Full Strength MS Basal Medium Research Articles

English

In vitro regeneration of plants is influenced to a great extent by genotype, the type of explant used, media composition and plant growth regulators. This research was devised to study the effect of explant source and different hormonal combinations on the in vitro regeneration of Heracleum candicans. Two different explants viz; leaf and petiole were used to investigate their morphogenic response on MS medium supplemented with different concentrations of auxins (2, 4- dichlorophenoxyacetic acid and indole 3-acetic acid) and cytokinins (6-benzyl amino purine and kinetin) either individually or in combinations. Petiole explants were most responsive for callus production as well as shoot regeneration. 2,4-D at 3 mg/L proved to be the best concentration for callus induction. For shoot regeneration, MS medium supplemented with BAP at 3 mg/l and IAA at 2 mg/l proved to be effective in production of 21±0.7 mean number of shoots in 90% of cultures. Regenerated shoots were separated and rooted on full strength MS basal medium producing 6.9 average numbers of roots per shoot in 90% of cultures. The present research yielded a suitable explant and optimum concentrations of different plant growth regulators for in vitro regeneration of this important medicinal herb. Key words: Heracleum candicans, leaf, petiole, callus, shoot regeneration.

Encapsulation, short-term storage, conservation and molecular analysis to assess genetic stability in alginate-encapsulated microshoots of Ocimum kilimandscharicum Guerke

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Ocimum kilimandscharicum Guerke. The best gel complexation for encapsulation of shoot tips was achieved using 3 % sodium alginate and 75 mM calcium chloride. Re-growth ability of encapsulated shoot tips explant was evaluated after storage at 4 and 25 °C for 30, 45, 60, 75 and 90 days. Preservation at 25 °C being significantly more resistant compared to that stored in 4 °C. Maximum shoot re-growth (83.33 %) was recorded on 0.7 % agar solidified full strength MS basal medium supplemented with 1.0 mg/l BA. Micro shoots, recovered from encapsulated shoot tips (capsule) were best rooted on ½ strength MS medium containing 1.5 mg/l IBA. Complete plantlets (with shoot and root) were transferred to plastic pots containing soil: vermiculite (1:1) showing 79.53 % survival rate in field condition. The genetic stability of synseed-derived plantlets (following 4 and 25 °C of storage) was assessed and compared with mother plant using two PCR based molecular markers namely random amplified polymorphic DNA and inter-simple sequence repeat. Both types of markers revealed high degree of monomorphism, similar to those of the mother plants.

Molecular and phytochemical analyses to assess genetic stability in alginate-encapsulated microshoots of Ocimum gratissimum L. following in vitro storage

An efficient protocol was developed for short term storage and conservation of Ocimum gratissimum L., using encapsulated micro shoots. The encapsulation of micro shoots was significantly affected by the concentrations of sodium alginate and calcium chloride. A gelling matrix of 3 % sodium alginate and 75 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Re-growth ability of encapsulated shoot tips explant was evaluated after storage at 4 and 25 °C for 30, 45, 60, 75 and 90 days. Preservation at 25 °C being significantly more resistant compared to that stored in 4 °C. Maximum shoot re-growth (98.62 %) was recorded on 0.8 % agar solidified full strength MS basal medium supplemented with 1.0 mg/l BA. Micro shoots, recovered from encapsulated shoot tips (capsule) were best rooted on ½ MS medium containing 1.5 mg/l IBA. Complete plantlets (with shoot and root) were transferred to plastic pots containing soil: vermiculite (1:1) showed 83.31 % survival rate in field condition. The genetic stability of synseed-derived plantlets (following 4 and 25 °C of storage) was assessed and compared with mother plant using two PCR based molecular markers namely random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). Both types of markers revealed high degree of monomorphism, similar to those of the mother plants. Essential oil composition of the synseed-derived plants and the mother plant were determined by GC-FID analysis and insignificant differences were found in the oil composition, thereby confirming the stability of plants derived from synthetic seeds following short term storage.

English

A reproducible protocol for direct shoot organogenesis of Cleome viscosa, an important medicinal weed herb was developed. The seed explants were collected primarily from field grown mature plants for in vitro germination on different strength of MS basal medium with or without selection of GA3 at various concentration (0.1 to 1.0 mg/L). The highest rate of seed germination (55.3%) was noticed on full strength MS basal medium fortified with 0.5 mg/L GA3 after 30 days of culture. The excised 7 to 10 days old cotyledonary leaf, cotyledonary node and hypocotyls explants cultured on MS medium fortified with different concentration of individual cytokinin (BA/KIN/TDZ) alone or BA+KIN or TDZ+KIN or TDZ in combinations with different auxins (IBA/NAA/IAA) influenced the frequency of adventitious microshoot formation. The rate of shoot multiplication was greatest (100%) in cotyledonary leaf explants cultured on 3.0 mg/L TDZ and 0.3 mg/L IBA tested medium after 45 days of culture. The individual microshoots were elongated well in 0.3 mg/L TDZ and 0.1 mg/L GA3 treated medium, but more number of adventitious micro roots were developed on half strength MS medium fortified with 0.1 mg/L NAA. The regenerated healthy plants were hardened in pots containing soil mix and well established into complete state similar to that of field grown plants under greenhouse condition.   Key words: Cleome viscosa L, medicinal weed plant, shoot multiplication, direct organogenesis.

Organogenesis from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis -a vulnerable species of Western Ghats

An efficient system was developed for indirect plant regeneration from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis. Maximum percentage of the organogenic callus was obtained on MS medium supplemented with NAA (1.0 mg/l) and 2,4-D (2.0 mg/l). The best shoot bud induction was observed on MS medium with BA (1.0 mg/l) +IBA (0.5 mg/l). The coconut water (15%) was better, resulting in a differentiation of the shoot initials in to well-developed shoots. The elongated shoots (› 3cm long) were rooted on a full strength MS basal medium, supplemented with 0.2 mg/l of IBA. Finally, the rooted plants were transferred to the soil with 80% success rate. This protocol was utilized for the in vitro propagation of this endangered plant species.

In Vitro Response of Cotyledon Explants of Cashew (Anacardium occidentale L.) Cultured in Different Concentrations of BAP

This experiment was carried out to study the embryogenic response of cashew ( Anacardium occidentale L.) cotyledon explants cultured in different concentrations of BAP under in vitro conditions. Sterilized cotyledon segments (10 mm long, 5 mm width) were cultured on full strength MS basal medium containing various concentrations (0-10 mg/L) of BAP. The result revealed the medium without BAP exhibited profuse root organogenesis which decreased with the increase in the concentration of BAP. Greenish or yellowish white nodular structures appeared on the edge of the surface concentrated towards the convex side of the explants. Consequently, these structures gave rise to somatic embryoids and the degree of embryoid formation increased with the increasing concentration of BAP. MS medium with 8 mg/L BAP was the most optimal concentration for the induction of somatic embryos among the tested. Cytological study confirmed the development of somatic embryoids directly from cultured cotyledon explants. Journal of Food and Agriculture 2010 Vol. 03, No. 1 & 2, Page 7-13 DOI: http://dx.doi.org/10.4038/jfa.v3i1-2.5165

Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local

A protocol for plant regeneration by somatic embryogenesis was developed in guava cv. Banarasi local by using immature zygotic embryo explants. Best induction of somatic embryogenesis was achieved from 10-week-old zygotic embryos on MS medium supplemented with 2,4- d (4.52 μM) and 5% sucrose. Maximum number of somatic embryos was produced when zygotic embryo explants were transferred to growth regulator free full strength MS basal medium after 8 days treatment with 2,4- d. Full strength MS basal medium containing 5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of conversion and normal plantlet production were recorded from elongated torpedo stages of somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted shoots survived acclimatization.

Bioguided Fractionation and Isolation of Free Radical Scavenging Components from in Vitro Propagated Chinese Medicinal Plants Dendrobium tosaense Makino and Dendrobium moniliforme SW

This study was performed to investigate the free radical scavenging active components from in vitro propagated medicinal herbs of the genus Dendrobium, namely, Dendrobium tosaense Makino and Dendrobium moniliforme SW, using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical antioxidative assay. Seeds of the capsules derived after 12 weeks of hand-pollination germinated asymbiotically (50-74%) on half-strength Murashige and Skoog's (MS) basal medium with 3% sucrose and solidified with 0.9% Difco agar. Active growth in the germinated seedlings was achieved by reculturing on full-strength MS basal medium supplemented with 8% banana homogenate, 8% potato homogenate, 8% coconut water, 1.5% sucrose, and 0.9% Difco agar. Healthy plantlets transferred to plastic trays containing moss or moss and tree fern successfully acclimatized (84-100%) in the greenhouse. Extracts were prepared from plants grown in the greenhouse for a period of 6 months. Methanolic extracts of D. tosaense and D. moniliforme scavenged DPPH at 95.9 and 83.4%, respectively, at a concentration of 0.4 mg/mL. Therefore, methanolic solubles of D. tosaense and D. moniliforme were subjected to bioguided fractionation and separation by column chromatographic methods individually. After chromatographic separation of these crude extracts, the obtained fractions (Dm 1, Dm 2, Dm 3, Dt 1, Dt 2, and Dt 3) were tested for their activity. Among them, fractions Dm 2 and Dt 1 showed significant antioxidant activity by DPPH radical antioxidative assay. Active fractions were purified further by column chromatography and resulted in identification of the antioxidant components alkyl ferulates from D. moniliforme and quercetin from D. tosaense.

Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium.

Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets. The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in a greenhouse when they were planted in soil.

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l-1), BA (0.1 mg l-1) and GA3 (0.1 mg l-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.