Axillary Shoot Research Articles

Rootstock effect on growth, branch morphology and flowering precocity of young monoaxis and biaxis trees of ‘Scilate’ and ‘Fuji supreme’ apple cultivars

ABSTRACT The New Zealand Institute for Plant and Food Research has developed three semi-dwarfing apple rootstocks: PFR2, 4 and 5 with pest and disease resistance/tolerance. Nursery performance and flowering of ‘Scilate' and ‘Fuji Supreme' grafted onto PFR rootstocks and grown as monoaxis or biaxis trees were evaluated in comparison with ‘Malling 9' (dwarf) and ‘G.202' (semi-dwarf) at Hawkes' Bay research centre. For monoaxis ‘Scilate' trees, PFR rootstocks promoted primary, axillary and total shoot length/scion by (11–15%), (54–76%) and (22–37%), respectively compared with ‘M.9', but the same were lower by (4–11%), (25–62%) and (17–42%) compared with ‘G.202'. On PFR2 and PFR5, ‘Scilate' produced 6 axillary shoots/tree, compared with ‘M.9' (4) and ‘G.202' (7). ‘Scilate' on PFR2, 4 and 5 produced 74%, 62% and 93% of axillary shoots with moderate (30°–45°) crotch angle compared with ‘M.9' (30%) and ‘G.202' (79%). On biaxis trees, the total shoot length was higher on PFR2, 4 and 5 compared with ‘M.9', but not different from ‘G.202'. Flowering precocity was not affected by rootstocks. Overall, PFR2, 4, 5 and ‘G.202’ produced high-grade young trees expressing greater potential for early canopy development.

A Novel Solid Media-Free In-Planta Soybean (Glycine max. (L) Merr.) Transformation Approach

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation.

Limited genetic diversity found among genotypes of the Entada landrace (Ensete ventricosum, (Welw.) Chessman) from Ethiopia.

The Entada landrace of enset (Ensete ventricosum (Welw.) Chessman) is probably the most unique indigenous crop in Ethiopia, being maintained and utilized by the Ari people in the South of Ethiopia. Here we describe genetic diversity, selection signatures and relationship of Entada with cultivated and wild enset using 117 Entada genotypes collected from three Entada growing regions in Ethiopia (Sidama, South and North Ari). A total number of 1,617 high-quality SNP markers, obtained from ddRAD-sequences, were used for the diversity studies. Phylogenetic analysis detected a clear distinction between cultivated enset, Entada and wild enset with Entada forming a completely separated clade. However, extremely short branch lengths among the Entada genotypes indicate very little molecular evolution in the Entada lineages. Observed and expected heterozygosities were high, 0.73 and 0.50, respectively. Overall, our results strongly indicate that the Entada genotypes we have studied originated from one or a few clonal lineages that have been propagated and spread among farmers as clones. Prolonged clonal propagation of heterozygous genotypes from a single or few founding lineages has led to populations with very little or no diversity between genotypes, and high heterozygosity within genotypes. Signatures of directional selection were identified at eight loci based on an FST outlier analysis. Four candidate genes detected are involved in axillary shoot growth and might be involved in controlling natural sucker formation in Entada.

Influences of Thidiazuron Concentrations and Exposure Duration on Axillary Shoot Proliferation of Faba Bean [Vicia faba (L.)] Genotypes

Background: Faba bean is one of the most important feed grain legume crops in the world. However, this crop is a recalcitrant species for in vitro regeneration. Methods: In this study, in vitro shoot proliferation of ten faba bean genotypes, that are classified according to their seed size into small, medium and large, were investigated in response to different light regimes and thidiazuron (TDZ) treatments. Result: Incubation under different light regimes, explant type as well as genotypes influenced seedling growth and axillary shoot multiplication of faba bean. The explants with single cotyledon and incubated under continuous darkness for 6 days resulted higher number of shoot than nodal explants. Medium size seeded genotype (ILB4347) produced the highest shoot and root length and shoot proliferation as compared with large (Luz) or small (Triple White) seeded genotypes. The effects of TDZ concentrations (2-10 mM/L) and exposure duration (10 and 20 days) on axillary shoot multiplication were investigated for the ten faba bean using cotyledonary explants. The overall mean of TDZ time exposure showed that treatment of 20 days exposure had higher number of shoot compare to 10 days exposure treatment with 3.77 shoot per explant. On the basis of genotype used, the three most responsive genotype in tissue culture were medium sized seeds genotypes (ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot per explant, respectively). ILB4347 were the most responsive genotype in tissue culture. Treatment of 8 mM TDZ after 20 days followed by 2 weeks on MS medium without PGRs generated the highest proliferation with 6.63 shoot per explant. These findings contribute towards effective in vitro propagation of faba bean.

Study of Shoot Tip Necrosis Problems of Fegra Fig (Ficus palmata Forssk.) In Vitro in Saudi Arabia

This work studied the micropropagation of fegra fig (F. palmata Forssk.) during which we experienced the incidence of shoot-tip necrosis (STN). STN was evident during the shoot elongation stage, which was regenerated on Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine. To alleviate SNT, we conducted a series of experiments and supplemented the medium with calcium chloride (40, 80, and 120 mg/L), ascorbic acid (50, 100, and 150 mg/L), silver nitrate (1, 2, and 3 mg/L), and boric acid (9.3, 12.4, and 15.5 mg/L). Results showed that all the treatments controlled STN at varying levels, and supplementation of medium with 3 mg/L silver nitrate reduced the incidence of STN from 80% to 24%. The regenerated shoots were rooted on the same medium with incubation of cultures in the dark for 3 weeks and subsequent 4 weeks of incubation under 16/8-hour light/dark photoperiod. The growth parameters (number of shoots and roots, length of the main shoot and root, fresh and dry weights), photosynthetic pigments (chlorophylls and carotenoids), and relative water content of plantlets were restored with the application of 3 mg/L silver nitrate to the medium. Incubation of cultures initially in the dark followed by 16/8-hour light incubation facilitated axillary shoot elongation. On the basis of our findings, it is recommended to culture the regenerated axillary shoots of fegra fig onto MS medium containing 3% sucrose, 1.5 mg/L activated charcoal, and 3 mg/L silver nitrate to manage STN effectively.

Multiplication of apical and axillary shoots of coffee arabica using various types of cytokinins

Abstract In-vitro shoot multiplication presents a promising avenue for replicating the genetic characteristics of parent plants, commonly utilized in various plant biotechnological techniques such as mutation replication, protoplast fusion, anther culture, and genetic engineering. The primary aim of this research was to assess the impact of different cytokinins on the proliferation of apical and axillary shoots in Arabica coffee multiplication. A Completely Randomized Factorial Design was employed, considering two factors: explant source and cytokinin type. Explants were sourced from both apical and axillary branches of AS2K Arabica coffee plantlets. Three cytokinins, namely 6-Benzyl adenine purine (BAP), isopentenyl adenine (2-IP), and kinetin, were incorporated into the Murashige and Skoog (MS) 1962 basal medium at concentrations of 0, 1, 2, and 3 mg/l. The findings revealed significant effects of all three cytokinins on shoot number, plantlet height, and leaf growth, albeit without influencing root development. Notably, axillary shoot-derived explants demonstrated a more pronounced effect on shoot count compared to those from apical shoots. Among the cytokinin treatments assessed, BAP at a concentration of 2 mg/l in the MS medium yielded optimal results for propagating the AS2K variety of Arabica coffee, thus representing the recommended medium for this purpose.

Cost Effective Methods of in Vitro Multiple Shoot Production in Two Commercial Diploid Bananas of Kerala

Cost-effective methods for in vitro propagation of two commercial diploid bananas of Kerala, namely Musa (AB) ‘ Njalipoovan’ and Musa (AA) ‘ Nivedyakadali’ were carried out to reduce unit production cost. Sword sucker, peeper sucker and eye bud explants were found to be equally effective concerning the number of axillary shoots from a single explant in both cultivars. Sucrose 3.0 per cent and table sugar 2.0 per cent produced the maximum number of multiple shoots in Njalipoovan. In the case of Nivedyakadali, table sugar at 2.0 per cent recorded the maximum number of multiple shoots. Half strength of vitamins in full MS medium observed more multiple shoots in cv. Njalipoovan. Half a tablet of vitamin B complex (2.0mgl-1) in the medium resulted in more shoots in the case of cv. Nivedyakadali. Agar 0.7 per cent recorded a significantly higher number of multiple shoots per culture in both cultivars. The treatments involving different types of paper support (filter paper, brown paper and ordinary paper) in liquid media were inferior to agar 0.7 per cent treatment.

Comprehensive analysis of little leaf disease incidence and resistance in eggplant

BackgroundLittle leaf disease caused by phytoplasma infection is a significant threat to eggplant (also known as brinjal) cultivation in India. This study focused on the molecular characterisation of the phytoplasma strains and insect vectors responsible for its transmission and screening of brinjal germplasm for resistance to little leaf disease.ResultsSurveys conducted across districts in the Tamil Nadu state of India during 2021–2022 showed a higher incidence of phytoplasma during the Zaid (March to June), followed by Kharif (June to November) and Rabi (November to March) seasons with mean incidence ranging from 22 to 27%. As the name indicates, phytoplasma infection results in little leaf (reduction in leaf size), excessive growth of axillary shoots, virescence, phyllody, stunted growth, leaf chlorosis and witches’ broom symptoms. PCR amplification with phytoplasma-specific primers confirmed the presence of this pathogen in all symptomatic brinjal plants and in Hishimonus phycitis (leafhopper), providing valuable insights into the role of leafhoppers in disease transmission. BLAST search and phylogenetic analysis revealed the phytoplasma strain as “Candidatus Phytoplasma trifolii”. Insect population and disease dynamics are highly influenced by environmental factors such as temperature, relative humidity and rainfall. Further, the evaluation of 22 eggplant accessions revealed immune to highly susceptible responses where over 50% of the entries were highly susceptible. Finally, additive main effect and multiplicative interaction (AMMI) and won-where biplot analyses identified G18 as a best-performing accession for little leaf resistance due to its consistent responses across multiple environments.ConclusionsThis research contributes essential information on little leaf incidence, symptoms, transmission and resistance profiles of different brinjal genotypes, which together ensure effective and sustainable management of this important disease of eggplants.

Meta-Topolin as an Effective Benzyladenine Derivative to Improve the Multiplication Rate and Quality of In Vitro Axillary Shoots of Húsvéti Rozmaring Apple Scion.

In vitro mass propagation of apple plants plays an important role in the rapid multiplication of genetically uniform, disease-free scions and rootstocks with desired traits. Successful micropropagation of apple using axillary shoot cultures is influenced by several factors, the most critical of which is the cytokinin included in the culture medium. The impact of medium composition from single added cytokinins on shoot proliferation of apple scion Húsvéti rozmaring cultured on agar-agar gelled Murashige and Skoog medium fortified with indole butyric acid and gibberellic acid was investigated. The optimum concentration for efficient shoot multiplication differs according to the type of cytokinin. The highest significant multiplication rate (5.40 shoots/explant) was achieved using 2.0 μM thidiazuron while the longest shoots (1.80 cm) were observed on the medium containing benzyladenine at a concentration of 2.0 μM. However, application of either thidiazuron or benzyladenine as cytokinin source in the medium resulted in shoots of low quality, such as stunted and thickened shoots with small leaves. In the case of benzyladenine riboside, the 8 μM concentration was the most effective in increasing the multiplication rate (4.76 shoots/explant) but caused thickened stem development with tiny leaves. In the present study, meta-topolin was shown to be the most effective cytokinin that could be applied to induce sufficient multiplication (3.28 shoots/explant) and high-quality shoots along with shoot lengths of 1.46 cm when it was applied at concentrations of 4 μM. However, kinetin was the least active cytokinin; it practically did not induce the development of new shoots. The superior cytokinin for in vitro axillary shoot development of apple scion Húsvéti rozmaring with high-quality shoots was the meta-topolin, but it may be different depending on the variety/genotype under study.

Micropropagation and Genetic Fidelity of Fegra Fig (Ficus palmata Forssk.) and Grafting Compatibility of the Regenerated Plants with Ficus carica.

Ficus palmata is an important fig species that produces edible and nutritious fruit and possesses several therapeutic uses. This study reports an effective method for the micropropagation of F. palmata using nodal explants. In vitro shoots were cultured for 7 weeks onto MS medium fortified with different concentrations of cytokinins, light intensities, sucrose concentrations, and light/dark incubation treatments. Optimal axillary shoot proliferation (10.9 shoots per explant) was obtained on a medium containing 30 g/L sucrose and supplemented with 2 mg/L 6-benzylaminopurine (BAP) under 35 μmol/m2/s light intensity. Dark incubation limited the foliage growth but favored shoot elongation and rooting compared with light incubation. Elongated shoots, under dark conditions, were rooted (100%; 6.67 roots per explant) onto MS medium containing 1 mg/L indole-3-acetic acid (IAA) and 1.5 g/L activated charcoal. The micropropagated plantlets were acclimatized with a 95% survival rate. In this study, the genetic fidelity of micropropagated F. palmata clones along with their mother plant was tested using randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and start codon targeted (SCoT) molecular markers. The genetic similarity between the micropropagated plantlets and the mother plant of F. palmata was nearly 95.9%, assuring high uniformity and true-to-type regenerated plants. Using micropropagated F. palmata plantlets as a rootstock proved appropriate for the grafting F. carica 'Brown Turkey'. These findings contribute to the commercial propagation and production of the fig crop.

Synthesis and Accumulation of Phytocompounds in Field-, Tissue-Culture Grown (Stress) Root Tissues and Simultaneous Defense Response Activity in Glycyrrhiza glabra L.

Harsh climates, i.e., drought, extreme temperatures, and toxic gases, pose issues to agriculture by altering plants’ growth and yield. Biotechnology with biochemical defense approaches is beneficial for generating new plants/varieties with extra resilience to adverse conditions. In response to stress, cultures show an enriched level of secondary metabolite synthesis. Here, an efficient in vitro propagation method using axillary shoot proliferation, along with callus formation, was established in Glycyrrhiza glabra L. The phytochemical composition of in vitro and in vivo grown tissues was analyzed using a gas chromatography–mass spectrometry (GC–MS) technique, and the biochemical attributes were measured and compared in different investigated tissues. Callus formation from root explants was achieved with a frequency of 88.89% on MS medium containing 2.0 mg/L BAP and 0.5 mg/L 2,4-D. Axillary shoot proliferation was obtained from dormant buds when cultured onto MS supplemented with BAP alone, or in combination with, IAA. The maximum shoot proliferation (94.44%) was recorded on MS with 1.0 mg/L BAP with an average shoot length of 10.5 cm. The regenerated shoots were subcultured and transferred to the root induction medium, supplemented with various concentrations of IAA/IBA, wherein 2.0 mg/L IBA resulted in the best rooting frequency (88.89%). The GC–MS-based phytocompounds analysis of the methanolic extracts of root-derived callus and in vivo- and in vitro- grown root tissues was conducted. These samples revealed the presence of more than 35 therapeutically important bioactive compounds, such as methylglabridin, sitosterol, lupeol, squalene, stearic acid, linoleic acid, etc. The biochemical parameters, like total phenolic content, flavonoid content, DPPH scavenging activity, superoxide dismutase, and peroxidase activity were also measured. All the biochemical attributes were found to be higher in in vitro derived roots than the callus and in vivo grown root (donor) samples. These findings demonstrated that callus (root derived) and in vitro roots are a stable and potent source of multiple phytocompounds, encompassing medical significance with wide applications. This study may serve as an alternative opportunity in the sustained and continuous synthesis of important compounds without harming natural vegetation and normal environment.

Optimization of In Vitro Regeneration of Pinus peuce (Gris.)

Pinus peuce (Macedonian pine) is considered a valuable ornamental tree that is frequently planted in parks and gardens, especially in Western Europe. This endemic pine is one of the most valuable conifer species in its native range, which currently consists of only two disjunct populations restricted to small mountainous areas of the Balkans and is listed as a near-threatened species. The reproduction of Macedonian pine by seed is limited, so in vitro propagation methods have emerged as a promising tool for large-scale propagation. The objective of this study was to develop an improved system for the micropropagation of P. peuce from juvenile plant material using a short-term liquid cytokinin pulse. For that, explants derived from 4-week-old seedlings were pulse-treated with different concentrations of N6-benzyladenine (BA) for 1 or 2 h to stimulate the induction of axillary buds. The highest axillary shoot formation was achieved with 222 µM BA pulse treatment, with an average number of ~six shoots per explant. Elongated shoots (≥10 mm) were detached from the explants and pulse-treated with 0.27 or 1.08 mM α-naphthaleneacetic acid (NAA) or 0.25 or 0.98 mM indole-3-butyric acid (IBA) for 1 or 2 h. IBA was more effective than NAA and led to a maximum rooting percentage (up to 40%) and the highest number of acclimatized plants (15–20%). Rooted plants were successfully transferred to ex vitro conditions.

Scion–Rootstock Combination Determines Pruning Responses in Young Almond Trees

Almond growers are seeking ways to reduce costs but maintain yield. Intensive planting systems with greater planting densities using trees on growth-controlling rootstocks, combined with mechanical pruning and shake-and-catch harvesting are becoming popular. In this study we examined the responses of six almond cultivars with distinctive architecture grafted onto five rootstocks with varying degrees of vigor control. Trees were planted in 2018 in a nursery row and left to grow without pruning until Winter 2021. Pruning involved a rudimentary hedging treatment akin to mechanical pruning. Branching and tree structure were recorded in 2020, before pruning, and again at the end of 2021, after one season’s growth following pruning. A rating system was developed to record qualitative data on central leader dominance and the number, length, basal diameter, and, in some cases, branching angle of axillary shoots and including scaffold branches. Relatively few changes were recorded in the basic growth habit of these trees in response to pruning. Before pruning, the most common rootstock effect was on axillary shoot production. After pruning, the most common rootstock effects were on scaffold branching and the length of subterminal axillary shoots. Further studies are required to determine how these differences produced by the interaction between pruning and rootstock may affect the productivity of fruit-bearing trees. Although in this study with young trees we were not able to record crop yield, the results highlight that it is mainly the scion–rootstock combination, with or without pruning, that determines the potential productivity of fruiting canopies. Scion–rootstock combinations that produce narrow upright canopies naturally with strong central leader dominance and highly branched canopies are preferred for superintensive growing systems with or without use of mechanical hedging.

Optimization of Tissue Culture Medium for Little-leaf Mockorange (Philadelphus microphyllus A. Gray) by Adjusting Cytokinin and Selected Mineral Components

Little-leaf mockorange is a native plant species with desirable characteristics for landscape use. The need to conserve specific genotypes and the difficulty of seed propagation and stem cutting propagation make axillary shoot micropropagation a good option for this species. A series of experiments were completed individually with the goal to improve in vitro propagation protocols by evaluating different types of cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), and thidiazuron (TDZ)] at 0, 1.1, 2.2, 4.4, or 8.8 µM. Selected minerals (0 to 60 mM or 0 to 45 mM N, 0 to 500 µM or 0 to 100 µM Fe, 0 to 3 mM Ca, 0 to 1.5 mM Mg, or 0 to 1.25 mM P) were also tested separately in the tissue culture medium; the base medium was ½ strength MS in these mineral experiments. At the end of each experiment (8 or 12 weeks), plant growth characteristics including number of axillary shoots, shoot height, and dry weight were determined. Of the six cytokinins tested, Zea produced the largest increase in shoot growth. Supplementation with 1.1 µM Zea resulted in the most shoot dry weight, almost 2.5-fold more than control shoots. Shoots on 0.55, 1.1, or 2.2 µM Zea were at least 64% taller than control shoots. Shoots placed on regular ½ strength MS basal salts, described above, and/or media lacking the nutrient of interest, were used as a positive and negative control treatments. For each separate mineral tested, the best concentration for optimum shoot growth was the concentration of that mineral used in ½ strength MS medium. A medium containing mineral concentrations of 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, and 0.625 mM P, and 1.1 µM Zea should be used to produce the optimum in vitro shoot growth of little-leaf mockorange.

Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems.

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors.

Axillary shoot proliferation and regeneration of red rose [pisiformis (christ.) D. Sosn. An under thread extinction species endemic to Turkey

In vitro propagation of red rose [Rosa pisiformis (Christ.) D. Sosn.] has great importance in rapid proliferation of species with medicinal features and in culture of healthy plants free from deseases. After a succesfully pre-sterilization procedure, experiments were maintained by two-phase: multiplication phase and rooting of microshoots phase. Micropropagation of Rose was improved, using its nodal segments under different combination of BAP (0, 1.0, 2.0, 0.6 mg/l), NAA (0, 0.1, 0.2, 0.01 mg/l) on Driver and Kuniyuki (DKW) and MS medium in multiplication phase and using distinct compositions of IBA (0, 1.0, 3.0, 5.0 mg/l) and DKW medium in rooting phase. The maximum number of shoots/per plant (22.6) with the highest number of leaves (222.6) were obtained in DKW medium supplemented with 0.6 mgl-1 BAP and 0.01 mg/l NAA. The leaf explants were also used to occur callus in MS with different combinations of BAP and NAA. Good callus formation was obtained with 1:2 ratio of BAP:NAA combination in MS medium. Furthermore, the highest root induction (100%) was achieved in DKW medium consisting of 1 mg/l IBA. The rooted plantlets were transferred to the field after acclimatized in greenfield conditions. The present investigation presents an in vitro protocol for R. pisiformis. Bangladesh J. Bot. 52(4): 949-958, 2023 (December)

Comprehensive Analysis of Highbush Blueberry Plants Propagated In Vitro and Conventionally.

In vitro culture allows the production of numerous plants with both desirable and undesirable traits. To investigate the impact of the propagation method on highbush blueberry plants, an analysis was performed on four groups of differentially propagated plants: in vitro with axillary (TC-Ax) or adventitious shoots (TC-Ad), conventionally (SC) and using a mixed method (TC/SC). The analysis included plant features (shoot length and branching, chlorophyll and fluorescence and DNA methylation) and fruit properties (antioxidant compounds). The data obtained indicated significant differences between plants propagated conventionally and in vitro, as well as variations among plants derived from in vitro cultures with different types of explants. SC plants generally exhibited the lowest values of morphological and physiological parameters but produced fruits richest in antioxidant compounds. TC/SC plants were dominant in length, branching and fluorescence. Conversely, TC-Ax plants produced fruits with the lowest levels of antioxidant compounds. The methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect molecular differences. TC-Ad plants showed the highest methylation level, whereas SC plants had the lowest. The overall methylation level varied among differentially propagated plants. It can be speculated that the differences among the analysed plants may be attributed to variations in DNA methylation.

Evaluation of Stevia rebaudiana Leaf-Axillary Shoot Formation, Cultured in MS Medium Supplemented with IAA-BAP and MS Medium Supplemented with Kinetin

Stevia rebaudiana leaves can be used as a sweetener alternatives because they contain steviol glycoside derivative compounds, including steviosides and rebaudioside-A. Propagation of Stevia is more optimally carried out using in vitro culture when compared to conventional propagation through seeds or cuttings. This study aimed to evaluate the formation and growth of Stevia shoots and leaves in MS medium containing a mixture of IAA and BAP with MS medium containing kinetin only, as well as evaluating the use of a liquid medium containing kinetin. Stevia was initiated from apical shoot then grown in MS medium containing a mixture of IAA and BAP with MS medium containing kinetin only. Stevia was subcultured every 4 weeks. Several parameters measured were number of axillary shoots and number of leaves. It was transferred into a liquid medium for 7 days. The results showed that the formation and growth of axillary buds and leaves at the initiation stage were better in medium containing IAA and BAP compared to medium containing single hormone kinetin. At the stage of shoot multiplication and maintenance, cultivation in semi-solid medium containing kinetin showed more leaves and axillary shoots compared to that cultivated in semi-solid medium with the addition of IAA and BAP. Plants acclimatized in liquid medium supplemented with 1 ppm kinetin showed fast plant growth but were not accompanied by sturdy stem growth. The presence of brownish color on certain parts of the plant such as in some leaves and stems was also observed.

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