MS Medium Research Articles

In Vitro Micropropagation of Eria Lasiopetala (Willd.) Ormerod- A Native Medicinal Orchid in Bangladesh

The present study focuses on the asymbiotic germination of seeds through in vitro culture of Eria lasiopetala, a native medicinal orchid in Bangladesh. Out of the four basal media, MS was found to be the most effective medium, where 93.58±0.374% seed germinations were recorded. Different plant growth regulators (PGRs), e.g. BAP, Kin, NAA and were used for secondary protocorm proliferation, plantlet development, and multiplication of shoot buds through in vitro conditions. The maximum number of secondary protocorms (12.80±0.115) were developed that were derived from primary protocorms on MS medium fortified with 0.5 mg/l BAP and 1.0 mg/l NAA. Data revealed that 0.5 mg/l BAP + 0.25 mg/l NAA showed the best combination for height elongation of plantlets (3.12±0.012 cm) in MS medium. While for multiple shoot buds (MSBs) 1.0 mg/l BAP + 0.5 mg/l NAA showed better (6.8±0.127) performance than other combinations of PGRs. The single shoots were isolated from the multiple shoots and sub-cultured on ½MS medium supplemented with NAA (0.5 - 2.0 mg/l), IAA (0.5 - 2.0 mg/l) and IBA (0.5 - 2.0 mg/l) for root induction. The maximum number of roots (7.53±0.176) and its growth (4.61±0.01 cm) were found in ½MS medium supplemented with 1.0 mg/l IAA. The well rooted plantlets were hardened and successfully transferred after acclimatization in pots containing the mixture of coconut husk, charcoal and brick pieces in the ratio of 2:1:1 and eventually established under natural condition. J. Bio-Sci. 32(1): 45-60, 2024

Clonal propagation of rare orchid species Paphiopedilum spp. (Orchidaceae) to save Indonesian biodiversity

Paphiopedilum spp. is one of the terrestrial orchids that is interesting as an ornamental plant. This orchid flower has a distinctive characteristic, namely its labellum resembles a sac, so it is sought after by ornamental flower collectors because of its uniqueness. This orchid is included in the Appendix I group according to CITES, namely a critical species that is threatened with extinction, so it needs to be conserved both in situ and ex situ. Ex situ conservation can be done by propagating orchids using in vitro culture techniques. The objective of this research is to carry out mass propagation of 3 species of Paphiopedilum spp orchids: P. supardii, P. primulinum, P. glaucophyllum through somatic embryogenesis and to characterize the existence of embryo gene RKD4 homolog in Paphiopedilum orchids. The method is to culture the nodes slices in dark conditions for 2 weeks, and then changed to photoperiod mode 16:8 hours (dark:light) on ½ MS media plus a combination of 2,4-D or NAA with TDZ. The somatic embryo were analyzed anatomically. This method is an improvement over previously reported methods for Paphiopedilum spp. due to its efficiency and reproducibility for conservation. We concluded that ½ MS media with a combination of NAA or 2,4D with TDZ were able to induce somatic embryogenesis. The result show that 3 mg L−1 NAA was the best PGRs for PLBs induction in P. supardii (50 %), 1 mg L−1 2,4-D + 0.5 mg L−1 TDZ was the best PGRs for callus embryogenic induction in P. primulinum (20 %), and 1 mg L−1 NAA was the best PGRs for PLBs induction in P. glaucophyllum (33 %).

An Overview of Pharmaceutical Applications and In vitro Micropropagation Techniques for Rare and Endangered Plant Species

Many rare and endangered plant species possess valuable secondary metabolites with pharmacological applications. These bioactive compounds are often integral to traditional medicine systems, highlighting the cultural significance of these plants. The health benefits of many medicinal species are not fully validated by contemporary scientific research, and some may be facing extinction due to habitat loss, overharvesting or climate change. This situation highlights the urgent need for effective conservation strategies of the species and sustainable cultivation methods. Micropropagation is a valuable technique for producing large numbers of plants from a single explant, significantly aiding in the conservation and commercial cultivation of rare species. Among the various types of explants, shoot tips and nodal segments have been identified as the most effective explants for micropropagation. These explants can be induced to generate multiple shoots in Murashige and Skoog (MS) medium containing Benzyl aminopurine (BAP). Thidiazuron (TDZ), Kinetin (KIN), or 2,4-Dichlorophenoxyacetic acid (2,4-D) are commonly used in MS medium to promote shoot and root development in both direct and indirect organogenesis processes. Rooting of the plantlets was typically achieved using MS medium either supplemented with Indole-3-butyric acid (IBA) or devoid of auxins, depending on the species and the specific requirements for rooting.

DAYA MULTIPLIKASI EKSPLAN KENTANG AR 8 PADA BERBAGAI KONSENTRASI BENZIL AMINO PURIN (BAP) DAN EKSTRAK BAWANG MERAH DALAM MEDIA DASAR MURASHIGE DAN SKOOG (MS) SECARA IN VITRO

The technique of propagating potato seeds through tissue culture has long been used to produce quality and disease-free seeds. This study aims to test the effect of the combination of BAP and Shallot Extract on the growth of potato plant explants on MS media (Murshige & Skooge). Research in experimental form uses a completely randomized design (CRD) in a factorial pattern consisting of two factors. As the first factor, the growth hormone BAP consists of 4 levels, namely: 0 ppm, 2 ppm, 3 ppm and 4 ppm. Meanwhile, the second factor is: shallot extract consists of 4 treatment levels, namely: 0%, 0.2%, 0.4% and 0.6%. Thus, there were 16 treatment combinations which were repeated 6 times so that the total experimental units were 96. The ABA treatment with a concentration of 2 ppm/liter gave the best effect on the shoot formation time and root formation time of AR 8 potato plant explants. However, the treatment without ABA had a good effect on Shoot length and root length of explant potato plant varieties AR 8. Shallot extract treatment with a concentration of 20% had the best effect on shoot formation time, root formation time, shoot length and number of leaves. However, the treatment with a concentration of 40% had a better effect on the parameters of the number of shoots and the number of explant segments of the AR 8 variety of potato plants. The interaction of the ABA treatment with a concentration of 2 ppm and the shallot extract with a concentration of 20% had the best effect on the time of emergence of shoots and roots on the AR 8 Potato Explants. However, the interaction without ABA (0ppm) and shallot extract had a better effect on the parameters of shoot length, number of leaves, and number of explant segments in the AR 8 potato variety.

Sexual and Asexual Propagation of Wild Lime (Zanthoxylum fagara L. Sarg.), a Native Florida Plant with Ornamental and Ecological Value

Abstract A series of four propagation studies were implemented to better understand the seed biology of wild lime (Zanthoxylum fagara L. Sarg. [Rutaceae]) and its adventitious rooting response to auxin treatments. Plant origin (north central vs south Florida ecotypes) did not affect initial seed viability but significantly influenced germination response to seasonal temperatures. Fifty-five days after sowing north central Florida seeds, germination was similar among spring, summer and fall treatments (28.9 to 41.1%), but was reduced by the winter temperature (10.7%). South Florida seeds showed greatest germination under the fall temperature (71.2%), and the least germination under the summer temperature (30.2%). Additional seed treatments including applications of gibberellic acid (GA3) with kinetin nominally improved germination by 1.2 times compared to non-treated seed. Seeds tolerated cryopreservation treatments, including combinations of a plant vitrification solution, liquid nitrogen, phloroglucinol and precooling, suggesting long-term storage capability. As an alternative to seed propagation, cutting propagation was found to be a reliable means of reproducing wild lime with 91.0% rooting success when softwood cuttings were treated with 8,000 mg·kg−1 (0.13 oz·lb−1) indole-butyric acid (IBA), compared to 3,000 mg·kg−1 (0.05 oz·lb−1) IBA (86.3%) or the non-treated control (71.2%). Species used in this study: Wild lime, Zanthoxylum fagara (L. Sarg). Chemicals used in this study: 2,3,5-triphenyl-2H-tetrazolium chloride (TZ); sodium hypochlorite solution (Clorox bleach); gibberellic acid (GA3); kinetin (kinetin), plant vitrification solution 2 (PVS2); liquid nitrogen (LN); phloroglucinol (phloroglucinol); glycerol (glycerol); dimethyl sulfoxide (DMSO); MS media (Murashige and Skoog media), sucrose (sucrose); 15N-5P-15K liquid fertilizer (Peters Excel Cal-Mag Special); indole-3-butyric acid (IBA); 14N-14P-14K slow-release fertilizer (Osmocote).

IN VITRO REGENERATION OF STRIPE EBONY (DIOSPYROS FRUTESCENS BLUME)

The Stripe ebony tree (Diospyros frutescens Blume) is considered a species in danger of extinction due to overexploitation. Therefore, there is an urgent need to regenerate and conserve the species for future generation. In this study micropropagation protocol of Diospyros frutescens Blume was developed using nature zygotic embryos. Zygotic embryos isolated from germinated on MS medium containing 0.4mg L-1 Kinetin + 1.0mg L-1 BA. There were (83.11±0.79%) of explants induced shoots after 30 days of culture. The highest number of shoot on 1/2 MS medium supplemented with 2.5mg L-1 BA + 0.6mg L-1 IBA was 5.84±0.035 shoots per explant after 45 days of culture. The percentage of shoot produced roots the highest 85.52±0.48% after 45 days culture on 1/2 MS medium contain 0.8mg L-1 IBA.

Callus of Mundulea sericea as potential anti-diabetic agents; an in silico approach

Mundulea sericea, a rare plant of the fabaceae family, has high medicinal value but a low regeneration rate in its natural habitat. The present study aims to conduct in vitro propagation and screen the potential bioactive compounds in the callus through in silico analysis. The explants were collected, sterilized, and inoculated in a standardized MS media with growth regulators in vitro to obtain sterile plants. The experiment resulted in the formation of a greenish-brown callus at a rate of 82.41 % on MS medium supplemented with BAP (6.66 µM L−1). The callus was powdered and subjected to GC–MS analysis. A total of 31 compounds were identified in the methanolic extract, along with their corresponding peak values. The compounds underwent ADME analysis for toxicity predictions. Eight compounds were chosen further based on pharmacokinetic and pharmacodynamic properties. The selected ligands were docked with anti-diabetic targets α-amylase and α-glucosidase. As a result, the compound CID 5283663 exhibited a strong binding affinity of −9.3 with the α-amylase receptor. The ligand CID 33010 exhibits a strong interaction of −7.5 with the α-glucosidase receptor. Glibenclamide and acarbose were used as standards. This study is the first attempt to identify compounds in the callus extract that could inhibit α-amylase and α-glucosidase enzymes. The findings from molecular docking analysis suggest these compounds could become potent treatments for diabetics, showcasing the promise of natural sources in drug discovery.

Effects of Nitrogen Content and pH on Micropropagation of Rudraksha (Elaeocarpus ganitrus Roxb.)

This study evaluated the effects of pH and inorganic nitrogen sources (KNO3 and NH4NO3) on the regeneration of Elaeocarpus ganitrus Roxb. The pH and nitrogen concentration in the medium were crucial for the successful micropropagation of E. ganitrus. The concentration of nitrogen in the medium significantly affected the development and growth rate of cultures. As a basal medium, Murashige and Skoog's medium (MS) was employed together with various nitrogen concentrations and plant growth hormones. After 4-5 weeks of inoculation, BAP (0.5 mg/l) and NAA (0.1 mg/l) produced the maximum number of shoots (4.42 ± 2.17) among all the growth hormones tested. To obtain the maximum number of shoots and shoot length, different strengths of KNO3 (500-3500 mg/l), NH4NO3 (500-3500 mg/l), and pH (5-7.5) were adjusted in the medium. The largest number of roots was contained in MS medium with 1.0 mg/l NAA (4.47 ± 1.38) after root induction was carried out on MS with NAA, IBA, and IAA (0.2-2.0 mg/l). When the medium contained 1500 mg/l KNO3 and 2000 mg/l NH4NO3 (3.02 ± 1.60), the highest root initiation was observed. The rooting medium with a pH of 6.0 had the most influence and produced the maximum number of roots (4.8 ± 0.48). The plantlets were allowed to harden and acclimate in a greenhouse. A 76% survival rate was noted in the field.

A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

Tea (Camellia sinensis L. O. Kuntze) callus development based on morphological and phytochemical traits applied by variations in plant growth regulators (PGRs)

A variation of the clonal propagation system for tea has been established through in vitro methods, utilizing shoots and leaves derived from primary explants of mature field-grown plants. The research aimed to assess the diversity of tea calluses based on morphological and phytochemical traits derived from leaf and shoot explants on MS media induced by the application of plant growth regulators, including auxin and cytokinin. The research was conducted at the Seed Technology Unit Tissue Culture Laboratory, Faculty of Agriculture, Jatinangor, from April 2022 to February 2023. The experiment employed MS-modified multiplication media and PGR, with BAP at 9 mg/L, TDZ at 1.0 mg/L, Zeatin at 0.1 mg/L, NAA at 0.01 mg/L, and 1 mg/L. Results indicated that applying PGR, auxin, and cytokinin with MS media influenced morphological and phytochemical traits of tea's callus derived from leaves and shoots. Specifically, the treatment of 1 mg/L NAA + 1 mg/L BAP showed the best performance on callus weight and total phenolic and flavonoid contents compared to other treatments., i.e. 9.038% % and 0.05% %, respectively. The success of clonal propagation in tea through in vitro methods is expected to increase by carefully selecting the appropriate source and type of explant, planting media, PGR, and addressing potential contaminants.

In vitro seed propagation of Dendrobium moschatum as influenced by different plant growth regulators

This study aimed to develop an effective in vitro protocol for germinating and multiplying Dendrobium moschatum seeds by identifying optimal concentrations of plant growth regulators for shoot proliferation and root formation. Five different MS media formulations were utilized, including a control (M?) and those supplemented with varying concentrations of kinetin and NAA (M?, M?, M?) and BAP (M?). Results showed significant variation in seed germination time across media, with the longest duration observed in M? (76 days) and the shortest in M? (55 days). Protocorm formation and plantlet development were quickest in M?, while M? and M? exhibited the longest duration. The initiation of leaves or shoots occurred earliest in M? (103 days) and latest in M? (149 days). Notably, leaf and plantlet growth differed between three- and five-month intervals, with M? demonstrating optimal growth over the longer term. These findings underscore the importance of carefully balanced plant growth regulator concentrations in achieving successful seed germination and subsequent growth in D. moschatum.

Molecular Diversity Analysis of in vitro and Irradiated Tomato (Lycopersicon esculentum Mill) Grew Under Salt Stress Expressed by SCoT and ISSR Markers

Tomato buds of cv. Idkawy were cultured in vitro on solid MS medium with 0.2 mg-l BAP. The plantlets that were produced were exposed to different doses of gamma radiation, ranging from 100 to 200 Gy. Afterward, single pieces of nodes were cut and moved to a fresh MS medium with 0.2 mg-l of BAP. The gamma radiation caused a mortality rate of 18.75% to 52.5% among the explants. The surviving plantlets were then cut into single node pieces and transferred to an MS medium containing 0.2 mg-l of BAP, with added NaCl concentrations of either 50 or 100 mM. There was increased mortality of the vegetative buds on the explants with increased salt concentrations. It was shown that the all gamma radiation doses caused reduced the percentage of survival at saline levels. The genetic diversity was assessment by using ten primers for each SCoT and ISSR markers to six irradiated treatments grew under salt stress (100 Gy x 50 mM, 150 Gy x 50 mM, 200 Gy x 50 mM, 100 Gy x 100 mM, 150 Gy x 100 mM, 200 Gy x 100 mM). It was showed that the polymorphism percentage mean of SCoT marker (29.56%) is higher than the ISSR marker (26.78%). The average of PIC values for both markers SCoT and ISSR were 0.197 and 0.288 (PIC ˂0.5), as well as, MI values were 0.077 and 0.081, respectively. In contrast, when considering the number of alleles (Ne), Nei's genetic diversity (H), and Shannon's information index (I) parameters, it was observed that the greatest genetic variation was caused by the combined treatment of 200 Gy x 50 mM NaCl using the SCoT marker. On the other hand, with the ISSR marker, the highest induced genetic variation was seen with the combined treatment of 150 Gy x 50 mM NaCl. The obtained results demonstrate that SCoT marker was more accurate and efficient than ISSR marker for distinguishing and genetic variation analysis of irradiated tomato plantlets grew under salt stress. The relationships within treatments were estimated through cluster analysis (UPGMA) based on SCoT and ISSR analysis.

Nutrient requirements shape the preferential habitat of Allorhizobium vitis VAR03-1, a commensal bacterium, in the rhizosphere of Arabidopsis thaliana.

A diverse range of commensal bacteria inhabit the rhizosphere, influencing host plant growth and responses to biotic and abiotic stresses. While root-released nutrients can define soil microbial habitats, the bacterial factors involved in plant-microbe interactions are not well characterized. In this study, we investigated the colonization patterns of two plant disease biocontrol agents, Allorhizobium vitis VAR03-1 and Pseudomonas protegens Cab57, in the rhizosphere of Arabidopsis thaliana using Murashige and Skoog (MS) agar medium. VAR03-1 formed colonies even at a distance from the roots, preferentially in the upper part, while Cab57 colonized only the root surface. The addition of sucrose to the agar medium resulted in excessive proliferation of VAR03-1, similar to its pattern without sucrose, whereas Cab57 formed colonies only near the root surface. Overgrowth of both bacterial strains upon nutrient supplementation inhibited host growth, independent of plant immune responses. This inhibition was reduced in the VAR03-1 ΔrecA mutant, which exhibited increased biofilm formation, suggesting that some activities associated with the free-living lifestyle rather than the sessile lifestyle may be detrimental to host growth. VAR03-1 grew in liquid MS medium with sucrose alone, while Cab57 required both sucrose and organic acids. Supplementation of sugars and organic acids allowed both bacterial strains to grow near and away from Arabidopsis roots in MS agar. These results suggest that nutrient requirements for bacterial growth may determine their growth habitats in the rhizosphere, with nutrients released in root exudates potentially acting as a limiting factor in harnessing microbiota.

Cryopreservation of zygotic embryos derived from BBTV resistant accessions of wild banana Musa acuminata

The wild banana species Musa acuminata encompasses numerous subspecies, some of which serve as ancestors of edible bananas. Preserving these subspecies is critical for food security, especially in regions where bananas are staple crops. While cryopreservation of M. acuminata subsp. burmanicoides zygotic embryos using air dehydration have shown high success rates, this method has not been effective for other subspecies. With at least 15 subspecies displaying various morphological traits, including seed morphology, alternative cryopreservation methods warrant exploration. In a recent study, successful cryopreservation using the droplet-vitrification method was achieved for mature zygotic embryos of accession of M. acuminata subspecies tomentosa, rutilifes, and malaccensis, all of which are resistant to the Banana bunchy top virus (BBTV). Using this approach, subjecting embryos to dehydration for 20 min in PVS2 solution and employing a regeneration medium comprising MS medium supplemented with benzyladenine and thidiazuron resulted in a success rate of 77.4 % (86 % survival rate × 90 % regrowth rate) for subsp. tomentosa and 50.4 % (70 % survival rate × 72 % regrowth rate) for subsp. malaccensis. Conversely, for subsp. rutilifes, dehydration for 10 min led to a success rate of 15.6 % (30 % survival rate × 52 % regrowth rate). Subspecies emerged as the primary factor influencing embryo regrowth, suggesting the need for protocol optimizations tailored to other subspecies. These findings highlight the potential of droplet-vitrification as a long-term preservation method for certain subspecies, while underscoring the necessity for further optimization for the other subspecies of M. acuminata.

An effective in vitro propagation of Coelogyne mossiae Rolfe: An endangered endemic orchid of Western Ghats, India

Coelogyne mossiae Rolfe, an unexplored taxon given its red-listed status by IUCN was selected for in vitro propagation studies due to its conservational significance. The mature pods harboring viable seeds were subjected to inoculation on eight different nutrient media, revealing full-strength Murashige and Skoog (MS) media as highly efficient for seed germination (96.65 %) and subsequent stages of seedling development. Protocorm explants are used for micropropagation on MS media supplemented with various plant growth regulators (PGRs). α- Naphthaleneacetic acid (NAA) in the concentration of 0.5 mg/L facilitated optimal shoot induction resulting in an average of 2.39 multiple buds and 2.46 cm shoot length while 1.0 mg/L 6-Benzyl amino purine (BAP) augmented cultures provided 2.32 multiple shoot buds with an average 1.92 cm height. Half macro-MS augmented with 0.5 mg/L BAP promoted pseudobulb elongation (0.73 cm) while 0.5 mg/L NAA supported root formation (8.33 roots with an average length of 1.12 cm). The resultant plants were acclimatized in pots containing coco peat and brick pieces, exhibiting a survival rate of 63.23 %. The anatomical characteristics of the hardened plants provided insight into factors conducive to their adaptability. Also, the 3-month-old hardened plants demonstrated significantly greater total photosynthetic pigment (0.4915±0.00 mg g-1), total carbohydrate content (72.33±1.69 mg SSE/g), and total protein content (40.26±0.53 mg BSAE/g) when compared to in vitro plants. These findings underscore the potential of in vitro propagation techniques in the conservation and sustainable utilization of Coelogyne mossiae.

Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions

The Sideritis genus includes over 150 species primarily found in the Mediterranean basin, including the S. clandestina subsp. pelopponesiaca from the Peloponnese and S. scardica from North and Central Greece. In vitro seed germination has proven effective for conserving and amplifying the genetic diversity of endangered species such as Sideritis. This study aimed to optimize in vitro germination and seedling growth of S. scardica and S. clandestina subsp. pelopponesiaca under different lighting conditions at 22 °C, including white fluorescent lamps (WFL-BG-40) and LEDs (LED-BGYOR-40, LED-BR-40, LED-BR-80, LED-BR-120) all under a 16-h light/8-h dark photoperiod (WFL: white fluorescent light, B:blue, G:green, Y:yellow, O:orange, R:red, 40–80–120 μmol m−2 s−1), along with a 24-h dark treatment. The results indicated that LED-BR-80 combined with 250 mg L−1 GA3 in the MS medium promoted best germination (40%, day 55) and shoot proliferation in S. clandestina subsp. pelopponesiaca. Conversely, 5-year-old cold stratified S. scardica seeds showed higher germination rates (80%) and robust seedling growth under LED-BGYOR-40 with 250 mg L−1 GA3, particularly thriving in LED-BR-120 for increased shoot height and root number. This is the first report of the efficacy of LED technology in optimizing in vitro conditions for Sideritis species, crucial for their conservation and sustainable commercial cultivation.

Optimized In vitro Propagation, Acclimatization and Reintroduction of Endemic Rhododendron inaequale Hutch. from Northeast India

Rhododendron inaequale Hutch., an endemic plant with high economic potential and susceptibility, requires urgent conservation measures. This study aimed to develop an optimized in vitro propagation protocol for this species. In vitro seed germination was conducted using Anderson medium, Woody Plant Medium (WPM), and MS Medium. WPM supplemented with 8 mg L⁻¹ 2iP [N6-(2-Isopentenyl) adenine] yielded the highest multiple shoot induction (3.55 ± 0.14 shoots, 0.77 ± 0.03 cm shoot length). For rooting, WPM with 0.5 mg L⁻¹ IBA [Indole-3-butyric acid] produced the maximum number of roots (3.40 ± 0.43) with a mean root length of 1.84 ± 0.12 cm. Successfully acclimatized plants were reintroduced into natural habitats in various locations in Meghalaya and Nagaland. Additionally, 200 micropropagated plants were distributed for further conservation initiatives. This study provides a crucial protocol for the conservation and reintroduction of R. inaequale, aiding in the preservation of this vulnerable species.

Elicitation of potato plants to increase their resistance against Soft Rot Pectobacteriaceae bacteria

AbstractThe investigation aimed to assess three methods for inducing resistance in Solanum tuberosum L. (potato) plants against Soft Rot Pectobacteriaceae (SRP) bacteria. The approaches involved the incorporation of elicitors into the in vitro nutrient medium during plant cultivation, pre-planting of seeds soaked in water-containing elicitors, and irradiating in vitro cultivated potato plants with 21 kJ/m2 of ultraviolet C light (UVC). The research validated the authors’ hypothesis, that the use of specific elicitors and UV-C light could induce resistance in potato plants against SRP bacteria, identifying the most effective elicitation technique for potato plants under experimental conditions. Two elicitors, specifically 50 μM salicylic acid and 250 μg/mL proline, were determined to be the most potent under experimental conditions. Supplementation of the Murashige and Skoog medium (MS) with 50 μM salicylic acid resulted in 21% of infected plants exhibiting no symptoms of infection on the 14th-day post-inoculation, and all plants treated with 10, 30, 35, 40, 45, or 50 μM of salicylic acid displayed a diminishing rate of infection progression. Plants growing with 25 µM SA were visually characterized by faster growth rates and higher vegetative mass relative to both control and test variants. Similarly, the incorporation of 250 μg/mL proline into the MS medium caused a response with 37.5% of plants showing no disease symptoms on the 14th day post-inoculation.

Effect of BA on Callus Induction, Shoot and Root Development from Callus of Nepenthes ampullaria through In Vitro Culture

Nepenthes ampullaria Jack. or pitcher plant is a carnivorous plant in the Nepenthaceae family. They are in danger of extinction because of the less forest area, volatile weather, and pollution. This study aimed to investigate the influence of BA (6-benzyl adenine) on callus induction of Nepenthes sp. through In vitro culture. The experiment was divided into three parts; (1) Callus induction; shoot of a pitcher plant were cultured on solid MS medium (Murashige & Skoog, 1962) supplemented with BA (6-benzyladenine) at 0, 1, 2, and 4 mg/l for 70 days. The experiment design was completely Randomized Design (CRD) (4 treatments X 10 replicates). It was found that MS medium supplemented with 4 mg/l BA proved to be a suitable medium for callus induction (70%). (2) Shoot induction from callus; callus were cultured on MS medium supplemented with 0, 2, and 4 mg/l BA (CRD, 3 treatments X 10 replicates) for 45 days. The highest average number of regenerated shoots (19.7±0.3 shoots) was observed on MS medium without BA. (3) Rooting induction; the young shoots, 1-2 cm in length, were cultured on MS medium containing 0, 1, 3, and 5 mg/l NAA combination with 0 and 3 mg/l IBA for 40 days (CRD, 8 treatments X 10 replicates). The results showed that the MS medium supplemented with 1 mg/l NAA and 3 mg/l IBA was suitable for root induction with the average of 7.4±1.6 roots.

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.