Shoot Organogenesis Research Articles

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.

In vitro Propagation and Caulogenesis of Dracaena draco Plants

Dracaena draco is a monocot from family Asparagaceae. The dragon tree is a subtropical plant and features many botanic and Mediterranean gardens worldwide. D. draco has been categorized as vulnerable in Europe and endangered worldwide in the IUCN Red List of Threatened Species (IUCN, 1998). The aim of this study is to develop an efficient protocol for rapid in vitro propagation and caulogensis of D. draco Plants via enhanced axillary bud proliferation from single nodal explants cultured on full strength MS medium, 30g/l sucrose, 4g/l gelrite augmented with various concentrations of plant growth regulators as BA, NAA, IBA and their combinations. In general, the present study concluded that the best results for the initiation stage were recorded on MS medium fortified with NAA and BA at 2.00 and 1.00 mg/l, respectively. Meanwhile, supplemented medium with BA and NAA at 4.00 and 1.00 mg/l, respectively, gave rise to the best results for the multiplication stage. Regarding rhizogenesis stage, the best results were recorded when the neoformed shoots of the multiplication stage were divided singly and cultured on MS medium plus IBA and NAA at 1.00 and 0.50 mg/l, respectively which led to the highest mean number of roots formed per propagule. For callus induction, the internodal segments cultured into MS medium supplemented with BA and NAA at 0.50 and 2.00 mg/l, respectively, gave rise to the best results for the percentage of explants formed callus and callus size. Concerning the percentage of direct organogenesis and number of shoots formed per explants best results obtained from BA and NAA at 0.250 mg/l and 0.00 mg/l, respectively.Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in a mixture of perlite and peat moss at (1:1) or (2: 2) or (2:3) and (3:3) consecutively, in addition to fixed volume (1 portion) of sterile sand; which resulted in the highest mean value of survival percentage/plant (100%) and showed true-to-type plants ex vitro.

English

A bract is a non-inflorescence structure that exists in many plant species. In daylilies, the bract is biologically functional and is the object of the present tissue culture study. After sterilization, bract explants were treated with 6-benzylaminopurine (BA) and thidiazuron (TDZ) that were used individually or in combination in Murashige and Skoog (MS) nutrients medium, under room environmental conditions, to study its capacity to induce shoots in vitro. The results were successful. Both direct and indirect shoot organogenesis were observed. Although variably, all nineteen cultivars that were investigated induced multiple shoots. TDZ was the most potent chemical stimulus for shoot organogenesis. The results also showed no significant correlation between shoot conversion potential and genotype or treatment. Key words: Daylily, tissue culture, plant organogenesis, bract, Hemerocallis, plant regeneration, thidiazuron.

Cytokinin Signaling and De Novo Shoot Organogenesis.

The ability to restore or replace injured tissues can be undoubtedly named among the most spectacular achievements of plant organisms. One of such regeneration pathways is organogenesis, the formation of individual organs from nonmeristematic tissue sections. The process can be triggered in vitro by incubation on medium supplemented with phytohormones. Cytokinins are a class of phytohormones demonstrating pleiotropic effects and a powerful network of molecular interactions. The present study reviews existing knowledge on the possible sequence of molecular and genetic events behind de novo shoot organogenesis initiated by cytokinins. Overall, the review aims to collect reactions encompassed by cytokinin primary responses, starting from phytohormone perception by the dedicated receptors, to transcriptional reprogramming of cell fate by the last module of multistep-phosphorelays. It also includes a brief reminder of other control mechanisms, such as epigenetic reprogramming.

Polyamine Metabolism Is Involved in the Direct Regeneration of Shoots from Arabidopsis Lateral Root Primordia.

Plants can be regenerated from various explants/tissues via de novo shoot meristem formation. Most of these regeneration pathways are indirect and involve callus formation. Besides plant hormones, the role of polyamines (PAs) has been implicated in these processes. Interestingly, the lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems by exogenous cytokinin application. In this system, no callus formation takes place. We report that the level of PAs, especially that of spermidine (Spd), increased during meristem conversion and the application of exogenous Spd improved its efficiency. The high endogenous Spd level could be due to enhanced synthesis as indicated by the augmented relative expression of PA synthesis genes (AtADC1,2, AtSAMDC2,4, AtSPDS1,2) during the process. However, the effect of PAs on shoot meristem formation might also be dependent on their catabolism. The expression of Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) was shown to be specifically high during the process and its ectopic overexpression increased the LRP-to-shoot conversion efficiency. This was correlated with Spd accumulation in the roots and ROS accumulation in the converting LRPs. The potential ways how PAO5 may influence direct shoot organogenesis from Arabidopsis LRPs are discussed.

Enhanced somatic embryogenesis and plantlet regeneration in Cenchrus ciliaris L.

A highly reproducible plant regeneration protocol through somatic embryogenesis and shoot organogenesis has been developed for Cenchrus ciliaris. Three explants (seeds, shoot apices, and immature inflorescences) of four genotypes (IG-3108, IG-718, IG-74, and DBC15-8/32/10) were used for callus induction and plant regeneration. The highest rate of callus formation was found using Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 benzylaminopurine (BA) and 3.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The largest number of somatic embryos was generated with the addition of 400 mg L−1 L-proline, 400 mg L−1 L-glutamine, and 300 mg L−1 casein hydrolysate. Somatic embryos were successfully germinated on MS medium with 3.0 mg L−1 BA and 0.25 mg L−1 2,4-D. In vitro plant regeneration was accomplished through somatic embryogenesis using all three explants. Ultra-structural features of somatic embryos confirmed proper formation and ontogeny.

Overexpression of miR319 in petunia (Petunia × hybrida) promotes de novo shoot organogenesis from leaf explants

MicroRNA319 (miR319) plays an important role in leaf development. Although leaf discs are frequently used for plant transformation, little is known about the function of miR319 in shoot regeneration from leaf explants. To investigate the role of miR319 in de novo shoot organogenesis from leaf explants, the MIR319 gene was overexpressed in petunia (Petunia × hybrida), under the control of the 35S promoter. Petunia leaves overexpressing miR319 (miR319-OX) showed several morphological abnormalities, such as rounded, uneven, and curled leaf blade, larger leaf size, and smaller and rounded abaxial surface cells, which were differences compared to wild-type (WT) leaves. Additionally, miR319-OX leaf explants demonstrated enhanced shoot organogenesis on a growth medium containing 6-benzylaminopurine (BA) at shoot-inducing concentrations, and cytokinin-independent shoot regeneration on hormone-free medium compared with WT explants. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression of petunia NO APICAL MERISTEM (PhNAM) and SHOOT MERISTEMLESS (PhSTM) genes was markedly increased in miR319-OX explants compared with WT explants under shoot induction conditions, whereas the expression of petunia cytokinin-responsive type-A response regulator genes, PhRR2 and PhRR3, showed no differences in expression between WT and miR319-OX leaf explants. These results suggest that miR319 regulates the formation of shoot meristems from leaf explants in petunia during in vitro culture.

Pre- and Postzygotic Barriers Associated with Intergeneric Hybridization between Aronia melanocarpa (Michx.) Elliott x Pyrus communis L. and ×Sorbaronia dippelii (Zabel) CK Schneid. x Pyrus communis

Intergeneric hybridization between Aronia and Pyrus may provide a pathway for developing novel fruit types with larger, sweeter fruits, while maintaining the high levels of biologically health-promoting compounds present in Aronia fruits. Here we describe a deleterious genetic incompatibility, known as hybrid necrosis or hybrid lethality, that occurs in intergeneric F1 hybrids of Aronia melanocarpa x Pyrus communis and ×Sorbaronia dippelii x Pyrus communis. Pollination experiments revealed that maternal A. melanocarpa and ×S. dippelii pistils are compatible with pollen from P. communis. Controlled pollinations using different mating combinations resulted in varying levels of fruit and seed set. Because every combination produced at least some viable seeds, prezygotic incompatibility does not appear to be present. We attempted to recover putative intergeneric progeny via either in vitro germination or in vitro shoot organogenesis from cotyledons. Progeny of putative hybrids from A. melanocarpa x P. communis only survived for a maximum of 14 days before succumbing to hybrid lethality. Regeneration of ×S. dippelii x P. communis was successful for two seedlings that have been maintained for an extended time in tissue culture. These two seedlings have leaf morphologies intermediate between the two parental genotypes. We also confirmed their hybrid status by using AFLPs and flow cytometry. Putative intergeneric hybrids were grown out ex vitro before showing symptoms of hybrid necrosis and dying after 3 months. Eventually micrografts failed, ultimately showing the same symptoms of hybrid necrosis. These results show that intergeneric hybridization is possible between Aronia and related genera in the Rosaceae, but there are postzygotic barriers to hybridity that can prevent the normal growth and development of the progeny.

Immunolocalization of some arabinogalactan protein epitopes during indirect somatic embryogenesis and shoot organogenesis in leaf culture of centaury (Centaurium erythraea Rafn)

Common centaury (Centaurium erythraea Rafn) is a medicinal plant of great importance for both pharmaceutical and food industries. Distribution of arabinogalactan proteins (AGPs) with specific epitopes recognized by seven monoclonal antibodies (mAbs) was investigated during indirect somatic embryogenesis (ISE) and shoot organogenesis (ISO) in centaury leaf culture. Dynamic changes were observed in localization of JIM4, JIM8, JIM13, JIM15, LM2, LM14, and MAC207 epitopes during somatic embryo (SE) and adventitious bud (AB) development. AGPs responded to all tested mAbs and expressed in numerous meristematic centers that were formed on leaf explants. In globular SEs, the distribution pattern of JIM4, JIM13, JIM15, LM2, and MAC207 epitopes was observed, while during the progression of somatic embryo development, the number of detected AGPs decreased. During formation of ABs, the number of detected AGPs also decreased. In fully formed ABs, only JIM4 and MAC207 were detected. The present study suggests JIM13 antibody as a marker for ISE in centaury leaf culture. These results implicated that AGPs were developmentally regulated during centaury ISE and ISO.

Shoot organogenesis from leaf discs of the African ginger (Mondia whitei (Hook.f.) Skeels), an endangered medicinal plant

The African ginger (Mondia whitei) is an endangered, endemic African medicinal plant, widely used as an aphrodisiac and to manage gastrointestinal problems and anorexia. Due to high demand, it is over-utilized from the wild without replenishment and destroyed by inappropriate harvesting methods. Therefore, there is a need to develop an efficient conservation system for the African ginger. This study focused on developing an in vitro propagation procedure for the African ginger through indirect shoot organogenesis. Optimal callus formation was induced from leaf explants cultured on Murashige and Skoog (MS) basal medium fortified with 1.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The 1.5 mg L−1 6-benzylaminopurine (BAP) concentration was optimal with 50% of the callus-forming buds. For shoot formation, media with 2.0 mg L−1 BAP and 1.0 mg L−1 NAA resulted in 60% of the buds developing shoots. Regenerated shoots were rooted on woody plant medium (WPM) fortified with 0.1 mg L−1 BAP and no rooting hormone included. The survival rate of weaned and acclimatized plants was 70% and up to 80% respectively. The study demonstrates an in vitro propagation route for the African ginger which could support its mass production for medicinal use, availing of planting materials, conservation, and commercialization purposes.

Use of Thidiazuron for High-Frequency Callus Induction and Organogenesis of Wild Strawberry (Fragaria vesca).

Strawberry, belonging to the Fragaria genus, is an important crop that produces popular fruits globally. F. vesca, known as wild strawberry, has great characteristics, such as a robust and powerful aroma, making it an important germplasm resource. The present study aims to establish an efficient regeneration method for the in vitro propagation of F. vesca. Firstly, leaf explants were used to induce callus formation on a Murashige and Skoog medium with combinations of cytokinins (thidiazuron (TDZ) and 6-benzylaminopurine (BA)) and auxin (2,4-dichlorophenoxyacetic acid (2,4-D)). Among them, 0.45–4.54 µM TDZ combined with 0.45–4.53 µM 2.4-D exhibited a high induction rate after 4 weeks of culturing. Different explants were examined for their ability to form a callus, and whole leaves on the medium containing 2.27 µM TDZ and 2.27 µM 2,4-D showed the highest callus induction rate at 100% after 2 weeks of culturing in darkness. The highest shoot regeneration ability through organogenesis from the callus was obtained at 0.44 µM BA. After 2 weeks of culturing, the shoot regeneration rate and shoot number per explant were 96% and 19.4 shoots on an average, respectively. Rooting of shoots was achieved using indole-3-butyric acid (IBA) or an α-naphthaleneacetic acid (NAA)-containing medium, and the resulting plantlets were acclimatized successfully and formed flowers eventually. In this report, we demonstrated that shoot organogenesis was derived from the meristematic cells of calli and by transferring the induced calli to a 0.44 µM BA medium, the regeneration period can be shortened greatly. A protocol for the efficient regeneration of wild strawberry was established, which might be useful for their large-scale propagation or obtaining transgenic plants in the future.

Clonal bulblet regeneration and endophytic communities profiling of Lycoris sprengeri, an economically valuable bulbous plant of pharmaceutical and ornamental value

Lycoris Herb. is valuable as a natural source of starch, broad-spectrum fungicides, and natural compounds with antimicrobial, anticancer and is an antiviral abilities, and for ornamental and urban landscaping plant. The unlimited massive exploitation of Lycoris bulbs has led to a considerable threat to its wild resources and natural habitats. In this study, an efficient protocol for clonal bulblet regeneration of Lycoris sprengeri via seed explants was developed for resource protection and to satisfy the needs of the pharmaceutical and ornamental industries. Contamination-free probulbs were obtained at a rate of approximately 88.7% by using a combination of treatment with HgCl2 for 10 min and NaClO for 5 min to achieve surface sterilization. The endophytic community profiling of seed explants provided a comprehensive overview of changes in the Lycoris endophytes before and after effective surface sterilization. Additionally, the survival of the majority of endophytes without causing secondary contamination suggests the potential utilization of endophytes for the production of novel natural products in vitro. Well-rooted probulbs obtained from Murashige and Skoog’s (MS) medium containing 3% sucrose and 0.2 mg l-1 α-naphthaleneacetic acid (NAA) were selected as secondary explants for multiplication. Cross-cutting without complete separation was superior to complete separation for bulblet multiplication. Maximum bulblet multiplication was achieved in 4.0 mg l−1 6-benzyladenine (BA) and 1.0 mg l-1 NAA with 3% sucrose via direct shoot organogenesis. We were able to transplant approximately 95% of the rooted regenerated bulblets in a natural field after acclimatization. Genetic stability assessment of the in vitro regenerated bulblets was performed using simple sequence repeat (SSR) markers and flow cytometry analysis, with no observed difference compared to seed-germinated probulbs.

Tissue Culture, In Vitro Organogenesis and Regeneration of Plantago lanceolata

Introduction: Plantago lanceolata is one the most important species of the Plantago genus and has valuable medicinal secondary metabolites. Materials and Methods: The effect of different factors on germination of P. lanceolata seeds was studied and leaf and root explants of in vitro growth seedling were cultured on Murashige and Skoog (MS) medium supplemented with combination of 6-benzylaminopurine (BAP) or thidiazuron (TDZ) (0, 0.5, 1, 1.5, 2 and 3 mg/L) and auxins: α-naphthaleneacetic acid (NAA) (0, 0.2, 0.5, 1 mg/L), Indole-3-acetic acid (IAA) (0.1, 0.5, 1, 1.5 mg/L) or Indole-3-butyric acid (0.5, 1, 1.5, 2 mg/L) (IBA) at different concentrations. Results: The results showed that cold pre-treatment, daylight and 1/8 MS salt concentration are more suitable for high germination. The best shoot organogenesis rate (95%) in leaf explants was observed in 1:1 mg/L, and 1.5:1 mg/L TDZ: IBA. The highest percentage of shoot organogenesis (100%) was observed in most of the plant growth regulator (PGR) treatments in root explants. About 58.67 and 60 shoot numbers obtained with 2 mg/L TDZ in leaf and 1:1.5 mg/L BAP: IBA in root explants, respectively. Conclusions: It can be suggested that the best shoot organogenesis and proliferation medium is MS basal medium containing cytokinin TDZ and auxin IBA in comparison with other hormone compounds on leaf and root explants. The result behind this fact is high callus induction and regeneration potential of explants in all concentrations.

Histological assessment of regenerating plants at callus, shoot organogenesis and plantlet stages during the in vitro micropropagation of Asparagus cochinchinensis

In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect organogenesis using leaf segments cut from seedlings of in vitro-germinated seeds. We obtained 85% callus induction from the leaf segments within 4–5 weeks when grown on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP, 1.0 mg/L) and 1-naphthaleneacetic acid (NAA, 0.5 mg/L). We found that MS media supplemented with Kn (1.0 mg/L) in combination with NAA (1.0 mg/L) and BAP (1.0 mg/L) combined with NAA (0.5 mg/L) were the most effective in promoting shoot regeneration, yielding plantlets with 6.72 and 6.48 shoots per culture, respectively. When cultured on PGR-free half-strength MS medium, regenerated plants developed root systems with an average 11.0 roots per shoot cluster and an average length of 36.14 mm at 9 weeks. During acclimatization, regenerated plantlets showed 96.4% survival and exhibited normal growth characteristics and morphology. We also made an attempt to directly regenerate A. cochinchinensis from shoot apices but it was futile. Histological analyses revealed the presence of crystal idioblasts in young leaves from the early stages of leaf differentiation. The leaf-based plant regeneration technique developed herein could be employed for large-scale propagation of the plants over a short time period, thereby substantially contributing to the germplasm preservation and rapid propagation of A. cochinchinensis. A repeatable in vitro micropropagation protocol for Asparagus cochinchinensis was developed based on indirect organogenesis. Histological analysis revealed crystal idioblasts for the first time in leaf primordia of this species.

PEG 6000-Stimulated Drought Stress Improves the Attributes of In Vitro Growth, Steviol Glycosides Production, and Antioxidant Activities in Stevia rebaudiana Bertoni.

This study addresses the favourable effects of drought stress imposed by polyethylene glycol (PEG) 6000 on the micropropagated shoots of Stevia rebaudiana. Various concentrations, i.e., 0, 0.5, 1, 2, and 4% of PEG 6000 were applied to the nodal shoot explants for four weeks, and the influence produced on shoots growth parameters, bioactive steviol glycosides (rebaudioside A and stevioside), and nonenzymatic antioxidant activities (total phenolic content (TPC), total flavonoid content (TFC), total antioxidant capacity (TAC), total reducing power (TRP) and 1,1-diphenyl-2-picrylhydrazyl(DPPH)-free radical scavenging activity (FRSA)) was elucidated. The significantly highest yield (92.4% direct shoot organogenesis) and secondary metabolites (2.94% Reb A, 2.52% ST, 95.3% DPPH-FRSA, 15.0% TPC, 13.0 µg/mg TFC, 22.3 µg/mg TAC, and 19.8 µg/mg TRP) production in response to abiotic stress elicitors was obtained in Murashige and Skoog (MS) medium treatment provided with 4% of PEG 6000. The overall trend was significant enhancement of growth dynamics and pharmaceutical compounds from control to 4% of PEG 6000 concentration as a defensive response against reactive oxygen species (ROS) produced in excess by water deficit. This is a very promising study to be employed in bioreactors to get markedly enhanced content of compounds of medicinal importance in the pharmaceutical market.

Expression of some Epigenetic-Related Genes in Regenerated Shoots of Prunus persica

Establishment of an efficient adventitious shoot regeneration system for mature embryo of Prunus persica via shoot organogenesis has been achieved. Three different combinations of plant growth regulators (PGRs) have been applied in the current study; 6-Benzyl adenine (BA) or thidiazuron (TDZ) with Indole acetic acid (IAA) or Indole butyric acid (IBA). The high regeneration frequency was illustrated by 1.5 mg/l BA with 0.5 mg/l IAA combinations. Quantitative expression of eight epigenetic regulatory genes; Chromomethylase 3 (CMT3), Domains Rearranged Methyltransferase2 (DRM2), Fertilization Independent Endosperm (FIE2) and Chromodomain Protein Like Heterochromatin Protein1 (LHP1), Pickle (PKL), Proliferating Cell Nuclear Antigen (PCNA), Shoot Meristemless (STM) and DICER-LIKE1 (DCL1) have been estimated in the peach regenerated shoots. We found that the auxins and cytokinins in different combinations especially BA effect on the expression of some epigenetic genes in relation to the mature embryo.

Morphological and anatomical insights into de novo shoot organogenesis of in vitro ‘Delite’ rabbiteye blueberries

Blueberries are valued for their taste and their high nutritional benefits, including their antioxidant and anti-inflammatory properties. In vitro culturing is an alternative method for clonal propagation, and has been used in many biotechnological studies. Most blueberry research is concentrated on highbush and lowbush taxa (Vaccinium corymbosum and Vaccinium angustifolium respectively), with only limited investigations of rabbiteye cultivars (Vaccinium virgatum) that are more suitable for subtropical climates and regions with warmer winters as a result of climate change. There is therefore a need to determine in vitro protocols for that species and group of cultivars. We examined here adventitious shoot regeneration in the ‘Delite’ rabbiteye blueberry cultivar. Leaf explants were cultured in vitro in Woody Plant Medium (WPM), and the effects of different thidiazuron (TDZ) concentrations, the orientation of the leaf (adaxial or abaxial surface in contact with the medium), and two portions of the leaf segment (basal or apical) were examined. De novo shoot development was studied using light and scanning electron microscopy. All concentrations of TDZ used showed similar survival and regeneration rates; 0.5 μM TDZ showed high efficiency in regenerating adventitious shoots (100%, with 57 adventitious shoots/explant), as did the adaxial surface in contact with the medium using either the apical or the basal portion of the leaf (97% shoot regeneration, 47.5 adventitious shoots/explant). Anatomical analyses showed direct and indirect organogenesis. The shoots developed leaf primordia with stomata, trichomes, and well-developed vascular tissues, with further elongation and rooting of the plants. We therefore describe here a high-efficiency regeneration method through de novo shoot organogenesis using TDZ in foliar explants of rabbiteye blueberry, with direct and indirect organogenesis.

Effect of silver nitrate and growth regulators on direct shoot organogenesis and in vitro flowering from internodal segment explants of Alternanthera sessilis L.

Abstract The present report deciphers a rapid method for efficient shoot regeneration via direct organogenesis from internodal segment explants of Alternanthera sessilis. Internodal segment explants were placed on MS medium containing various doses of BAP and/or KIN (0.5–10.0 mg L 1) for shoot bud initiation. High frequency shoot bud induction (89%) was recorded on MS medium containing 2.5 mg L 1 BAP. Of the two cytokinin combinations tested, highest percent of plant regeneration (100%) with 81.1 shoots/culture was obtained on medium fortified with 2.5 mg L 1 BAP along with 50 mg L 1 adenine sulphate (AdS) and 0.5 mg L 1 silver nitrate (AgNO3) combination. Interestingly, BAP at 2.5 mg L 1 exhibited in vitro flowering from elongated shoots. Elongated shoot buds were carefully excised from the multiple shoot clumps and transferred onto half-strength MS medium without and/or fortified with different doses of NAA/IBA (0.1–1.0 mg L 1) for root initiation. IBA at 0.5 mg L 1 was found to be the best concentration for maximum percent of rooting (95.7%) over NAA. Well rooted plantlets were transferred into field conditions where they were successfully survived with 95%. It is noteworthy to mention that the random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker analysis between mother (control) and in vitro derived plantlets was found to be identical (monomorphic banding pattern) and no variability was observed with ISSR marker while RAPD marker showed 1.19% polymorphism. Hence, molecular results confirm that the in vitro regenerated plantlets were found to be the true-to-type in nature. In conclusion, the suitable growth hormonal combinations were identified for large scale production of genetically identical plantlets from internodal segment explants of Alternanthera sessilis.

Natural Variation in Plant Pluripotency and Regeneration.

Plant regeneration is essential for survival upon wounding and is, hence, considered to be a strong natural selective trait. The capacity of plant tissues to regenerate in vitro, however, varies substantially between and within species and depends on the applied incubation conditions. Insight into the genetic factors underlying this variation may help to improve numerous biotechnological applications that exploit in vitro regeneration. Here, we review the state of the art on the molecular framework of de novo shoot organogenesis from root explants in Arabidopsis, which is a complex process controlled by multiple quantitative trait loci of various effect sizes. Two types of factors are distinguished that contribute to natural regenerative variation: master regulators that are conserved in all experimental systems (e.g., WUSCHEL and related homeobox genes) and conditional regulators whose relative role depends on the explant and the incubation settings. We further elaborate on epigenetic variation and protocol variables that likely contribute to differential explant responsivity within species and conclude that in vitro shoot organogenesis occurs at the intersection between (epi) genetics, endogenous hormone levels, and environmental influences.

Productive method for commercial propagation through direct embryogenesis and organogenesis in Ledebouria revoluta—an underutilized medicinal plant with cardioprotective properties

The bulbaceous herb Ledebouria revoluta (family Asparagaceae) is an important cardioprotective plant. Two different pathways of in vitro direct regeneration system consisting of somatic embryogenesis and shoot organogenesis have been established for high-frequency micropropagation. A rectangular piece (6 × 10 mm2) of leaf lamina was used as an explant to study the morphogenetic regulation by exogenously supplied auxins, cytokinins, and polyamine within controlled in vitro environment. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose and various concentrations and combinations of plant growth regulators (PGRs), as per experimental needs. Both β-naphthoxyacetic acid and α-naphthalene acetic acid (NAA) auxins individually induce somatic embryogenesis. Combined application of auxins and cytokinins further improves the embryonic response. The synergistic effect of NAA (2.0 mg L–1), kinetin (1.0 mg L–1), and spermidine (SDN, 217.88 mg L–1) provided the best result with 96.7 ± 1.8 % response and highest 63.4 ± 0.8 embryos per explant. The highest (94.5 ± 2.5 %) conversion of embryos to complete plantlets was noted in presence of 1.0 mg L–1 kinetin. The morphogenic path altered from embryogenic to organogenic when auxin was totally withdrawn, and a higher concentration of cytokinin was applied. The synergism between 6-benzylaminopurine (3.0 mg L–1) and SDN (217.88 mg L–1) proved the best PGR combination for the direct shoot (or bulblet) organogenesis with maximum 96.6 ± 2.2 % response and highest 48.6 ± 0.5 bulblets per explant. Total withdrawal of auxin was essential for direct bulblet organogenesis because the presence of any auxin always shifts the morphogenic path from organogenic to embryogenic. Histological study confirms the bipolar structure of somatic embryo and unipolar bulblets. Highest 12.7 ± 0.54 roots per bulblet were induced on ½ strength MS medium containing 2.0 mg L–1 indole-3-butyric acid. Finally, the plantlets were acclimatized with 96.7 % survival rate and all of the survived plants produced bulbs and 92.5 % of them flowered normally in field conditions. This in vitro system permits a high-frequency regeneration through embryogenic and organogenic paths, which can be used for large-scale commercial micropropagation for round-the-year supply of this important cardioprotective medicinal plant.