Increased taxane accumulation in callus cultures of Taxus cuspidata and Taxus × media by some elicitors and precursors

In Taxus cuspidata callus, vanadyl sulfate (10 mg l^–1) induced a high (146 g g^–1 dry wt) production of 10-deacetylbaccatin III in comparison to 7 g g^–1 dry wt of the control. The content of paclitaxel in this species increased from 16 g g^–1 to 74 g g^–1 dry wt when 20 mg phenylalanine l^–1 was used. In T. media, p-aminobenzoic acid induced the highest content of 10-deacetylbaccatin III (481 g g^–1 dry wt) versus 181 g g^–1 in the control. Paclitaxel increased from 89 to 139 g g^–1 dry wt after adding chitosan (20 mg l^–1) to the cultures.     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species

The aim of this work was to assess whether agroforestry species have the ability to acquire P from pools unavailable to maize. Tithonia diversifolia(Hemsley) A. Gray, Tephrosia vogelii Hook f., Zea mays and Lupinus albusL. were grown in rhizopots and pH change and depletion of inorganic and organic P pools measured in the rhizosphere. Plants were harvested at the same growth stage, after 56 days for maize and white lupin and 70 days for tithonia and tephrosia, and the rhizosphere sampled. The rhizosphere was acidified by tithonia (pH change –0.3 units to pH 4.8) and lupins (–0.2 units to 4.9), alkalinised by tephrosia (+0.4 units to pH 5.4), and remained unchanged with maize growth. Concurrent with acidification in the rhizosphere of tithonia there was a decline in resin-P (0.8 g P g^–1). However, there was also a decline in NaOH extractable inorganic P (NaOH-P_i) (5.6 g P g^–1 at the root surface) and organic P pools (NaOH-P_o) (15.4 g P g^–1 at 1.5 mm from the root), which would not be expected without specific P acquisition mechanisms. Alkalinisation of tephrosia rhizosphere was accompanied by changes in all measured pools, although the large depletion of organic P (21.6 g P g^–1 at 5 mm from the root) suggests that mineralisation, as well as desorption of organic P, was stimulated. The size of changes of both pH and P pools varied with distance away from the rhizoplane. Decline of more recalcitrant P pools with the growth of the agroforestry species contrasted with the effect of maize growth, which was negligible on resin-P and NaOH-P_i, but led to an accumulation of P as NaOH-P_o (14.2 g P g^–1 at 5 mm from the root). Overall the depletion of recalcitrant P pools, particularly P_o, suggests that the growth of tithonia and tephrosia enhance desorption and dissolution of P, while also enhancing organic P mineralisation. Both species appear to have potential for agroforestry technologies designed to enhance the availability of P to crops, at least in the short term.     

Phytoextraction of selenium from soils irrigated with selenium-laden effluent

This two-part study compared the efficacy of different plant species to extract Se from soils irrigated with Se-laden effluent. The species used were: Brassica napus L. (canola), Brassica juncea Czern L. and Coss (Indian mustard), and Hordeum vulgare L. (barley). In Study 1 we irrigated the plants with a saline effluent containing 0.150 mg Se L^–1, while in Study 2, the same species were planted in a saline soil selenized with 2 mg Se L^–1. Plants were simultaneously harvested 120 days after planting. In Study 1, there were only slight effects of treatment on dry matter (DM) yield. Plant Se concentrations averaged 21 g Se g^–1DM for the Brassica species, and 4.0 g Se g^–1 DM for barley. Total Se added to soils via effluent decreased by 40% for Brassica species and by 20% for barley. In Study 2, total DM decreased for all species grown in saline soils containing Se. Plant Se concentrations averaged 75 g g^–1 DM for Brassica species and 12 g Se g^–1 DM for barley. Total Se added to soils prior to planting decreased by 40% for Brassica species and up to 12% for barley. In both studies, plant accumulation of Se accounted for at least 50% of the Se removed in soils planted to Brassica and up to 20% in soils planted to barley. Results show that although the tested Brassica species led to a significant reduction in Se added to soil via use of Se-laden effluent, additional plantings are necessary to further decrease Se content in the soil.     

Utilisation of soil organic P by agroforestry and crop species in the field,western Kenya

A field experiment in western Kenya assessed whether the agroforestry species Tithonia diversifolia (Hemsley) A. Gray, Tephrosia vogelii Hook f., Crotalaria grahamiana Wight & Arn. and Sesbania sesban (L) Merill. had access to forms of soil P unavailable to maize, and the consequences of this for sustainable management of biomass transfer. The species were grown in rows at high planting density to ensure the soil under rows was thoroughly permeated by roots. Soil samples taken from beneath rows were compared to controls, which included a bulk soil monolith enclosed by iron sheets within the tithonia plot, continuous maize, and bare fallow plots. Three separate plant biomass samples and soil samples were taken at 6-month intervals, over a period of 18 months. The agroforestry species produced mainly leaf biomass in the first 6 months but stem growth dominated thereafter. Consequently, litterfall was greatest early in the experiment (0–6 months) and declined with continued growth. Soil pH increased by up to 1 unit (from pH 4.85) and available P increased by up to 38% (1 g P g^–1) in agroforestry plots where biomass was conserved on the field. In contrast, in plots where biomass was removed, P availability decreased by up to 15%. Coincident with the declines in litterfall, pH decreased by up to 0.26 pH units, plant available P decreased by between 0.27 and 0.72 g g^–1 and P_o concentration decreased by between 8 and 35 g g^–1 in the agroforestry plots. Declines in P_o were related to phosphatase activity (R²=0.65, P<0.05), which was greater under agroforestry species (0.40–0.50 nmol MUB s^–1 g^–1) than maize (0.28 nmol MUB s^–1 g^–1) or the bare fallow (0.25 nmol MUB s^–1 g^–1). Management of tithonia for biomass transfer, decreased available soil P by 0.70 g g^–1 and P_o by 22.82 g g^–1. In this study, tithonia acquired P_o that was unavailable to maize. However, it is apparent that continuous cutting and removal of biomass would lead to rapid depletion of P stored in organic forms.     

Effects of Cd and Zn on the development of annual xylem rings of young Norway spruce (Picea abies) plants

Three-year-old spruce (Picea abies) saplings were planted and cultivated for 2 years in pots with 3 1 substrate, consisting of a homogenized mixture of sand, peat and forest soil with a high organic content (volume ratio 11.52). This substrate was amended with 10–180 mol Cd [kg soil dry weight (DW)]^–1, 50–7500 mol Zn (kg soil DW)^–1 (determined with 1 M ammonium acetate extracts) or combinations of both elements. Annual xylem growth rings in stems of plants treated with 50 mol Cd (kg soil DW)^–1 or 7500 mol Zn (kg soil DW)^–1 were significantly narrower than in control plants. Growth reductions were more pronounced in the second year of the experiment. The contents of Cd and Zn in stem wood and needles were positively correlated with the substrate concentrations. The Mg contents of the spruce needles were inversely correlated with soil concentrations of Cd and Zn. Root development was impeded at moderate concentrations of Cd (50 mol kg^–1) or Zn (1000 mol kg^–1) in the substrate. The adverse effects of potentially toxic trace elements, like Cd or Zn, on xylem growth of spruce plants are discussed with regard to possible growth reductions in forest trees under field conditions.     

Effect of the insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam

A study of the effects of two selected organophosphorus insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam was made. The insecticides had concentrations of 10 to 300 g g^–1. The presence of methylpyrimifos at concentrations of 100 to 300 g g^–1 or chlorpyrifos at concentrations from 10 to 300 g g^–1 significantly decreased aerobic dinitrogen fixing bacteria and dinitrogen fixation. Nitrifying bacteria decreased at concentrations of 200 and 300 g g^–1 of methylpyrimifos. The presence of 10 to 300 g g^–1 of chlorpyrifos decreased the total number of bacteria. However, fungal populations and denitrifying bacteria were not affected as a consequence of the addition of the organophosphorus insecticides to the agricultural soil, showing that these microorganisms can tolerate high amounts of those insecticides.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Growth effects of nagilactone E on cultured cells of lettuce

Nagilactone E, a norditerpene dilactone isolated from a gymnosperm, Podocarpus nagi (Podocarpaceae), was able to stimulate the growth of cultured cells of Lactuca sativa cv. Grand Rapids at 0.1 g ml^-1 but not at 0.01 or 1 g ml^-1. Cell wet weight/unit time and cell number/unit wet weight were increased when they were recorded at the end of the 14-day incubation period. Also, the population of cells treated with that concentration of nagilactone E had a higher percentage of cells with shorter cell length compared to the control (untreated).     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

Genotypes of lucerne (Medicago sativa L.) show differential tolerance to manganese deficiency and toxicity when grown in bauxite residue sand

The physical and chemical characteristics of bauxite residue sand (BRS) affect the availability of a number of nutrients to plants, especially manganese (Mn). Lucerne (Medicago sativa L.) has been chosen as a BRS revegetation species because of its deep-rooting habit and tendency to tolerate moderately alkaline and saline soils, even though it is still prone to Mn deficiency stress. Sixteen commercially available lucerne genotypes were grown in BRS after addition of 5, 50 or 500 g Mn g^–1 BRS in a glasshouse. Manganese deficiency and toxicity symptoms were observed in 5 and 500 g g^–1 treatments, respectively. Symptom expression varied in severity between genotypes. Relative tolerance to Mn deficiency was defined by shoot dry weight at 5 g Mn g^–1 as a percentage of shoot dry weight at 50 g Mn g^–1. Salado, a genotype tolerant to Mn deficiency, and Sirosal, a genotype intolerant to Mn deficiency, were then grown with 0, 10, 20, 50, 100, 200 or 800 g Mn g^–1 BRS and found to have critical shoot Mn concentrations of 17.7 (Salado) and 26.6 g g^–1 (Sirosal). The use of genotypes with high relative Mn deficiency tolerance is recommended to help improve sustainability of BRS revegetation as well as to improve productivity on Mn-fixing agricultural soils.     

Phytoavailability of Cd and Zn in soil estimated by stable isotope exchange and chemical extraction

The distribution of labile Cd and Zn in two contrasting soils was investigated using isotopic exchange techniques and chemical extraction procedures. A sewage sludge amended soil from Great Billings (Northampton, UK) and an unamended soil of the Countesswells Association obtained locally (Aberdeen, UK) were used. ¹¹⁴Cd and ⁶⁷Zn isotopes were added to a water suspension of each soil and the labile metal pool (E-value) determined from the isotope dilution. Samples were obtained at 13 time points from 1h to 50 days. For the sewage sludge amended soil, 29 g Cd g^–1 (86% of total) and 806 g Zn g^–1 (65% of total) were labile and for the Countesswells soil the value was 8.6 g Zn g^–1 (13% of total); limits of detection prevented a Cd E-value from being measured in this soil. The size of the labile metal pool was also measured by growing plants for 90 days and determining the isotopic content of the plant tissue (L-value). Thlaspi caerulescensJ. & C. Presl (alpine penny cress), a hyperaccumulator of Zn and Cd, Taraxacum officinale Weber (dandelion) and Hordeum vulgare L. (spring barley) were used. L-values were similar across species and lower than the E-values. On average the L-values were 23±0.8 g Cd g^–1 and 725±14 g Zn g^–1 for the Great Billings soil and 0.29±0.16 g Cd g^–1 and 7.3±0.3 g Zn g^–1 for the Countesswells soil. The extractable metal content of the soils was also quantified by extraction using 0.1 M NaNO₃, 0.01 M CaCl₂, 0.5 M NaOH, 0.43 M CH₃COOH and 0.05 M EDTA at pH 7.0. Between 1.3 and 68% of the total Cd and between 1 and 50% of the total Zn in the Great Billings soil was extracted by these chemicals. For the Countesswells soil, between 6 and 83% of the total Cd and between 0.1 and 7% of the total Zn was extracted. 0.05 M EDTA and 0.43 M CH₃COOH yielded the greatest concentrations for both soils but these were less than the isotopic estimates. On the whole, E-values were numerically closer to the L-values than the chemical extraction values. The use of isotopic exchange provides an alternative estimate of the labile metal pool within soils compared to existing chemical extraction procedures. No evidence was obtained that T. caerulescens is able to access metal within the soil not freely available to the other plants species. This has implications for long term remediation strategies using hyperaccumulating plant species, which are unlikely to have any impact on non-labile Cd and Zn in contaminated soil.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid