Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca²⁺ ions, but not by Ba²⁺, Cu²⁺or Fe²⁺. Among the metals tested, only Zn²⁺ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca²⁺ or Zn²⁺. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca²⁺ complex showed cross-resistance to inhibition by dipicolinic acid: Zn²⁺. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca²⁺, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca²⁺ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca²⁺ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca²⁺ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca²⁺ ions in the medium and (iii) Mg²⁺ requirement for the mutant increased three fold. Higher requirement of the Mg²⁺ could be partially relieved by Ca²⁺ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca²⁺complex.     

Killing bacterial spores by organic hydroperoxides

Killing of wild-type spores of Bacillus subtilis by t-butyl hydroperoxide, cumene hydroperoxide and peracetic acid was not through DNA damage, as shown by the absence of mutations in the survivors and the identical sensitivity of spores of strains with or without a recA mutation. In contrast, B. subtilis spores (termed α⁻β⁻) lacking the DNA protective α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to t-butyl hydroperoxide and cumene hydroperoxide, and their killing was in large part through DNA damage, as shown by the high frequency of mutations in the survivors and the greater sensitivity of α⁻β⁻ recA spores. Analysis of t-butyl hydroperoxide-treated spores showed that generation of DNA damage in α⁻β⁻ spores was more rapid than in wild-type spores; α/β-type SASP also protected against DNA strand breakage in vitro caused by t-butyl hydroperoxide. α/β-Type SASP appeared to play no role in protection of spores from killing by peracetic acid; wild-type and α⁻β⁻ spores exhibited identical peracetic acid sensitivity and their killing by this agent appeared to be not through DNA damage. Received 17 December 1996/ Accepted in revised form 13 March 1997     

α,ω-Dicarboxylic acid accumulation by acyl-CoA oxidase deficient mutants of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

α,ω-Dicarboxylic acid accumulation from alkanes and alkane degradation intermediates was investigated using Yarrowia lipolytica wild type strain W29 as well as a double, a triple and a quadruple POX-deleted strains. Six genes, POX1 through POX6, encode six acyl-CoA oxidase isozymes in Y. lipolytica. All the strains accumulated dodecanedioic acid (5–20 mg ml⁻¹) from the diterminal functionalised 1,12-dodecane diol and 12-hydroxdodecanoic acid. The quadruple-deleted strain was the only strain that was able to accumulate dioic acids from C16 alkanol and monocarboxylic acid as well as from C12, C14 and C16 alkanes (maximum 8 mg ml⁻¹ from dodecane).     

Degradation of veratric acid and other lignin-related aromatic compounds by the white-rot fungus Pycnoporus cinnabarinus

Metabolism of veratric acid and other aromatic compounds has been studied in two strains of Pycnoporus cinnabarinus. In non-agitated cultures which contained cellulose as an additional carbon source, veratric acid was demeth(ox)ylated to vanillic acid which accumulated in the medium. Under these conditions, ¹⁴CO₂ evolution from [4-O¹⁴CH₃]-veratric acid preceded that from [3-O¹⁴CH₃]-veratric acid in the case of both strains. ¹⁴CO₂ evolution was markedly accelerated and increased when 100% oxygen was employed instead of air. Oxygen had not so strong effect on the decarboxylation of ¹⁴COOH-labelled vanillic and p-hydroxybenzoic acid but it did increase decarboxylation of ¹⁴COOH-labelled veratric acid, indicating the effect of oxygen on the preceding demeth(ox)ylation. There were indications, for example rapid demethylation of veratric acid in early stages of growth when apparent phenol oxidase (laccase) activity was zero, for an existence of a separate demethylase enzyme. However, the participation of phenol oxidases in demeth(ox)ylation cannot be ruled out. Degradation pattern of vanillic acid was basically similar in P. cinnabarinus compared to Sporotrichum pulverulentum (Phanerochaete chrysosporium). Also the effect of carbon source was similar: cellulose as a carbon source enhanced degradation of vanillic acid through methoxyhydroquinone whereas in glucose medium, vanillic acid was reduced to the respective aldehyde and alcohol.Non-standard abbreviations CBQ cellobiose: quinone oxidoreductase - MHQ methoxyhydroquinone     

TRANSPORT OF IAA AND ACC IN FLORAL ORGANS OF IPOMOEA NIL (CONVOLVULACEAE)

The involvement of the stamens as transporters of plant growth regulators in flowers was examined by measuring the movement of ¹⁴C-indole-3-acetic acid (IAA) and ^l4C-l-aminocyclopropane-1-carboxylic acid (ACC) through floral organs of Ipomoea nil. During the transport of ¹⁴C-IAA through isolated filament segments, the polar accumulation of ¹⁴C-IAA in receiver blocks increased with time during filament development, which correlated with polar efflux rates at older stages of filament development. An inhibitor of polar IAA transport, 2,3,5-triiodobenzoic acid, disrupted the polarity of auxin transport by reducing the movement of ¹⁴C- IAA from filaments into receiver blocks. Transport of both ¹⁴C-IAA and ^l4C-ACC through filaments into other floral organs also was monitored in isolated flower buds in the laboratory and intact buds in the greenhouse. In isolated and intact buds 21 hr before anthesis, substantially higher levels of isotope were recovered in corolla tissue when ¹⁴C-ACC was transported through the filaments than when ¹⁴C-IAA was transported from the filaments. In isolated buds, substantial levels of both isotopes accumulated in the pistil (69 hr and 45 hr before anthesis), but minimal amounts were observed in receptacle and calyx tissues (69 hr to 21 hr before anthesis). In intact buds, high levels of both isotopes were recovered in receptacle, calyx, and pistil tissues (69 hr to 21 hr before anthesis). The results from this study support the hypothesis that Ipomoea stamens are transporters for ACC and IAA to regulate ethylene production in the corolla and other floral tissues.     

Purification by silica gel chromatography using dialysis tubing and characterization of sophorolipids produced from <Emphasis Type="Italic">Candida bombicola</Emphasis> grown on glucose and arachidonic acid

The yeast Candida bombicola (ATCC 22214) grown on primary carbon source glucose (100 g l⁻¹) and secondary carbon, arachidonic acid (2 g l⁻¹) produced mixture of sophorolipids up to 1.44 g l⁻¹. The crude product was a heterogeneous mixture of sophorolipids, which are glycolipids of sophorose linked to the fatty acid through glycosidic bond between ω and ω−1 carbon of arachidonic acid. The derived sophorolipids were isolated by silica gel chromatography using dialysis tubing. The purified sophorolipids were characterized by ESI-MS and FT-IR. Acid hydrolysis of the resolved sophorolipids were characterized by ESI-MS for the presence of 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (20-HETE) and 19-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (19-HETE), compounds of pronounced pharmaceutical importance.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

A kinetic study of acetic acid production by liquid-surface cultures of Acetobacter aceti

Summary The kinetics of acetic acid production by liquid-surface cultures of Acetobacter aceti strain M7 was investigated. Specific rates of acetic acid production (q _pf , g acetic acid/ cm² microbial film per hour), ethanol consumption and oxygen uptake were estimated on the basis of liquid-surface area: these were virtually constant, irrespective of the thickness of the microbial film. The effect of acetic acid concentration (P) on q _pf was found to be expressed from a series of experiments, as follows: q _pf = 0.0227(1-P/93)^2.204. A simple mathematical model was proposed to describe quantitatively the acetic acid production rate as a function of specific surface area and dilution rate. The mathematical model could reasonably well approximate to experimental data in the literature. The oxygen transfer rate through the air-liquid interface of a reactor in the presence of a microbial film was 3.6 times larger than that in the absence of the microbial film. Offprint requests to: K. Toda     

Hybridization of Gossypium species through in ovulo embryo culture

An interspecific hybrid of the sexually incompatible species G. hirsutum cv. Laxmi and G. arboreum cv. Jyoti was obtained through in ovulo embryo culture. Eightto twelve-day-old ovules were excised and cultured on Beasley and Ting's medium supplemented with Indol-3 acetic acid (5×10^-6 to 7×10^-6 M), Kinetin (5×10^-6 to 5×10^-8 M), Gibberellic acid (5×10^-7 to 5×10^-9M), Ammonium chloride (5 to 15mM) and Casein hydrolysate (50 to 200mg/l) added individually and in various combinations along with sucrose. No single medium was adequate to ensure complete development of the fertilized ovules to plantlets, thus necessitating a sequential five step transfer to different media. Cytological studies confirmed the hybrid nature of the plants.Abbreviation IAA Indol-3 acetic acid - Kn Kinetin - GA₃ Gibberellic acid - CH Casein hydrolysate - NAA -Naphthalene-acetic acid - BT Beasley and Ting's basal medium - MS Murashige and Skoog's basal medium - W White's basal medium NCL Communication number 3823.     

Analysis of the sensing and transducing processes implicated in the stomatal responses to carbon dioxide in Commelina communis L.

It has been previously debated whether CO₂ would depolarize the guard cell plasma membrane through malate‐mediated activation of the anion channel. Moreover, it has been assessed that the CO₂ signal would be transduced via cytosolic free Ca^{2 +} . In the present study, the CO₂ sensing and transducing processes were reinvestigated in Commelina communis (L.) mainly by studying how L ‐(–)‐malic acid and Ca^{2 +} flux modulators affected different CO₂ stomatal responses. L ‐(–)‐malic acid (1 m M ) inhibited by about 50% both CO₂‐induced stomatal closing and CO₂‐triggered inhibition of the stomatal opening response to the auxin indolyl‐3‐butyric acid. Stomatal closing in response to atmospheric CO₂ was strongly inhibited by the 1,4 dihydropyridines SDZ‐202 791 R(–) (SDZ (–)) and nifedipine, and this inhibition was attenuated by the 1,4 dihydropyridines SDZ‐202 791 S( + ) and S‐(–)‐BAY K8644. Suboptimal concentrations of the slow anion channel blockers 5‐nitro‐2,3‐phenylpropyllamine benzoic acid and anthracene‐9‐carboxylic acid increased the 50% inhibition of the CO₂ closing response by the Ca^{2 +} flux modulators SDZ (–) and 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N ′ N ′ ‐tetraacetic acid in a stronger manner than an additive one. Together, these results support the view that CO₂ is sensed through reducing proton extrusion. Moreover, they might suggest that the CO₂ signal is transduced through Ca^{2 +} signalling arising from depolarization‐mediated activation of a putative plasma membrane voltage‐gated L‐type Ca^{2 +} channel and for which the plasma membrane slow anion channel is a potential target.     

Isolation, identification and quantitation of hydroxycinnamic acid conjugates, potential platform chemicals, in the leaves and stems of Miscanthus × giganteus using LC-ESI-MSn

Miscanthus × giganteus is a source of platform chemicals and bioethanol through fermentation. Cinnamates in leaves and stems were analysed by LC–ESI-MSⁿ. Free phenols were extracted and separated chromatographically. More than 20 hydroxycinnamates were identified by UV and LC–ESI-MSⁿ. Comparative LC–MS studies on the leaf extract showed isomers of O-caffeoylquinic acid (3-CQA, 4-CQA and 5-CQA), O-feruloylquinic acid (3-FQA, 4-FQA and 5-FQA) and para-coumaroylquinic acid (3-pCoQA and 5-pCoQA). Excepting 3-pCoQA, all were also detected in stem. 5-CQA dominated in leaf; a mandelonitrile–caffeoylquinic acid dominated in stem. Three minor leaf components were distinguished by fragmentation patterns in a targetted MS² experiment as dicaffeoylquinic acid isomers. Others (M_r 516) were tentatively identified as hexosylcaffeoyl-quinates. Three positional isomers of O-caffeoylshikimic acid were minor components. p-Hydroxybenzaldehyde was also a major component in stem. This is the first report of the hydroxycinnamic acid profile of leaves and stems of M. × giganteus.     

Absorption,Translocation and Metabolism of Nicotinamide in Some Plants

The seedlings of rice, eggplant and tomato at the 5th leaf stage of growth readily absorbed exogenous ¹⁴C-nicotinamide through the root and the foliage in water culture. Within the 24 hr period after the bigining of cultivation, the radioactivity gradually translocated from the part treated with ¹⁴C-nicotinamide to the whole plant body. This compound was rapidly metabolised in the plants to at least six metabolites, in which three compounds were identified as nicotinic acid, NAD and NADP. ¹⁴C-Nicotinic acid was also taken up quickly through the root of rice and its metabolism showed a similar pattern to that of ¹⁴C-nicotinamide. The incorporation of radioactivity into NAD and NADP from ¹⁴C-nicotinamide added to cultivating solution at a concentration of 0.21 ppm was decreased to 10~20% by the simultaneous addition of unlabeled nicotinic acid at a concentration about 1000 times higher than that of the labeled one. It was concluded that the biosynthesis of these pyridine nucleotides from nicotinamide was chiefly via nicotinic acid. The formation of ¹⁴C-nicotinamide in the ¹⁴C-nicotinic acid metabolism suggested a breakdown of NAD. Three unknown compounds observed in both the metabolisms described above were not intermediates in the pyridine nucleotide biosynthesis.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Isolation and characterization of polysaccharides of a hybrid mushroom (backcross mating between PfloVv12 and Volvariella volvacea)

Three polysaccharide fractions (PS-I, PS-II, and PS-III) were isolated from the aqueous extract of a hybrid mushroom obtained through backcross mating of a somatic hybrid mushroom PfloVv12 (Sterile line) with Volvariellavolvacea. PfloVv12 was obtained through protoplast fusion of Pleurotusflorida and V. volvacea. PS-I was identified as 1,6-β glucan. PS-II and PS-III were identified as mannoglucogalactan but differing in molecular weights only. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR experiment (¹H, ¹³C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) the structures of these polysaccharides were established as;     

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Sphagnum plantlets, cultivated in continuous-feed bioreactors, are characterised by high levels of free endogenous phenolics and a pronounced excretion of some phenolics into the effluent culture medium. The transfer of Sphagnum fallax, precultivated in continuous-feed bioreactors, to batch cultures resulted in an increased flux through phenylpropanoid metabolism and an accumulation of p-coumaric acid to 0.1 μM and of trans-sphagnum acid up to 0.5 μM in the external medium [³H]-labelled L-phenylalanine (7.7 GBq mol^?1) was rapidly taken up, resulting in an enhanced synthesis and excretion of p-coumaric and trans-sphagnum acid. Specific activities were 6.9 and 5.4 GBq mol^?1, respectively, for these cinnamic acids excreted into the external medium. Endogenous pools of trans-cinnamic and p-coumaric acid did not increase and no labelling could be detected in these compounds. Cell wall-bound activity amounted to ca 14% of the applied activity after 48 h of incubation, 59% of which was recovered in dioxane/2 M HCl extracts of the cell wall. Exogenously applied trans-cinnamic acid (0.1 mM) was taken up to 46% and resulted in a transient endogenous accumulation of trans-cinnamic acid, the level of free endogenous p-coumaric and trans-sphagnum acid was found to have decreased. The concentrations of p-coumaric and trans-sphagnum acid in the culture medium rose to 17 and 2.4 μM, respectively, after 48 h of incubation in 0.1 mMtrans-cinnamic acid. Exogenously applied p-coumaric acid (0.1 mM) was taken up to 79% from the incubation solution but not stored endogenously, as metabolic products trans-sphagnum acid and an unknown p-coumaric acid-conjugate accumulated in the external medium and endogenously. These results give evidence for the biosynthetical route from phenylalanine to sphagnum acid and a channelling of pathway intermediates by the enzymes L-phenylalanine ammonia-lyase (EC 4.3.1.5) and cinnamic acid 4-hydroxylase (EC 1.14.13.11).     

1H-NMR study on ganglioside amide protons: evidence that the deuterium exchange kinetics are affected by the preparation of samples

The kinetics of H/²H chemical exchange of the amide proton has been suggested as one of the tools available for investigating hydrogenbond stabilizing interactions in gangliosides.The amide proton/deuterium (NH/²H) exchange rates in GM2 ganglioside were studied by¹H-NMR spectroscopy on 12 samples prepared following different procedures. In samples passed through a sodium salt Chelex-100 cation exchange resin column prior to being analysed theN-acetylneuraminic acid NH exchange occurred in less than 10 min and that of ceramide NH in 30 min. TheN-acetylgalactosamine acetamido NH exchange was slower, the half-life of the signal ranging from 15 min to 3.5 h. Contact of the Chelex-treated GM2 samples with water, through a dialysis process, modified the NH/²H exchange rate values, theN-acetylgalactosamine acetamido NH exchange becoming faster than that of ceramide NH and similar to that ofN-acetylneuraminic acid NH. Our results indicate that the deuterium/proton exchange rate strongly depends on sample preparation (ion content and minor contaminants present in water). The three-dimensional model involving theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group hydrogen-bonding, which is supported by experimental evidence, cannot be confirmed by NH-exchange measurement.     

Studies on the Proteolytic Action of Dairy Lactic Acicl Bacteria

Aseptic rennet curd prepared under the aseptic conditions and Str. cremoris- and L. helveticus-cheese prepared by sandwiching the cell pellets of Str. cremoris and L. helveticus between aseptic rennet curd, respectively, were ripened at 10°C for desired period.

Water soluble nitrogen (WSN) contents of both aseptic rennet curd and two kinds of cheese were determined. Gradual increase of WSN content of aseptic rennet curd was recognized all through the ripening preiod. WSN contents of both Str. cremoris- and L. helveticus-cheese were remarkably higher than those of aseptic rennet curd after 12 days ripening. This tendency was more remarkably recognized after 60 or 70 days ripening. α^s-Casein was mainly hydrolyzed by these lactic acid bacteria during ripening. α^s-Casein in two kinds of the cheese was more easily degradated by these lactic acid bacteria than that in aseptic rennet curd by rennet.

Judging from the results in previous and present reports, it was estimated that lactic acid bacteria used as a starter began to autolyze after 12 days ripening and that intracellular proteases released from their cells mainly hydrolyzed α^s-casein contained in Ca-paracaseinate of aseptic rennet curd to water soluble substances. This hydrolysis was also estimated from the viscous texture observed by scanning electron micrography.     

In vitro regeneration of Trifolium glomeratum

In vitro regeneration of Trifolium glomeratum, a leguminous forage species, was attempted through leaf, petiole, cotyledon, hypocotyl, collar and root explants and two media combinations. Root and collar explants showed no callus induction. Medium with 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA) and 0.10 mg dm⁻³ N⁶-benzyladenine (BA) was more effective for hypocotyl explant whereas cotyledon and petiole explant were more responsive to 5.0 mg dm⁻³ NAA and 1.0 mg dm⁻³ BA. Friable, green calli obtained from petiole explant on this medium showed organogenetic potential. Modified root-inducing medium having 0.21 mg dm⁻³ indole-3-acetic acid and 2.5 % sucrose was successful for root induction and plantlets were successfully transferred to field after hardening and Rhizobium inoculation.     

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Summary Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N⁶-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N⁶-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N⁶-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N⁶-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.