Cytokinin modification of metabolism of p-coumaric acid by a cell suspension of soybean (Glycine max (L.) Merrill)

Cells of a soybean tissue strain suspended in an aerated liquid medium caused the disappearance of p-coumaric acid from the medium. The rate of disappearance was modified by cytokinins. When the coumarate and the cytokinin were added to the medium simultaneously, disappearance was increased if the cytokinin was used in the concentration range from 0.05 to 50 M; higher concentrations inhibited the disappearance. If, however, the cytokinin was added at the beginning of the shaking period (for aeration) and the coumarate added 1 h later, the results were more complex. With this procedure, cytokinins at concentrations from 0.0005 to about 1 M inhibited, at 50 M they promoted, and at higher concentrations they inhibited the coumarate disappearance. The promotion was elicited by zeatin, ribosylzeatin, kinetin, 6-benzylaminopurine (BAP), by BAP substituted at the 9-position by methyl, methoxymethyl, cyclohexyl or tetrahydropyran-2-yl groups, by adenine with the amino group substituted by methyl, dimethyl, n-propyl, n-pentyl or n-hexyl groups, by 1,3-diphenylurea and nicotinamide, all at about 50 M. Adenine and benzimidazole were not effective. The promotion was detected in as little as 12 min. The delayed inhibitory effect required the presence of the cytokinin during the 1 h of shaking before the coumarate was added. This effect was elicited by zeatin, ribosylzeatin, kinetin, BAP, the aforementioned 9-substituted-BAP compounds, 9-glucosyl-BAP, 7-glucosyl-BAP, and 6-isopentenylaminopurine and its ribonucleoside. It was not caused by adenine, cis-ribosylzeatin, diphenylurea, benzimidazole, 6-methylaminopurine, 6,6-dimethylaminopurine or nicotinamide. The chemical specificity for this effect was much the same as that known for promotion of cell division in the soybean tissue.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid     

Uptake and metabolism of benzyladenine during shoot organogenesis in Petunia leaf explants

Benzyladenine (BAP) uptake and metabolism were characterized during the key stages of shoot organogenesis in leaf explants of Petunia MD1. Using leaf explant transfer experiments, it was shown that exposure to 2.2 M BAP for 6, 8 or 10 days induced shoot formation on 27, 80 and 100% of the explants respectively, with a concomitant increase in the number of shoots per explant. BAP uptake and metabolism were characterized in leaf explants after 1, 3, 6 or 10 days exposure to [³H]BAP or 10 days exposure plus an additional 2 days on basal medium (10+2). BAP and 9--D-ribofuranosyl-BAP ([9R]BAP) were detected at days 1 and 3 only. Therefore, the BAP free base was not detectable during the shoot induction period between days 6 and 10, as defined by leaf transfer experiments. The BAP ribotide pool was largest on day 1 and decreased to day 10+2. It is possible that the BAP ribotide pool provided either the active cytokinin itself or acted as a short-term storage form for the active cytokinin in petunia shoot organogenesis. Other metabolites detected in petunia leaf tissue included 7--D-glucopyranosyl-BAP ([7G]BAP), 9--D-glucopyranosyl-BAP ([9G]BAP) and an unidentified metabolite C.Abbreviations BAP benzyladenine - [7G]BAP 7--D-glucopyranosyl-BAP - [9G]BAP 9--D-glucopyranosyl-BAP - [9R]BAP 9--D-ribofuranosyl-BAP - [9R-5P]BAP 5-monophosphate of [9R]BAP - [9R-5PP]BAP 5-diphosphate of [9R]BAP - [9R-5PPP]BAP 5-triphosphate of [9R]BAP - TEA Triethylamine This research was supported in part by NSF Grant DCB-8917378 to J.D.C. and USDA-CRGO Grant 89-37261-4791 to T.J.C.     

The response of different genotypes of Fragaria × ananassa and their seedling progenies to in vitro micropropagation and the effects of varying the concentration of 6-benzylaminopurine in the proliferation medium

Two experiments are reported. In the first, six different strawberry genotypes were each grown in vitro on media containing five different concentrations of 6-benzylaminopurine (BAP) ranging from 0.5 M to 8.0 M. Responses of the genotypes to different BAP levels varied and the optimum levels for maximum proliferation were between 1 M and 8 M. In the second experiment the six genotypes were split into two groups of three and all crosses between the two groups were made. Seedlings from the nine progenies were then grown on media with the same five levels of BAP. Of the 12% of seedlings that did not proliferate, most were on media with the two lowest BAP levels (0.5 and 1.0 M). Seedlings from crosses involving the cv. Bogota proliferated least, while the three progenies with the breeding line ES652 as a parent had the highest mean proliferation rates. One cross, Bogota × Providence, had a particularly low mean proliferation rate. It was concluded that to optimize proliferation rate the medium must be tailored to the genotype. The best medium could not be reliably predicted from knowledge of the pedigree of a genotype.     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.     

Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)

The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5-monophosphate (ZR5P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

Morphological development of normal and phyllody expressing Rosa hybrida cv. Motrea flowers

A large number of soybean (Glycine max L. Merr.)flowers and young pods abscise rather than develop into mature pods. Flower andpod drop or abortion accounts for the majority of total reproductive abscissionand influences potential soybean yield. The objectives of this study were todetermine the patterns of flower, pod and seed development under treatmentswiththe growth regulators, 2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and6-benzylaminopurine (BAP), applied at the early reproductive stages, and toexamine the association of reproductive abscission with growth characteristicsand agronomic traits, including seed yield and seed weight. Small seeded [cvPungsan (11.1±0.4 g100-seed^–1)] and large seeded [cv Manlee(21.0±0.5 g 100-seed^–1)]genotypes were separately planted in the greenhouse and field, and treated witheither 2,4-DP or BAP. 2,4-DP (a synthetic auxin) and BAP (a syntheticcytokinin)were each applied at three concentrations (i.e. high, intermediate or low):0.12mM, 0.08 mM, 0.04 mM, and 1.5mM, 1 mM, 0.5 mM respectively. High andlow concentrations were employed for greenhouse experiments to examine thenumber of flowers per plant in pots. With the exception of low BAP (0.5mM) treatment in Pungsan, all treatments increased total podnumberwith various numbers of seeds per pod. Low 2,4-DP (0.04 mM) inbothgenotypes or BAP (0.5 mM) in Manlee significantly reduced flowerabortion and delayed abscission of pods in both genotypes, resulting inincreased pod setting rates. Under field conditions using intermediateconcentrations, 1 mM BAP significantly increased 100-seed weightto22.3 g at R1 in Manlee and 11.9 g at R3 in Pungsan.BAP (1 mM) at R3 in Pungsan significantly improved seed yield(40.1g plant^–1). Maturity was not significantlyaffected by either application in Manlee, but was significantly affected by BAPin Pungsan. In Pungsan, 2,4-DP increased pod number, plant height and nodenumber, but decreased 100-seed weight in Pungsan treated at R1, causing nosignificant change of seed yield. This study suggested that exogenousregulatorssignificantly influenced reproductive and growth characteristics, andconsequently seed yield, but increase of pod number was not always beneficialfor seed yield.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Effects of temperature on phyllody expression and cytokinin content in floral organs of rose flowers

Formation of leaf-like organs known as phylloids in Rosahybrida cv. Motrea flowers was promoted by exposure of plants toelevated temperatures. At a day/night temperature regime of26°C/21°C respectively theproportion of malformed flowers exhibiting phyllody was four times higher thanthat in flowers of plants grown at21°C/15°C. The number ofpetals in phyllody-expressing flowers was higher than that in normal flowers.The total content of endogenous cytokinins in young flower buds of plantsexposed to the lower temperature was six times higher than that at the highertemperatures. The effects of the reduced temperature were pronounced on all thegroups of cytokinins examined. However, the proportion of the various cytokiningroups remained similar at both temperature regimes. In contrast to thecytokinins in the flower buds, the content of all cytokinin types in youngleaves increased following exposure to the higher temperature and was reducedbythe lower temperatures. After 11 weeks at the lower temperature, about18% of the flowers remained malformed, whereas at the higher temperatureabout 20% of the flowers still remained normal. All thephyllody-exhibiting flowers were formed on vigorously grown basal shootscharacteristic to Rosa hybrida plants, whereas the normalflowers at the elevated temperatures were formed on lateral shoots which weremost distal to the plant base. However, irrespective of the season, thepresenceof normal and malformed flowers was observed on plants kept growing at standardconditions of 30°C/17°C inthegreenhouse. This phenomenon led us to examine the cytokinins in floral organsofnormal and malformed cv. Motrea flowers grown in the greenhouse as well as inflowers of a complete rose mutant known as a 'Green Rose(Rosa chinensis viridiflora). The highest content ofcytokinins was found in the pistils and stamens of normal 'Motreaflowers. On the other hand, the content of cytokinins in leaf-like style-tubesin the malformed flowers as well as in partially malformed ovaries at the baseof phylloids was significantly lower. A low content of cytokinins was alsopresent in petals of both normal and phyllody-exhibiting flowers and the lowestcontent has been found in the phylloids of the 'Green Rose. Apossibility of mutant deviations in metabolism of cytokinins in rose plants isdiscussed.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 M thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 M -naphthaleneacetic acid or 1.0–2.0 M indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.Abbreviations BAP 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) by organogenesis

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 M) in combination with naphthaleneacetic acid(0.05 M) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 M) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 M), indole-3-butyric acid (1.2 M) and benzyladenine (0.4 M). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - GD Gresshoff and Doy (1972) nutrient media - IBA Indole-3-butyric acid - KN Kinetin - NAA -Naphthaleneacetic acid - 2iP 2-isopentenyl adenine - ZN Zeatin     

Effects of some growth regulators on in vitro tuberization in Dioscorea alata L. ‘Brazo fuerte’ and D. abyssinica Hoch.

Summary Nodal cuttings of Dioscorea alata L. Brazo fuerte and D. abyssinica Hoch. were cultured in vitro to assess the influence of NAA on the production of microtubers. In D. alata, high concentrations of NAA (27 and 54 M) favored the production of large microtubers, whereas the highest number of microtubers was obtained with 2.7 M. D. abyssinica was found to be more sensitive to NAA since concentrations higher than 0.27 M promoted the growth of callus on the root system. In this species, the production of the largest microtubers was obtained at 2.7 M whereas the number of microtubers was not affected by any concentration tested. In D. alata, the effects of ABA and BAP were also evaluated. The weight of the microtubers increased with increasing concentrations of ABA. This effect, however, was observed only on expiants cultured under 8 h photoperiod, but not on those cultured under 16 h. Finally the presence of BAP at concentrations as low as 0.22 M adversely affected the survival of the explants.Abbreviations ABA abscissic acid - BAP benzyl aminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine