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.

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.

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol.

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.

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).

Influence of cell wall degrading enzymes on colonization of N2 fixing bacterium, Azorhizobium caulinodans in rice.

In rice, nodule like structures were formed by inoculation of A. caulinodans combined with growth regulators and enzymes. Among the treatments, combination of cell wall degrading enzyme mixture and NAA with A. caulinodans induced more number of paranodules in rice. Total nitrogen content also increased in treated plants compared to uninoculated control.

Vegetative propagation of Cecropia obtusifolia (Cecropiaceae)

Cecropia is a relatively well-known and well-studied genus in the Neotropics. Methods for the successful propagation of C. obtusifolia Bertoloni, 1840 from cuttings and air layering are described, and the results of an experiment to test the effect of two auxins, naphthalene acetic acid (NAA) and indole butyric acid (IBA), on adventitious root production in cuttings are presented. In general, C. obtusifolia cuttings respond well to adventitious root production (58.3 of cuttings survived to root), but air layering was the better method (93 of cuttings survived to root). The concentration of auxins used resulted in an overall significantly lower quality of roots produced compared with cuttings without auxin treatment. Future experiments using Cecropia could benefit from the use of isogenic plants produced by vegetative propagation.

Optimization of some additives to improve protease production under SSF.

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.

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.

Propagation of Coriandrum sativum L. through somatic embryogenesis.

A highly embryogenic callus was obtained from hypocotyl segments of Coriandrum sativum L. when cultured in the medium consisting of MS + H vitamins (MSH). Induction of somatic embryos required 2,4-dichlorophenoxyacetic acid or napthalene acetic acid. Germination of fully developed embryos was accomplished by subculture on half strength MSH medium containing benzylamino purine 0.05 mg/L. Plantlets developed from somatic embryos were transferred to soil and were successfully flowered.

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.

Somatic embryogenesis in Vigna radiata (L.) Wilczek.

Somatic embryogenesis was achieved from immature cotyledon derived callus of mungbean, V.radiata (L.) Wilczek in MS liquid medium. Embryogenic callus was induced on MS medium with NAA (5 mg/L). Differentiation of somatic embryos was observed when embryogenic callus was transferred to MS liquid medium containing 2,4-D (1.5 mg/L) and L-proline (50 mg/L). The torpedo shaped embryos were transferred to MS liquid medium with BAP and ABA (1 mg/L each) for maturation and germination. Fifty per cent of torpedo shaped embryos were converted into tiny plants (8-9 plants out of 17) after one week of culture. The germinated embryos were isolated and transferred to MS half strength basal (solid) medium for further development.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.

Morphogenesis in short-term and long-term anther derived calli of Oenothera hookeri de vries

Anther culture of O. hookeri on Murashige and Skoog (1962) medium supplemented with 2 mg l-1 2,4-dichlorophenoxyacetic acid and 2 mg-1 1-naphthaleneacetic acid produced callus formation. When subcultured onto medium lacking auxin, the callus regenerated through the organogenic pathway. Non-organogenic and organogenic callus was observed using histological methods after 2, 3 and 24 weeks in culture. Three types of calli were recognized: non-organogenic friable calli, organogenic friable calli with roots and organogenic hard calli with shoots. The microscopical sections showed striking differences in tissue organization among friable and compact calli. Vascular bundles were prominent in compact calli, but were not found in friable calli. Calli sections showed at light microscopy cells at two developmental stages; differentiated highly vacuolated cells and meristematic small isodiametric cells with densely stained cytoplasm. At electron microscopy level abnormal chloroplasts were present in non-organogenic calli, while chloroplasts were well developed in organogenic hard calli. Peroxisomes with paracrystalline protein bodies were abundant in both types of calli.

Micropropagation of Sapindus mukorossi Gaertn.

Bud break and multiple shoots were induced in apical and axillary meristems derived from one month old seedlings of S. mukorossi on Murashige and Skoog (MS) medium supplemented with benzylamino purine (BAP) 0.4 microM or 0.8 microM alone. A combination of BAP and gibberellic acid (GA3) 0.4 microM and 2.8 microM produced elongated multiple shoots from both types of explants. Excised shoots were rooted on MS medium respectively with indole-3-butyric acid (IBA) 3.4 microM or 2.4 microM. The regenerated plantlets were successfully acclimatized and transferred to soil.

Micropropagation of terrestrial orchids, Anoectochilus sikkimensis and Anoectochilus regalis.

Two horticulturally important jewel orchids of the genus Anoectochilus were successfully micropropagated. Isolated nodes of A. sikkimensis collected from Sikkim in Eastern Himalayas and subsequently reared under nursery conditions and A. regalis collected from Western Ghats in Southern India were cultured for 12 weeks on Woody Plant Medium (WPM) to produce a maximum of 4.8 and 5.6 callus--free axillary shoots respectively at 95 and 98% efficiency. During reculture of the explants from in vitro raised shoots under the same conditions, the total number of shoots obtained from the nodes (21.4) and shoot tips (8.2) of A. regalis were significantly higher than those hardy and slow growing shoots of A. sikkimensis (12.3 and 4.3) respectively. Shoots (4-6 cm) were rooted in medium containing NAA (2.70 microM) and activated charcoal (0.2%). The rooted plants established at 95-98% rate in community pots after hardening. After 6 months, green house adapted community potted plants of A. regalis were transferred to natural forest habitat locally with 95 and 70% survival respectively after 12 months. The plants, established in community pots and native forest habitat were free from any morphological and growth defects.

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots.

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.