Liquid Murashige And Skoog Medium Research Articles

Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.

A two step method for accelerated mass propagation of Dendrocalamus asper and their evaluation in field

An accelerated protocol for large-scale propagation of Dendrocalamus asper, an edible bamboo, has been described. Seven axillary shoots were induced in vitro from each excised tender node (15-20mm in length) containing single axillary bud when nodal segments were inoculated in semisolid Murashige and Skoog (MS) medium fortified with 5mg/l 6-benzylaminopurine (BAP). Maximum multiple shoot formation (14) was observed when in vitro generated axillary shoots were transferred to liquid MS medium containing 5mg/l BAP and 40mg/l adenine sulphate. A maximum of 93.33% shoots were effectively rooted when transferred to liquid MS medium supplemented with 1mg/l indole-3-biutyric acid (IBA). A simple acclimatization procedure of 55days, primarily in cocopeat for 20days and finally in a blend of sand, soil and farm yard manure (1:1:1 v/v), ensured a very high survival rate within next 35days. After acclimatization, rooted plantlets were further multiplied by splitting of rhizomes, formed in vivo within 90days of growth. After 90 and 180days of acclimatization, plants were successfully transferred to the field and maintained in an unirrigated condition with the initial supplementation of farm yard manure @ 10kg/pit; where around 85% survivability with 25 culms per bush attaining an average height of 4.5m was recorded up to four years.

Micropropagation of Bambusa balcooa Roxb. through axillary shoot proliferation

Bambusa balcooa is one of the most commercially important bamboo species. Regeneration of this species by sexual means is impossible because no seeds are set after flowering. Vegetative propagation is hindered due to bulky propagules, low rooting ability of the culm and branch cuttings, and seasonal specificity. This makes in vitro-based methods of regeneration important. This paper describes an efficient micropropagation protocol for multiplication of B. balcooa from nodal explants. Nodal segments were surface sterilized with 0.1% mercuric chloride for 10 min, and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP), 2.32 μM kinetin (Kn), and gelled with 0.2% w/v gelrite. Eighty-five percent of explants could be established in vitro with 90% of these achieving bud break. In vitro-formed shoots were successfully multiplied in MS liquid medium supplemented with 6.6 μM BAP, 2.32 μM Kn, 2.5% v/v coconut water, and 100 mg l−1myo-inositol. Subculturing shoots every 3 wk yielded a consistent proliferation rate of 4.11-fold without decline in vigor. Shoot clusters, containing 5 to 8 shoots, were rooted with 87.5% success in 1/2 MS supplemented with 5.71 μM indole-3-acetic acid (IAA), 4.9 μM indole-3-butyric acid (IBA), and 5.37 μM naphthaleneacetic acid (NAA) within 3 wk. Plants regenerated in this manner were acclimatized in the greenhouse and under a shade net with 88% success.

Micropropagation of a Thai medicinal plant for women’s health, Curcuma comosa Roxb., via shoot and microrhizome inductions

We studied the effects of explant types, plant growth regulators, and sucrose concentrations on shoot and microrhizome inductions of Curcuma comosa Roxb., an important Thai medicinal plant for women's health. Explant types significantly affected shoot induction of the plant. The maximum shoot multiplication rate of 11.82 ± 1.03 shoots/responding explant was obtained when culturing terminal bud explants on semi-solid Murashige and Skoog (MS) medium supplemented with 18.16 μM thidiazuron for 8 weeks. Subsequently, they were transferred to a semi-solid MS medium without plant growth regulators for 4 weeks. The regenerated shoots produced roots spontaneously. Rooted plantlets were successfully transferred to the soil. Microrhizome induction was significantly influenced by sucrose concentrations, but not by 6-benzyladenine (BA). Liquid MS medium with a combination of 17.76 μM BA and 50 g L(-1) sucrose was optimal for microrhizome induction of C. comosa. After 12 weeks of culture, the microrhizome induction rate was 3.36 ± 0.44 microrhizomes/responding explant. Starch accumulation in microrhizomes increased with higher sugar concentration and with longer duration of culture. The microrhizomes were allowed to germinate under greenhouse conditions and further developed into normal plants. The protocols established will be used for the production of uniform plantlets suitable for field plantation for the herbal industry.

Establishment of Cell Suspension Cultures and Plant Regeneration in White Dandelion (Taraxacum coreanum NAKAI.)

In this study, we established a novel somatic embryogenesis and plant regeneration system through cell suspension culture of white dandelion (Taraxacum coreanum NAKAI.). Embryogenic calli could be initiated from leaf and root explants of sterile seedlings on solid Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 3-week cultures. To proliferate embryogenic calli rapidly, cell suspension culture was performed with transferred to liquid MS medium with various combinations of plant growth regulators (PGRs) including 2,4-D, <TEX>${\alpha}$</TEX>-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), <TEX>$N^6$</TEX>-benzylamino purine (BAP), thidiazuron (TDZ), and kinetin. During suspension cultures, embryogenic calli not only greatly proliferated, but shoot organogenesis also simultaneously occurred from the surface of somatic embryos. Among them, TDZ at lower concentration, 0.1 mg/L produced the highest efficiency of somatic embryo formation and shoot organogenesis. Rooting of embryogenic calli with adventitious shoots was done on solid MS medium containing 0.1 mg/L NAA and 0.3% activated carbon. Nearly 80% of embryogenic calli with shoot organogenesis could be rooted normal. Well-rooted plantlets were transferred into pots under a greenhouse condition, and plants derived from this system appeared phenotypically normal.

A novel approach for rapid micropropagation of maspine pineapple (Ananas comosus L.) shoots using liquid shake culture system

Maspine ( Ananas comosus L.) is currently the most preferred pineapple variety in Malaysia due to its pleasant aroma and applicability in caning. Large quantities of plant materials are needed to fulfill the market demand which could not be obtained from the conventional breeding method. Hence, in vitro procedure was developed as an alternative method to improve the multiplication rate of this special variety. Sterilized explants were cultured on solidified Murashige and Skoog (MS) medium supplemented with various combinations of 6-benzylaminopurine (BAP) (1 to 5 mg/l) and ∝-naphthaleneacetic acid (NAA) (1 to 5 mg/l) hormones. Pineapple plant cultures required 5 mg/l BAP to significantly increase the shoot development during the in vitro stage. In addition, explants were subsequently sub-cultured on medium with 1 mg/l BAP which produced highest number of proliferated in vitro plantlets. The optimization of the conditions for shoot propagation was carried out in both liquid and solid medium by supplementing with 1 or 5 mg/l of BAP. MS liquid medium supplemented with 1 mg/l BAP produced the highest number of shoots (31) after 4 weeks. The number of shoots formed was increased to 204 after third sub-culture in liquid medium. Shoot proliferation was increased up to nine-fold in liquid medium when compared to the cultures maintained on solid medium. This improved method of Maspine in vitro multiplication will serve as an alternative source of planting materials of this cultivar for subsistence and large-scale pineapple farmers. Key words : Pineapple, in vitro, 6-benzylaminopurine, ∝-naphthaleneacetic acid, liquid medium.

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l−1 sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l−1 resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l−1 sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.

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.

Transformation through agroinfection on decapitated shoot apex of field-growing Phyllanthus amarus

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A4M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A4M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A4M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.

Micropropagation and root culture of Turkish endemic Astragalus chrysochlorus (Leguminosae)

Efficient micropropagation and root culture protocols were developed for the endemic Astragalus chrysochlorus Boiss. & Kotschy. A high frequency of shoot formation (100%) and maximum multiplication (13 shoots per hypocotyl explant) were achieved on Murashige and Skoog (MS) media supplemented with 0.5 mg/L trans-Zeatin riboside (ZR). By using single regenerated hypocotyl explants, rooting was achieved at a rate of 93% on MS medium containing 2% sucrose without growth regulators. High frequency callus initiation and growth were achieved when single hypocotyl explants were inoculated on MS medium supplemented with 0.5 mg/L 2,4-Dichlorophenoxyacetic acid. Plant regeneration through indirect organogenesis was achieved on MS medium supplemented with 0.5 mg/L ZR. Root cultures were successfully established in liquid MS medium containing 0.5 mg/L \alpha-Naphthaleneacetic acid. The optimised in vitro propagation, callus culture, and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies on Astragalus L. species.

High frequency shoot regeneration through cotyledonary node explants of Bauhinia tomentosa L., a woody leguminous tree

SummaryAn efficient in vitro method has been established for the production of whole plantlets of Bauhinia tomentosa using cotyledonary node (CN) explants excised from 15 d-old aseptic seedlings. Proliferating shoot cultures were obtained by placing CN explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), kinetin (Kn), and 2-isopentenyl adenine (2-iP), singly or in combination, with either of the auxins, indole-3-acetic acid (IAA) or αnaphthalene acetic acid (NAA). MS medium augmented with 5.0 µM BA was found to be most effective in providing the highest frequency (84%) of shoot regeneration, associated with the maximum number of shoots (9.0 ± 0.6) per explant. The highest efficiency of shoot proliferation was observed using a combination of 5.0 µM BA and 0.5 µM NAA, where the number of shoots increased to 16.2 ± 0.2 per explant after 8 weeks of culture. Improved shoot multiplication and elongation rates were achieved when 20.0 mg l–1 adenine sulphate (AdS) was added to the combined treatment of 5.0 µM BA and 0.5 µM NAA, where an average shoot number of 17.0 ± 0.6 and an average shoot length of 5.8 ± 0.3 cm were obtained.The maximum frequency of root formation (70%) and greatest root lengths (2.3 ± 0.4 cm) were obtained on filter-paper bridges in liquid MS medium augmented with 2.5 µM IBA and 5.0 µM chlorogenic acid (a phenolic compound) after 4 weeks of culture. Regenerated plantlets were successfully acclimatised in a growth room and subsequently transferred to a greenhouse.This is the first report on in vitro plantlet regeneration in B. tomentosa using CN explants.

Developing a model system in vitro to understand tracheary element development in Douglas-fir (Pseudostuga mensziesii)

Callus cells were initiated on cambial strips obtained from 4 to 8 y old Douglas-fir (Pseudostuga menziesii) trees, cultured on solidified Murashige and Skoog (MS) medium supplemented with 2,4- dichlorophenoxyaceticacid (2,4-D) and benzylaminopurine (BA). The cultures could be maintained by sub-culturing on fresh medium every four weeks. When the callus cells were subsequently transferred to liquid MS medium supplemented with different phytohormones, suspension cultures could be initiated and maintained by periodic sub-culture. Approximately 65% of the callus cells cultured on liquid MS medium supplemented with 2,4-D, when maintained for 6-7 weeks without sub-culture, differentiated to tracheary element (TE) like cells. The formation of TE like cells was confirmed histochemically by staining with phloroglucinol-HCl. Secondary thickening of the cell walls were confirmed by polarized light microscopy, which showed strong birefringence of the cell wall due the presence of crystalline cellulose. The presence of lignin was determined by pyrolysis- GC-MS and FTIR spectroscopy. The lignin content in differentiated cell wall samples was quantified at 21% by the lignothioglycolic acid assay. Analysis of monosaccharide composition of cell wall samples after acid hydrolysis showed that the percentage of glucose, xylose and mannose had increased in the differentiated cell walls. These increases correspond to the formation of cellulose, glucomannan and xylan, primarily associated with secondary cell walls.

Shoot regeneration from petiole explants of Simmondsia chinensis (Link) Schneider

SummaryA method has been developed for in vitro shoot regeneration from in vivo and in vitro petiole explants of Simmondsia chinensis. Efficient shoot regeneration could be initiated on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). Shoot regeneration was influenced significantly by the size of the leaf attached to the petiole and the orientation of the in vivo explant (i.e., vertical or horizontal). Petioles obtained from pruned branches of plants responded better than those from normal plants (unpruned). Explants placed horizontally on MS medium supplemented with 2.76 µM TDZ resulted in 70.3% regeneration. When in vitro leaves were placed on MS medium containing filter-sterilised 0.46 µM TDZ, regeneration was 71%.The efficiency of regeneration of in vitro leaves was not influenced by the size of the leaf attached to the petiole, but the orientation of the explant significantly influenced regeneration. Explants which responded positively were transferred to MS medium supplemented with various concentrations of TDZ and αnaphthaleneacetic acid (NAA) for shoot elongation and proliferation. The highest numbers of shoot buds (four-tofive) were achieved on MS medium supplemented with 1.38 µM TDZ and 1.62 µM NAA. Shoots were pulse-treated in half-strength liquid MS medium supplemented with 24.5 µM indole-3-butyric acid (IBA) and 5.40 µM NAA for 8 d, and then transferred to half-strength MS medium supplemented with 2.0% (w/v) sucrose, 0.02% (w/v) activated charcoal, 0.65% (w/v) agar for rooting. Rooted plants were hardened and established in soil with a ≥ 90% success rate.

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).

In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation

This communication describes for the first time an efficient and reproducible protocol for large-scale multiplication of Bambusa nutans. Nodal segments collected from field-grown clumps and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kin) gelled with 0.2% gelrite yielded 80% aseptic cultures with 100% bud-break. The in vitro-formed shoots obtained after bud-break were successfully multiplied in MS liquid medium supplemented with 13.2 μM BA, 2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every 3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-fold. Shoot clusters containing three to five shoots were successfully rooted with 100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA, 2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthaleneacetic acid (NAA), and 3% sucrose. Plantlets grown in vitro were acclimatized and subsequently transferred to the field. Inter-simple sequence repeat analysis has confirmed the genetic uniformity of the tissue-cultured plants up to 27 passages.

Hairy roots production of transgenic Catharanthus roseus L. plants with Agrobacterium rhizogenes under in vitro conditions.

Hypocotyl explants of Catharanthus roseus L. gave rise to callus at a frequency of 90% when cultured on Murashige and Skoog (MS) basal medium supplemented BA (31.1 µM) and NAA (5.4 µM) on dark conditions. The calli (8 weeks old) were placed onto half strength liquid MS medium that had been polluted by bacterial clones of Agrobacterium rhizogenes. Transgenic of calli regenerated hairy roots after 10 days of inoculation and they have grown after 8 weeks. Hairy roots of in vitro C. roseus L. plant which produces several important terpenoid indole alkaloid by infection with A. rhizogenesis. Transformed hairy root increased and produced alkaloids, including vinblastin and vincristine, which are extracted and analyzed by high performance liquid chromatography (HPLC) and used for anticancer and antimicrobial researches. Key words: Catharanthus roseus, Agrobacterium rhizogenes, genetic transformation, hairy roots.

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg’s B5 (B5) medium, supplied with 0.1 mg/L indole-3-butyric acid (IBA) and 20 g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3 mg/L IBA and 30 g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026 mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4 g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07 mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.

Sustainable use of &lt;i&gt;Cryptocoryne wendtii&lt;/i&gt; and &lt;i&gt;Echinodorus cordifolius&lt;/i&gt; in the aquaculture industry of Sri Lanka by micropropagation

In the aquaculture industry of Sri Lanka, Cryptocoryne and Echinodorus species are important aquatic plants. In the absence of a regular supply due to lack of effective propagation methods, Cryptocoryne species are indiscriminately harvested from the wild to supply to the export market. The threat on the species is further compounded by the loss of their rainforest habitats. Out of the ten endemic Cryptocoryne species that occur in Sri Lanka, nine are classified as Highly Threatened species in the Red List of International Union for the Conservation of Nature. Echinodorus cordifolius is not found in the wild but is popular in aquaria. In order to overcome the problems of species loss and inadequate supply to the local and foreign markets, an in vitro micro-propagation method was developed for both these species. Due to the difficulty in obtaining axenic cultures from these species, rhizome segments were used to induce axillary bud growth and subsequent shoot multiplication. Shoot buds were induced from rhizome segments of C. wendtii cultured on a Murashige and Skoog (MS) basal medium supplemented with 11 – 133 µM benzyladenine (BA) and 13.4 µM naphthalene acetic acid (NAA). The induced shoots were separated after 21 days and sub-cultured twice every 14 days on MS medium, which increased the shoot multiplication. E. cordifolius responded positively to a combination of 24.6 µM N-isopentenyladenine (2iP) and 2.68 µM NAA, while 2iP alone did not induce shoots. Two sub-cultures at 14 day intervals increased the shoot multiplication. Rooting was induced in both species by culturing the shoots in ½ MS liquid medium with indole butyric acid (IBA). Acclimatization was done in a humid growth chamber for one week and the plantlets were gradually transferred to the green house. All the plantlets rooted and survived in the green house. DOI: 10.4038/sljas.v12i0.2216 Sri Lanka J. Aquat. Sci. 12 (2007): 89-101

THE EFFECTS OF PROLINE AND COCONUT WATER ON CALLUS INDUCTION OF CUCUMBER (CUCUMIS SATIVUS L.)

Callus was initiated from shoot tips and embryonic axes of cucumber (Cucumis sativus L.) in a Murashige and Skoog (MS) medium supplemented with 1 mg/L naphthalene acetic acid (NAA) and 0.5 mg/L kinetin in the presence of 0, 10 and 20 mM proline. Earlier callus induction was observed with embryonic axes (3 days after initiation), compared to shoot tips (7 days after initiation) with callusing frequency of 100%. Moreover, the average size of embryonic axis-derived calli was 1.3- to 1.9-fold larger than those derived from shoot tips at 2 weeks of culture. No effect of proline was found on percentage of callus induction of both explants. Addition of proline also did not affect the growth of callus from shoot tips. However, inhibitory effect was found on the growth of embryonic axis-derived calli (1.3-fold reduction in comparison with control (without proline)). When the effect of coconut water on callogenesis was evaluated on embryonic axes, callus formation was substantially retarded. However, coconut water significantly promoted hypocotyl elongation. Germination of seeds either in liquid MS medium supplemented with 0.1 mg/L thidiazuron (TDZ) or 0.1 mg/L kinetin prior to callus initiation slightly delayed callus induction but did not significantly affect callus size. However, at 2 weeks of culture kinetin significantly decreased the length of hypocotyls.

Production of withanolide-A from adventitious root cultures of Withania somnifera

Withanolides are biologically active secondary metabolites present in roots and leaves of Withania somnifera. In the present study, we have induced adventitious roots from leaf explants of W. somnifera for the production of withanolide-A, which is having pharmacological activities. Adventitious roots were induced directly from leaf segments of W. somnifera on half strength Murashige and Skoog (MS) semisolid medium (0.8% agar) with 0.5 mg l−1 indole-3-butyric acid (IBA) and 30 g l−1 sucrose. Adventitious roots cultured in flasks using half strength MS liquid medium with 0.5 mg l−1 IBA and 30 g l−1 showed higher accumulation of biomass (108.48 g l−1FW and 10.76 g l−1 DW) and withanolide-A content (8.8 ± 0.20 mg g−1 DW) within five weeks. Nearly 11-fold increment of fresh biomass was evident in suspension cultures and adventitious root biomass produced in suspension cultures possessed 21-fold higher withanolide-A content when compared with the leaves of natural plants. An inoculum size of 10 g l−1 FW favoured the biomass accumulation and withanolide-A production in the tested range of 2.5, 5.0, 10.0 and 20.0 g l−1 FW. Among different media tested [Murashige and Skoog (MS), Gamborg’s (B5), Nitsch and Nitsch (NN) and Chu’s (N6)], MS medium favoured both biomass accumulation and withanolide-A production. Half strength MS medium favoured the biomass accumulation and withanolide-A production among the different strength MS medium tested (0.25, 0.5, 0.75, 1.0, 1.5 and 2.0). The current results showed great potentiality of adventitious roots cultures for the production of withanolide-A.