Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase

The carboxypeptidase gene from rice and corresponding cDNA clones were isolated. The SalI 11.2 kb fragment of DNA cloned from a size-fractionated genome library contained eight introns and an open reading frame that encoded 500 amino acids (M _r 55445). The structure deduced for the carboxypeptidase from rice was very similar to those of type III serine carboxypeptidases from barley and wheat. The extent of homology of the amino acid sequence to that of these carboxypeptidases from barley and wheat was 92.3% and 87.2%, respectively. The accumulation of mRNA for the rice carboxypeptidase was conspicuous in germinating endosperms that contained aleurone layers, but levels were lower in leaves and roots. The abundance of the mRNA in endosperms was enhanced by gibberellic acid (GA) and accumulation of the mRNA was inhibited by abscisic acid (ABA). The rice gene for carboxypeptidase contained some pyrimidine boxes (_T ^CCTTTT_T ^C), in the 5 flanking region, which are a characteristic of a GA-responsive gene.     

Structure of a rice β-glucanase gene regulated by ethylene,cytokinin, wounding,salicylic acid and fungal elicitors

A rice -glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots expressGns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogenSclerotium oryzae or from the non-pathogenSaccharomyces cereviseae. Shoots also expressGns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The -glucanase shows 82% amino acid similarity to the barley 1,3;1,4--D-glucanases, and from hybridization studies it is the -glucanase gene in the rice genome closest to the barley 1,3;1,4--glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G+C in the third position of the codon in the mature peptide coding region, but only 61% G+C in the signal peptide region.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - EtBr ethidium bromide - GA gibberellic acid - n.p. nucleotide position in gene sequence - PCR polymerase chain reaction - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄, 1 mM EDTA pH 7.4     

Partitioning of leaf nitrogen with respect to within canopy light exposure and nitrogen availability in peach (Prunus persica)

Summary Relationships between leaf nitrogen content and within canopy light exposure were studied in mature nectarine peach trees (Prunus persica cv. Fantasia) that had received 0, 112, 196, 280 or 364 kg of fertilizer nitrogen per hectare per year for the previous 3 years. The relationships between light saturated leaf CO₂ assimilation rates and leaf nitrogen concentration were also determined on trees in the highest and lowest nitrogen fertilization treatments. The slope of the linear relationship between leaf N content per unit leaf area and light exposure was similar for all nitrogen treatments but the y-intercept of the relationship increased with increasing N status. The slope of the relationship between leaf N content per unit leaf area and light saturated CO₂ assimilation rates was greater for the high N trees than the low N trees, but maximum measured leaf CO₂ assimilation rates were similar for both the high and low N treatments. A diagrammatic model of the partitioning of leaf photosynthetic capacity with respect to leaf light exposure for high and low nitrogen trees suggests that the major influence of increased N availability is an increase in the photosynthetic capacity of partially shaded leaves but not of the maximum capacity of highly exposed leaves.     

Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

Heavy metal-hormone interactions in rice plants: Effects on growth,net photosynthesis,and carbohydrate distribution

The effect of external applications of gibberellins (GA₃) and abscisic acid (ABA) on the growth, carbohydrate content, and net photosynthesis of heavy metal-stressed rice plants (Oryza sativa cv. Bahía) was investigated. Treatment with cadmium (0.1 mm) and nickel (0.5 mm) inhibited rice growth and stimulated carbohydrate accumulation, especially in seeds from which seedlings were developing, stems, and first leaves. The addition of GA₃ (14 m) to the rice culture solution together with Cd or Ni partially reversed the effects of heavy metals, stimulating growth as well as mobilization of carbohydrate reserves in seeds from which seedlings had developed. GA₃ increased the sugar content in roots and second and third leaves and also modified the carbohydrate distribution pattern compared with heavy metal-treated plants. In contrast to GA₃, ABA (19 m) supplied to rice cultures potentiated the effect of heavy metals, inhibiting the growth of young leaves and the translocation of storage products from source to sink organs. In addition, sugars were accumulated in roots and second leaf but not in the third leaf, the extension in length of which was also inhibited by the treatment. Net photosynthesis rates recovered transitorily in Cd-treated plants after the addition of hormones. The possible relationship between growth and carbohydrate distribution, as well as the involvement of hormones, in the response of plant to heavy metal stress is discussed.Abbreviations 5DT 5 days after treatment - 10DT 10 days after treatment - ABA abscisic acid - GA₃ gibberellic acid - TMC total metabolizable carbohydrates     

Pipecolic acid as an indicator of abnormal protein metabolism in diseased plants

Summary During the study of the free amino acid spectrum of diseased rice, soybean, potato and tobacco leaves, an unknown compound was detected, the occurrence of which was correlated with an abnormal physiological condition of the plants or with some disturbance in amino acid or protein metabolism. On chromatograms this substance stains blue following fixation with cupric nitrate. After purification with the aid of bacteria, this blue substance was identified as pipecolic acid.Since pipecolic acid could be found in the leaves of rice sprayed with maleic hydrazide, but not in control plants, it would seem that it is a metabolic product of growth inhibition.     

Amino acid composition of fractions of ‘Kentucky-31’ tall fescue as affected by N fertilization and mild water stress

Summary Environmental and management factors can influence the protein concentration of forages, significantly altering specific amino acid content. Drought, high rates of fertilizer N and the presence of a fungal endophyte have been associated with significant alterations in plant N metabolites and animal performance problems on tall fescue. A controlled environment study was conducted to examine the influence of N fertilization (10 and 100 gN/g) and water regime (low and adequate soil water availability) upon the distribution and concentration of amino acids in endophyte infected tall fescue (Festuca arundinacea, Schreb.) herbage. Tall fescue tissue was collected from three replicates of each treatment, quick frozen in liquid N and lyophilized. Two insoluble (R_I, structural residue; R_II, membrane residue) and two soluble (S_I, soluble protein; S_II, low molecular weight N compounds) fractions were collected. Amino acid analyses of acid hydrolysates of fractions showed that application of 100 N significantly increased the concentration (per unit dry weight) of all amino acids in the entire plant, with an average increase of about 55%. Application of 110 N increased the concentrations of most amino acids in fractions R_I, R_II, and S_I, but only aspartate-asparagine, glutamate-glutamine, alanine, threonine, serine, valine and proline in fraction S_II. Fraction R_I contained about 65% of total amino acids under 10 N and 55% under 110 N even though N level did not alter dry matter distribution among fractions. While the amount of dry matter was least in S_I, amino acids in the fraction ranged from 8% (leucine, 10 N) to 20% (lysine, 110 N) of the total amount of specific amino acids recovered. Significant increases in proline, glutamate, aspartate, serine, valine, threonine, alanine and phenylalanine concentration occurred under low soil-water availability compared with adequate water conditions. Basic amino acids including histidine, arginine and lysine increased with increased N and with water stress at each N level. Application of N increased amounts, and water stress influenced distribution of amino acids among the fractions of tall fescue herhage. Nitrogenous components, such as non-protein amino acids which could influence plant nutritive quality, were increased in fraction S_II by increased N and water stress.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

The effect of nitrogen nutrition on growth and biomass partitioning of annual plants originating from habitats of different nitrogen availability

Summary The hypothesis was tested that faster growth of nitrophilic plants at high nitrogen (N) nutrition is counterbalanced by faster growth of non-nitrophilic plants at low N-nutrition. Ten annual plant species were used which originated from habitats of different N-availability. The species' preference for N was quantified by the N-number of Ellenberg (1979), a relative measure of nitrophily. The plants were cultivated in a growth cabinet at five levels of ammonium-nitrate supply. At low N-supply, the relative growth rate (RGR) was independent of nitrophily. At high N-supply, RGR tended to be higher in nitrophilic than in non-nitrophilic species. However, the response of RGR to N-supply was strongly and positively correlated with the nitrophily of species. Increasing N-supply enhanced partitioning to leaf weight per total biomass (LWR) and increased plant leaf area per total biomass (LAR). Specific leaf weight (SLW) and LWR were both higher in non-nitrophilic than in nitrophilic species at all levels of N-nutrition. NAR (growth per leaf area or net assimilation rate) increased with nitrophily only under conditions of high N-supply. RGR correlated positively with LAR, irrespective of N-nutrition. Under conditions of high N-supply RGR correlated with SLW negatively and with NAR positively.     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.     

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 g per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S₁-, S₂- and S₃-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.     

Effect of parboiling and bran removal on aflatoxin levels in Sri Lankan rice

Commercial parboiling of rice in Sri Lanka and many south Asian countries provides ideal conditions for the occurrence of aflatoxins because the rice is steeped (allowing fermentation) thus providing ideal conditions for growth of toxigenic Aspergillus species. However the traditional cottage method of parboiling rice, which does not involve steeping, appears to reduce Aspergillus growth even after long storage periods. Preferential infection of parboiled rice by Aspergillus flavus was observed. Aflatoxin contents in inoculated rice produced by commercial parboiling (AFB₁ 60–92 mg/kg) were significantly higher than that in inoculated cottage processed rice (AFB₁ 12–29 g/kg). The steeping (precooking/ soaking) process in commercial parboiling appears to increase the susceptibility of rice grains to fungal infection. Aflatoxin content in grains increased considerably with the increase in duration of soaking. However, the addition of 10 ppm calcium hypochlorite (bleach) to soaking water appreciably reduced A. flavus contamination and subsequent aflatoxin content in parboiled rice. No significant reduction in aflatoxin levels were observed after bran removal of contaminated rice.     

Potential pathogenicity of Cladosporium tennuisimum,Phaeoisaria clematidis and Ramichloridium subulatum in a mouse model

In Sri Lanka, rice is the main staple which is mostly processed into parboiled rice. The levels of aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) in parboiled and raw milled rice collected from major rice producing areas and rice consuming townships were estimated. In almost all the samples of parboiled rice examined, the AFB₁ and AFG₁ contents were significantly higher than in raw milled rice. The highest AFB₁ content was 185 µg/kg and AFG₁ content 963 g/kg. These samples were collected from a major rice producing/milling district where the mean relative humidity is 78% and mean annual temperature 27 °C which is the highest amongst the rice growing areas in Sri Lanka. Raw rice was either free of aflatoxins or when toxins were detected, they occurred in less than 10% of the samples. The frequency of occurrence of surface fungal flora (Aspergillus/Penicillium) and aflatoxin content in market samples was closely related. Brownish or greenish moldy rice samples with fermented odour contained over 1000 g/kg of AFB₁.     

Kinetics of inhibition in propionic acid fermentation

The inhibitory effect of propionic acid P and biomass concentration X is studied in batch and continuous fermentations with cell recycle.In batch fermentations, the specific growth rate decreases and cancels out at a critical propionic acid concentration Pc ₁; the formerly decreasing specific production rate becomes constant after Pc ₁ and cancels out when a second critical propionic acid concentration Pc ₂ is reached.In continuous fermentation with cell recycle, a similar inhibition is observed with biomass. The specific rates decrease and become constant at a critical biomass concentration Xc. They cancel out at different high biomass concentrations.In both cases, the specific production rate can be related to the specific growth rate by the Luedeking and Piret expression: =+, [1], where the constants and are determined by the fermentation parameters.List of Symbols t h time - X kg/m³ biomass concentration - P kg/m³ propionic acid concentration - A kg/m³ acetic acid concentration - S kg/m³ lactose concentration - dX/dt kg/(m³h) instantaneous rate of cell growth - dP/dt kg/(m³h) instantaneous rate of propionic acid production - h^–1 specific growth rate - h^–1 specific propionic acid production rate - D h^–1 dilution rate     

Effect of phosphoros nutrition in peat on tomato plant growth and fruit development

Summary The effect of P nutrition on the growth of tomato plants in peat was examined. Initially, plants received an adequate supply of P and then received either nil, 0.78 or 2.34 kg superphosphate per m³ in combination with either 50 g N/ml (N₁) or 300 g N/ml (N₂) as ammonium nitrate in a liquid feed. Vegetative growth was restricted in the lower P treatmentsi.e. inhibited shoot growth, reduced duration of leaf expansion phase, thinner stems and reduced vegetative dry wt. Plants receiving N₂ showed a greater restriction in growth compared with N₁ plants when the P supply was limiting. P deficiency disrupted protein metabolism in the leaves, in that soluble leaf protein was reduced and trichloroacetic acid-soluble N accumulated. Flower development was accelerated by low P applications but the final numbers of flowers and the fruit-setting efficiency were reduced. Lowering the N supply reduced the fruit yield by 36 per cent while an intermediate P level reduced yields by about 15 per cent. Maximum fruit yields and good vegetative growth occurred when plants contained 0.4 per cent P or above in the mature leaves, and this value was achieved by adding the highest level (2.34 kg/m³) of superphosphate to the peat.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.     

Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.)

Summary Two semidwarfism-related proteins, SRP-1 and SRP-2, were detected as major spots in a long-culm rice cultivar, Norin 29 and its semidwarf near-isogenic line, SC-TN1, respectively, by two-dimensional gel electrophoresis (2D-PAGE). The testcross showed that SRP-1 and SRP-2 are controlled by codominant alleles, Srp-1 and Srp-2, respectively, at a single locus Srp. This locus was considered to be closely linked with the semidwarfing gene locus sd-1. SRP-1 and SRP-2 were separated by 2D-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and sequenced by a gas-phase protein sequencer. The N-terminal amino acid sequences, however, could not be determined due to the blockage of the N-terminals of these proteins. After removal of the N-terminal residue with pyroglutamyl peptidase given to the membrane, the amino acid sequence in the N-terminal region was determined. The N-terminal and internal amino acid sequences of SRP-1 and SRP-2 were highly homologous with those of the glutelin -subunits of seed endosperm storage protein, which were deduced by the cDNA sequences. In the seed endosperms of Norin 29 and SC-TN1, a total of eight glutelin -subunits was identified by 2D-PAGE. The amino acid sequences in the N-terminal and internal regions of these proteins were determined. This experiment confirmed that SRP-1 and SRP-2 are almost identical in structure with the glutelin _5a- and _5b-subunits, respectively, which were identified in several organs such as endosperms, embryos, and leaves, unlike the other glutelin -subunits.     

How does plant population density affect the biomass of Ravenna grass?

Ravenna grass, Tripidium ravennae (L.) H. Scholz, is known to produce an abundance of biomass, but how plant density affects its biomass potential remains unknown. The objectives were to determine the effects of plant density on biomass yield; plant growth traits; biomass?carbon, nitrogen, and ash concentrations; heating value; nitrogen removal; and sucrose concentration in leaves and culms. The treatments consisted of five plant densities (1,250; 2,500; 5,000; 10,000; and 20,000 plants per hectare) in a randomized complete block design with four blocks. Plots were nonirrigated, unfertilized, and harvested once during the dormant season each year. Data were collected from 2015?2019. Dependent variables that varied with plant population density (p < .05) were biomass yield, number of reproductive culms per plant, reproductive culm diameter, reproductive culm sucrose concentration, and nitrogen removal with biomass. Biomass yield ranged from 5.6 to 16.3 Mg/ha for plant densities of 1,250–20,000 plants per hectare, respectively. Combined over years, nonlinear regression of the data showed the equation for biomass yield to plateau at 16.2 Mg/ha at a plant density of 10,640 plants per hectare. As plant density increased, the number of reproductive culms per plant, culm diameter, and culm sucrose concentration significantly decreased. At 1,250 plants per hectare, the number of reproductive culms per plant, culm diameter, and culm sucrose averaged 70, 10.2 mm, and 63.2 g/kg, respectively. Nitrogen removed with biomass significantly increased as biomass yield increased with plant density. At a density of 10,000 and 20,000 plants per hectare, the amount of nitrogen removed annually in the harvested biomass averaged 88 kg/ha. The data suggest that 10,000 plants per hectare would produce the greatest annual biomass yields; however, research is needed to determine the nutrient requirement for Ravenna grass to sustain biomass production at that density.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.