Half-strength MS Medium Research Articles

Role of season, source of explant, media concentration and sterilants in obtaining aseptic cultures for initiating in vitro tuberization in sweet potato

ABSTRACT In vitro tuberization in sweet potato is a potential means for germplasm exchange in sweet potatoes, though not exploited due to the lack of a reliable protocol. The initiation of in vitro cultures and in vitro tuberization in sweet potato is limited due to fungal contamination and poor response of cultures. In order to obtain aseptic cultures, the effect of season was studied in khariff, rabi and summer seasons, and effect of source of explants studied using explants from storage root sprouts vs. field grown plants. Two sterilants, sodium dichloroisocyanurate and mercuric chloride, were compared on explants from storage root sprouts. The effect of media concentration on plant growth parameters was studied using full and half-strength MS medium. Significant difference was obtained in the results. Summer season was found to be best for the collection of field explants, storage root sprouts were better to field explants and sodium dichloroisocyanurate was assessed as an effective sterilant in terms of low contamination and better survival percentage. A single-node culture gave rise to shoots with 23 nodes after 20 weeks on full-strength MS media, which could be used as source of aseptic explants for initiating in vitro tuberization without using sterilants, thus improving in vitro response.

In Vitro Regeneration of Bambusa Vulgaris Var. Striata Using Nodal Explants

The current study aimed to develop an effective micropropagation protocol for Bambusa vulgaris var. striata, through direct organogenesis using nodal explant. The maximum axillary bud break (84%) with the greatest number of shoots per explant (5.20 ± 0.37) was achieved using liquid MS medium containing 2.5 mg/l BAP and 1.5 mg/l TDZ, while the maximum length of shoots (7.12 ± 0.27 cm) was induced on 2.0 mg/l BAP. Shoots were multiplied in stirred liquid MS medium with 3.0 mg/l BAP, resulting in the highest number of shoots per culture (13.20 ± 0.58) with the shoot length of 7.32 ± 0.48 cm. BAP in combination with TDZ resulted in a feeble response to shoot proliferation; although in some cases, the presence of TDZ with BAP was inhibitory. The shoots progressively increased in number and length over the first four subculture cycles in the aforementioned medium, but declined in the 5th sub-culture cycle. Following the 4th sub-culture cycle, each culture produced 16.60 ± 0.60 shoots with an average length of 8.96 ± 0.61 cm. Adding 10% coconut water to the medium resulted in excellent shoot growth and development, with an average of 18.40 ± 1.44 shoots per culture, measuring 8.56 ± 0.79 cm. Half-strength MS medium with 3.0 mg/l IBA resulted in the highest rooting percentage (85%) and the most roots per culture (8.00 ± 0.71). Successful acclimatization of well-rooted clumps of 3-4 shoots was achieved in a mixture of soil, sand, and compost (2:1:1) with 85% survival rate. Bangladesh J. Bot. 53(4): 939-947, 2024 (December)

Standardization of Sucrose Concentration in Micropropagation of Valuable Aquascape Plant Echinodorus grisebachii

Aims: To standardize the optimum sucrose concentration in shooting and rooting medium in micropropagation of valuable aquascape plant Echinodorus grisebachii. Study Design: CRD. Place and Duration of Study: Department of Floriculture and Landscaping, College of Agriculture Vellayani 2022-2024. Methodology: In this study, eight treatments were evaluated separately for their effectiveness in shooting and rooting medium. For the shooting medium, 2 mgL-1 BAP (6-Benzylaminopurine) and 3 sucrose concentrations (10, 20,30) gL⁻¹ were used in the MS medium and half-strength MS medium. For rooting medium 3.0 mgL⁻¹ IBA (Indole-3-butyric acid), three concentrations of sucrose as in shooting medium were tried. Results: The result of the study showed that out of 8 treatments tried for shooting medium MS + 2mgL-1 + 30 gL⁻¹ sucrose was best resulted in Shoot initiation (100 %), Minimum days to shoot emergence (6.17), Maximum number of shoots (9.83), Longest shoot (10.33) and maximum number of leaves (31). For rooting medium ½ MS + 3.0 mgL⁻¹ IBA + 30 gL⁻¹ sucrose was best resulted in minimum number of days in root emergence (12.58) and maximum root initiation percentage (99.25). Conclusion: An effective concentration of sucrose was standardized for Echinodorus grisebachii.

Effects Of 2,4-D, BAP, and Sucrose Concentrations in The Callus Induction of White (Clitoria ternatea var. Albiflora) and Blue Butterfly Pea (Clitoria ternatea)

The blue butterfly pea (Clitoria ternatea) and white butterfly pea (Clitoria ternatea var. Albiflora) belong to the Fabaceae family. Both are locally known as “bunga telang” and native to the Southeast Asian regions. The blue flowered variety is traditionally used to treat headaches, fever, and diabetes and is renowned scientifically for its memory-enhancing properties due to the presence of novel pentacyclic triterpenoids. However, farming of C. ternatea is challenged by inconsistent yields of novel secondary metabolites, especially under changing environmental conditions. Callus and cell suspension cultures, on the other hand, offer an alternative for the consistent production of these metabolites. The current study aims to optimize the treatments of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and sucrose concentrations for friable callus formation from seedling explants. Sterile cotyledon explants of in vitro seedlings from both types of butterfly pea were subjected to half-strength MS medium supplemented with different concentrations and combinations of 2,4-D and BAP, with sucrose at 15 g/L and 30 g/L. The highest friable callus fresh weight from the white butterfly pea explants (0.064 ± 0.010 g) was achieved in treatments of 0.40 mg/L 2,4-D and 0.50 mg/L BAP. In contrast, the highest fresh weight of friable callus for the blue variety (0.025 ± 0.016 g) was induced in 0.25 mg/L of 2,4-D. Both varieties showed the highest friable callus weight in 15 g/L sucrose supplemented with 1.00 mg/L of 2,4-D (0.146 ± 0.032 g) and 0.25 mg/L of 2,4-D (0.245 ± 0.075 g) for the white and blue variety respectively. The morphology of calli for both varieties were yellowish, watery, and sticky. This study provides an essential basis the establishment of cell suspension cultures, as an efficient alternative to harness the secondary metabolites associated with the mammalian neuroprotective properties.

In-vitro Regeneration of An Endangered Medicinal Plant Anodendron paniculatum (Apocynaceae)

Objectives: The present study aims to establish a standardized and efficient regeneration protocol for Anodendron paniculatum A. DC. (Apocynaceae) by optimizing the effect of various plant growth regulators as it is categorized globally as an endangered medicinal plant. Methods: MS medium fortified with 3% sucrose (w/v) and different concentrations of cytokinins 6-benzylamino purine (BAP), kinetin, thidiazuron either alone or in combination with auxins, indole 3-acetic acid (IAA), naphthalene acetic acid (NAA) and GA3. For the in vitro root induction, individual and healthy shoots (3-4 cm in length) were excised in the 7th week and were cultured onto basal and half-strength MS medium supplemented with various concentrations of auxins and incubated in the culture room. Findings: MS medium supplemented with thidiazuron (2.5 𝜇M) in combination with indole 3-acetic acid (2.5 𝜇M) and GA3 (1.25 𝜇M) was most effective, providing multiple shoot regeneration for 79.33% of cotyledonary leaf explants associated with a high number of shoots per explant (20.00 ± 1.15) and length of shoot (2.66 ± 0.08 cm). Whereas, the frequency of response, mean number of shoots, and respective lengths were varied with different concentrations of 6-benzylamino purine when used in combination with IAA or NAA and BAP (5.0 𝜇M) along with IAA (2.5 𝜇M) at the frequency of 47.66%. Whereas, the addition of GA3 (1.25 𝜇M) in combination with BAP (5.0 𝜇M) and IAA (2.5 𝜇M) has shown a maximum number of multiple shoot formation/explant (12.33) with length of shoot (1.53 ± 0.08 cm). MS medium supplemented with 10 𝜇M 6-benzyl amino purine and 2.5 𝜇M naphthalene acetic acid recorded the highest response (53.66%) with more number of the shoot (9.66) and length of the shoot (1.83 ± 0.03 cm) per explant. The maximum percentage of response (84.33%) was recorded at 1 2 strength MS medium with 5.0 𝜇M NAA, whereas the highest number of roots per shoot (6.66 ± 0.33) was induced at MS + 5.0 𝜇M NAA. The highest length of roots (3.66 cm) was recorded at basal MS medium fortified with 5.0 𝜇M NAA and 7.5 𝜇M NAA. Novelty: The standardized protocol method is of first of its kind, a quick and suitable approach for the high quantity production of Anodendron paniculatum with appropriate concentrations of plant growth regulators that meet thecommercial demand of endangered medicinal plants. Keywords: Anodendron paniculatum; Endangered plant; Plant growth regulators; Regeneration; Protocol standardization

In vitro Propagation of Leucas biflora (Vahl) Sm. - A Vulnerable Medicinal Herb

A rapidly well-developed in vitro regeneration system has been established for Leucas biflora using nodal segments as explants and MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRS). MS medium containing 2.0 mg/l BAP and 0.5 mg/l NAA showed the highest response (95.99%) in induction of shoot buds. This media combination also produced the maximum number (11.20 ± 0.49) of multiple shoot bud per explant after 3 subcultures at an interval of 14-days. Microshoots showed the maximum elongation (4.70 ± 0.07 cm) in the same medium along with subcultures. These elongated shoots were further grown on rooting media, producing vigorous and healthy root systems. The best response (95.99 ± 1.63%) for rooting was obtained when half-strength MS medium was supplemented with 0.5 mg/l IAA + 0.5 mg/l IBA in 21 days of culture. In this combination, the average number and length of roots were 5.20 ± 0.33 and 4.80 ± 0.30 cm, respectively. The seedlings were removed from culture tubes and transplanted into the pots via several stages of acclimatization. Finally, 98% of the seedlings were found to survive under natural condition. Plant Tissue Cult. & Biotech. 34(1): 49-54, 2024 (June)

In vitro mass propagation of Dendrocalamus asper (Giant bamboo) through direct organogenesis

Dendrocalamus asper (giant bamboo) is a clumping type of bamboo belonging to the Poaceae family. Due to its economic and environmental value, demand for this species has increased tremendously. Conventional propagation methods have limitations due to low seed viability and the lack of healthy clumps. Therefore, an in vitro mass propagation protocol was developed to provide healthy plants for large-scale plantations. Seeds were used as the explant and they were surface sterilized and cultured on MS medium free of growth regulators. Nodal segments taken from in vitro germinated seedlings were used for shoot initiation. The best medium for shoot induction (MS medium supplemented with 0.0–2.5 mg/L BAP), best medium for multiple shoot induction (MS medium supplemented with 0.0–5.0 mg/L BAP), effect of shoot cluster size (shoot clusters containing 1–4 shoots) and effect of physical state of the medium (semisolid and liquid media) on multiple shoot induction were determined using shoots per node, mean shoot length and mean number of leaves per shoot after 6 weeks of incubation. Elongated shoots were transferred into a rooting medium and the best medium for root induction (MS medium supplemented with 0.0–5.0 mg/L IBA and IAA) and effect of cluster size (shoot clusters containing 1–4 shoots) on rooting were determined using the number of roots and root length after 6 weeks of incubation. All the cultures were maintained under a 16-hour photoperiod. Well-developed plantlets were transferred to coir pellets and after four weeks transferred into different potting mixtures containing different combinations of sand, compost and coir dust. Unless otherwise mentioned there were at least twenty replicates in all treatments. The highest mean number of shoots per node (16.87±0.52), mean shoot length (4.12±0.27 cm) and mean number of leaves per shoot (4.80±0.33) were observed in the MS medium supplemented with 1.0 mg/L BAP. The MS medium supplemented with 2.0 mg/L BAP was the best for multiple shoot induction, shoot cluster with 3 shoots was the best cluster size and the liquid medium had a better effect on shoot multiplication. The half-strength MS medium supplemented with 2.0 mg/L IBA was the best for in vitro root induction with the highest mean number of roots (7.15±0.77) and a mean root length of 10.79±1.11 cm. Shoot clusters with 3 shoots was the best cluster size for root induction. A sand: compost: coir dust (1:1:1) mixture was the best potting mixture giving 100% survival. These findings provide a reliable micropropagation protocol for D. asper, which holds great promise for meeting the growing demand for bamboo resources and promoting sustainable bamboo cultivation.

Influence of Iba Concentrations on in Vitro Rooting of Cucumber Varieties

In vitro root induction was observed in cucumber as influenced by varieties and IBA concentrations. Four cucumber varieties viz. Shila, Green Field, Shital and Shahi-50 and three IBA concentrations viz. 0.0 (Control), 1.0, and 2.0 mg l-1 were used in this investigation. The Shital variety with in maximum number and the highest percentage of shoot produced roots. No root induction was observed when medium was auxin free. Higher concentration of IBA resulted in higher number (3.94) and percentage (98.65 %) of root. Half strength MS medium when fortified with 2.0 mg l-1 IBA took minimum time (6.88 days). Combined effect of varieties and IBA concentrations were found significant in all the parameters studied. The highest number (19.54) of roots were obtained from variety Shital when they were interacted with higher IBA concentration (2.0 mg l-1) followed by the interaction of same variety with lower concentration (19.18). The highest root length (8.80 cm) was found in the interaction of Shila x 2.0 mg l-1 IBA and the lowest root length (6.31 cm) was found in the interaction of Shahi-50 x 1.0 mg l-1 IBA. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):01-10, 2012

Mass Scale Micropropagation Of Andrographis Paniculata (Burm. F.) Wall Ex Nees

A reliable and efficient protocol was developed for in vitro propagation of Andrographis paniculata (Burm. f.) Wall ex Nees with the use of nodal and shoot tip explants. Nodal segments and shoot apex of in vitro grown seedlings were cultured on agar solidified MS medium supplemented with different concentrations and combinations of plant growth regulators (PGRs). Both the explants underwent direct organogenesis and produced multiple shoot buds. In case of shoot apex, maximum number of multiple shoot buds were formed when cultured on MS medium containing 1.5 mg/l Kn + 1.0 mg/l IAA. But nodal segments produced highest number of shoot buds on MS medium fortified with 1.5 mg/l BAP + 1.0 mg/l IAA. Multiple shoot buds underwent rapid elongation on elongation media and maximum elongation took place on MS medium fortified with 2.0 mg/l Kn + 1.5 mg/l IAA. Elongated shoot buds, on further culture in rooting media, produced strong and stout root systems. Half strength MS medium fortified with 2.0 mg/l IAA was most effective for induction and proliferation of roots. The healthy complete plantlets were successfully acclimatized and established in outside pots through successive phases of acclimatization. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):121- 130, 2012.

Endogenous ureides are employed as a carbon source in Arabidopsis plants exposed to carbon starvation conditions

Ureides, the degraded products of purine catabolism in Arabidopsis, have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1, Ataln, and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1, Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth.

Effect of half-strength MS medium supplemented with plant growth regulators on in vitro response in ‘Bird of Paradise’ (Strelitzia reginae L.)

Effect of half-strength MS medium supplemented with plant growth regulators on in vitro response in ‘Bird of Paradise’ (Strelitzia reginae L.)

Rapid Multiplication of Geographical Indication (GI) Tagged Mysore Mallige (Jasminum azoricum L.) Using Single Node through In vitro Culture

Aims: The present study was undertaken to rapid multiplication of (GI) tagged Mysore Mallige (Jasminum azoricum L.) using single node through in vitro culture. Study Design: The present study follow Completely Randomized Design (CRD). Place and Duration of Study: The study was conducted at the Plant Tissue Culture Laboratory, Department of Horticulture, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bengaluru, from 2020 to 21. Methodology: Single node cutting from the young shoots of field growing plants were used as explants to conduct the experiment. The explants were sterilized and placed on MS medium supplemented with different concentrations and combinations of growth regulators, namely BAP and Kinetin. Results: Among the different combinations of growth regulators, the combination of BAP 1.5 mg L-1 and Kin 1.5 mg L-1 produced maximum number of shoots from single node. Very low intensity of callus formation was observed from the cut ends of single node explants that were cultured on half strength MS medium consisting of 1.5 mg L-1 BAP + 0.0 mg L-1 Kin and 1.5 mg L-1 BAP + 1.0 mg L-1 Kin. Conclusion: The use of growth regulators such as BAP and KIN is reliable for shoot regeneration even when the field explants are used.

Development of robust in vitro propagation protocol and cyto-genetic fidelity assessment of Siraitia grosvenorii (monk fruit)

Siraitia grosvenorii (Swingle) C. Jeffrey of the family Cucurbitaceae is a unique and precious plant of medicinal and economic importance in the international market. Due to the huge market potential and to narrow down the gap between demand and supply, an efficient in vitro propagation system using nodal explants was established. Shoot nodal segments were cultured in medium supplemented with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kn 1.0–7.5 µM). A two-stage culture is recommended for micropropagation of S. grosvenorii. The highest shoot multiplication was observed in BAP-supplemented medium (5.0 µM), whereas rooting was observed in half-strength MS medium supplemented with 8.75 µM of indole-3-butyric acid (IBA). Fully developed plantlets were acclimatized with 100 % survival in sterilized sand. These hardened 15-day-old plants were successfully established under shade net conditions in fields. In vitro raised plants were found genetically stable using molecular markers, including RAPD and ISSR. The 2C nuclear DNA content using flow cytometry was observed to be 0.73–0.76 pg. Hence, the protocol developed for in vitro propagation of S. grosvenorii is cost-effective and can be efficiently used for the industrial production of this natural sweetener and pharmaceutically important plants.

Ex-situ Conservation of Bulbophyllum leopardinum, A Threatened Medicinal Orchid of Nepal

A successful micropropagation method was developed via the in-vitro seed germination and seedling growth of the epiphytic and/or lithophytic orchid Bulbophyllum leopardinum, a species having horticultural and therapeutic significance. To enhance seed germination, several quantities and combinations of naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), indole acetic acid (IAA), gibberellic acid (GA3), and coconut water (CW) were added to 0.8% (w/v) agar-solidified MS medium. Half-strength MS medium has been experimented with alone and in combination with BAP, Kinetin (Kn), and GA3 to promote shoot development. In-vitro-developed healthy shoots were chosen to establish roots in a half-strength MS (HMS) medium supplemented with various auxins. The best and earliest seed germination with the greenest protocorms (96.3±0.5% in 7 weeks) was achieved on HMS medium fortified with 15% CW (H15C). Further tests for the shoot as well as root development were continued with an H15C medium. H15C with 1 mg/l BAP and 1.5 mg/l Kinetin was most effective for early in vitro development and differentiation into seedlings with the many long shoots (9.3±0.1 shoots and 2.4±0.1 cm per culture) within 12 weeks of sub-culture. The most suitable rooting hormone was 1 mg/l NAA (4.2±0.26 roots per culture). This medium also produced the longest roots (1.9±0.09 cm per culture). By successfully developing a protocol for the mass propagation of B. leopardinum, this research has enhanced both the cultivation and the commercialization potential of this species.

Overexpression of AtMYB12 transcription factor simultaneously enhances quercetin-dependent metabolites in radish callus

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g−1 dry weight), flavonoid content (463 mg g−1 dry weight), and quercetin content (48.24 mg g−1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.

Invitro germination and optimization of basal media for protocorm-like bodies proliferation in Dieniaophrydis(J. Koenig) Seidenf

Dienia ophrydis is an endangered terrestrial orchid used for its ornamental and therapeutic purposes. This orchid possesses an anti-malarial alkaloid known as Malaxin. There is ongoing exploitation in the wild due to illegitimate herbal collection and climate change. To control the potential risk of extinction, there are no conservational measures or in vitro protocols established in this genus. The study focuses on addressing a germination protocol and enhancement of protocorm-like bodies (PLB) proliferation by optimizing the basal media and vitamins. It also aims to improve the misinterpretation of tetrazolium viability results in orchid testa along with a permeability test. Further, the seeds were subjected to 1% TTC staining to perform embryo viability test and 0.4% trypan blue (TB) dye for testa permeability. Ten basal media such as Gamborg's medium, Mitra medium, Thomale GD medium, Vacin and Went medium, Knudson C medium, Knop's medium, MS medium and ½ MS medium, ¼ MS medium, and modified MS medium (MMS) were tested for seed germination. The Half-strength MS medium responded with 92% of the highest germination and T-GD medium is not suitable for seed germination. The seed viability and permeability result showed 93% of permeability, 72% viability, and 9% dead seeds. Protocorms were transferred to the modified mMS medium containing half-strength macronutrients, full micronutrients, and 10 ppm of thiamine in vitamin formulation. PLB proliferation was observed and 12 shoots emerged from a single protocorm explant. Subsequently, the seedlings were acclimatized with 8-0-24 ratios of nitrogen, phosphorus, and potassium (NPK) as determined by soil testing. The study contributes in experimenting with seed germination of terrestrial orchid seeds asymbiotically and to establishing a protocol for conservation programs, especially in this genus.

Anther-derived microspore embryogenesis in pepper hybrids orobelle and Bomby

BackgroundTraditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby.ResultsWe achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds.ConclusionOur findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.

In Vitro Seed Germination and Seedling Development of Mimusops laurifolia (Forssk.) Friis.: An Endangered Plant Species

Mimusops laurifolia (Forssk.) Friis is an endangered medicinal plant and natural propagation of this plant through seeds is found to be very difficult due to its hard seed coat. To conquer this problem, an efficient in vitro seed germination as well as subsequent in vitro regeneration technique has been developed in conserving this endangered plant species. Seed germination was achieved on half strength of MS (HMS), full strength MS (FMS) and MS medium supplemented with different concentration and combination of Plant Growth Regulators (PGR) like BA, Kn and GA. The highest seed germination rate (18%) was obtained from water soaking de-coated seeds inoculated on full strength of MS (FMS), on the other hand, 12% germination of seeds was recorded on HMS medium, but both the conditions required more than 90 days to germinate. A similar rate of germination was also found on MS with PGRs, but it required 55-60 days. Best responses towards seed germination with elongated shoots (2.8 cm and 3.0 cm) were obtained when seeds were inoculated on MS with 3.0 mg/l BA, 0.5 mg/l Kn and 0.2 mg/l GA. After a long period of period of 8 months following germination, seedlings produced elongated tap roots only. Rooted seedlings were successfully transferred to soil for their further growth and development. Apart from this, for in vitro regeneration of shoots, explants of nodal segments and shoot tips from the natural plant were cultured on woody plant medium (WPM) supplemented with various combination and concentration of BAP, Kn and TDZ. In these cases, there was no positive response recorded towards the initiation of shoots from nodal segment and shoot tips. Plant Tissue Cult. & Biotech. 33(2): 143-153, 2023 (December)

Efficient in vitro Regeneration of Brassica napus L. Var. BARI Sorisha-18

Brassica is one of the most nutritious and significant oil yielding crop plants around the world, including in Bangladesh. However, the development of a smart variety with a short maturity duration and high yield is a need of the present day. To achieve that, both genetic transformation and genome editing can be promising technologies for improvement, and establishment of suitable regeneration systems is a pre-requisite to deploy such technique. For this reason, an efficient and reproducible in vitro regeneration protocol for Brassica napus L. var. BARI Sorisha-18 was developed using MS medium supplemented with various concentrations and combinations of 2, 4-D, BAP and NAA. The highest frequency of in vitro regeneration of multiple shoots was obtained on MS medium supplemented with 1.0 mg/l BAP and 0.1 mg/l NAA from both cotyledonary leaf attached with petiole and hypocotyl explants. The elongation of shoots was also achieved with the same media composition. Half strength of MS medium supplemented with 0.2 mg/l IBA was found to be more effective in producing effective roots than other hormonal supplements applied. Following the development of effective roots, in vitro raised plantlets were successfully transplanted into the soil. Plant Tissue Cult. & Biotech. 33(2): 107-114, 2023 (December)

Direct Shoot Regeneration of Plumbago zeylanica Linn. through Tissue Culture Technology

ABSTRACT: Chitrak is scientifically known as Plumbago zeylanica Linn. belongs to Plumbaginaceae. Root shows medicinally valuable drug in pharmacological enterprise as the humanity is certainly prone to numerous contagious ailments due to ecological imbalance triggered by the current technology development. One of its remarkable phytoconstituents is plumbagin, a naphthoquinone molecule used to treat skin conditions, tumours, and tenacious chronic rheumatoid arthritis. The objective was to standardize the procedure of Plumbago zeylanica direct-organogenesis micropropagation. The solution contained various concentrations and combinations of auxin and cytokinins, and the nodal explant was used for quick micropropagation. The maximum multiple shoots (18.24 ± 0.51) per explant with shoot length 4. 2cm ± 0.20 cm was produced as the MS medium supplemented with B5 vitamins together with 2mg/l 6- Benzyleaminopurine (BAP) and 1mg/l Indole-3- acetic acid (IAA). The regenerated shoots were rooted in half strength MS medium with B5 vitamins that contained indole-3-butyric acid (IBA). The highest rooting frequency (100%) was seen in the 1.5 mg/l IBA-containing rooting media. The extreme roots were 9.2±0.14 per explant. The average root length was 4.2± 0.10 cm. The in vitro rooted Shoots were then transplanted in the field showing a 100 percentage survival rate.