bull) CV. ‘Violet’ and ‘Scarlet Bronze Leaf’

New Guinea Impatiens (Impatiens hawkeri W. Bull) is an eye-popping landscaping plant of bright and colorful blooms. A highly efficient in vitro plant regeneration system via direct shoot organogenesis was established for the first time from hypocotyl with partial cotyledons of New Guinea Impatiens. Our results showed that the explant sterilization method, basic medium type, AgNO3, sucrose and plant growth regulators have significantly influenced in vitro morphogenesis. The regeneration rate in regeneration media that MS supplemented with 0.5 mg·L−1TDZ and 0.1 mg·L−1NAA was acceptable, the induction rate of ‘Violet’ was 86.67%, and its proliferation coefficient was 5.27, while the induction rate of 'Scarlet Bronze Leaf' was 83.33%, and its proliferation coefficient 5.13. PIC could not induce clumped sprouts; however, it had a better effect on callus induction. We also included a shoot multiplication stage using regeneration New Guinea Impatiens medium that MS supplemented with 0.8 mg·L−16-BA, 0.5 mg·L−1TDZ and 0.05 mg·L−1NAA. Reducing sucrose concentration to 20 g·L−1 or adding 1 mg·L−1AgNO3 could alleviate the hyperhydricity phenomenon in the process of tufted bud proliferation. The optimal root culture medium for the regenerated seedlings of ‘Violet’ and 'scarlet bronze leaf' of New Guinea Impatiens was MS supplemented with 0.05 mg·L−1IBA, the rooting rate reached 100%.The study examined the micropropagation responses of New Guinea Impatiens in the presence of various growth regulators, which provide a simple and more suitable protocol adapted for the mass propagation of clones.

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

Micropropagation of pokeweed (Phytolacca americana L.) and comparison of phenolic, flavonoid content, and antioxidant activity between pokeweed callus and other parts.

BackgroundPokeweed (Phytolacca americana L.) is regarded as an invasive plant in many parts of the world but possesses therapeutic characteristics used for antitumor and rheumatism treatment. This study investigated the effects of auxins and four explants on pokeweed callus induction. The effects of cytokinins and combinations between cytokinins and NAA on shoot and root induction were also studied. TPC, TFC and antioxidant activity of calli were screened and compared with other pokeweed plant parts.MethodsFour explants were used to induce callus using 2,4-D and IBA at 1, 2, 3 and 4 mg/l for each auxin. Direct shoot organogenesis from nodal explants was investigated using BAP, kinetin and TDZ (1, 2 and 4 mg/l for each cytokinin). Combined effects between cytokinins and NAA at 0.1, 0.2 and 0.3 mg/l were further simultaneously estimated with root induction. Calli derived from the leaves were compared with other plant parts for TPC, TFC and antioxidant activity using the Folin-Ciocalteu, AlCl3 colorimetric assay and DPPH assays, respectively.ResultsResults showed that MS medium containing 2 mg/l 2,4-D induced callus formation on leaf explants that provided highest fresh and dry weights. Three types of synthetic cytokinins as kinetin, TDZ and BAP were used for direct shoot organogenesis from pokeweed nodes. MS medium containing 2 mg/l kinetin was effective in stimulating normal shoots, with the largest number of shoots and leaves and the longest shoots. The combination between cytokinins and NAA showed no positive effect on shoot and root induction from pokeweed nodal explants. For TPC and TFC determination, pokeweed seeds and leaves possessed the highest phenolic and flavonoid contents, respectively. Highest phenolic content of pokeweed seeds led to lowest IC50 by DPPH assay. Phenolic content was higher than flavonoid content.ConclusionResults suggested promising conditions for callus induction. Leaf explants cultured on MS medium with 2 mg/l 2,4-D and nodal explants cultured on MS medium with 2 mg/l kinetin provided the largest number of normal shoots and leaves. NAA did not show positive effects on shoot and root induction when combined with cytokinins. Chemical constituent screening indicated that seeds and leaves provided highest TPC and TFC, respectively, while pokeweed calli contained higher phenolic than flavonoid content. This is the first report describing chemical constituent screening and antioxidant activity of calli and other parts of the pokeweed plant. Results provided significant information to further enhance bioactive compound contents of pokeweed calli using elicitation methods.

High-frequency adventitious shoot organogenesis from in vitro stem explants of Scutellaria araxensis Grossh.

The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.

Plant regeneration via somatic embryogenesis and direct shoot organogenesis of a C4 bioenergy crop Pennisetum pedicellatum Trin.

Pennisetum pedicellatum Trin. is a C4 tropical perennial forage grass known as Deenanath grass holding great potential to address vital, puzzling issues such as bio-economy, human health and the environment. Genetic complexity not only is a big barrier in selection and breeding practices but also restricts harnessing of the full potential of this multipurpose fodder crop. By optimizing a rapid, effective and consistent in vitro regeneration protocol through callus mediated and direct shoot organogenesis, we can multiply these plants for producing a large amount of biomass. Calli were induced from mature seeds and shoot apices of two genotypes on Murashige and Skoog (MS) medium containing different levels of 9.0–22.5 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) and a constant level of 2.22 µM N6-benzyladenine (BA). Seventy-one percent of the calli turned embryogenic when 2,4-D (2.25 µM) was reduced in the medium. Somatic embryogenesis was accomplished on MS medium with an increased level of either BA (4.4–17.6 µM) or kinetin (4.6–18.4 µM) along with 0.56 µM 2,4-D. Ultra-structural and histological studies indicated globular, scutellar and coleoptilar somatic embryos. A maximum of 82.7±1.6% plant regeneration was accomplished from seed explants on MS medium containing 8.8 µM BA and 0.56 µM 2,4-D. Multiple shoots were induced on MS media containing either BA (4.4–18.0 µM) or kinetin (4.6–18.4 µM) along with 0.56 µM 2,4-D using shoot apices. A maximum of 22.1±0.45 shoots per shoot apex was produced on MS medium containing 8.8 µM BA and 0.56 µM 2,4-D. Successful rooting of healthy shoots generated through both the pathways could be optimized on ½MS medium containing 0.4% charcoal. Complete plants could be hardened in plastic pots and were well established on field conditions. In vitro regeneration was established for the first time successfully, which would be very useful for genetic improvement of this apomictic species through transgenic approach.

In-vitro Regeneration from Direct and Indirect Organogenesis of Crescentia alata Kunth an Important Multipurpose Medicinal Tree

In this present work, an in-vitro regeneration protocol for Crescentia alata (C. alata) was developed using various explants on Murashige and Skoog (MS) medium augmented with different concentrations and combinations of plant growth regulators (PGRs) for direct and indirect regeneration. The direct organogenesis was established from nodes and internodes on MS medium supplemented with cytokinins and auxins. The indirect organogenesis via callus phase was obtained from leaf, nodes and internodes on MS medium supplemented with different concentrations of PGRs. The high frequency shoot organogenesis were achieved directly from nodal explants were cultured on MS medium supplemented with 3.0 mg/L BAP+0.5 mg/L KIN +1.0 mg/L NAA. Indirect organogenesis callogenic frequency was optimized at the concentration of MS medium containing 1.0 mg/L BAP + 5.0 mg/L IAA. The callus was obtained from all the explants were used, among these explants internodal explants gave best result on MS medium supplemented with different concentrations of cytokinins and auxins for indirect organogenesis experiment. Indirect organogenesis the highest number of shoot regeneration was obtained in MS Basal Medium with 4.0 mg/L BAP + 0.5 mg/L KIN + 2.0 mg/L NAA from internodal explants. For root formation the regenerative shoots which were sub cultured on MS medium containing different ratios of auxins. The rooted plantlets were transferred successfully to the pots containing sterilized soil and were successfully hardened at greenhouse condition for 20 days then exposed to the natural environment. This is the first successful micropropagation report of an efficient and rapid in-vitro clonal propagation protocol for C. alata by direct and indirect shoot organogenesis through various explants, which can be employed for conservation of this important medicinal tree species as well as the utilization of an biologically important active biomolecules. This protocol can be very useful to obtain plants from various explants, without the requirement of meristematic regions, enabling the obtainment of a higher number of plants in short period.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

SUMMARYCowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium‐mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium‐mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas‐mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.