Cotyledonary Node Research Articles

Role of biosynthesized silver nanoparticles in mitigating hyperhydricity in micropropagated watermelon (Citrullus lanatus Thunb.) and assessment of genetic fidelity using RAPD and SCoT markers

Watermelon (Citrullus lanatus) is one of the five most consumed fresh fruits and is a commercially important horticulture crop. The consumer demand for seedless watermelons has considerably increased due to their superior taste and ease of consumption. Seedless watermelon plants are triploids hence plant tissue culture technology which is the cultivation of plant cells, tissues, or organs on specially formulated nutrient media can facilitate the rapid multiplication of seedless watermelon varieties. However, hyperhydricity has been reported as a frequent problem for in vitro growth and development of watermelon. Hence, the present study highlights the application of silver nanoparticles biosynthesized from Parthenium hysterophorus (PHAgNPs) for reversal of hyperhydricity and enhancing in vitro shoot production in watermelon. The biosynthesized PHAgNPs was characterized using UV–vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis and Transmission electron microscopy. Cotyledonary nodes used as explant and cultured in three types of cytokinins (BAP, TDZ and KIN) became hyperhydric, losing their ability to regenerate at high cytokinin concentrations (> 1.5 mg/L) irrespective of the cytokinin type. The optimum concentration of BAP (1.0 mg/L) fortified with PHAgNPs (15.0 mg/L) exhibited an improved response of 91 % and induced a maximum of 32.33 shoots with a maximum shoot length of 5.66 cm after 6 weeks. The incorporation of PHAgNPs reduced the percentage of hyperhydricity from 29 % to 1.33 % in regenerated shoots, which was confirmed by the reduction in relative water content (RWC) from 93.33 % to 80.33. Silver ions reduced H2O2 content by 50 % and increased photosynthetic pigments (chlorophyll a, b and carotenoids) by 50 %, which contributed to the formation of healthy regenerated shoots, with opened stomata compared to closed in hyperhydric shoots. Compared with those of hyperhydric shoots, the gene expression patterns of 1-aminocyclopropane-1-carboxylase synthase (ACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) decreased after the retroversion of shoots to 15 mg/L PHAgNP medium. The relative gene expression profiles of ACS1 and ACO1 in hyperhydric shoots were 8.8- and 1.8-fold greater than those in normal shoots, respectively. Profuse root growth was observed in MS basal media supplemented with IBA (1.0 mg/L) and PHAgNPs (10.0 mg/L), which resulted in the greatest number of roots (12.33), with an average root length of 4.33 cm and a response of 89.33 %. The genotoxic effect of PHAgNPs evaluated using RAPD and SCoT molecular markers, showed that the addition of low levels of silver nanoparticles to the medium did not cause any genetic variation. The efficient regeneration protocol standardized in this study provides a solution to solve the problem of hyperhydricity in watermelon regeneration.

Development of efficient and scalable regeneration tissue culture method for Cannabis sativa

Large scale production of uniform disease-free plants is crucial for Cannabis sativa biotechnology. Existing micropropagation protocols rely heavily on shoot multiplication from existing meristems via direct organogenesis. Such protocols do not allow multiplication of plant material through continuous sub-culturing. Protocols that use indirect regeneration are usually not efficient enough and have very low multiplication rates. In the present study, an efficient protocol that uses a combination of direct organogenesis and callogenesis to induce multiple shoot development cultures is developed. Callogenesis was induced from various explants cultured on the media having various combinations of thidiazuron (TDZ) and naphthaleneacetic acid (NAA); best callogenesis and shoot regeneration was achieved from hypocotyl explants cultured on TDZ 0.4mgl-1 NAA 0.2mgl-1. Hypocotyls with cotyledonary node and shoot apical meristem were significantly better for shoot regeneration than explants without it. Shoots obtained from multiple shoot cultures were successfully rooted and then acclimatized under greenhouse conditions to develop into adult cannabis plants.

Development of In Vitro Regeneration Protocol for Sweet Pepper (Capsicum annuum L.) using Cotyledon as Explant

Plant tissue culture techniques offer a promising avenue for the efficient propagation of sweet pepper (Capsicum annuum L.), a valuable crop with significant agricultural importance. An effective and repeatable in vitro plant regeneration protocol was developed for the CKN-1 (Lemon yellow) and CKN-8 (Red) genotypes of sweet pepper. Utilizing cotyledonary leaf and cotyledonary node explants, various concentrations and combinations of growth regulators were tested for shoot rejuvenation. For both explants, MS medium supplemented with 8 mg/L BAP, 0.02 mg/L NAA, and 0.5 mg/L IAA was found to be most effective for achieving high rates of response and shoot induction. Notably, cotyledonary node explants outperformed cotyledonary leaf explants, displaying a remarkable 80% response within 11-12 days. Additionally, the supplementation of 1 mg/L IBA with MS medium significantly improved root initiation, with cotyledonary node explants exhibiting the highest responsiveness at 83.77%. Throughout all stages, the genotype CKN-8 (Red) consistently outperformed CKN-1 (Lemon yellow), including in the percentage of regenerated plant establishment. J Bangladesh Agril Univ 22(3): 310–316, 2024

Features of cellular selection of tomato plants, resistance against salining

Aim. For selection at the cellular level, nutrient media are offered, which, although not completely, would partially correspond to natural stress conditions. Methods. As explants, immature embryos, cotyledon nodes with meristematic activity, and hypocotyls of tomato plants of the Lagidnyi, Khoryv, Borivskyi. Results. An agar medium of general mineral composition with different concentrations of growth regulators was used for the cultivation of isolated tissues the generally accepted method. Conclusions. When creating in vitro new forms of tomato plants with increased resistance to chloride salinization of the soil, it is necessary to select resistant cell lines using the stepwise selection method according to the scheme, which includes several consecutive actions of a selective agent in different concentrations at the stages of induction – proliferation, morphogenesis of callus cultures. An increase in the content of hydrogen peroxide in callus structures and stimulation of the activity of antioxidant enzymes superoxide dismutase and peroxidase were also found.

Optimization of stable genetic transformation protocol in castor (Ricinus communis L. cv. TMV 5) using beta glucuronidase reporter gene for pioneer of desirable genes

The simple and stable protocol was standardised for castor (Ricinus communis L. cv. TMV 5) genetic transformation using Agrobacterium tumefaciens strain LBA4404 harbouring the binary plasmid pBAL2 (18.8 kb). Cotyledonary nodes from ten days old, in vivo seedlings were utilized as target cells for Agrobacterium mediated transformation. Explant pre-culture studies were carried out at 2, 4, 6, 8, 10 and 12 day intervals. The 4th day old explants cultivated on mMS medium (MS medium+B5 Vitamins) using plant growth regulators had the highest response percentage (50.6%). Kanamycin (0-175 mg/L) and Hygromycin (0-13 mg/L) sensitivity in well-developed shoots was investigated. Of the two antibiotics, Kanamycin 50 mg/L and Hygromycin 3 mg/L was found optimum. Different levels of acetosyringone (0-200 mg/L) were used in the co-cultivation medium to study the transformation efficiency of castor. Among the different concentrations, maximum number of explants showed GUS expression at 100 mg/L of acetosyringone in the co-cultivation medium at 2 days of co-cultivation period and the Cotyledonary node produced multiple shoots development and plantlet establishment in 0.3 mg/L TDZ, 0.6 mg/L PF-68, kanamycin 50 mg/L, 0.3 mg/L GA3, 1.5 mg/L IBA and 0.6 mg/L AgNO3. The rooted shoots were successfully acclimatized. Histochemical GUS assay was used to monitor T-DNA delivery into the target cells. PCR and Southern hybridization were used to confirm the transformants with the NPT II and GUS gene. A very high frequency (29.3%) of β-glucuronidase (GUS) gene expression was obtained through Agrobacterium-mediated gene transfer into cotyledonary node explants of Castor. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of Cator with desirable gene of agronomic importance.

Plantlet regeneration of Cucumis melo L. Glamour cv. using different types of cytokinin and explants

Plantlets or clones’ regeneration in Cucurbitaceae species can be performed in vitro by applying the tissue culture techniques. Hypocotyls, cotyledons, leaves, cotyledonary nodes and petioles can be used as the explants or starting materials to initiate the regeneration under laboratory condition. BAP (Benzyl aminopurine) and TDZ (Thidiazuron) were among the well-known cytokinin used in tissue culture as plant hormones to regulate the plant growth in vitro. The effectiveness of BAP and TDZ was investigated in this study to determine the effect of different types of cytokinin hormone on C. melo explants since both hormones might show differences in their activity towards C. melo explants. This study found that BAP was superior at all used concentrations compared to TDZ during most of the growing stage of the explants (cotyledons and hypocotyls). Based on this finding, the experiment was repeated by using different types of explants which were nodal explants, petioles and young shoots along with cotyledons and hypocotyls, by using BAP that showed excellent plantlet regeneration. Cotyledon and nodal explants successfully regenerated plantlets with excellent height and significant number of shoots compared to the rest of the explants. The plantlets from cotyledons and nodal explants were regenerated via direct regeneration without any formation of callus. The direct shoot regeneration was the preferred method for mass production via tissue culture because the chances of somaclonal variation is low. Contrarily, the hypocotyls, petioles and shoot tips mostly developed callus and only several treatments formed very small plantlets.

Optimization of in vitro Regeneration of Cowpea (Vigna unguiculata L.) using Computational Models

Cowpea (Vigna unguiculata L.) is the most preferable cultivated crop in the World and can grow on poor soil with water scarcity. More than 80.0% of its cultivation is confined to the Sub-Saharan African continent. However, it is now one of the most preferable crops in Asia and Europe due to its low nutritional and water demand during cultivation under field conditions. In this study, the Bodrum Red cultivar of cowpea was used for in vitro regeneration. The 9-0 days old half cotyledonary node explant was used and placed on different concentrations of Benzylaminopurine (BAP), Indole-butyric acid (IBA), and Naphthalene acetic acid (NAA). The results were analyzed by ANOVA and response surface methodology (RSM). The ANOVA analysis revealed the maximum shoots from the medium enriched with 1.0 mg/L BAP with shot counts ranging from 1.17-3.11. The results were further analyzed by surface regression analysis which revealed the significant impact of NAA on callus formation. The results were also analyzed through Pareto charts and normal plots which confirmed the significant role of BAP and NAA on in vitro regeneration. The use of a response optimizer revealed the maximum shoot counts of 3.0 per explant from the medium supplemented with BAP. Results revealed the significance of RSM for optimizing input factors precisely for multiple input factors simultaneously. Advanced biotechnological approaches like genetic engineering and marker-assisted selection could enhance cowpea resilience to environmental conditions, ensuring its sustainability and adaptability across diverse agroecological zones with the aid of RSM

Direct shoot regeneration from cotyledonary node of Uraria picta (Jacq.) Desv. ex DC., an important plant of dashmula drugs, and assessment of genetic fidelity, metabolic profiling, and anti-diabetic activity

Uraria picta root is used in the polyherbal product ‘Dashmula’. Its exploitation for formulation preparation has depleted its availability, leading to medicine adulteration. Direct shoot regeneration from the cotyledonary node holds promise as a source of raw material. This study aimed to develop a regeneration protocol for U. picta and validate its genetic and metabolic fidelity. The seeds of U. picta showed low germination rates, prolonged dormancy, and poor viability. Root exploitation in the wild poses a threat to its availability in nature. Seedling’s derived cotyledonary nodes cultured on B5 medium supplemented with BAP (0.5–3 mg L−1), Kinetin (0.5–3 mg L−1), Thidiazuron (0.01–1 mg L−1), and meta-Topolin (0.1–4 mg L1). To address hyperhydricity in regenerated shoots, cotyledonary nodes were cultured on high-agar concentration media. Microshoots were exposed to IBA solution (50–800 mg L−1) pulse treatment for rooting. Tissue-cultured plants genetic fidelity was assessed using ISSR and SCoT markers, while metabolic fidelity was studied with HRMS. The chlorophyll content, antioxidant, and antidiabetic activity of micropropagated plants were evaluated. The highest shoot regeneration frequency, with a maximum of 6.57±0.278 shoots per explant, was achieved using 2 mg L−1meta-Topolin. The shoots were elongated, had expanded leaves, and were hyperhydrated. BAP (2 mg L−1) induced a maximum of 9.83±0.333 shoot buds per explant. BAP caused explant browning, profuse callus formation, dwarfing, and hyperhydric shoots. Hyperhydricity was alleviated with a higher agar concentration (1 %). IBA (400 mg L−1) induced a maximum of 2.18±0.090 roots per shoot and a root length of 9.23±0.033 cm. Tissue-cultured and mother plants exhibited clonal fidelity, similar metabolite and chlorophyll content, strong antioxidant activity, and equal efficacy for inhibiting α-amylase and α-glucosidase. This method can propagate elite clones of U. picta and offer its improvement via genetic transformation.

In vitro morphogenesis and micro-morpho-anatomical developments in Moringa concanensis Nimmo.: An endemic tree of Indian sub-continent

Moringa concanensis Nimmo. is a regionally important and underutilized medicinal tree of the Indian subcontinent. An in vitro morphogenesis protocol for Moringa concanensis has been developed using seedling-derived explants. The cotyledonary node explants were found to be the most suitable exhibiting cent-percent bud break with each regenerating 2.33±0.76 shoots of 1.32±0.69 cm length within 15 days on 1.0 mg/L 6-benzyl aminopurine (BAP) supplemented modified Murashige and Skoog Medium (MMS) media. The rate of shoot multiplication increased up to 10.17±2.54 shoots/inoculum by sub-culturing of regenerated shoots on 0.5 mg/L BAP along with 0.1 mg/L Indole acetic acid (IAA). The in vitro cloned shoots were rooted using both in vitro and ex-vitro methods by applications of root-inducing auxins, indole butyric acid (IBA), and naphthalene acetic acid (NAA). IBA proved to be more effective and induced roots in all in vitro raised shoots. It produced 17.72±4.83 roots/shoot with 2.12±0.88 cm length on 1/4th strength MMS media fortified with 1.0 mg/L of IBA. Ex vitro rooted plantlets with concurrent acclimatization were achieved by pre-treating shoot bases with IBA at 250 ppm for 5 min. Comparative micro-morpho-anatomical evaluation of leaf and stem revealed differentiation of non-glandular trichomes, anomocytic stomata, and vascular tissue development under applied in vitro conditions, which suggests the survivability of plantlets. Sixty percent of the in vitro regenerated plants survived in the field. It is the foremost report on in vitro morphogenesis of Moringa concanensis protocol using cotyledonary nodal explant and ex vitro rooting method. The tissue culture methods defined and developed can be used for large-scale multiplications of Moringa concanensis as non-conventional approaches.

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

N6-benzyladenine (BAP)-Based Seed Preconditioning Enhances the Shoot Regeneration of Seedling-Derived Explants for Subsequent Indirect Gene Transfer in Soybeans (Glycine max [L.] Merrill.)

This study evaluated the effects of N6-benzyladenine (BAP) seed preconditioning and seedling-derived explants on in vitro plant regeneration potential in soybeans (Glycine max [L.] Merrill.). The findings showed that seed preconditioning with 2.55 mg/L BAP prior to germination significantly influenced seedling establishment and the development of shoots, shoot elongation, and rooting on MS media supplemented with BAP and TDZ, compared to the negative (MS-NC) and positive (MS-NP) controls. The results also showed significant differences based on the genotypes, with Dundee recording 91.0% germination over a minimum of 5 days, compared to 74.2% with Peking, followed by 87.5% and 80.0% overall shoot induction frequency in these genotypes, respectively. Regenerated shoots were successfully elongated on MS medium supplemented with 0.5 mg/L BAP plus 0.6 mg/L GA3 and rooted on hormone-free medium, for 3‒4 weeks, and then hardened in the acclimatization growth room under elevated light levels. Overall, this study revealed that BAP preconditioning of seeds enhances the frequency of bud initiation and shoot proliferation, mostly in whole-seedling and cotyledonary node explants subcultured on MS-E and MS-A media supplemented with BAP in combination with TDZ.

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

In vitro propagation and DNA barcoding of the rare near endemic Plantago sinaica (Barnéoud) plant in Saint Katherine, Sinai

Plantago sinaica is a rare perennial shrub near-endemic to Egypt and found in Saint Katherine Protectorate in Sinai. The first successful in vitro propagation protocol was conducted to protect the plant outside its natural reserves. Shoot tip, stem node section, cotyledonary node, and root explants separated from in vitro germinated seedlings were cultured in vitro on Murashige and Skoog (MS) medium enriched with different concentrations and types of cytokinins. It was found that 6-benzyl adenine (BA) is the most efficient cytokinin. MS medium containing 3.33 µM BA and 0.54 µM α-naphthalene acetic acids (NAA) produced 10.25 and 11.30 shoots/explant using shoot tip and stem node section, respectively. Conversely, MS medium + 2.22 µM BA + 0.54 µM NAA produced 13.25 shoots from root explants. Surprisingly, the cotyledonary node explants favored MS medium free from plant growth regulators (PGRs), which produced only 4.25 shoots/explant. The multiplied shoots were rooted successfully with a 100% rooting percentage on half MS medium containing 1.23 or 2.46 µM indole-3-butyric acid (IBA). In vitro, rooted plantlets were efficiently transferred to the greenhouse with a 90% survivability. Finally, the plant was identified using three DNA barcodes; 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), plastid photosystem II protein D1 intergenic spacer region (psbA–trnH), and Internal Transcribed Spacer (ITS) barcodes. Additionally, psbA–trnH and ITS were novel and submitted to the GenBank databases for the first time for Plantago sinaica. Our study supports the United Nations Sustainable Development Goal number 15, which is to preserve, restore and reinstate sustainable usage of terrestrial ecosystems and to stop biodiversity loss.

An efficient approach of in vitro plant regeneration and propagation of Mungbean [Vigna radiata (L.) Wilczek

Mungbean [Vigna radiata L. (Wilczek)] is a valuable legume crop in Bangladesh. However, it faces severe difficulties forming viable roots during the growth stage. The successful root production of mungbean through an efficient tissue culture system has not been well established. Therefore, this study aims to investigate the problem of unsuccessful root formation in mungbean plants and to develop a very efficient method of in vitro regeneration using micropropagation. The BARI Mung-3 mungbean variety was used as an explant. Several shoots were produced from cotyledonary node (CN) explants obtained from 3-day-old seedlings that were germinated in vitro. The plant samples were cultivated on MSB5 medium enriched with 5.0 μM BAP. Shoot generation efficiency per plant was 5.36 ± 0.56 (80.08%). However, the viable root generation in a regulated setting was unsuccessful despite employing multiple combinations of rooting media, including full and half-strength MSB5 medium with different concentrations and combinations of auxins. To resolve this issue, a micrografting approach was applied with scion 3.0 cm in length and 14-day-old mungbean rootstocks. This system resulted in efficient shoots where the viable root generation efficiency rate was 55%. Interestingly, the micro-grafted plantlets successfully produced viable seeds. The successful micropropagation with viable root generation in mungbean plants successfully overcomes the difficulties in rooting and offers an efficient method for successful mungbean production. These new findings open new options for efficient plant generation with mass propagation for ready-smart mungbean production.

Genetic transformation of GmFBX322 gene and salt tolerance physiology in soybean.

Soybean is one of the most important food crops, breeding salt-tolerant soybean varieties is of great significance to alleviate soybean shortage. In this study, the F-box protein family homologous gene GmFBX322 was cloned from the soybean variety Williams 82 and overexpressed in the Shennong 9 soybean variety to further study and explore the physiological mechanism of soybean salt tolerance. GmFBX322 was constructed on the vector pTF101:35S, and integrated into the genome of Shennong 9 soybean variety by Agrobacterium EHA101-mediated cotyledonary node transformation technology, and 4 overexpressed transgenic lines were obtained, molecular assays were performed on the transformed plants. The expression of GmFBX322 was detected by qRT-PCR and it was found that the leaves of the 4 transgenic lines increased by 2.49, 2.46, 2.77, 2.95 times compared with the wild type; after salt treatment for 12 hours, it was found that the expression of wild type Shennong 9 Inducible expression of GmFBX322. After 72 hours of salt treatment, the leaves of wild-type Shennong 9 soybean plants showed obvious wilting and chlorosis, while the leaves of GmFBX322 plants overexpressing GmFBX322 showed no obvious changes. The leaves were taken at 0, 6, 12, 24, and 48 hours of salt stress to determine the antioxidant activity. Ability and osmotic adjustment level, etc. The results showed that the catalase activity in the leaves of the transgenic lines 2265, 2267, 2269, and 2271 was 2.47, 2.53, 3.59, 2.96 times that of the wild-type plant after 48 hours of salt treatment; the soluble sugar content was 1.22, 1.14, and 1.22 of the wild-type plant. 1.14, 1.57 times; the proline content is 2.20, 1.83, 1.65, 1.84 times of the wild type. After comparing the physiological indicators determined by the experiment, the transgenic lines performed better than the control group, indicating that overexpression of GmFBX322 can enhance the salt tolerance of soybean plants. To verify the function of GmFBX322 gene related to stress resistance, add it to the candidate gene of stress resistance, and provide scientific basis for the selection and breeding of salt-tolerant varieties.

Establishment of an Effective in vitro Regeneration System for Sugar Beet (Beta vulgaris L.)

Plant regeneration and dedifferentiation are known to be difficult for sugar beet (Beta vulgaris L.) due to its recalcitrant nature. This leads to low transformation efficiency and thus successful application of plant molecular techniques is limited in sugar beet for its genetic enhancement. A prolific regeneration method has been established by modulating several plant growth regulators on the in vitro regeneration of Beta vulgaris var. V6 KWS Serenada. Several types of explants excised from young seedlings of this variety were used for both direct and indirect regeneration of shoots. The highest response towards direct shoot formation and callus induction were obtained from cotyledonary nodes and hypocotyls, respectively. Explants were cultured on MS medium supplemented with different concentrations of BAP, GA3 and 2, 4 -D for callus induction as well as formation of shoots. Hypocotyls responded well for callus induction on MS medium containing 0.1 mg/l BAP + 2.0 mg/l 2, 4-D, while cotyledonary nodes exhibited the highest responses towards shoot formation on MS medium containing 1.0 mg/l BAP and 1.5 mg/l GA3. MS medium containing 2.0 mg/l IBA produced the highest number of roots per shoot. The in vitro raised rooted plantlets were successfully transferred to soil for acclimatization. Plant Tissue Cult. & Biotech. 33(2): 181-188, 2023 (December)

Micropropagation from cotyledonary explants of grapefruit (<i>Citrus</i> <i>paradisi</i> Macf.) by indirect and direct organogenesis

Grapefruit (Citrus paradisi Macf.) is an economic fruit crop worldwide cultivated for its nutritional value, and aromatic flavours. However, the development of new varieties and high production of quality seedling constitutes a limit to improving grapefruit yields. An efficient regeneration protocol could be of great interest for grapefruit transformation, micropropagation, and germplasm conservation. In vitro plant regeneration systems of Citrus paradisi have been studied by using cotyledonary explants. Different concentrations of plant growth regulators were used to explore the organogenic potential of cotyledonary explants under laboratory conditions. For indirect organogenesis using cotyledon segment explants, a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) lead to the formation of white callus, while a high concentration of 2,4-D induced brown callus and adventitious root differentiation. The best response for callogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg/L of 2,4-D. Only brown callus cultured on MS added with 2 mg/L 6-benzylaminopurine (BAP) has regenerated shoots, but the percentage of callus producing shoots was very low (6.66 ± 2.68%). Regarding direct organogenesis from the cotyledonary node explants, the best response for shoot proliferation was observed on MS supplemented with 4 mg/L BAP (86.66 ± 12.58% of explants regenerating shoots, and an average number of 5.45 ± 0.45 shoots per explant). Low percentages of In vitro rooting of regenerated shoots were observed with different concentrations of α-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). Nevertheless, the highest rooting percentage (13.33 ± 2.88%) was obtained on MS containing 2 mg/L NAA. These results provide new information for mass regeneration and the production of healthy seedlings, and can be applied in grapefruit improvement programs.

Pharmacognostic, Physicochemical and Preliminary Phytochemical Investigation of Trapa natans Linn. root

Background: Water caltrop (Singara), a plant belonging to the Trapaceae or Lytraceae family is an essential medicinal herb used in Ayurvedic preparations. Due to its unique characteristics and therapeutic properties. It is one of the most widely consumed vegetables in Asian countries. Taiwan, China and areas of Southeast Asia are home to Singara. Objective: To study the pharmacognostic, physicochemical and preliminary phytochemical investigation of Trapa natans Linn. root to establish the salient diagnostic features. Methods: Macroscopical, microscopical, physicochemical evaluation, detection of inorganic constituents, fluorescence analysis, extractive value and phytochemical analysis were performed. Extraction was performed by using different solvent such as petroleum ether, chloroform, acetone, methanol and distill water. Results: Macroscopically roots were mostly 10–65 mm long and less than 1 mm in diameter, except for the first adventitious roots arising from the cotyledonary nodes, which were much longer and often descended into the substrate river bottom. Microscopically the stele of roots is typically tetrarch, but hexarchy and pentarchy occur in large roots and triarchy in some roots. Central metaxylem vessel elements with a discontinuity of mature xylem elements between protoxylem and metaxylem are common. The cortex of Trapa natans Linn. root is characterized by aerenchyma in its middle by schizogeny but it often has asymmetrically larger lacunae in the inner cortex. The result of physicochemical parameters such as foreign matter, loss on drying (moisture content), total ash, acid insoluble ash, water soluble ash, water soluble extractive value, alcohol soluble extractive value, foaming index were found to be 0.3%w/w, 2.04%w/w, 9.5%w/w, 3.5%w/w, 5.0%w/w, 10.2%w/w, 5.2%w/w, less than 100 respectively. Qualitative tests of inorganic elements confirmed the presence of calcium, sodium, potassium, iron, sulphate, phosphate and chloride. The result of fluorescence analysis showed that in visible light, the root powder exhibit various shades of green and brown fluorescence. Various shades of green, brown and black were found under UV light. Preliminary phytochemical investigation of various extracts using different solvents confirmed the presence alkaloids, amino acid, glycosides, flavonoids, carbohydrate, steroids, triterpenoid compound, tannins, phenolic compounds, mucilage and saponins respectively. Conclusion: The results of this study concluded that the presences of various phytoconstituents in Trapa natans Linn. root may be medicinally useful. Also the results can be helpful in setting some diagnostic indices for identification, authentication and preparation of the monograph of the plant.

Molecular identification and in vitro propagation of the endangered medicinal Cleome droserifolia plant of Saint Katherine

The protectorate of Saint Katherine considers one of the biggest protected areas in Egypt characterized by a unique location and environment. It supports a high number of rare and endangered species. Cleome droserifolia (Rorida droserifolia) is an endangered plant found in Saint Katherine, Sinai, Egypt. It has been known for its medicinal uses so it becomes rare due to over-grazing and over-collection for use in traditional medicine, research and trade home use. DNA barcoding analysis and in vitro culture tools were conducted for saving the plant. This research supports the goal number 15 of the United Nations Sustainable Development (SDGs); aimed at preserving, restore and reinstate sustainable usage of terrestrial ecosystems and halting biodiversity deterioration. Two pairs primers of three DNA markers: [ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL), maturase K (matK) and trnH-psbA] were used for PCR amplification and sequencing. BLAST results and phylogenetic analysis proved a correct authentication of Cleome droserifolia on both; species and genus levels and confirms the involvement of Cleome droserifolia in Cleome genus. For in vitro propagation, cotyledonary node explants of germinated seedlings were cultured on Murashige and Skoog (MS) medium fortified with 0.27 µM α-naphthaleneacetic acid (NAA), and 2.32 µM of kinetin (Kin). The highest mean number of axillary shoots was 8.16 shoots/explant. 85% of multiple shoots were successfully rooted on ¼ MS medium fortified with 7.42 µM indole-3-butyric acid (IBA). The successfully rooted plants were transferred to a mixture of sand, soil, and peat moss (1: 1: 1) and efficiently acclimatized in the greenhouse.

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023