Strength Murashige And Skoog Basal Medium Research Articles

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.

Improved protocol for micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cardamomum Maton)

Cardamom (Elettaria cardamomum Maton) is a highly valuable commercial spice crop, originating from the Western Ghats of southern India. It has importance both as a spice and for its medicinal properties. An efficient and reliable micropropagation protocol using shoot tips as explants is presented which can be utilized for development of elite planting material. Growth patterns of cardamom shoots were analyzed on 45 media treatments and best shoot proliferation was recorded on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.32 μM kinetin (Kn) with 5.83 shoots regenerated per explant. The longest shoots (6 cm) were observed on 0.44 μM BAP and 2.32 μM Kn containing medium. Regenerated shoots rooted well on full strength MS basal medium, generating 3.50 roots per explant with an average root length of 4.33 cm within 4 wk. Successful hardening with a field survival percentage of 80% was achieved after acclimatization of well-developed plantlets. Genetic stability of the micropropagated plants was confirmed using 13 ISSR markers. This study reports an improved micropropagation protocol for cardamom with an enhanced rate of shoot multiplication leading to the production of healthy shoots which can be easily rooted and successfully transferred to the field.

Effect of cytokinin and MS medium composition on efficient shoot proliferation of Nyctanthes arbor-tristis L. through cotyledonary node explant and evaluation of genetic fidelity and antioxidant capacity of regenerants

Nyctanthes arbor-tristis L. is an important member of family Oleaceae. An efficient shoot proliferation response (21.53±0.58 shoots/explant) from cotyledonary node was achieved on Murashige and Skoog (MS) medium supplemented with 22.19 µM 6-benzyl amino purine (BAP). To optimize the role of ammonium and nitrate on shoot proliferation, the explants were cultured on modified MS media (MS-1 to MS-12). Among these media, MS-3 and MS-9 were found most suitable for shoot-proliferation than others. The microshoots after indole 3-butyric acid (IBA) pulse treatment (1968.11 µM) for 30 min, when sub-cultured on ½ strength MS basal medium were successfully rooted with a maximum frequency of root induction (77.66±1.45), the maximum number of roots/ shoot (10.66±0.88) and maximum root length (1.60±0.05 cm). In-vitro rooted plants were acclimatized and transplanted outside into field with 80 percent survival-rate after 90 days. The genetic-fidelity was confirmed by monomorphic nature of inter-simple sequence repeat (ISSR) marker pattern. The ethanol extract of leaves of regenerated plants contained good amount of phenol (118.00±0.82 µg/mg gallic acid equivalent) and flavonoid (17.34±0.64 µg/mg rutin equivalent) and possess strong antioxidant activity. The EC50 value for 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, superoxide radical (SOD) scavenging activity and lipid peroxidation (LPO) inhibition activity was 339.98±3.27, 755.80±75.50 and 689.67±2.46 µg/ml respectively. Hence, these findings suggest that regenerated plants can be used as a therapeutic and inhibitory agent against free radical-induced disease progression.

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.

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.

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.

Direct plant regeneration from nodal explants of Balanites aegyptiaca L. (Del.): a valuable medicinal tree

This report describes in vitro shoot induction and plant regeneration from nodal segments of Balanites aegyptiaca on Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA), thidiazuron (TDZ) and kinetin (Kin) (0.5–20.0 μM). MS medium supplemented with BA (12.5 μM) was the most effective in inducing bud break and growth and also in initiating multiple shoot proliferation. However, the optimal level of TDZ supplementation to the culture medium was 5.0 μM. Shoot cultures were established by repeatedly subculturing the original nodal explants on the same medium. Highest number of shoots (11.5 ± 0.7) and shoot length (5.0 ± 0.2 cm) were achieved when cultures were subcultured on MS medium supplemented with 12.5 μM BA and 1.0 μM α-naphthalene acetic acid (NAA). The shoots regenerated from TDZ supplemented medium when subcultured to hormone free MS basal medium considerably increased the rate of shoot multiplication and shoot length by the end of fifth subculture. Rooting of the shoots was achieved on MS medium augmented with 1.0 μM indole-3-butyric acid (IBA) plus 0.5% activated charcoal followed by their transfer to half strength MS basal medium. The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 70% survival rate. The results of this study provide the first successful report on in vitro direct plant regeneration of B. aegyptiaca.

Plantlet regeneration from leaf derived callus of Vanilla planifolia Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0±1.0 shoots with an average length of 3.6±0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3±0.14 plantlets with an average height of 3.6 cm±0.10 cm. All plantlets produced profuse rooting within 35-40 d after transfer to half- strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.

Multiple Shoot Regeneration from Young Shoots of Kenaf (Hibiscus cannabinus)

A method was developed to initiate multiple shoots from the young shoot of kenaf. Young shoots along with the cotyledons were excised from ten-day old aseptically germinated seeds and pre-cultured for two weeks in Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or a combination of BA and kinetin. After two weeks in culture, elongated shoots were excised above the cotyledonary nodes and cultured on fresh medium of the same composition. Multiple shoots were initiated within eight weeks. The number of shoots varied among cultivars. The highest number of shoots (11/explant) occured in cultivar Tainung 2 (T2) cultured in MS medium supplemented with 8.8 μM BA. Concentrations of BA higher than 8.8 μM had a negative effect on the number of shoots. Furthermore, callus growth was initiated from which morphologically abnormal shoots were induced. Kinetin had a significant effect only on cultivar Everglades 41 (E41). Shoot elongation and rooting were obtained simultaneously in half strength MS basal medium with no plant growth regulators. About 98% of the rooted plants were grown to maturity under greenhouse conditions. This method was successful with all four genotypes tested. However, significant genotypic variations were observed among the genotypes.