In vitro callus induction and estimation of plumbagin content from Plumbago auriculata Lam.

The medicinal plant Plumbago contains a very potent secondary metabolite, plumbagin having many therapeutic properties. Callus culture was induced using explants, leaf, stem and shoot apex, from P. auriculata. Murashige and Skoog media fortified with various growth hormones like NAA, IAA, IBA and 2, 4-D individually and in various combinations were checked for callus induction. Among the growth hormones used, 1 mg/L 2, 4-D showed best callusing. The hormonal combinations of 1 mg/L IAA and 1.5 mg/L NAA in the media exhibited best callus induction using stem internode as an explant. Plumbagin content from root, stem, leaf and callus was analyzed by using thin layer chromatographic technique. The callus derived from stem showed comparable plumbagin content to the in vivo plant parts. Quantitative spectrophotometric analysis of plumbagin from plant samples and callus indicated that plumbagin content was maximum in roots which was followed by callus, stem and leaf samples respectively. Generation of in vitro sources for plumbagin, for therapeutic applications will serve as a continuous supply and will contribute to preserve the natural plant recourses.

In vitro rhizogenesis: histoanatomy of Cedrela odorata (Meliaceae) microcuttings

Cedrela odorata (Meliaceae) is considered as one of the most valuable forest tree in the tropics. Clonal propagation of this species provide an alternative method to propagate superior genotypes, being the production of good quality adventitious roots one of the most important steps in micropropagation techniques. The sequence of anatomical changes that takes place during the formation of adventitious roots in shoots of Cedrela odorata cultured in vitro is described in this study. Eigth-week-old shoots, from multiplication cultures, were rooted in Murashige and Skoog´s medium (1962) with half- strength macronutrients and with 0 or 1mg/l indole-3-butyric acid (IBA). Between 12 and 24h after the start of rooting, some cambium, phloem and interfascicular parenchyma cells became dense cytoplasm, nuclei with prominent nucleoli and the first cell divisions were observed, especially in shoots treated with auxin (dedifferentiation phase). After 3-4 days, the number of dedifferentiated cells and mitotic divisions increased considerably, and the formation of groups of some 30-40 meristematic cells (meristemoids) was observed (induction phase). The first primordial roots developed from the 4th-5th day. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 6. Development of the primordial roots was similar in the control shoots and shoots treated with 1mg/l IBA, although there were more roots per explant in the latter. Rev. Biol. Trop. 59 (1): 447-453. Epub 2011 March 01.

Cedrela odorata (Meliaceae) es una especie tropical de gran valor económico. La propagación in vitro de esta especie ofrece una vía alternativa para la clonación de genotipos superiores, siendo la formación de un buen sistema radical uno de los pasos claves en la micropropagación. En este trabajo analizamos la secuencia de cambios anatómicos que tienen lugar durante la formación de raíces adventicias en microestaquillas de Cedrela odorata. Para el enraizamiento se utilizó el medio MS con los macronutrientes reducidos a la mitad, suplementado con AIB 0 ó 1mg/l. A partir de las 12-24 horas del comienzo del enraizamiento, se observaron los primeros cambios en las células del cambium, del floema y del parénquima interfascicular (fase de diferenciación). Después de 3-4 días, aparecen grupos de células meristemáticas (fase de inducción). Los primordios se desarrollan después de 4-5 días, siendo visibles al exterior a partir del sexto día (fase de emergencia). El desarrollo de las raíces fue similar en ambos tratamientos, pero la presencia de AIB aumenta el número de raíces.

Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings

The effects of plant growth promoting rhizobacteria (PGPR) on the rooting and root growth of semi-hardwood and hardwood kiwifruit stem cuttings were investigated. The PGPR used were Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19. All the bacteria showed indole-3-acetic acid (IAA) producing capacity. Among the PGPR used, the highest rooting ratios were obtained at 47.50 percent for semi-hardwood stem cuttings from Bacillus RC03 and Bacillus simplex RC19 treatments and 42.50 percent for hardwood stem cuttings from Bacillus RC03. As well, Comamonas acidovorans RC41 inoculations indicated higher value than control treatments. The results suggest that these PGPR can be used in organic nursery material production and point to the feasibility of synthetic auxin (IBA) replacement by organic management based on PGPR.

Distribution of linker histone variants during plant cell differentiation in the developmental zones of the maize root, dedifferentiation in callus culture after auxin treatment

Although several linker histone variants have been studied in both animal and plant organisms, little is known about their distribution during processes that involve alterations in chromatin function, such as differentiation, dedifferentiation and hormone treatment. In this study, we identified linker histone variants by using specific anti-histone Hl antibodies. Each variant's ratio to total Hl in the three developmental zones of maize (Zea mays L.) root and in callus cultures derived from them was estimated in order to define possible alterations either during plant cell differentiation or during their dedifferentiation. We also evaluated linker histone variants' ratios in the developmental zones of maize roots treated with auxin in order to examine the effects of exogenous applied auxin to linker histone variant distribution. Finally, immunohistochemical detection was used to identify the root tissues containing each variant and correlate them with the physiological status of the plant cells. According to the results presented in this study, linker histone variants' ratios are altered in the developmental zones of maize root, while they are similar to the meristematic zone in samples from callus cultures and to the differentiation zone in samples from roots treated with auxin. We propose that the alterations in linker histone variants' ratios are correlated with plant cell differentiation and dedifferentiation.

A new approach for enhanced multiplication of arbuscular mycorrhizal fungi and isolation of ITS regions from Glomus deserticola and Laccaria fraterna.

Rootlets induced from the petiole base of L. purpureus, using IAA and kinetin was used for enhanced multiplication of arbuscular mycorrhizal (AM) fungus, G. deserticula. Using conserved short arbitrary oligonucleotides, as specific primers, we amplified the ITS-region, a molecular marker for fungal identification, from the genomic DNA extracted from cultured spores of G. deserticola, and genomic DNA extracted from the mycelium of L. fraterna. The capacity of fungal colonization and subsequent spore formation of G. deserticola, compared with the natural root system was evaluated. This technology would provide a simple way to multiply AM fungi and to produce spores without microbial contamination useful for further molecular characterization.

Endophytic colonization and in planta nitrogen fixation by a diazotrophic Serratia sp. in rice.

Nitrogen fixing endophytic Serratia sp. was isolated from rice and characterized. Re-colonization ability of Serratia sp. in the rice seedlings as endophyte was studied under laboratory condition. For detecting the re-colonization potential in the rice seedlings, Serratia sp. was marked with reporter genes (egfp and Kmr) using transposon mutagenesis. The conjugants were screened for re-colonization ability and presence of nif genes using PCR. Further, the influence of flavonoids and growth hormones on the endophytic colonization and in planta nitrogen fixation of Serratia was also investigated. The flavonoids, quercetin (3 microg/ml) and diadzein (2 microg/ml) significantly increased the re-colonization ability of the endophytic Serratia, whereas the growth hormones like IAA and NAA (5 microg/ml) reduced the endophytic colonization ability of Serratia sp. Similarly, the in planta nitrogen fixation by Serratia sp. in rice was significantly increased due to flavonoids. The inoculation of endophytic diazotrophs increased the plant biomass and biochemical constituents.

In vitro cloning and homestead cultivation of primitive Musa cultivars.

Two primitive diploid Musa cultivars, Matti and Chemmatti from the extreme southern part of the Western Ghats were multiplied by in vitro culture of sucker-derived shoot apices. Decontaminated corm explants (1 cm x 1 cm) having shoot apex (approximately 0.3 cm) cultured for 1 month in Murashige and Skoog basal agar medium was cut vertically into eight segments and each segment having a part of shoot meristem was cultured in presence of 6-benzylaminopurine (BAP) and combinations of BAP and indole-3-acetic acid (IAA) or indole-3-butyricacid (IBA) to produce multiple shoots. After 12 weeks of culture, maximum number of shoots (32) in both the cultivars were produced in approximate 60% of the explants in presence of BAP and IAA each at 1.5 mg/l(-1) (Matti) and 40% of the explants in 2.5 mg/l(-1) of BAP and 1.5 mg/l(-1) of IAA (Chemmatti). Buds were formed from the base of the subcultured shoots and somewhat more number (34) of shoots were obtained in Matti than in Chemmatti (31) after 8 weeks. Difference in the concentration of cytokinin required for shoot initiation and multiplication, persistence of exudation through the subculture and red colouration of the early formed sheathing leaf bases in the shoots in Chemmatti indicated possible genotypic differences between the two cultivars. Multiple shoot proliferation achieved through five subcultures of the isolated shoots without any decline. Transfer of shoots (4-5 cm) into MS basal medium favoured rooting in 4 weeks and rooted plants (9 cm) were hardened and established (80-95%). Mericlones of Matti cultivated in homesteads produced bunches of uniform characters in 13 months.

Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats.

Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins. Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production. Berberine was synthesized and released continuously into the liquid medium. Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis. On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth. Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D. IAA and IBA were not favourable for cell growth and berberine synthesis.

In vitro control of fasciation in proliferating nucellar embryos of Mangifera indica L. var totapari red small for cloning.

Nucellar tissue contained in ovular halves of young fruits of Mangifera indica L. totapari red small, a dwarfing rootstock, differentiated fasciated embryonal structures in presence of 6-benzylaminopurine [BAP(0.15 mg l(-1))], 6-(gamma-gamma-dimethylallylamino) purine [2iP(0.15 mg l(-1))] and indole-3-acetic acid [(IAA(0.5 mg l(-1))] incorporated in the semisolid medium during 50-60 days. Due to embryonal fasciation, hardly 2-3 well-formed embryos could be obtained per culture of proliferating embryos. Of the 3 ethylene inhibitors [L-alpha-(2-aminoethoxyvinyl)-glycine-HCl (AVG), AgNO3 and salicylic acid (SA)] used, embryonal fasciation and necrosis of intervening tissue was completely controlled by 3-4 subcultures of fasciated mass of embryos under the influence of AVG (0.05 mg l(-1)) in presence of adenine sulphate [AdS (50 mg l(-1))] incorporated in the same medium. Almost synchronized development of isolated embryos, measuring ca 2 cm in length, was observed in a different medium used in liquid stationary state and supplemented, particularly with stress-producing substances [abscisic acid (ABA, 0.01 mg l(-1)); and polyethylene glycol (PEG, 100 mg l(-1))] besides certain other modifications. About 34% convertibility of processed embryos was obtained during a period of 90 days. The plantlets had well-developed roots along with laterals which were longer than leafy shoots. In vitro raised plants survived ex vitro for about 2 months.

In vitro micropropagation of Baliospermum montanum (Willd.) Muell-Arg--a medicinal plant.

Micropropagation of B. montanum was achieved on Murashige and Skoog's (MS) medium augmented with BAP using nodal segments. Maximum number of shoots (3.4 +/- 0.25) were found in MS medium fortified with BAP (3.10 microM). In vitro raised shoots were rooted on half strength MS medium augmented with various concentrations and combination of auxins viz.. IAA, IBA and NAA. Maximum number of roots were observed on half strength MS medium fortified with IBA (9.84 microM) combined with NAA (5.37 microM).

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng.

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.

Micropropagation of Terminalia arjuna Roxb. from cotyledonary nodes.

Cotyledonary node explants excised from 21 day old seedlings of T. arjuna produced multiple shoots when cultured on full strength MS or modified MS (1/2 strength major salts and Fe-EDTA) medium supplemented with different concentrations (0.1-1.0 mg/l) of BAP. Maximum 8.9 shoots/explant could be recorded after 30 days of inoculation on modified MS medium supplemented with BAP (0.5 mg/l). A proliferating shoot culture was established by reculturing the original cotyledonary nodes (2-3 times) on shoot multiplication medium after each harvest of the newly formed shoots. Shoots (each having 2-3 nodes/shoot) thus obtained were also used as a source of nodal explant that gave rise to 1-2 shoots when cultured on modified MS+BAP (0.5 mg/l) medium. Thus, 45-55 shoots could be obtained after 60 days of culture initiation from a single cotyledonary node. About 88% shoots rooted well after 15 hr pulse treatment with IBA (1 mg/l) in liquid MS medium followed by transfer to modified MS medium without IBA. About 80% of these plantlets were successfully acclimatized in plastic pots containing sand and soil mixture and 70% plantlets transferred in the field those survived even after 6 months of transplantation.

In vitro propagation of Lagerstromia parviflora Roxb. from adult tree.

A micropropagation protocol based on axillary bud proliferation has been developed from mature Lagerstromia parviflora adult tree. Nodal segments cultured on woody plant medium supplemented with 5.0 microl. BAP and 0.25 microm IAA gave maximum (86.9%) morphogenetic response. Proliferated shoots (10.7 per explants) were elongated to 3.9 cm within 6 weeks. In vitro produced micro-shoots were subjected to an IBA treatment (500 ppm for 2 min. dip) and placed under misting conditions for rooting. Misting beds were prepared with sand: soil (3:1) for 80.6% rooting and was acclimatized. Shoot length seems to be important to induce adventitious roots. The highest (91.7%) rooting was recorded on shoots ranging a length between 3.1-4.0 cm. Rooted and hardened plants were later transferred to poly bags and maintained in shadenet house. The protocol has the realizes capacity to produce 260 plants from a single explants within 10 months multiplication cycle.

In vitro regeneration of Hypericum patulum Thunb.--a medicinal plant.

A protocol was developed for high frequency plant regeneration in H. patulum by shoot-tip culture. H. patulum plants were collected from a wild source growing at high altitude in the eastern Himalayas. Multiple buds were initiated from shoot-tips cultured on Murashige and Skoog's medium supplemented with BAP, kinetin. Addition of thiamin HCI, Ca-pantothenate and biotin enhanced multiple shoot formation. Upon transfer to phytohormone free liquid medium following a brief exposure to auxin, root formation occurred from the micro shoots . Rooted plants were hardened and transferred to soil. Regeneration potentiality was found to be constant throughout the year in long term cultures.

Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae), a medicinal woody climber.

Nodes, shoot tips, internodes and leaf bases (approximately 1.0 cm) excised from young vines of the flowering woody climber, Celastrus paniculatus WilId. sub. sp. paniculatus (Celastraceae) were cultured in Murashige and Skoog (MS) medium containing agar (0.6%), sucrose (3%) and varied concentrations of 6-benzyl aminopurine (BAP) and kinetin. All the explant types were regenerative and maximum number (3.6) and frequency (94%) of axillary shoot formation of (5.08 cm long) was recorded in the nodes cultured in BAP (1 mg L(-1)) after 6 weeks. Combinations of BAP (1 mg L(-1)) and indole-3-acetic acid/l-naphthalene acetic acid (0.01-1 mg L(-1); IAA/NAA) tested with nodes induced formation of less number (3 and 2.2) of shoots at same frequency (94%). All the explant types viz. node, shoot tip, internode and leaf base of in vitro derived shoots responded earlier and better in lower concentrations of BAP (0.5-2 mg L(-1)) with formation of 8, 3.1, 6.4 and 1.8 shoots respectively during the same period. In spite of the advanced and increased caulogenic responses, differences in cytokinin requirements between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiole segments responding best at 0.5, 1 and 2 mg L(-1) BAP, respectively. The repeated reculture up to 10 cycles of the nodes from the shoot cultures each at 6-week intervals enabled multiplication and stocking of shoots without decline. Rooting of 3-7 cm shoot cuttings was induced in half-strength MS liquid medium containing IAA (1 mg L(-1)) with formation of 7.25 roots of 2.41 cm length within 6 weeks. Rooted plants were established at 84-96% rate in community pots without hardening, the least value (84%) being obtained with NAA- induced thick and calloid rooted plants. Four month old community potted plants were reintroduced into native forest habitats at 95% efficiency and 8 months after restoration, the plants were uniform in morphological, growth, cytological and peroxidase and esterase isozyme characteristics.

Auxin regulated poly(A)polymerase activity in Cicer arietinum epicotyls.

There was a linear increase in poly (A+) polymerase activity in the C. arietinum epicotyls during germination. Six-day-old auxin treated seedlings showed about 3-4 fold stimulation in enzyme activity, accompanied with 3- fold rise in the relative abundance of poly (A+) RNA levels. Actinomycin D, cycloheximide, cordycepin and amino acid analogues caused dramatic decline in poly (A+) polymerase as well as poly (A+) RNA levels. It seems that auxin induced a de novo synthesis of this enzyme.

In vitro propagation of Acorus calamus Linn.--a medicinal plant.

High frequency in vitro propagation protocol was standardized from rhizome explants of A. calamus. Maximum shoot multiplication frequency was obtained on Murashige and Skoog's media supplemented with 4 mg/l 6-benzyl amino purine and 0.5 mg/l indole-3-acetic acid. Regenerated shoots were rooted in vitro or directly transferred to sterile soil and well developed roots were observed within two weeks. The rooted plants were successfully established in the field.

Nitrosomethylurea induced streptomycin resistance in Lycopersicon esculentum Mill.

High frequency of streptomycin resistant variants of Lycopersicon esculentum were isolated on selective shoot regeneration medium supplemented with IAA (0.5 mg/L), zeatin (1.5 mg/L) and streptomycin sulphate (500 mg/L). Nonmutagenized (controls) and NMU treated cotyledons were placed on shoot regeneration medium supplemented with antibiotic streptomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. Shoot regeneration occurred from the nodular tissues developed at the cut ends of cotyledons. Resistant shoots developed into complete plantlets on rooting medium containing selective concentration of antibiotic. Stability of streptomycin resistance was confirmed by leaf assay and reciprocal crosses between streptomycin-resistant and sensitive plants.

Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation.

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius. On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced.

Inter-conversion of free sugars and accumulation of galactomannan in response to supplied metabolic mediators during pod filling in guar.

Detached inflorescences of guar (Cyamopsis tetragonoloba), each bearing 4 uniformly-developing pods at 42 days post anthesis (DPA), were cultured for 6 days in complete liquid medium manipulated with a fixed concentration of mannose and varying concentration of myo-inositol. Such inflorescences, but with 2 pods, were also maintained in the solutions of (i) glucose(U-14C) containing myo-inositol or phytohormones, and (ii) mannose(U-14C) containing galactose for 36 hr. Effect of such exogenously supplied metabolic mediators on interconversion of free sugars in pod wall, endosperm and cotyledons and galactomannan accumulation in endosperm was studied. Myo-inositol decreased, over control, the relative proportion of invert sugars in pod wall, endosperm and cotyledons and at lower concentration (27.75 mM) it decreased the level of free sugars in pod wall and galactomannan in endosperm. In all pod tissues, 14C from both glucose and mannose got incorporated into myo-inositol as well as various sugars and maximum incorporation occurred in sucrose. High concentration of total free sugars and their 14C activity in pod wall indicated that this pod tissue was a potent accumulator of free sugars. With myoinositol, the relative proportion of 14C from glucose into raffinose sugars of pod wall and endosperm increased with a simultaneous decrease in this incorporation into galactomannan of the latter. Accompanying this, relative proportion of 14C into hexoses and myo-inositol decreased in pod tissues. Galactose increased 14C incorporation from mannose into total free sugars, sucrose and galactomannan with a concomitant decline in the labelling of hexoses. IAA and ABA enhanced 14C incorporation from glucose into total free sugars and this enhancement was much higher with IAA than ABA. The latter inhibited 14C incorporation into galactomannan. Based on these results, it was suggested that myo-inositol at lower concentration was inadequate to mediate the metabolism of sugars and, thereby, galactomannan synthesis. Galactose and mannose exhibited a mutual beneficial effect on their transportation to pods. Phytohormones stimulated the accumulation of sucrose in pod wall for its obligatory unloading into the seed.