Direct Shoot Research Articles

Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant

SR4 genotype of rice is high altitude Himalayan rice prone to various abiotic stresses such as cold stress and therefore gives a poor yield. An efficient protocol for callusing and regeneration via direct and indirect means was established using mature seeds as an explant which can be utilized for molecular studies for genetic advancement of Himalayan rice genotype SR4 through transformation. Highest frequency (96.6%) of callus induction was obtained on MS media 3.0 mg/L 2, 4-D. While maximum regeneration frequency (100%), number of shoots with maximum length 9.14 ± 0.204 (cm) from callus was recovered from MS media amended with 5.0 mg/L BAP in combination with 0.5 mg/L NAA with highest number of shoots having an average shoot length 9.14 ± 0.204 (cm) after four weeks of culture. Direct multiple shoot regeneration from seed explants was obtained using various concentrations of TDZ and BAP with highest regeneration frequency was observed on MS media fortified with 6 mg/L of TDZ with maximum number of shoots. The shoots developed roots on MS media supplemented with 0.6 mg/L IBA.

Influence of Plant Growth Regulators on Glandular Trichome Density and Steviol Glycosides Accumulation in Stevia rebaudiana.

With the advent of modern lifestyles, diabetes-related comorbidities attributed the importance of low-caloric natural sweetener plants such as Stevia rebaudiana. This plant is the viable source of steviol glycosides (SGs) and other economically important secondary metabolites. Glandular trichomes (GTs) play the role as a reservoir for all secondary products present in the plant species. Therefore, the present study was carried out to evaluate the influence of different plant growth regulators (PGRs) on GT density and its impact on the SG content. The direct shoot regeneration system was developed on Murashige and Skoog (MS) + benzyl aminopurine (BAP) (1.0 mg/L) + naphthaleneacetic acid (NAA) (0.5 mg/L), and MS + BAP (1.5 mg/L) + NAA (0.5 mg/L) from nodal and leaf explants, respectively. Among the combination of PGRs used, MS medium fortified with BAP (1.0 mg/L) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg/L) played a significant role in increasing the GT density on leaf and stem tissues of S. rebaudiana. Furthermore, high-performance thin-layer chromatography and gas chromatography–mass spectrophotometry data confirmed a notable rise in SGs and other valuable secondary metabolites. Thus, the protocol developed can be used for the propagation of stevia with an improved metabolic profile at a large scale.

Micropropagation, encapsulation, physiological, and genetic homogeneity assessment in Casuarina equisetifolia.

Casuarina equisetifolia is an important tree of the forest, cultivated in tropical and subtropical regions, providing fuelwood, land reclamation, dune stabilization, paper production, and nitrogen fixation. We have developed a systematic in vitro propagation protocol in C. equisetifolia using nodal segments (NS). Murashige and Skoog (MS) medium augmented with BA (5.0 μM) and NAA (0.5 μM) gave rise to a maximum of 32.00 ± 0.31 shoots per explant (S/E) with shoot length (SL) of 3.94 ± 0.02 cm, and a maximum of 70% regeneration potential (RP) was recorded after 8 weeks of post inoculation. For root induction, in vitro derived shoots were transferred to the nutrient medium consisting of a half-strength (½) MS medium augmented with 2.5 μM NAA, which produced a maximum of 12.68 ± 0.33 roots/shoot (R/S) with 3.04 ± 0.50 cm root length (RL) in 60% of culture after 6 weeks. Micropropagated plants with healthy shoots and roots were successfully acclimatized in vermicompost + garden soil + sand (1:2:1) and a maximum survival percentage of 95.1% was recorded. NS was taken from a 6-weeks-old in vitro derived plant of C. equisetifolia for synthetic seed production, and it was reported that CaCl2 · 2H2O (100 mM) + Na2-alginate (4%) resulted in clear and uniform beads. Furthermore, the maximum conversion of synthetic seeds into plantlets occurred over a period of 4 weeks of storage at 4°C. Scanning Electron Microscopy (SEM) revealed the formation of direct shoot buds without any intermediate callus formation. In addition, the chlorophyll and carotenoid contents of the direct regenerated and mother plant were compared. Similarly, RAPD and ISSR primers were used for genetic homogeneity assessment of the direct regenerated plants, where a total of 18 and 19, respectively, clear and reproducible bands with 100% monomorphism were recorded. The developed micropropagation protocol can certainly be used for large-scale multiplication and germplasm preservation of C. equisetifolia. It will also help in meeting the growing demands of C. equisetifolia in the forest industry.

Micropropagation of Pogostemon paniculatus

Plant regeneration potential from node & leaf explants of Pogostemon paniculatus through direct shoot proliferation was studied on Murashige and Skoog medium supplemented with various concentrations of cytokinins & auxins. As the seed germinability of the plant is low in vitro, we have tried these explants for micropropagation. Among different concentrations of individual hormones tested for nodal explants, benzylaminopurine (BAP) at 5µM & kinetin (Kn) at 0.5µM showed 4 & 2 mean number of shoots/explant respectively & combination of Kn (0.5 µM) + BAP (1.5 µM) showed 15 mean number of shoots/explant. For leaf explant combination of Kn (0.5 µM) + BAP (1.5 µM) showed 20 mean number of shoots/explant. Other hormones tested at all concentrations showed callus formation without the shoot development. Roots were established on the isolated shoots on naphthaleneacetic acid (NAA) at 0.25µM. Rooted plants were acclimatized. This is the first report of Pogostemon paniculatus micropropagation.

Study on in vitro micropropagation of Rosa sp.

Tissue culture has long been recognized as an ideal experimental paradigm for studying the mechanisms governing plant cell growth, division, and physiological and biochemical processes. The present investigation was undertaken to establish a standard method for callus induction, shoot and root regeneration of rose at Genetic Engineering and Biotechnology Laboratory, University of Rajshahi, Bangladesh in 2010. Shoot tip and nodal segments were used as experimental materials excised from field grown plants for callus induction. The direct shoot was observed from both shoot tip and nodal segments for shoot regeneration. In vitro regenerated shoot cuttings were used for root regeneration. Proper manipulation of auxin (2, 4 -D and NAA) was used to induce callus from different explants. Different growth regulators (BAP, KIN and GA3) were casted-off in combination for shoot regeneration and proliferation. Again, different concentrations of auxins (IBA, NAA, and IAA) were applied for root initiation. Among the hormonal supplements used, 2, 4 -D was found best in all respect of callusing response for all types of explants. The highest percentage (90%) of callus induction was observed in media having MS (Murashige and Skoog) + 4.0 mg/L 2, 4 -D. In case of shoot regeneration, 100% of cultured explants regenerated shoot in media with MS + 2.0 mg/L BAP + 0.5 mg/L KIN. While, 2.0 mg/L BAP + 0.5 mg/L KIN + 0.1 mg/L GA3 induced 100% shoot proliferation. Moreover, for root induction, ½ MS + 1.0 mg/L IBA + 1.0 mg/L NAA proved to be the best (80%) from in vitro regenerated shoot cuttings, and the highest mean number of roots was 5.0. Rooted shoots were acclimatized and successfully established in a natural condition where 60% of the transplanted plants survived. Bangladesh J. Agri. 2022, 47(1): 66-74

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 Mass Propagation of Withania Somnifera (L.) Dunal An Important Medicinal Plant of Bangladesh

An efficient in vitro regeneration system was developed for Withania somnifera (L.) Dunal through direct and indirect organogenesis from nodal and leaf segment explants. Highest direct shoot regeneration was recorded when nodal explants were cultured on MS with 2.0 mg/l BAP and 0.5 mg/l NAA where the average number of shoots per explants was 10.5. For indirect regeneration remarkable results on shoot initiation were observed when node and leaf segment callus were cultured on MS +1.0 mg/l BAP +0.5 mg/l NAA. It was observed that node callus showed best response for multiple shoot/explants (28.9) on MS +1.0 mg/l BAP +0.5 mg/l NAA. But the mean of shoots from the callus of leaf segment explants was found to be 15.1 on the same media. The best response towards root induction was observed on MS with 2.5 mg/l IBA. The well rooted plants were successfully acclimatized and transferred to soil. Bangladesh J. Bot. 51(2): 191-197, 2022 (June)

Assessment of somaclonal variation occurrence in micropropagated Mansonia altissimia (A. Chev.) A. Chev. using molecular marker

Plant tissue culture that offers limited space and short time for clonal propagation of plant species can sometimes be discouraging if true to type clones are not generated. This study investigated direct shoot induction and genetic stability of Mansonia altissimia using molecular markers in order to ascertain that the in vitro propagated plantlets were true to type. Nodal cutting was used as explant, and Murashige and Skoog (MS) medium was supplemented with 6-benzyl aminopurine (BAP) (µM) + naphthaleneacetic acid (NAA) (µM), BAP (µM) + Gibberellic acid (GA3) (µM), Kinetin (KIN) (µM) + NAA (µM), KIN (µM) + GA3 (µM), thidiazuron (TDZ) (µM) + NAA (µM), TDZ (µM) + GA3 (µM), for direct micropropagation. PCR amplification of wild plant and direct micropropagation of Mansonia altissimia were done using 15 RAPD and 6 ISSR primers. The highest shoot height (4.77 cm) and number of leaves (5.33) were obtained from 2.5 µM BAP + 3.0 µM GA3. Moreover, the polymerase chain reaction amplification results showed 100% monomorphic and 1.0 level of similarity coefficients, which indicates that there is no occurrence of somaclonal variation.

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.

Impact of Stevia rebaudiana Culturing in Liquid Medium: Elevation of Yield and Biomass, Mitigation of Steviol Glycosides

An efficient micropropagation system is developed by culturing nodal segments containing the axillary shoot buds on liquid Murashige and Skoog (MS) medium. Direct shoot and root formation are highly effective in both solid and liquid MS media without any plant growth regulators (PGRs). Interestingly, a significant difference in yield is obtained between solid and liquid cultures. It is revealed that a relatively higher amount of plant biomass is obtained after culturing for 4 weeks in a liquid MS medium. However, the shoots produced on solid MS medium produce a remarkable decline in all physiological parameters. On contrary, the bioactive steviol glycosides (rebaudioside-A and stevioside) content is higher in shoots grown in solid MS on a comparative basis, which could be compensated by higher yield. After hardening off, all the regenerants are effectively grown in the field with a negligible loss (<1 %), and steviol glycosides spectra is again obtained by conducting high-performance liquid chromatography (HPLC) analysis after 10 weeks of the plantation. This method has great potential to be applied on large scale in bioreactors.

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.

In Vitro Cultures and Volatile Organic Compound Production in Chiliadenus montanus (Vhal.) Brullo.

Callus and microshoot cultures were established for Chiliadenus montanus (Vhal.) Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 μM Gibberellic Acid (GA3) and 4.4 μM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3 followed by culturing on MS media supplemented with 8.8 μM BAP and 0.5 μM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and β-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-β-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.

In Vitro Plant Regeneration via Adventitious Shoot Regeneration from Cotyledon and Leaf Explants of Diplocyclos Palmatus

ABSTRACT Micropropagation protocol for Diplocyclos palmatus through direct shoot induction from isolated cotyledon and leaf explants was established. Cotyledons were isolated from 2-week-old seedlings and cultured in the Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP, 0.5–2.0 mg L−1) alone or in combination with α-naphthalene acetic acid (NAA, 0.2–1.0 mg L−1) for shoot induction. The highest frequency of response (89%) and the mean number of shots (6.8) were observed in an MS medium supplemented with 1.5 mg L−1 BAP and 0.5 mg L−1 NAA. Leaves at various stages of development were isolated from in vitro grown shoots and cultured in an MS medium supplemented with different concentrations of BAP (0.5–2.5 mg L−1), Thidiazuron (TDZ, 0.5–2.5 mg L−1) alone and in combination with NAA (0.2 mg L−1) for shoot induction. The highest shoot induction frequency of 91% and shoot number of 7.8 shoots per explant were observed when 15-d-old- leaves were cultured in an MS medium enriched with 2.0 mg L−1 TDZ and 0.5 mg L−1 NAA after 45 d. The isolated cotyledon and leaf-derived shoots showed optimum rooting on ½ MS medium fortified with 4.0 mg L−1 indole-3-butyric acid (IBA). The rooted plants were transferred to the soil with 90% success.

Effect of silver nanoparticles on sterilization of different explant sources of Gerbera jamesonii cultured in vitro

In this study, silver nanoparticles effects on the sterilization of different sources of explants (young leaves, young flower stalks and young flower buds) of Gerbera as well as on the in vitro morphogenesis and their growth were investigated. The explants were sterilized and cut transversally (1 mm) with the flower stalk, square (0.5 × 0.5 cm) for the leaf sample, longitudinally (0.5 mm) for the flowers (removed the petals) and cultured on MS medium; then, the explants (contamination-free or no browning/necrosis) were transferred into MS medium supplemented with 0.02 mg/L TDZ plus 0.8 mg/L adenine, 10% coconut water, 30 g/L sucrose and 8 g/L agar in 15 days. The results showed that AgNPs at the appropriate concentration and duration treatment was effective in explant sterilization of flower bud (0.02% AgNPs and 20 min), flower stalks (0.02% AgNPs and 30 min) and young leaves (0.05% AgNPs and 20 min) after 15 days of culture. In addition, 3 types of morphogenesis including callus induction, somatic embryogenesis and direct shoot regeneration of explants derived from sterilization by AgNPs were improved as compared to that of HgCl2. In addition, research on the optimal medium for shoot multiplication, rooting as well as evaluation of acclimatization and the growth at the greenhouse were also studied. Results showed that MS medium supplemented with 2 mg/L NAA combined with 0.5 mg/L BA and 2 mg/L KIN is suitable for shoot multiplication; meanwhile, MS medium supplemented with 2 mg/L NAA improved rooting ability as well as quality of plantlets and to improving survival rate and acclimatization of Gerbera cultured in vitro.

Cryo-Technologies for Ex Situ Conservation of Rosa Germplasm.

In this study, we compare two rapid cryopreservation (−196 °C) procedures, droplet-vitrification and encapsulation-dehydration for rose (Rosa × hybrida L., cultivars ‘Ioana’, ‘Mariana’, ‘Vulcan’). Significant factors for cryopreservation, such as sucrose concentration during osmoprotection, treatment duration with plant vitrification solution 2 (PVS2) in droplet-vitrification, duration of air desiccation and moisture content of alginate beads in encapsulation-dehydration, were investigated. In addition, the morphogenetic response to in vitro culture and to liquid nitrogen storage and the content in photosynthetic pigments have been assessed. The in vitro cultures were initiated from plant material originating from field collection. The highest regeneration frequencies were obtained for cv. ‘Vulcan’ in both of the cryopreservation procedures tested, 72% in droplet-vitrification and 65% following encapsulation-dehydration. The morphogenetic response (multiplication index and height of shoots) to liquid nitrogen storage was direct multiple shoot formation per initial shoot tip for all genotypes. The content in chlorophyll a and b was statistically comparable in plant material resulting from cryopreserved and non-cryopreserved shoot tips in all cultivars. The findings expand the information on Rosa‘s response to in vitro culture conditions and cryopreservation, providing protocols with a high regeneration capacity for the storage of genotypes with high ornamental value.

High-frequency direct shoot induction from leaf explants of Pogostemon quadrifolius (Benth.) F. Muell.: an ethnomedicinal herb

High-frequency direct shoot induction from leaf explants of Pogostemon quadrifolius (Benth.) F. Muell.: an ethnomedicinal herb

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.

Regulation of WOX11 Expression Represents the Difference Between Direct and Indirect Shoot Regeneration.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under in vitro culture condition, shoot meristem can be formed directly or indirectly, depending on the absence or presence of callus as the intermediate status. However, the difference of regulatory mechanisms between the two regeneration types remains unknown. In this study, we established a bi-directional system in which shoots regenerated directly from lateral root primordia (LRP) and indirectly from hypocotyl-derived callus simultaneously. The results based on this system revealed that regulation of WOX11 expression represents the difference between the two regeneration types in two aspects. Firstly, number of founder cells expressing WOX11 is tightly associated with regeneration types. Relatively more founder cells gave rise to callus and produce larger meristem, whereas less founder cells produce LRP that regenerate smaller meristem. Secondly, non-CG DNA methylation specifically regulated WOX11 transcription in LRP and promoted direct shoot regeneration, but had no influence on indirect regeneration. The results provide new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.

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.

Standardization of protocol for shoot regeneration in Bacopa monnieri (L.) Pennell using leaf explants

Bacopa monnieri (L.) Pennell commonly called ‘Brahmi’ is used as a medicinal herb since ages. It is known for its revitalizing and nootropic actions in Ayurvedic medicine as it strengthens memory as well as intellect. This activity of the plant has been accredited to a mixture of triterpenoid saponins, sterols and alkaloids. Of these saponins, Bacoside-A and Bacoside-B have been found to be the most important. Present work aimed to establish direct and indirect shoot regeneration protocol in Bacopa monnieri (L.) Pennell using leaf explants. It was found that MS medium supplemented with 1.0 mg/l 2, 4-D and 0.5 mg/l BAP was best for callus formation (53.33%). Leaf explants showed both direct and indirect regeneration and MS medium supplemented with 6.0 μ mol/ was found to be best for shoot regeneration.