Sitakanta Pattnaik

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

SummaryAn efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N6-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l−1, 4.4 µM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl−1, 1.1 µM). Gibberellic (GA3) at 0.4 mg·l−1 (1.2 µM) added to the medium along with BA (1.0 mg·l−1, 4.4 µM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l−1 (5.0 µM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.

In vitro propagation of the medicinal herbs Ocimum americanum L Syn O. canum Sims (hoary basil) and Ocimum sanctum L (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA3) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

Abstract A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoogs medium containing either N6-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil.

Morphogenic response of the alginate-encapsulated axillary buds from in vitro shoot cultures of six mulberries

Axillary buds obtained from in vitro shoot cultures of six mulberries (Morus alba L., M. australis Poir., M. bombycis Koidz., M. cathyana Hemsl., M. latifolia Poir., and M. nigra L.) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog (1962) nutrients (MS) and 4.4 μM benzyladenine (BA). Morphogenic response of encapsulated buds to various planting media such as MS medium + 4.4 μM BA, MS basal medium, soilrite mix + half-strength MS medium, garden soil + half-strength MS medium, soilrite mix + tap water and garden soil + tap water was evaluated. Encapsulated buds of M. alba, M. bombycis, M. latifolia and M. nigra exhibited shoot development in each of the six media tested whereas that of M. australis and M. cathyana responded only to the first four media. Analysis of variance revealed that the planting medium exhibited the greatest influence on shoot development. Of the six planting media evaluated, shoot development was highest in MS medium containing 4.4 μM BA and lowest in garden soil moistened with water. Of the six Morus species studied, one-step regeneration, i.e. both shoot and root formation, was recorded in M. alba, M. bombycis and M. latifolia. Rooted shoots were retrieved from encapsulated buds of these species on all planting media tested except the one that contained BA. Root development was significantly affected by the planting medium and the plant species with planting medium contributing the maximum amount (82%) of the total variation observed. Of the five planting media tested, the percentage of root development was highest in MS basal medium. Of the six Morus species studied, the best shoot and root development was observed in M. alba. Encapsulated buds of M. bombycis, M. latifolia and M. nigra stored for 90 days and those of M. alba, M. australis and M. cathyana for 60 days at 4 °C still regenerated shoots. Plants regenerated from the encapsulated buds were hardened off and transferred to soil.

Rapid clonal propagation of three mulberries, Morus cathayana Hemsl., M. lhou Koiz. and M. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.

Micropropagation of Curry Leaf Tree [Murraya koenigii (L.) Spreng.] by axillary proliferation using intact seedlings

Abstract An efficient and reproducible procedure for the large scale propagation of Murraya koenigii (L.) Spreng. (Curry Leaf Tree) is described. High-frequency direct shoot proliferation was induced in intact seedlings of M. koenigii on modified Murashige and Skoog (1962) (MS) medium supplemented with 5.0 mg/l benzyladenine. Shoot buds originated from the region adjacent to the apex of the primary shoot and the epicotyledonary node of the intact seedling. Shoots elongated following transfer to MS medium without plant growth regulators. The shoot-forming capacity of intact seedlings was influenced by explant orientation. Maximum shoot proliferation was obtained when the shoot-forming region was in direct contact with the medium surface or slightly embedded into the medium. Proliferating shoot cultures were established by repeatedly subculturing mother seedlings on fresh medium of the same composition after excising all newly formed shoots. Roots were formed on excised shoots when they were transfered to half-strength MS containing 1.0 mg/l indole-3-butyric acid. Plantlets were acclimatized and established in soil where they exhibited normal growth.

Adventitious shoot organogenesis and plant regeneration from cotyledons of Dalbergia sissoo Roxb., a timber yielding tree legume

A procedure is outlined to induce adventitious shoot organogenesis from semi-mature as well as mature cotyledons lacking the embryonic axis of Dalbergia sissoo Roxb., an economically important leguminous tree. Shoot buds were induced in the proximal region of the semi-mature cotyledons on Murashige and Skoogs (MS) medium supplemented with 4.44 μM 6-benzyladenine (BA) and 0.26 μM ∝-naphthaleneacetic acid (NAA). These buds elongated into shoots following transfer to a similar medium containing half-strength macro-nutrients. Adventitious shoot bud formation was also induced in the mature cotyledons. However, unlike the semi-mature explants, the mature cotyledons exhibited shoot bud differentiation on MS medium containing 22.20 μM BA without NAA. Pre-culture of mature cotyledons in liquid MS medium containing 8.88 μM BA for a duration of 48 h improved shoot bud regeneration up to six-fold. Regenerated shoots, derived from semi-mature and mature cotyledons, rooted on half-strength MS medium containing 1.23 μM and 4.92 μM indole-3-butyric acid (IBA), respectively. Plantlets were acclimatized and established in soil.

ALGINATE ENCAPSULATION OF AXILLARY BUDS OF OCIMUM AMERICANUM L. (HOARY BASIL), O. BASILICUM L. (SWEET BASIL), O. GRATISSIMUM L. (SHRUBBY BASIL), AND O. SANCTUM L. (SACRED BASIL)

SummaryPropagation and conservation of four pharmaceutically important herbs, Ocimum americanum L. syn. O. canum Sims. (hoary basil); O basilicum L. (swett basil); O. gratissimum L. (shrubby basil); and O. sanctum L. (sacred basil) was attempted using synthetic seed technology. Synthetic seeds were produced by encapsulating axillary vegetative buds harvested from garden-grown plants of these four Ocimum species in calcium alginate gel. The gel contained Murashige and Skoog (MS) nutrients and 1.1-4.4 μM benzyladenine (BA). Shoots emerged from the encapsulated buds on all six planting media tested. However, the highest frequency shoot emergence and maximum number of shoots per bud were recorded on media containing BA. Of the six planting media tested, both shoot and root emergence from the encapsulated buds in a single step was recorded on growth regulator-free MS medium as well as on vermi-compost moistened with halfstrength MS medium. Rooted shoots were retrieved from the encapsulated buds of O. americanum, O. basilicum, and O. sanctum on these two media, whereas shoots of O. gratissimum failed to root. The encapsulated buds could be stored for 60 d at 4°C. Plants retrieved from the encapsulated buds were hardened off and established in soil.

High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.)

A complete protocol is presented for in vitro regeneration of pomegranate (Punica granatum L.), a tropical fruit tree, using cotyledonary nodes derived from axenic seedlings. Shoot development was induced from cotyledonary nodes on Murashige and Skoog (1962) (MS) medium supplemented with 2.3‐23.0 mM benzyladenine (BA) or kinetin (Kn). Both type and concentration of cytokinin significantly influenced shoot proliferation. The maximum number of shoots (9.8 shoots/explant) was developed on a medium containing 9.0 mM BA. Shoot culture was established by repeatedly subculturing the original cotyledonary node on a fresh batch of the same medium after each harvest of the newly formed shoots. In vitro raised shoots were cut into nodal segments and cultured on a fresh medium for further multiplication. Thus, from a single cotyledonary node about 30‐35 shoots were obtained in 60 days. Shoots formed in vitro were rooted on half-strength MS supplemented with 0.054‐5.4 mM naphthaleneacetic acid (NAA). However, a medium containing 0.54 m MN AA resulted in the highest per cent rooting of shoots and significantly higher number of roots than other concentrations. Plantlets were successfully acclimated and established in soil. # 2000 Elsevier Science B.V. All rights reserved.

Efficient plant retrieval from alginate-encapsulated vegetative buds of mature mulberry trees

Abstract Axillary vegetative buds of 3-year-old mature mulberry trees of three indigenous and two Japanese varieties from the open field were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 4% sodium alginate with 75 mM CaCl2.2H2O. Use of semi-solid MS medium resulted in 100% conversion of encapsulated shoot buds into plantlets, especially when buds were pretreated with 1.0 mg l−1 BA for 36 h. Although sprouting was faster on medium fortified with 1.0 mg l−1 BA and 0.3 mg l−1 GA3, one-step germination, i.e. both shoot and root induction, was better on a MS basal medium without added phytohormones. Among several plating substrates assessed for in vivo germination, the maximum response was elicited on artificial soil moistened with half-strength MS medium devoid of sucrose. A varietal difference was evident with respect to germination potential between encapsulated meristems under in vitro and in vivo situations. The encapsulated buds could be stored for 60 days at 4 °C without loss of viability only when the gel matrix contained MS nutrients, vitamins and sucrose. There was a negligible (