Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.     

An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Tylophora indica (Burm. f.) Merrill, via enhanced axillary bud proliferation from nodal explants collected from young shoots of a two-year-old plant. The physiological effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)], ascorbic acid (AA), different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.6 ± 0.71) of shoots and the maximum average shoot length (5.2 ± 0.31 cm) were recorded on MS medium supplemented with 2.5 μM BA, 0.5 μM NAA and 100 mg/l AA at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with 0.5 μM IBA. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with a 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long-term in vitro conservation of T. indica.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Interactive effect of light,temperature and TDZ on the regeneration potential of leaf discs of <Emphasis Type="Italic">Fragaria x ananassa</Emphasis> Duch

This is the first report where shoot regeneration in strawberry cultivar Chandler has been achieved simultaneously through both somatic embryogenesis and shoot bud formation. Direct somatic embryogenesis was observed in leaf discs which were cultured on medium containing MS salts + B₅ vitamins + 2% glucose + 18.16 μM thidiazuron (TDZ) and given both chilling and dark treatment for 2 wk at 4 ± 2°C followed by incubation at 25 ± 2°C under 16-h photoperiod for third wk. After 3 wk, these explants were then subcultured on medium containing MS salts + B₅ vitamins + 2% glucose and incubated under 16-h photoperiod at 25 ± 2°C for further growth and development. Direct regeneration via de novo shoot bud formation was observed in leaf disks which were given dark treatment and were cultured on medium containing MS salts + B₅ vitamins + 2% glucose supplemented with 9.08 μM TDZ. There was a synergistic effect of photoperiod, dark, and chilling treatments on somatic embryogenesis, whereas chilling treatment had an inhibitory effect on shoot organogenesis.     

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

An improved micropropagation of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm⁻³ BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

Oxidative events during in vitro regeneration of sunflower

The changes in the activity of some antioxidant enzymes and endogenous H₂O₂ level in zygotic sunflower embryos during organogenesis and somatic embryogenesis were monitored. Pathways of regeneration were induced on media differing with sucrose concentration 87 mmol dm⁻³ for shoot [shoot induction medium (SIM) medium] and 350 mmol dm⁻³ [embryo induction medium (EIM) medium] for somatic embryo induction. Water potential of the explants cultured on SIM increased, while the embryos maintained on EIM showed middle water deficit stress. The pattern of superoxide dismutase (SOD) isoforms was similar in organogenic and embryogenic culture; however, the intensity of MnSOD bands was higher on SIM than on EIM. Differences in catalase activity were observed: high activity on SIM predominated, whereas on EIM it was reduced. The activity of guaiacol peroxidase in the explants producing shoots and somatic embryos differed at the beginning of culture, but became comparable at the time of shoot and somatic embryo formation (day 5). H₂O₂ content was unchanged in organogenic culture, but on EIM it increased on day 1 followed by significant decrease. The results indicate that sugar concentration per se, or via induction of different developmental pathways influences the activity of antioxidant enzymes and also H₂O₂ level in cultured sunflower embryos.     

Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement: a review

Biotechnology can complement conventional breeding and expedite the mango improvement programmes. Studies involving in vitro culture and selection, micropropagation, embryo rescue, genetic transformation, marker-assisted characterization and DNA fingerprinting, etc. are underway at different centers worldwide. In vitro culture and somatic embryogenesis of several different genotypes have been achieved. The nucellus excised from immature fruitlets is the appropriate explant for induction of embryogenic cultures. High frequency somatic embryogenesis has been achieved in some genotypes; however, some abnormalities can occur during somatic embryo germination. Embryo rescue from young and dropped fruitlets can improve the hybridization success in a limited flowering season. Protocols for protoplast culture and regeneration have also been developed. In vitro selections for antibiotic tolerance and fungal toxin resistance have been very promising for germplasm screening. Genetic transformation using Agrobacterium tumefaciens has been reported. Genes that are involved with fruit ripening have been cloned and there have been attempts to deliver these genes into plants. DNA fingerprinting and studies on genetic diversity of mango cultivars and Mangifera species are also being conducted at several research stations. The purpose of this review is to focus upon contemporary information on biotechnological advances made in mango. It also describes some ways of overcoming the problems encountered during in vitro propagation of mango.     

MiniMax,a new diminutive <Emphasis Type="Italic">Glycine max</Emphasis> genotype with a rapid life cycle,embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.     

Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C. A. Meyer)

Summary Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Histological study of organogenesis and embryogenesis inCyclamen persicum Mill. tissue cultures: Evidence for a single organogenetic pattern

Summary Evidence is given thatCyclamen persicum callus culture give rise to structures such as shoot-buds, roots, unipolar tubers, bipolar tubers and embryos. Observations made on their structure by means of morphological and histological analyses are reported and discussed in relation to the anatomy of someCyclamen structures such as zygotic embryos and seedling tubers. Such analyses demonstrate that the bipolar structures including somatic embryos and bipolar tubers lack vascular connection with the callus whereas unipolar structures establish vascular strands with the callus. Here, the relationship between these regenerated structures is depicted. The organogenetic pattern inCyclamen persicum callus culture could well be a derived pathway for somatic embryogenesis. A theoretical diagram concerning the morphogenetic program is presented.     

A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts

Summary An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca²⁺-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.Abbreviations BA 6-benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ISA indole-3buryric acid - IPAR 6-(,-dimethylallylamino)purine riboside - NAA naphthaleneacetic acid - uidA ß-glucuronidase gene - GUS ß-glucuronidase enzyme - CaMV Cauliflower Mosaic Virus - nos nopaline synthase - MES 2[N-morpholino]ethane-sulfonicacid - PEG polyethylene glycol - X-gluc 5bromo-4-chloro-3-indolyl glucuronide - MUG 4-methyl umbelliferyl glucuronide - MU 4-methylumbelliferone     

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

A relationship between light conditions, auxin transport and adventitious root formation by hypocotyls of tomato seedling cuttings was demonstrated. Effective rooting of tomato seedling cuttings was observed under continuous white light (WL) irradiation. However, root formation was reduced in darkness or under red (RL) or blue light (BL). At least 3/4-day-long irradiation treatment with (WL) was necessary to increase the number of roots formed in comparison with control cuttings grown in darkness. Light was most effective if applied during the first half of the 13-day-long rooting period. The role of photoreceptor-dependent light perception in the light-regulation of rooting was tested using tomato photomorphogenic mutants: aurea (au) and high pigment (hp). When exposed to WL both mutants generated fewer roots then their isogenic wild type (WT). In darkness or under BL and RL less roots were formed on all plants and no difference was observed between mutants and WT plants. TIBA (2,3,5-triiodobenzoic acid) inhibited rooting in a dose-dependent manner both in darkness and under WL. However, although rooting was suppressed by 0.75 M TIBA in the dark, 8 M TIBA was necessary to block root formation in continuous WL. Inhibition of rooting by TIBA was most efficient when applied at the initial period of rooting, a 1-day-long treatment with TIBA being sufficient to suppress rooting if given during the first 2 days of culture. Later treatment had much less effect on the root formation.