Half-strength Murashige And Skoog Medium Research Articles

Micropropagation of Al-Taif Rose: Effects of Medium Constituents and Light on In Vitro Rooting and Acclimatization

Micropropagation facilitates the rapid production of roses. Nevertheless, in vitro rhizogenesis of essential oil roses has presented significant challenges, primarily attributed to low rates of rooting and poorer acclimatization compared to ornamental rose varieties. This study reports the optimization of in vitro rooting of Al-Taif rose (Rosa damascena f. trigintipetala (Diek) R. Keller) microshoots with the aim of increasing survival rate during acclimatization. We also investigated the effects of various parameters, including type and concentration of auxin (i.e., 2,4-Dichlorophenoxyacetic acid (2,4-D), indole acetic acid (IAA), indole butyric acid (IBA), and naphthaleneacetic acid (NAA) at concentrations of 0, 0.05, 0.1, 0.2, and 0.4 mg/L), salt strength (i.e., full- and half-strength Murashige and Skoog (MS) medium), sucrose concentration (i.e., 20, 30, 40, 60, and 80 g/L), light spectra (a 2:1 or 1:2 blue/red spectral ratio, cool or warm white light at a 1:1 ratio, and fluorescent light), light intensity (photosynthetic photon flux density (PPFD) values of 25, 50, and 100 µmol·m−2·s−1), and activated charcoal (i.e., 0 and 0.5 g/L) on the rooting and growth of in vitro regenerated Al-Taif rose axillary shoots. We found that half-strength MS medium supplemented with 0.2 mg/L NAA, 80 g/L sucrose, 0.5 g/L activated charcoal, and 50 μmol·m−2·s−1 PPFD were the optimal conditions for 100% induction of adventitious roots. Next, micropropagated Al-Taif rose plantlets were successfully transferred to a potting medium containing perlite/peatmoss (in a 1:1 ratio). We found that 98% of plants survived ex vitro conditions. The genetic fidelity of micropropagated Al-Taif rose clones along with their mother plant was tested using the inter-simple sequence repeats (ISSR) molecular marker. The genetic similarity between the micropropagated plantlets and the mother plant of Al-Taif rose plants was 98.8%, revealing high uniformity and true-to-type regenerated plants. These findings may therefore contribute toward the commercial micropropagation of Al-Taif roses.

A Comprehensive Study on the Micropropagation of Costus igneus: Media Composition, Growth, and Development

Costus igneus, commonly known as insulin plant, is an ornamental plant valued for its spiral ginger- like foliage and medicinal properties. Micropropagation is an efficient method for rapid multiplication of Costus igneus. This study aims to determine the optimal media composition and culture conditions for in vitro propagation of C. igneus. Axillary bud explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators including auxins (indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid) and cytokinins (6- benzylaminopurine and kinetin). Microshoots cultured on MS + 1.5 mg/L BAP showed the highest shoot proliferation rate (98%) and maximum number of shoots per explant (12.6). Elongated microshoots were rooted on half-strength MS medium supplemented with different auxins. The highest rooting percentage (95%) and maximum number of roots per shoot (9.8) were observed on medium containing 2.5 mg/L IBA. The regenerated plantlets were acclimatized and successfully transferred to pots with 80% survival rate. Morphological and phytochemical analysis showed no significant differences between the in vitro propagated and mother plants. This study demonstrates the potential of micropropagation for large scale production of quality planting material of C. igneus. Further studies on genetic and epigenetic stability are recommended to validate this protocol for commercial applications.

An efficient in vitro organogenesis protocol for the endangered relic tree species Bretschneidera sinensis and genetic fidelity assessment using DNA markers.

Bretschneidera sinensis is a monotypic species of rare and tertiary relic trees mainly distributed in China. B. sinensis is a potentially valuable horticultural plant, which has significant ornamental and research value, and is a crucial tool for the study of phylogeography. The artificial cultivation of B. sinensis is of great scientific value and practical significance. In this study, we developed a direct organogenesis process of B. sinensis using mature zygotic embryos as initial materials. The highest sterile germination induction (54.5%) from the mature zygotic embryo was obtained in a Murashige and Skoog (MS) medium with 2.0 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg·L-1 α-naphthaleneacetic acid (NAA). The highest percentage of shoot regeneration (90.37%) was attained using 1.0 mg·L-1 6-BA and 0.01 mg·L-1 NAA in the MS medium. The Woody Plant Medium (WPM) had the greatest adventitious shoot elongation rate of 93.33%. The most optimized rooting rate was 88.89% in a half-strength MS medium containing 2.0 mg·L-1 indole-3-butyric acid (IBA) and 1.0 mg·L-1 NAA. The genetic fidelity of in vitro regenerated plantlets was assessed using inter-simple sequence repeats and random amplified polymorphic DNA molecular markers, confirming the genetic uniformity and stability of regenerated B. sinensis plantlets. Our research presents an effective in vitro propagation system for B. sinensis, laying the groundwork for its germplasm conservation and large-scale production while maintaining high genetic integrity.

Otimização da micropropagação de maçã silvestre ‘Zi Dieer’ através da proliferação de brotos axilares

ABSTRACT: Malus is an important ornamental plant around the world and widely used in commercial production due to its diversity and wide adaptability. ‘Zi Dieer’ crabapple has good adaptability to a wide temperature range (high and low), disease resistance, and high commercial value. To satisfy the market demand, we established ‘Zi Dieer’ crabapple micro-regeneration system for large-scale reproduction. Induced buds were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP: 0.5, 1.0, 1.5 mg/L) and 1-naphthaleneacetic acid (NAA: 0.05, 0.1, 0.3 mg/L) for 35 days. The highest bud induction rate (93.3%) was observed on MS medium supplemented with 1.0 mg/L BAP and 0.1 mg/L NAA, with average micro-shoot length of 1.32 cm. The germinated buds (1 cm in length) were propagated and cultured on MS medium supplemented with BAP (1.0, 1.5, 2.0 mg/L) and NAA (0.1, 0.3, 0.5 mg/L), after 35 days, on the MS basal medium supplemented with 1.5 mg/L BAP and 0.3 mg/L NAA, the best average number of new shoots is 8.56±0.13 (P<0.05) and good shoot length (4.94±0.09 cm), reproduction coefficient higher than MS medium (0) without PGRs. In order to induce rooting, the shoots (length 2 cm) are inserted in half strength MS (1/2 MS) medium containing 0.1, 0.3 and 0.5 mg/L of 3-indole butyric acid (IBA) or NAA, and 1 g/L activated carbon (AC) was added on the basis of NAA 0.3 mg/L to study the effect of activated carbon on the rooting of crabapple. After 38 days of culture, compared with other treatments, among the explants treated with IBA, the rooting rate was higher in the 1/2 MS medium containing 0.1 mg/L (82.1%), and the highest root number (11.29±0.52) was observed, and the root length was the other treatments 2-3 times. Although, the highest rooting rate was observed on 1/2 MS medium containing 0.1mg/L NAA (87.8 %), but the bottom of these buds formed large callus, which was not conducive to survival, and the root length was short. In summary, a feasible and complete rapid propagation program for ‘Zi Dieer’ crabapple has been developed.

In Vitro Propagation of "Salsaparrilha" [Herreria salsaparrilha Mart. (Asparagaceae)]: An Antisyphilitic Medicinal Species.

An efficient procedure for in vitro propagation of Herreria salsaparrilha Martius was established from single-node explants (fourth and fifth nodes from apex to the base) derived from donor plants maintained under shading-house conditions. After surface sterilization, explants are inoculated in test tubes containing 15mL of Murashige and Skoog (MS) medium without growth regulators. Cultures are maintained under 35μmolm-2s-1 irradiance, a 16/8-h light/dark light regime, at 26±2°C. The subcultures are carried out under the same conditions, adding 6-benzyladenine 1.0mg/L and Phytagel® 2.8g/L. Shoots are elongated and rooted by transferring individual shoots to half-strength MS medium without growth regulators. After 25-30days, elongated rooted shoots are transferred to plastic pots containing 25-30mL of sterile distilled water, covered with a transparent plastic bag, and kept under the same growth room conditions for 2days. Plants are transferred to cups containing autoclaved and washed sand and kept in a shading house (50% light interception) for acclimatization. True-to-type adult plants were successfully recovered under ex vitro conditions.

In Vitro Micropropagation, Rooting and Acclimatization of Two Agastache Species (A. aurantiaca and A. mexicana)

Agastache aurantiaca ‘Sunset Yellow’ and A. mexicana ‘Sangria’ are aromatic plants with edible flowers characterized with good flavors, nutritional values and pleasant aromas. The aim of this study was to establish a successful protocol to ensure fast shoot propagation, rooting and in vivo acclimatization to obtain many vigorous plants for edible flower production. Micropropagation was tested with an agarized Murashige and Skoog (MS) medium enriched with different 6-benzylamino purine (BAP) levels. In vitro rooting trials were carried out using a half-strength MS medium comparing the traditional jar method with an agarized medium and the temporary immersion system (TIS) bioreactor with a liquid medium. After acclimatization in a greenhouse, the survival of plants and their development were recorded. Microscopical analyses were performed on both in-jar and in-TIS leaves at rooting stages and compared with in vivo leaves. In both species, the greatest number of new shoots was noted at 2.22 µM and 3.33 µM of BAP. In TIS, rooting started earlier, resulting in larger plants with more roots and longer roots than in the jar substrate. Any anatomical difference was observed in leaves collected from the jar and TIS during the rooting stage. The best acclimatization performances were recorded in plants coming from the TIS bioreactor.

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant
 Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and BAP (3.0 mg/L) in combination with IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively.
 Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded.
 Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period.

Development of an Efficient and Rapid Micropropagation Protocol for In Vitro Multiplication of Maerua crassifolia Forssk

The difficult propagation of shrub and tree species and their extensive exposure to grazing threaten their abundance and lead to the necessity to find alternative means of propagation for these species. In vitro micropropagation techniques, viz., tissue culture, offer a promising tool for the rapid, cost-effective, and efficient propagation of different plant species. In the current study, a rapid and efficient in vitro multiplication protocol was developed for the micropropagation of Maerua crassifolia Forssk. Our results revealed that Murashige and Skoog (MS) medium with 7.5 µM of 6-benzylaminopurine (BA) and 1.0 µM of 1-naphthaleneacetic acid (NAA) led to the highest shoot formation (13.9 shoots per explant in 85.7% of the cultivated hypocotyls) among all other treatments. The best in vitro root formation was obtained on half-strength MS medium with 1.0 µM of indole 3-butyric acid (IBA) as 94.1% of the cultivated shoots formed 6.8 roots per microshoot on average. Ninety percent of the rooted plantlets were successfully acclimatized and are currently growing in the botanical garden of the Botany and Microbiology Department, King Saud University, Riyadh, Saudi Arabia. The genetic fidelity of the micropropagated plants was authenticated via flow cytometry. The results of the current study explained a simple, cost-effective, and efficient protocol for the micropropagation of the endangered M. crassifolia trees.

Effects of Genotype, Light, and Plant Growth Regulators on Rhizome Browning, Proliferation, and Sprouting in Cymbidium

Chinese cymbidiums are important flowering ornamental plants. Traditional propagation via seed or division cannot satisfy growers’ demand for commercialization of new cultivars, and in vitro propagation has a low micropropagation efficiency due to the browning of rhizomes. In this study, rhizomes of Cymbidium ‘14-16-13’ and ‘14-16-5’ were cultured on half-strength Murashige and Skoog (MS) medium supplemented with 6-benzyl aminopurine (BAP), NAA (α-napthaleneacetic acid), or BAP with NAA under either the dark or light. The degree of browning was read, and rhizome proliferation or sprouting (sprout numbers) was evaluated. Results showed that there was significant difference in browning grade of rhizomes between ‘14-16-13’ and ‘14-16-5’ regardless of dark and light culture. Dark culture induced rhizome proliferation but failed to induce sprouts. Light culture slightly elevated the degree of browning but induced sprouting. Among the growth regulators evaluated, BAP was more effective for sprout induction. As rhizome browning appeared to be inevitable in micropropagation of the cymbidiums, a compromise between browning and sprout production could be a realistic approach. Our study showed that rhizomes cultured on half-strength MS medium supplemented with 1.5 mg·L−1 BAP were able to produce more than 16 sprouts per vessel even though browning occurred in the rhizomes. Thus, culturing rhizomes in this medium could be a practical solution for in vitro propagation of Chinese cymbidiums.

Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass.

The present study describes an efficient protocol for the micropropagation of Centratherum punctatum Cass. through transverse thin cell layers (tTCLs). The effect of plant growth regulators (PGRs), thickness of tTCLs, and source of tTCLs were evaluated. The tTCLs of varying thickness (0.5 to 5.0 mm) were excised from 10-d-old leaf and 45-d-old node and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of PGRs (BAP, TDZ, KN; 0.1 to 2.0 mg L−1), alone or in combination with NAA (0.1 to 1.0 mg L−1) for shoot induction. The leaf and node tTCLs responded with direct shoot regeneration. A significant effect of thickness or width on shoot induction was observed. For leaf, 1.0-mm-wide and for node 2.0-mm-thick tTCLs showed a maximum response. MS medium supplemented with 1.5 mg L−1 TDZ and 1.0 mg L−1 BAP in combination with 0.2 mg L−1 NAA was found to be optimum for shoot induction from tTCLs of leaf (96% response with 41.5 shoots per explant) and node (87% response with 11.9 shoots per explant), respectively. Rhizogenesis was obtained when micropropagated shoots were transferred to half strength MS medium supplemented with various concentrations (0.25 to 2.0 mg L−1) of IBA. The rooted plantlets were eventually acclimatized and transferred to soil. The clonal fidelity assessment by SCoT markers of the mother plant and micropropagated plants revealed their genetic similarity. Chlorophyll content estimation indicated lower levels in in vitro plants, which increased to a comparable level to that of field-grown plants after 3 mo of acclimatization, showing absence of stress during acclimatization. The procedure described here is a promising tool for micropropagation of C. punctatum as it produces high-frequency healthy shoots from a minimum explant source.

Optimization of Hormonal Compositions of Media for In vitro Propagation of Apple (Malus ×domestica Borkh.) Cultivars

Background: Apple (Malus × domestica Borkh.) is one of the most important fruit crops in the world. Traditionally, vegetative propagation methods (including cutting, budding, and layering) are time-consuming (about three years), with low production rates and low success in obtaining virus-free plants. Objective: The present study was planned to investigate the in vitro propagation of apple (M. domestica) cultivars from nodal segments. Methodology: The Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of plant growth regulators (PGRs) were used for shoot proliferation and root induction. The optimal concentrations of PGRs in the MS medium were assessed. The effect of full and half-strength MS medium on root induction was investigated. Results: Examination of the effects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin revealed that the significantly highest shoot response was recorded for the ‘Princess’ cultivar with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), and leaf number per shoot (6.15) after four weeks of culture. The root induction in micro shoots of three apple cultivars was observed after 20 days of culturing. The strength MS medium (full and half) containing 1.5 mg/L IAA significantly affected (at P<0.05, chi-square test) root induction in all three apple cultivars, especially rooting rate. However, there was no significant difference in root number and root length per micro shoot among the apple cultivars. Among the cultivars, significantly the highest rooting rate(48.30%), root number (6.25), and root length (4.15cm) were recorded for cultivar ‘Princess’ on full-strength MS medium. Conclusion: PGR combination of IAA (1.0 mg/L) and kinetin (3.0 mg/L) was found to be the best for shoot proliferation. The shoot responses were found to increase with an increase in kinetin concentration combined with IAA at 1.00 mg/L.

In Vitro Seed and Clonal Propagation of the Mediterranean Bee Friendly Plant Anthyllis hermanniae L.

Anthyllis hermanniae L. (Fabaceae) is a perennial Mediterranean shrub with the potential to be used as a bee-friendly ornamental plant in arid and semi-arid regions, valued for its tolerance of barren soils, winds, and strong temperature changes. With the aim of facilitating the introduction of the species into the horticulture industry, its in vitro seed and clonal propagation was investigated for the first time, to our knowledge. Seeds stored in the dark at room temperature for 4, 7, 9, 12, and 18 months germinated at percentages higher than 80% after scarification, when incubated in vitro in solid half-strength Murashige and Skoog (MS) medium at temperatures from 10 to 25 °C, while photoperiod (continuous darkness or 16 h light period/8 h dark) during incubation did not affect germination. Explants excised from in vitro grown seedlings established at higher percentages compared to explants from adult native plants, more efficiently in MS medium with 1.0 mg L−1 6 N benzyladenine (BA). During subcultures in the same medium, juvenile explants formed more and longer shoots than adult ones. Almost all adult explants formed shoots when subcultured in MS medium with 0.0 to 4.0 mg L−1 BA, zeatin, kinetin or 6-(γ,γ-dimethylallylamino)purine (2iP). ΒA at 0.5 to 2.0 mg L−1 induced many more shoots (17–21) per explant and much higher multiplication indices compared to all other cytokinins, while longer shoots were produced in a medium without hormones or with 0.5–1.0 mg L−1 2iP. Microshoots cultured in half-strength MS medium with 0.0–4.0 mg L−1 indole-3-butyric acid rooted at highest percentage (around 70%) in the medium containing 4.0 mg L−1 IBA, while microshoots of juvenile origin developed more and longer roots compared to adult ones. Micropropagated plantlets were successfully acclimatized ex vitro (>97%), regardless of their origin. The efficient micropropagation of A. hermaniae will facilitate its sustainable exploitation as a bee-friendly landscape plant, a forage plant for honeybees in Mediterranean areas, and a medicinal plant.

Assessment of Genetic Stability on In Vitro Propagation of Ardisia crenata var. bicolor Using ISSR Markers

Ardisia crenata var. bicolor is a multi-purpose plant and has important ornamental and medicinal properties. Conventional methods of propagating the species from seeds and cuttings have low efficiency because of the recalcitrant properties of seeds and low survival rate of high-quality cuttings. This work aims to study the in vitro regeneration protocol for direct organogenesis from nodal segments of A. crenata var. bicolor on Murashige and Skoog (MS) medium, with different combinations and concentrations of plant growth regulators (PGRs). The treatments used for the establishment and proliferation of shoots included MS medium supplemented with different concentrations of Benzyl-aminopurine (BAP) and indole-3-butyric acid (IBA). For rooting, IBA was used in combination with naphthaleneacetic acid (NAA) in full- and half-strength MS media. Maximum shoot establishment (76.67%) and the highest shoot length (6.6 cm) were observed on MS medium with 1.0 mg·L−1 BAP with 0.5 mg·L−1 IBA, while BAP at 1.0 mg·L−1 with 0.25 mg·L−1 IBA obtained the highest shoot proliferation (4.5 ± 1.53). The best rooting response (83.33%) was achieved on half-strength MS including 1.0 mg·L−1 IBA with 0.25 mg·L−1 NAA, and the maximum survival rate of 84.4% was observed after acclimatization under 75% shading. To define their genetic stability, using eleven primers of ISSR markers to assess the genetic stability of the unstable leaf color samples compared with their mother plant, the ISSR markers demonstrated a level of genetic polymorphism in plantlets, but without other morphological variations. This indicates the genetic resemblance to the mother plant and the reliability of this protocol for the efficient micropropagation of A. crenata var. bicolor.

STANDARDIZATION OF MICROPROPAGATION TECHNIQUE OF MULBERRY (MORUS ALBA L.)

A standardization of protocol for rapid multiplication of mulberry (Morus alba L.) was established in vitro using nodal explant excised from hydroponically grown mature mulberry plants. Shoot initiation was induced on Murashige and Skoog (MS) medium supplemented with different concentrations of (BAP) 6- Benzyl amino purine. Effect of different concentration of 6- Benzyl amino purine was studied on multiplication of shoot, revealed that elongated shoots (2-3 cm) were cultured on half strength MS medium supplemented with (IBA) indole-3-butyric acid. Higher percentage of shoot initiation (90%) was observed on MS medium supplemented with 2 mg/l 6-bezyl amino purine. Initiated shoots gave a higher average number of shoots and elongation was observed when it was subcultured on MS medium supplemented with 1.4 mg/l 6-bezyl amino purine. A maximum percentage of root formation (70%) was observed when elongated shoots were subcultured on half strength MS medium containing 1.3 mg/l indole-3-butyric acid. Rooted plants transferred to pots containing sterile FYM, garden soil and sand (1:1:1) for acclimatization in green house showed (75%) survival capacity.

In Vitro Propagation of the Mount Parnitha Endangered Species Sideritis raeseri subsp. Attica

Over the past few decades, both wildfires and human-sparked fires have ravaged Mount Parnitha, destroying the mountain’s unique natural ecosystem, applying pressure to its flora, and subjecting the vulnerable populations of Sideritis raeseri subsp. attica to excessive stress. The present study aims to establish an efficient micropropagation method starting from in vitro-grown seedlings. The in vitro germination study carried out during the production of seedlings revealed a higher germination rate (34.0% and 37.0%, respectively) at 20.0 °C and 25.0 °C. The in vitro-derived seedlings studied were used as the starting material for the establishment of various media. Murashige and Skoog (MS) media, hormone-free and containing 0.5 mg L−1 6-benzyladenine (BA), led to the satisfactory (84.0–89.0%) establishment of plantlets. During the multiplication phase, the study used BA in conjunction with α-naphthaleneacetic acetic acid and four different cytocinins (BA; kinetin (KIN); 6-(γ-γ-dimethylallylamino) purine; zeatin) at low concentrations (0.5 mg L−1). During the second subculture, a high multiplication index (7.3 and 6.4, respectively) was found for the hormone-free MS medium and the MS medium containing KIN at 0.5 mg L−1. Hyperhydricity took place on the media supplemented with hormones. Rooting occurred on the half-strength MS medium (51.0%). After two months, the plants’ survival rate stood at 100.0%, as did their ex vitro acclimatisation rate, which also registered at 100.0%. The present results could encourage not only the introduction of S. raeseri subsp. attica into the industry of floriculture as a new ornamental plant but also its ex vitro conservation.

In vitro</em></strong><strong> direct regeneration and <em>Agrobacterium tumefaciens</em> mediated <em>in planta</em> transformation of <em>Ocimum sanctum L.

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘ Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II ( npt-II) & β-glucoronidase ( uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

In vitro effect of phytosynthesised AgNPs to enhance plantlets and biomass production in Nyctanthes arbor-tristis L.

This work was aimed to establish an effective approach for direct regeneration of Nyctanthes arbor-tristis L. using nodal segment. Amongst the different combinations of plant growth regulators (PGR's), the maximum number of shoots/explant (32.94 ± 0.86) with 3.14 ± 0.08 cm shoot length was attained on Murashige and Skoog (MS) medium fortified with 3.5 mg /L 6-benzyladenine (BA) and 0.3 mg/L concentration of 1-phenyl-3-(1, 2, 3- thiadiazol-5-yl)- urea (TDZ). Besides, nodal segments of Nyctanthes grown on MS medium were used to analyze the effect of biogenic AgNPs (0,2,4,6,8 and 10 mg/L) on plant growth & multiplication in solid & suspension culture medium. After 35 days of in vitro shoot proliferation, the shoot number, shoot length, chlorophyll content, fresh & dry matter, guaiacol peroxidase (GPOX) and catalases enzyme (CAT) activities of plants were quantified. The administration of AgNPs (6 mg/L) along with the lower concentration of BA (1 mg/L) could enhance shoot multiplication from (1.29 ± 0.14) to (25.17 ± 0.45), chlorophyll content as well as the GPOX, and CAT enzyme activities in suspension culture medium. Optimized rooting response (12.15 ± 0.63) was noted on the half-strength MS medium supplemented with 0.5 mg/L concentration of Indole acetic acid (IAA), The maximum in vitro raised plantlets was hardened & acclimatized in a ratio of 6:2:2:1(garden soil: cocopeat: vermiculite: pearlite) potting mixture. The regenerated plantlets showed 80% survival rate under greenhouse conditions. Out of twenty PCR-based RAPD (Random Amplified Polymorphic DNA) molecular markers, 8 primers produced a total of 350 scorable bands which showed 90–99% similarity with mother plant.

Micropropagation and cytological studies of Aole vera Linn

Aloe vera Linn. is an essential medicinal plant. In this present research work, a protocol of in vitro regeneration and karyomorphological analysis of Aloe vera was developed using different concentrations and compositions of media. Shoot apices of field-grown plants were used as explant and aseptically cultured on Murashige and Skoog (MS) medium fortified with different concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP and Kn). The highest number of multiple shoot buds (4.36 ± 0.07) was obtained from MS + 2.0 mg/l BAP + 1.0 mg/l IAA and induced shoot buds underwent rapid elongation (4.24 ± 0.06 cm) on the same medium composition. Half strength MS media with 2.0 mg/l IBA was suitable for induction and proliferation (6.31 ± 0.05) of roots and 95% of plantlets were acclimatized to field conditions successfully. Somatic chromosome numbers of mother and in vitro grown plants were confirmed to be 2n = 14. Chromosome length ranged from 4.28 - 13.74 µm in the naturally grown plants and 4.46 - 14.1 µm for in vitro grown plants. The total form percent (TF%) of mother and in vitro grown plants was 41.69% and 42.23%, respectively. The karyotype formula of in vivo grown plants was 2n = 14 = 4Lsm + 6Mm + 4Sm, whereas that of the micropropagated plants was 2n = 14 = 4Lsm + 4Mm + 6Sm. The frequency of the chromosome having arm more than 2:1 was 0.08 for mother plants and 0.15 for in vitro grown plants. Therefore, the karyotype of both plants falls into the 2B symmetrical type based on Stebbins classification (1971).

Regeneration of Cotoneaster wilsonii Nakai through Indirect Organogenesis

The ornamental plant Cotoneaster wilsonii Nakai is a rare endemic species to the Ulleung Island in Korea. There is an urgent need to develop efficient propagation methods to preserve this endangered plant species. The cytokinin thidiazuron (TDZ) at a concentration of 0, 1.0, 2.0, or 3.0 mg·L−1 combined with 0, 0.5, 1.0, or 1.5 mg·L−1 a-naphthaleneacetic acid (NAA) was used to induce calli from young leaf explants on the Murashige and Skoog (MS) medium. The TDZ and/or 6-benzyladenine at a concentration of 0, 0.5, or 1.0 mg·L−1 combined with 0.2 mg·L−1 NAA was supplied to the MS medium to induce shoots from calli. Four auxins at 0, 0.2, 0.4, 0.6, 0.8, or 1.0 mg·L−1 were supplied to half-strength MS medium for root induction. The treatment of TDZ at 1.0 mg·L−1 combined with 1.5 mg·L−1 NAA not only had 100% callus induction ratio, but also induced the heaviest calli. The shoot induction ratio was the greatest with TDZ at 1.0 mg·L−1, and the greatest root induction ratio was obtained with IAA at a concentration of 0.8 mg·L−1. In conclusion, an effective indirect organogenesis system was established for C. wilsonii Nakai.

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.