In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.Abbreviations Kn Kinetin - BA 6-Benzyl adenine - 2iP Isopentyl adenine - NAA -Naphthalene acetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - GA Gibberellic acid - PVP Polyvinyl pyrrolidone     

Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L.

A protocol for multiple shoot bud induction and plant regeneration from leaf segment-derived callus of Ruta graveolens has been developed. Maximum organogenic callus induction frequency (70.6 ± 2.33%) was observed on Murashige and Skoog (MS) medium supplemented with 10 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus when transferred to shoot induction media (MS nutrients supplemented with 6-benzyladenine (BA), kinetin (Kn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) in various concentrations and combinations). The highest shoot multiplication (92.3%) was observed on MS medium with 7.5 µM BA and 1.0 µM NAA. Regenerated shoots were rooted in vitro on MS containing 0.5 µM IBA. Plantlets with well developed root and shoot systems were successfully acclimated (90%) and established in earthen pots containing garden soil; they exhibited normal morphology and growth characteristics.     

Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia: an important medicinal plant

An efficient in vitro regeneration protocol of a valuable medicinal plant, Vitex trifolia has been successfully established using nodal segments as explants. Three different cytokinins (BA, Kn, 2iP) and auxins (NAA, IAA, IBA) in different concentrations and combinations, evaluated as supplements to Murashige and Skoog’s medium showed to have a marked influence on the regeneration output. Among all the single cytokinin treatments MS medium supplemented with 5.0 μM BA produced the maximum number of shoots yielding 8.20 ± 0.37 shoots per explant with 4.8 ± 0.43 cm shoot length after 8 weeks of culture. Combined with low auxin concentrations, all the three cytokinins at their optimal concentrations synergistically enhanced the regeneration credentials. However, MS medium enriched with 5.0 μM BA and 0.5 μM NAA yielded the best possible regeneration in the species with a regeneration percentage of 97.33 ± 2.67 % and amounting to 16.80 ± 0.58 shoots per explant with 6.20 ± 0.25 cm mean shoot length at the end of 8 weeks in culture. Ex vitro rooting of in vitro derived microshoots was achieved by 20 min 500 μM IBA treatment followed by transfer to thermocol cups containing sterile soilrite. A 95 % plantlets survived acclimatization procedure to the field. Genetic conformity of the regenerated plants was established through RAPD. All the bands visualized on agarose gels were monomorphic with that of the donor plant indicating the clonal nature of the regenerants.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

High frequency plantlet regeneration from cotyledonary node cultures of <Emphasis Type="Italic">Aegle marmelos</Emphasis> (L.) Corr.

High frequency plantlet regeneration was achieved in cotyledonary nodes of Aegle marmelos. Cotyledonary nodes from 1 mo. old in vitro grown seedlings of A. marmelos were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) (0–8.8 μM), kinetin (KIN) (0–9.4 μM), and indole-3-acetic acid (IAA) (0–1.14 μM) either alone or in combinations. The highest regenerative response was observed on medium containing 6.6 μM BA + 1.14 μM IAA where approximately 86.6% of the cultures responded with an average shoot numbers of 487.5 per explant in 7-wk time. Cultures maintained on KIN-supplemented medium showed very poor response. In vitro responded shoots were transferred to root induction medium consisting of half-strength MS supplemented with auxins IAA, indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Rooting was best in medium supplemented with 14.7 μM IBA. Rooted plantlets were acclimatized and transferred to the field with 80% survival rate.     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 μM N⁶-benzyladenine (BA) + 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 μM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 μM α-naphthaleneacetic acid (NAA) to MS + 5 μM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 μM IBA.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

In vitro acclimation to prolonged metallic stress is associated with modulation of antioxidant responses in a woody shrub Daphne jasminea

Comparative effect of meta-topolin and other cytokinins was assessed to develop an efficient and reliable regeneration protocol for Tecoma stans, using mature nodal explants. The morphogenic effect of benzyl adenine (BA), kinetin (Kin), meta- topolin (mT) and 2-iP (2-iso pentenyl adenine) at various concentrations (1.0–10 µM) was studied individually or in combination with auxins (IAA, IBA or NAA). Superior multiplication rates were achieved on MS medium supplemented with mT and NAA. Of the tested combinations, maximum shoot regeneration (95%), mean shoot number (19.6?±?0.60) and length (5.26?±?0.73 cm) was recorded on MS medium supplemented with 7.5 µM mT?+?0.5 µM NAA after 8 weeks of incubation. Among the different auxins employed for in vitro root induction, 92.5% microshoots rooted on MS medium enriched with 1.0 µM IBA with 10.8?±?0.20 mean root number and 5.62?±?0.17 cm length after 4 weeks of incubation. The acclimatized plants grew well in green house with 90% survival rate. The gas chromatography–mass spectrometry (GC–MS) analysis of ethanol leaf extract of in vitro-raised plants yielded a higher number of compounds than control plant. The assessment of genetic fidelity among regenerants, using ISSR markers did not reveal any somaclonal variation. Therefore, the protocol developed appears to be simple and reliable for mass production of clones with higher diversity of secondary metabolites.

     

Influence of nutrient salts,auxins and cytokinins on the in vitro growth of Salvia greggii

Experiments were designed to determine the optimal MS salt concentration and the best auxin and cytokinin to use for shoot growth of Salvia greggii A. Gray. Full or 1/2 MS salts were superior to 1/4 MS salts based on number of shoots produced. There were no differences in the various auxins tested (IAA, NAA or IBA) as to their abilities to stimulate shoot production or increased fresh weight. BA, and BA + Kin stimulated the greatest shoot number among the cytokinins tested. A final experiment was designed to determine optimal BA and NAA concentrations for shoot growth. A medium containing 11.1M BA and no NAA produced the best growth of Salvia greggii in vitro. Shoots produced in vitro rooted and acclimatized readily in the green-house.Abbreviations MS Murashige and Skoog salts - IAA indoleacetic acid - IBA indolebutyric acid - NAA napthaleneacetic acid - BA benzyladenine - Kin kinetin - 2iP isopentenyl adenine     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Micropropagation and acclimatization of Hedeoma multiflorum

A method for the micropropagation of Hedeoma multiflorum Benth from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on half-strength Murashige and Skoog (1962) medium supplemented with 22.2 μM BA or 22.2 μM BA plus 0.05 μM NAA. Individual shoots were excised and transferred into rooting medium containing auxins (IBA, NAA or IAA). Rooting of shoots was better on half-strength MS medium containing 0.6 μM IAA than on half-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. Preconditioning at different sucrose concentrations prior to acclimatization had no effect on plant establishment, but influenced plant quality. This revised version was published online in June 2006 with corrections to the Cover Date.     

Assessment of factors affecting micropropagation and ex vitro acclimatization of Nyctanthes arbor-tristis L

Rapid differentiation of multiple shoots was observed in 94% of nodal explants of one year old Nyctanthes arbor-tristis L. plants. Shoot bud induction and multiplication took place on Murashige and Skoog (MS) medium supplemented with two cytokinins, i.e. Benzyladenine (BA) or Kinetin (Kn) either alone or in combination with different auxins, indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Between different media, pH levels and growth regulators tried, the optimum condition for maximum regenerative response was obtained on MS + Kn (2.5 μM) + N AA (0.5 μM) media at 5.8 pH, forming cultures with 23.26 ± 0.89 number of shoots and 6.36 ± 0.80 cm shoot length after 8 weeks of culture. Histological sections confirmed the formation of multiple buds from nodal explants. Rooting was achieved ex vitro by dipping the basal ends of microshoots in 200 μM IBA for 30 min followed by their transplantation in sterile soilrite. The plantlets with well-developed shoot and root system were successfully established in garden soil and grown outside in a greenhouse with a 80% survival rate.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.