Half-strength Murashige And Skoog Basal Medium Research Articles

Effect of Auxins on Growth Enhancement of Cell Suspension Culture of Tongkat Ali Hitam (Polyalthia bullata)

Polyalthia bullata, a Southeast Asian plant, is valued for its bioactive compounds with pharmaceutical potential. To prevent overharvesting and extinction, cell suspension culture offers a sustainable method for the mass production of these compounds. Despite its effectiveness, no studies on Polyalthia bullata cell suspension culture have been established. Therefore, this study aimed to establish the culture by evaluating growth and biomass production. To achieve the objective, leaf derived callus of Polyalthia bullata was multiplied on Murashige and Skoog (MS) + 30 µM dicamba medium. Subsequently, cell suspension initiation and multiplication were carried out using half-strength MS basal medium (½ MSO) supplemented with 5, 15, 25, and 30 µM of 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), respectively. In this study, suspension cells in the ½ MSO recorded the highest increment in fresh (4.455 ± 1.170 g FW) and dry weight (0.220 ± 0.033 g DW) but produced dark brown cells. Meanwhile, cells grown on ½ MS medium supplemented with 30 µM NAA recorded the highest increase in fresh weight (3.472 ± 0.694 g FW) and dry weight (0.190 ± 0.012 g DW), displaying a light yellowish-brown cell. Although the ½ MSO yielded the highest biomass, the cell suspension cultures supplemented with 30 µM NAA showed promising results, achieving higher biomass compared to other auxin treatments and exhibiting a light yellowish-brown cell. This suggests that 30 µM NAA is a more efficient auxin utilization in reducing the occurrence of dark brown cells. In conclusion, optimizing auxin concentrations is crucial for high-quality Polyalthia bullata cell suspension culture. This study can provide insight into sustainable cultivation practices for the plant, serving as a potential bio-factory for mass-producing bioactive compounds.

Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of Plectranthus amboinicus (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers

Plectranthus amboinicus (Lour.) Spreng is a medicinally important aromatic perennial herb used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache as well as a flavoring ingredient in traditional drinks, food, and meat stuffing. In this study, a high-performance in vitro propagation system of P. amboinicus through direct shoot organogenesis was developed using axillary node explants cultured on MS (Murashige and Skoog) medium augmented with 0.5, 2.5, 5.0, 7.5, and 10.0 µM of 6-benzyladenine (BA) or kinetin (Kin), alone or with 0.1, 0.5, 2.5, and 5.0 µM of indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). To optimize the regeneration potential of node explants, the effects of basal media strength and pH were also investigated. After 8 weeks of culture, explants cultured in full strength MS basal medium (pH 5.7) with 5.0 µM BA and 2.5 µM NAA exhibited the highest percentage (97.1%) of regeneration and the maximum number (19.3) of shoots per explant. Individual elongated shoots were rooted on half strength MS basal medium containing 0.25 µM indole 3-butyric acid (IBA) after 4 weeks of culture, producing 5.3 roots/shootlets with a root induction frequency of 93.7%. First time genetic stability of in vitro raised P. amboinicus plants was determined using SPAR markers, such as DAMD and ISSR, as well as flow cytometric tests, assuring the availability of authenticated raw materials for commercial production of the plant and its bioactive components.

Influence of different cytokinins on micropropagation of an important medicinal plant, Solanum erianthum D. Don, and assessment of the genetic fidelity of the regenerants

The present study describes the first successful report on an efficient in vitro protocol for direct and indirect organogenesis of an important medicinal and anticancerous plant, Solanum erianthum D. Don. An optimal response using leaf explants was achieved in half-strength Murashige and Skoog (MS)-medium supplemented with 5 mg L−1 naphthalene acetic acid for friable callus formation and 2 mg L−1 naphthaleneacetic acid and 0.5 mg L−1 6-benzylaminopurine amino purine for induction of compact callus. The maximum number of shoot buds (20.33 ± 11.55) was obtained from compact callus using indirect shoot organogenesis in MS medium supplemented with 0.5 mg L−1 thidiazuron among the four cytokinins tested, which included 6-benzyl amino purine, kinetin, 6-γ,γ-dimethylallylamino purine, and thidiazuron. Supplements of 3 mg L−1 6-benzyl amino purine played a crucial role in direct shoot bud formation from a leaf. The buds appeared as small protuberances, and finally developed into leafy shoots as the culture period progressed. Regenerated shoot buds were transferred to MS media that contained four cytokinins for shoot tip culture, in which 8 mg L−1 6-γ,γ-dimethylallylamino purine exhibited the best response (15 ± 2.52). Although efficient plant regeneration was achieved in the presence of all four tested cytokinins in the three organogenetic pathways, distinct differences exist in terms of shoot regeneration potential. Half-strength MS basal medium is recommended for root induction. An approximate 70% survival rate of regenerated plantlets was recorded following sequential transfer from culture conditions to the field. The genetic fidelity of the regenerated plants was evaluated using random amplified polymorphic DNA molecular markers, which showed a high number of uniform monomorphic bands.

Development of in vitro protocol for mass multiplication of open pollinated seedling of rose (Rosa × hybrida L.) cv. Rose Sherbet

An in-vitro protocol for promising open pollinated seedling of rose (Rosa × hybrida L.) cv. Rose Sherbet was developed for mass multiplication. Murashige and Skoog (MS) medium supplemented with 5.0 mg/l BAP + 0.1 mg/l NAA+ 0.5 mg/l GA3 was found most effective for culture establishment. The maximum number of micro shoots (36.67 shoots/explant) were induced on MS medium comprising 5.0 mg/l BAP + 0.1 mg/l NAA+ 0.5 mg/l GA3 along with 40 mg/l adenine sulphate and was found to be better for shoot proliferation. The highest shoot length after 15 days (1.90 cm), 30 days (3.25 cm) and 45 days (4.65 cm) was observed on MS medium supplemented with 1.0 mg/l GA3. Rooting of micro shoots was successfully induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (1: 1: 1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter plants were successfully transferred to the field with good survival.

Development of an effective in vitro Regeneration protocol for BARI Mash 2 (Vigna mungo L.) an important legume crop in Bangladesh

Black Gram (Vigna mungo L.), widely known as Mashkalai in Bangladesh is an important protein source used as human food as well as fodder. BARI Mash 2 is a popular black gram variety released by Bangladesh Agriculture Research Institute (BARI) which is cultivated throughout the country and very popular especially in the char areas. Establishment of a reliable regeneration system for BARI Mash 2 has been tried for further genetic improvement. A rapid, reproducible and efficient in vitro regeneration method was developed using hypocotyl and young leaf explants through callus formation. The frequency of callus formation was highest (75%) on Murashige and Skoog (MS) medium supplemented with a high concentration (31.66 ?M) of 2,4-Dichlorophenoxyacetic Acid (2,4-D) using the young leaf as explants’ source. Callus induction rate was less in hypocotyls in the same medium. No further progress was observed from those calluses. MS medium containing 16.11?M of ?- Naphthalene acetic acid (NAA) showed the 70% calli induction from hypocotyls segment. These calli were amenable to produce multiple shoots (5-6 shoot) in the medium containing 17.75 ?M of 6 Benzyl aminopurine (BAP) alone and the combination of BAP (17.75 ?M ) and NAA (2.68 ?M). Shoots were rooted most effectively (55%) in half strength MS basal medium containing 7.38 ?M of Indole-butyric Acid (IBA). Well rooted plantlets were successfully acclimatized, transferred to the soil and found to produce flowers and fruits. The efficient and reproducible regeneration protocol described here allows for successful in vitro regeneration of BARI Mash 2 that is vital for future genetic manipulation.Jahangirnagar University J. Biol. Sci. 6(1): 23-33, 2017 (June)

In Vitro Plant Regeneration by Organogenesis from Leaf Callus of Carnation, Dianthus caryophyllus L. cv. ‘Master’

An efficient indirect in vitro plant regeneration protocol for Dianthus caryophyllus L. cv. ‘Master’ was developed using leaf explants. This study revealed the morphogenetic potential of leaf explant as a source for micropropagation. Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l Naphthalene acetic acid (NAA) resulted in maximum (94.44%) callus induction. MS medium supplemented with 1.5 mg/l thidiazuron, 0.25 mg/l Kinetin and 0.25 mg/l NAA was found to be highest for average shoot regeneration (80.56%), average number of shoots (6.01) and average shoot length (1.93 cm). For in vitro multiplication of shoots, MS medium supplemented with 2.0 mg/l Kinetin and 0.25 mg/l NAA was found to be the best which resulted in 14.64 average number of microshoots. It was observed that the half strength MS basal medium supplemented with 1.5 mg/l Indole-3-butyric acid and 0.02% activated charcoal showed maximum rooting (98.19%) with 9.60 average number of roots per microshoot having 4.24 cm root length. The in vitro rooted plantlets were hardened gradually and were successfully acclimatized under ex vitro conditions. The genetic variation in the in vitro raised plants was confirmed using DNA based markers [Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats] for assessment of genetic stability of plants raised through indirect regeneration.

Somatic embryogenesis in Hypoxis hemerocallidea: An important African medicinal plant

An efficient in vitro plant regeneration system was established via somatic embryogenesis of Hypoxis hemerocallidea, an extensively utilized medicinal plant in South African traditional medicine. Embryogenic callus was obtained on Murashige and Skoog (MS) basal medium supplemented with 15μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5μM N6-benzyladenine (BA). Different developmental stages of somatic embryos (SEs: globular embryos, torpedo-shaped embryos and cotyledonary embryos) were obtained and further matured from the embryogenic callus by subsequent subculture on the same medium. Highest frequency of somatic embryos (30.56±1.15) was recovered after 6weeks. Scanning electron micrographs revealed the presence of somatic embryos. Mature somatic embryos germinated and developed into plantlets after 4weeks on half strength MS basal agar medium. The germination frequency reached 88.9% when using half strength MS basal agar medium supplemented with 1.44μM gibberellic acid (GA3). Rooted plantlets were successfully acclimatized in the greenhouse with a survival rate of 90%. This is the first report on the establishment of H. hemerocallidea somatic embryogenesis. The protocol developed would be helpful in reducing stress on natural populations, provide a system for germplasm conservation, regeneration of large numbers of high value clonal plants for commercial production, genetic transformational studies and provides a system for analysis of bioactive compounds.

Standardization of protocol for in vitro multiplication of rose (Rosa × hybrida) cv. Happiness

An efficient protocol for in vitro multiplication of Rosa × hybrida L. cv. Happiness was standardized using axillary bud segments. Out of different pre treatments for explants, the highest explant survival (80.25%) was obtained with T1 pre-treatment comprising 0.2% Carbendazim + 0.2% Mancozeb-45 +150 mg/l 8-HQC for 4 hragitation on a horizontal shaker (200 rpm). Sucrose concentration of 30g/l in the medium was found to be optimum for in vitro shoot multiplication. Murashige and Skoog (MS) medium supplemented with 2.5 mg/l BAP + 5.0 mg/l kinetin + 0.1 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment, however, MS medium comprising 2.5 mg/l BAP + 2.5 mg/l kinetin + 0.1mg/l NAA+ 0.5mg/l GA3 along with 40 mg/l adenine sulphate was found to be better for shoot proliferation with highest number of micro shoots (7.10 shoots/explant). Rooting of micro shoots was induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) and IBA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (2:1:1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter, plants were successfully transferred to the glasshouse with good survival.

English

Aseptic cultures of pineapple ‘smooth cayenne’ were established from shoot tip explants. They were initiated on Murashige and Skoog (MS) basal medium with vitamins supplemented with various combinations of 6-benzylaminopurine (BAP) (0, 0.1, 0.2, 0.3 and 0.4 mg L-1) and Gelrite [1.0 g L-1 (semi-liquid), 1.5 g L-1 (semi-solid) and 2.0 g L-1 (solid)]. Explants were transferred five weeks after in vitro initiation to semi-liquid medium containing higher concentrations of BAP (0, 0.5, 1.0, 1.5 and 2.0 mg L-1) for proliferation. Four sub-cultures were made at five weeks interval for twenty weeks in the proliferation medium. Established shoots were introduced into rooting medium containing either full (4.4 g L-1) or half (2.2 g L-1) strength MS basal medium with vitamins supplemented with 0, 0.5 mg L-1 BAP alone or in combination with 0, 0.9 and 1.8 mg L-1 α-naphthaleneacetic acid (NAA). Semi-liquid MS basal medium supplemented with 0.1 and 0.3 mg L-1 BAP gave the best regeneration results, producing the highest average shoot length (34.6 mm) and average number of shoot buds (2.4) per explants. Significant difference (p≤0.05) in average shoot number per explant was observed with semi-liquid MS basal medium supplemented with 1.5 mg L-1 BAP, producing the highest average shoot number (6.1) per explant at 5 weeks and this increased to 167.7 after 20 weeks. The half-strength MS basal medium without growth regulators or with 0.9 mg L-1 NAA gave the highest average root length (29.3 mm) and root number per shoot (7.9). The semi-liquid MS basal medium supplemented with low BAP (1.5 mg L-1) is a cost-effective method for in vitro propagation of pineapple. Half strength MS basal medium without hormones or with low NAA are also cost-effective methods for inducing roots in pineapple.   Key words: Gelrite, growth regulators, medium type, pineapple, propagation.

English

Yams (Dioscorea) are well known for their medicinal use as well as nutritional values. Dioscorea plants are rapidly vanishing from nature due to their over-exploitation by human beings. In order to conserve Dioscorea plants, the present investigation was carried out with a view to regenerate plantlet of Dioscorea alata L. through in vitro culture using full strength Murashige and Skoog (MS) medium and indole-3-acetic acid (IAA) with and without sucrose.  Nodal vine segments of D. alata were used as explants and nodal segments were cultured on MS (Murashige and Skoog’s) medium supplemented with different concentrations of auxin (IAA) for axillary bud proliferation. Best shoot proliferation was observed in MS medium containing 1.5 mg/L kinetin + 2 mg/L IAA with highest rate of shoot multiplication (average of 9.90 shoots/explants). Micro shootlets were inoculated in half strength MS basal medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L) of IAA and best rooting was observed in medium supplemented with 2.5 mg/L IAA with highest root length (8.14 cm). Regenerated plants were transplanted in hardening medium containing Brick bats + Charcoal + Dried moss + Leaf molds + Soil in 1: 1: 1:1:1 proportion. Maximum survival percentage was observed as 85 to 87% after one month of transfer in hardening medium. This work proposes an economic technique for the conservation of D. alata.   Key words: Yams, Dioscorea alata L., in vitro culture, growth regulators.

Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L.

Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4weeks and then transferred to full-strength MS liquid medium for 3weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200μM) of Cu for 7 and 14days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3±1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2μM benzyladenine (BA), 2.9μM indole-3-acetic acid (IAA), and 4.5μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7μM IAA and 2.5μM 2-isopentenyladenine (2iP) with 11.3±0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5cm in length) were treated for rooting and acclimatization in a moistened (5.7μM IAA and 2.5μM 2iP) vermiculite:sand (1:1v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.

Influence of aseptic seedling explants on in vitro shoot multiplication of Caralluma adscendens var. attenuata Wight

A protocol has been developed for in vitro shoot multiplication and plant establishment ofCaralluma adscendens var. attenuata. Aseptic seedlings raised on Murashige and Skoog (MS) basal medium were used as a source of explants. Among different seedling explants cultured on different basal media, nodal explant on MS basal medium supplemented with BA 2 mg/L gave best shoot regeneration frequency. Nodal explants cultured on MS basal medium supplemented with BA 2 mg/L + 2iP 0.5 mg/L produced an average of 4.56 shoots. Microshoots were rooted on half strength MS basal medium with and without plant growth regulators. The best rooting occurred on semisolid half strength MS basal medium containing 0.6% agar and 0.1 mg/L NAA. The plantlets developed were hardened and established in natural soil with 73% survival rate. Key words: Micropropagation, conservation, in vitro shoot multiplication, Caralluma adscendens var. attenuata.

Plant regeneration by somatic embryogenesis from mature seeds of Magnolia obovata

We attempted to develop a method for the regeneration of plantlets from mature seeds of medically important Magnolia obovata via the induction of somatic embryogenesis in vitro. We initially cultured halves of mature seeds on either Murashige and Skoog (MS) medium or B5 medium that contained 0, 1, 5 or 10 μM gibberellic acid (GA3) for 1 month and then transferred the half-seeds to half-strength MS basal medium or B5 basal medium for further culture in the absence of GA3. Proembryogenic masses (PEMs) were observed 1 month after the transfer of the halved mature seeds to the medium without GA3. The frequency of formation of PEMs was higher (28%) after initial culture in MS basal medium plus 1 μM GA3 than in other tested media (0 or 4%). Somatic embryos that had been developed from PEMs were cultured on half-strength MS basal medium or B5 basal medium for completion of maturation and then transferred to fresh aliquots of the same medium for initiation of germination. The frequency of germination, with the formation of normal primary leaves and roots, was above 80%. We transferred the somatic embryo-derived plantlets to soil for acclimatization and the plantlets continued to thrive.

High-frequency transgenic plant regeneration and plumbagin production through methyl jasmonate elicitation from hairy roots of Plumbago indica L.

High-frequency transgenic plant regeneration and production of plumbagin were accomplished from hairy roots induced by Agrobacterium rhizogenes strain A4M70GUS on Plumbago indica L. Of the two types of hairy roots developed on Murashige and Skoog (MS) basal medium, Type I was long and thick with lesser branches and root hairs, and Type II was highly-branched short and slender with tufts of root hairs. Of the different lines grown in half-strength MS liquid basal media, root line 5 (R5) of the Type I yielded the highest plumbagin (0.92% DW) and was significantly different to that of the in vitro control. R5 showed a stable production of plumbagin (1.09% DW) in subsequent cultures. Elicitation of R5 with 50 μM methyl jasmonate for 48 h increased the yield of plumbagin to 5.0% DW, and was superior to 100 μM acetylsalicylic acid (3.8% DW). Plumbagin yield was five times that of twoyear-old ex vitro roots. Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the transformation. Hairy root segments cultured on MS medium containing 8.8 μM benzyladenine and 2.5 μM indole-3-butyric acid induced a mean of 9.1 shoots. Subsequent culture of the isolated shoots developed more than 50 normal shoots per culture. The root-free shoots were rooted on half-strength MS basal medium. The plantlets transferred in field conditions grew normally and exhibited 90% survival. Transgenic plant regeneration and hairy root induction in P. indica serves as reliable source of plumbagin which in turn cut off the mass destruction of the plant species.

Development of a regeneration system via nodal segment culture in Veronica anagallis-aquatica L. – an amphibious medicinal plant

The present study describes an effective, reproducible and rapid regeneration protocol for in vitro shoot development of the medicinally important, semi-aquatic plant Veronica anagallis-aquatica L. through mature nodal segments on Murashige and Skoog (MS) medium. The most effective medium for shoot regeneration was MS medium fortified with 0.5 µM 6-benzyladenine (BA), on which the maximum number (43.7±1.85) of shoots per explant having a shoot length of 5.0±0.25 cm were produced. The addition of auxins along with optimal concentration (0.5 µM) of BA did not further improve the regeneration capacity of the explants; instead, it resulted in an adverse effect. Nodal segments cultured in MS liquid medium without agar provided a better and early response for efficient shoot regeneration, and simultaneous rooting as compared to solidified medium. The optimal liquid medium comprised of MS+BA (0.5 µM) on which a maximum 20.7±1.76 shoots per explant were produced and grew healthier. Both half strength MS and MS basal medium supplemented with α-naphthaleneacetic acid (NAA) responded equally for root induction. Rooted shoots were successfully transferred to field conditions with 80% survival. Histological studies revealed the de novo origin of shoots which would make this protocol suitable for transformation techniques.

High frequency plant regeneration system for Nymphoides coreana via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l−1 of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l−1 with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l−1 of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.

Direct adventitious shoot induction and plant regeneration of Embelia ribes Burm F.

An efficient micropropagation system via direct shoot organogenesis from hypocotyl segments of Embelia ribes Burm F. was developed. A high frequency (84%) of adventitious shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with additives (283.85 μM ascorbic acid [AA], 118.96 μM citric acid [CA], 142.33 μM cysteine, and 684.22 μM glutamine) and 1.13 μM of thidiazuron (TDZ) after 4 weeks following culture. Further development of shoot primordia into well-grown shoots of 4–5 cm in length was achieved by sub-culturing explants along with shoot primordia on MS medium supplemented with 0.44 μM benzyl adenine (BA) and 0.49 μM indole butyric acid (IBA) for three sub-culture periods with an interval of 15 days between them. The highest shoot multiplication was obtained when explants were incubated on MS medium supplemented with 2.2 μM BA and 0.49 μM IBA in 4 weeks. All in vitro developed shoots, 3–4 cm in length, rooted when grown on half-strength MS basal medium along with 2.47 μM IBA within 4 weeks. Moreover, 100% of shoots developed roots when these were treated with 4.93 μM IBA for 20 min and then transferred to pots containing soilrite mix and grown in the greenhouse. In vitro and ex vitro rooted plants showed a survival of 85 and 95% respectively, during hardening in the greenhouse for a 6-week period.

In vitro plantlets regeneration in Bambara groundnut [Vigna subterranea (L.) Verdc. (Fabaceae)] through direct shoot bud differentiation on hypocotyl and epicotyl cuttings.

In vitro regeneration system via direct organogenesis in Bambara groundnut (Vigna subterranea L.) using hypocotyl and epicotyl cuttings was studied. Basal Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) or Thidiazuron (TDZ) with or without
-naphthaleneacetic acid (NAA) were attempted. Multiple shoots were induced from both explants but regeneration efficiency was higher when epicotyl cuttings were used. The ability of hypocotyl and epicotyl cuttings to produce shoots varied depending on the culture medium. BAP (2 mg/l) gave the highest response (73.33 - 97.77%) with the regeneration of 3.7 shoots per explant with hypocotyl and 5.8 shoots per explant with epicotyl. Substitution of BAP at an equimolar concentration with kinetin and TDZ, and incorporation of NAA during shoot bud induction did not show any improvement over that obtained with BAP and promoted callusing to a different degree. The regenerated shoots were readily elongated on the same medium as used for induction and rooted on half-strength MS basal medium without any growth regulators. 62% of the plantlets were successfully acclimatized and potted plants were established in soil with 73% survival rate.