Showing papers on "Zeatin published in 1992"

Thidiazuron induces high-frequency shoot regeneration in intact seedlings of pea (Pisum sativum), chickpea (Cicer arietinum) and lentil (Lens culinaris)

Abstract: Axenic seedling cultures of chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.) and garden pea (Pisum sativum L.) were established by culturing mature seeds on Murashige and Skoog medium (MS) supplemented with thidiazuron (TDZ). Of various cytokinins or compounds with cytokinin-like activity (Kinetin, TDZ, Zeatin) tested for inducing shoot formation in pea seeds cultures, TDZ was found to be most effective. Pea seedlings exhibited a unique pattern of shoot formation which was accomplished in two distinct phases. Multiple shoots developed within a week, from the nodal and basal regions of the primary epicotyl in a medium that contained 5-50 μM TDZ. When these seedlings were exposed for a prolonged time period (3-4 weeks), to the same medium, numerous shoot buds emerged de novo from the base and/or from the upper part of multiple shoots. These shoots had no apparent vascular connection with parent tissues. The inductive capability of TDZ was then tested in several other genotypes of Pisum sativum and two other large-seeded grain legumes, Cicer arietinum, and Lens culinaris. In Cicer arietinum, and Lens culinaris, multiple shoots developed after 1 week of seed culture on media that contained 1-50 μM TDZ. However, de novo differentiation of shoot buds occurred in cultures exposed to TDZ for 4-6 weeks, only from nodal and subjacent areas. Secondary shoot formation occurred frequently in all of the species tested. Developing shoots were able to form roots and eventually whole plants on a modified MS medium containing 2.5 μM NAA. No genotypic difference for morphogenesis was observed.

Genotypic effects on tissue culture response of lettuce cotyledons

Abstract: A wide range of lettuce (Lactuca sativa L.) cultivars representing the four major morphological types have been screened for tissue culture aptitude, in order to identify genotypes especially amenable for genetic manipulation at the cellular and molecular level. The growth response of lettuce cotyledons showed marked genotypic differences on both callus initiation medium and subsequent transfer to a plant regeneration medium. A new regeneration medium consisting of SH salts and vitamins, 30 g/l sucrose, 7 g/l agar, 0.1 mg/l IAA, 0.5 mg/l kinetin and 0.05 mg/l zeatin, at pH 5.8 substantially improved callus induction and shoot regeneration on cotyledons in most cultivars. Lettuce genotypes exhibiting good regeneration include Greenfields and Summer Gem (Crisphead types), Bronze Mignonette and Green Mignonette (Butterhead types), Salad Bowl (Leaf Type) and Cos and Green Cos (Romaine types)

Convergent Induction of Osmotic Stress-Responses: Abscisic Acid, Cytokinin, and the Effects of NaCl

Abstract: In Mesembryanthemum crystallinum, salt stress induces the accumulation of proline and a specific isoform of the enzyme phosphoenolpyruvate carboxylase (PEPCase) prior to the switch from C3 to Crassulacean acid metabolism (CAM). To determine whether plant growth regulators initiate or imitate these responses, we have compared the effects elicited by NaCl, abscisic acid (ABA), and cytokinins using PEPCase and proline levels as diagnostic tools. Exogenously applied ABA is a poor substitute for NaCl in inducing proline and CAM-specific PEPCase accumulation. Even though ABA levels increase 8- to 10-fold in leaves during salt stress, inhibition of ABA accumulation does not affect these salt-induced responses. In contrast, the addition of cytokinins (6-benzylaminopurine, zeatin, 2-isopentyladenine) mimic salt by greatly increasing proline and PEPCase amounts. Endogenous zeatin levels remain unchanged during salt stress. We conclude: (a) The salt-induced accumulation of proline and PEPCase is coincident with, but is not attributable to, the rise in ABA or zeatin concentration. (b) For the first time, cytokinins and NaCl are implicated as independent initiators of a sensing pathway that signals leaves to alter PEPCase gene expression. (c) During stress, the sensing of osmotic imbalances leading to ABA, proline, and CAM-specific PEPCase accumulation may be mediated directly by NaCl.

Cytokinin Is Required to Induce the Nitrogen-Dependent Accumulation of mRNAs for Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaves.

Abstract: Previous studies with intact maize (Zea mays L.) plants indicated that phosphoenolpyruvate carboxylase (PEPC) levels are controlled by nitrogen (N) availability and that this regulation is presumably at the transcriptional level (B. Sugiharto, K. Miyata, H. Nakamoto, H. Sasakawa, T. Sugiyama [1990] Plant Physiol 92: 963-969; B. Sugiharto, T. Sugiyama [1992] Plant Physiol 98: 1403-1408). In the present study, detached maize leaves were used to investigate further the mechanism of N-dependent regulation of gene expression in C4 plants. PEPC and carbonic anhydrase (CA) mRNA levels decreased in leaves detached from maize plants. Addition of high nitrate did not prevent this decrease. However, the addition of zeatin to solutions bathing the cut ends of the detached leaves inhibited the decrease of PEPC and CA mRNA levels. Simultaneous addition of high nitrate and zeatin to leaves detached from N-deficient maize plants caused a large and rapid increase in PEPC and CA mRNA levels. Zeatin could be replaced by benzyladenine, but not by indoleacetic acid or abscisic acid. Both CA isozymes were effected and responded in an identical manner. We conclude that detached maize leaves provide an excellent experimental system to study the mechanism(s) of N-mediated regulation of PEPC and CA gene expression. However, zeatin is an essential component of this system.

Cytokinin biochemistry in relation to leaf senescence. VII. Endogenous cytokinin levels and exogenous applications of cytokinins in relation to sequential leaf senescence of tobacco

Abstract: The cytokinin complex in tobacco leaves of various maturities was characterized by radioimmunoassay and mass spectrometry. Zeatin was the major base, whereas zeatin riboside was identified as the main riboside. in leaves of all maturities studied. Relative to upper younger leaves, the basal yellow leaves had reduced levels of both cytokinin bases and ribosides. Exogenous applications of dihydrozeatin and zeatin to detached tobacco leaves in amounts sufficient to delay senescence, elevated cytokinin base and riboside levels 2–5 fold. Presenescent and senescent leaves of intact plants showed quantitatively similar changes in cytokinin content. which therefore appear to be of significance in control of senescence. When supplied exogenously, the principal cytokinin bases found to occur in tobacco leaves (zeatin and dihydrozeatin) were markedly more effective than auxins and gibberellic acid in retarding senescence. Localised application of cytokinins to leaf blades of detopped plants was much less effective than application to intact plants. The cytokinin induced senescence retardation in tobacco leaves was independent of effects on directed metabolite transport. Evidence that endogenous levels of active cytokinins in intact tobacco leaves are involved in control of sequential leaf senescence is discussed.

Changes in Cytokinin Activities and Mass Spectrometric Analysis of Cytokinins in Root Exudates of Rice Plant (Oryza sativa L.) Comparison between Cultivars Nipponbare and Akenohoshi

Abstract: Changes in exudation rate and cytokinin activities in the exudates were measured in two varieties of rice (Oryza sativa L.), cv Nipponbare (a Japanese normal cultivar) and cv Akenohoshi (a high-yielding cultivar). The exudation rates of Akenohoshi, the leaves of which remained green for a longer time, were higher than those of Nipponbare after the booting stage. Cytokinin activities in the exudates of Akenohoshi were higher than those of Nipponbare during the ripening period. Cytokinins in the exudates collected during the middle of the ripening stage were analyzed with mass spectrometry using deuterium-labeled standards. trans-Zeatin, trans-ribosylzeatin, and N6-isopentenyladenosine were detected as free cytokinins, and zeatin was detected in the hydrolysates of highly polar fractions (“conjugated zeatin”) in the exudates of both cultivars. Conjugated zeatin was the predominant cytokinin in both cultivars. Therefore, we suggest that conjugated zeatin is an important form of cytokinin during the ripening stage. The level of each of the cytokinins in Akenohoshi was higher than that in Nipponbare. Also, we discuss the correlation between the leaf senescence and cytokinin content in root exudates.

Cytokinin effects on root growth and possible interactions with ethylene and indole‐3‐acetic acid

Abstract: Etiolated pea seedlings (Pisum sativum L. cv. Weibull's Marma) were used to investigate the effects of exogenous cytokinins on root growth. Benzylaminopurine (BAP) added to the growth solution inhibited the elongation and formation of lateral roots and stimulated swelling of the root tips. Similar effects were obtained with zeatin. The effects were obtained over a wide concentration range down to 0.01 μM. Growth responses appeared only after treatment for several hours, and the duration of treatment had an important influence on the degree of the effects. BAP caused a moderate increase in ethylene production as measured in excised 10-mm-long root tips. Lowering ethylene production by treatment with cobalt ions counteracted both the inhibition and swelling caused by BAP. Treatment with silver ions also reversed the effect to some extent, indicating that ethylene is involved in the response of the roots to BAP. To further study the involvement of the increased ethylene production in the elongation and swelling response, the effects were compared with those obtained after application of 1-aminocyclopropane-1-carboxylic acid (ACC) in relation to the ethylene produced from this compound. This comparison showed that the increase in ethylene production caused by BAP was too low to explain the response of the roots. However, ACC treatment caused a considerable lowering of the content of indole-3-acetic acid (IAA) in the root tips, whereas BAP did not; instead, BAP increased the amount of IAA per root tip. It is concluded that cytokinins influence growth processes in roots via several mechanisms. A synergistic interaction between endogenous IAA, maintained at a high level by the cytokinin treatment, and the increased ethylene levels appears to explain most of the cytokinin effects during the first day of treatment.

Cytokinins in the xylem sap of desert-grown almond (Prunus dulcis†) trees: Daily courses and their possible interactions with abscisic acid and leaf conductance.

Abstract: summary Xylem sap samples were obtained from one- to four-year-old almond trees [Prunus dulcis (Miller) D A Webb] that had been grown in lysimeters of different volumes with different amounts of available water and subjected to an annual drying cycle The samples were analyzed for cytokinin patterns by ELISA during the growing season Free bases, ribosides and nucleotides of the N6-(Δ2-isopentenyl) adenine (iP) and the zeatin (Z) type could be identified and quantified Z-type cytokinins were always predominant In many cases the concentrations of the cytokinin fractions were rather constant during the day In a limited number of days, however, Z-type cytokinins showed peak concentrations in the morning and a rapid decrease in the afternoon A correlation between water status of the trees and the concentrations of cytokinins or their daily variations in the xylem sap could not be established When the concentration of abscisic acid in the xylem sap was not limiting leaf conductance, daily-variation of a cytokinin fraction preceded daily variation in leaf conductance We conclude that in almond trees, cytokinins may affect stomatal behaviour on a short-term basis This is an outcome of changes in their xylem sap concentrations during the course of a day Abscisic acid, on the other hand, acts as an opposing signal, the size of which reflects long-term water deficit

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Abstract: Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B5vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.

Cytokinin biochemistry in relation to leaf senescence. VIII: translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Abstract: Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When 3H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [14C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [14C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.

Cytokinin biochemistry in relation to leaf senescence. VI. Effect of nitrogenous nutrients on cytokinin levels and senescence of tobacco leaves

Abstract: The effect of nitrogenous nutrients on endogeneous cytokinins and senescence of tobacco leaves was investigated. Ammonium nitrate was the most effective in retarding senescence and its activity was attributed principally to NH4+ ions. Repeated applications or a continuous supply of ammonium nitrate was required for maximal retardation of tobacco leaf senescence. Ammonium nitrate solution supplied via the petioles reduced the senescence retarding effect of dihydrozeatin applied directly to the laminae of detached tobacco leaves. Ammonium nitrate also elevated the endogenous levels of cytokinins (especially zeatin and dihydrozeatin) particularly in growing tobacco leaves excised from near the apex of the plant. Ammonium nitrate induced retardation of leaf senescence may be mediated at least partly by its effect on foliar cytokinin content.

Role of cytokinin in enhanced productivity of maize supplied with NH4+ and NO3−

Abstract: Supplying both N forms (NH4 ++NO3 −) to the maize (Zea mays L.) plant can optimize productivity by enhancing reproductive development. However, the physiological factors responsible for this enhancement have not been elucidated, and may include the supply of cytokinin, a growth-regulating substance. Therefore, field and gravel hydroponic studies were conducted to examine the effect of N form (NH4 ++NO3 − versus predominantly NO3 −) and exogenous cytokinin treatment (six foliar applications of 22 μM 6-benzylaminopurine (BAP) during vegetative growth versus untreated) on productivity and yield of maize. For untreated plants, NH4 ++NO3 − nutrition increased grain yield by 11% and whole shoot N content by 6% compared with predominantly NO3 −. Cytokinin application to NO3 −-grown field plants increased grain yield to that of NH4 ++NO3 −-grown plants, which was the result of enhanced dry matter partitioning to the grain and decreased kernel abortion. Likewise, hydroponically grown maize supplied with NH4 ++NO3 − doubled anthesis earshoot weight, and enhanced the partitioning of dry matter to the shoot. NH4 ++NO3 − nutrition also increased earshoot N content by 200%, and whole shoot N accumulation by 25%. During vegetative growth, NH4 ++NO3 − plants had higher concentrations of endogenous cytokinins zeatin and zeatin riboside in root tips than NO3 −-grown plants. Based on these data, we suggest that the enhanced earshoot and grain production of plants supplied with NH4 ++NO3 − may be partly associated with an increased endogenous cytokinin supply.

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

Abstract: For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 μM zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 μM zeatin plus 0.2 μM indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 μM BA or 4.6 μM kinetin. This regeneration system may be useful for genetic transformation of chickpea.

Use of a cytokinin conjugate for efficient shoot regeneration from leaf sections of highbush blueberry

Abstract: Conditions for improving the efficiency of shoot regenration from leaf sections of highbush bluebeery (Vaccinium corymbosum L.) were investigated. Effectiveness of tissue culture medium supplemented with the cytokinin conjugate zeatin riboside or the cytokinin zeatin at 10, 20, or 30 μM was compared with medium supplemented with the optimum 2iP concentratioin of 15 μM. Use of 20 μM zeatin riboside resulted in the most shoots per leaf section, ≃6-fold higher than the number of shoots produced on 2iP medium. The number of shoots produced on medium supplemented with zeatin was not significantly higher than the number of shoots produced on 2iP medium []

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : I. Spatial distribution of endogenous cytokinins in relation to leaf growth.

Abstract: Immunoaffinity purification of zeatin, dihydrozeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adenosine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydrozeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spatial distribution of endogenous cytokinins in rapidly expanding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly expanding leaves was found to be correlated with the rate of leaf expansion which is high (∼40%/24 h) in the basal leaf tissue and low (∼10%/24 h) near the leaf tip. Applied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.

Amounts of Zeatin and Zeatin Riboside in Xylem Sap of Senscent and Nonsenescent Sorghum

Abstract: Xylem sap from decapitated vegetative and mature plants of nonsenescent ‘Tx2817’ and senescent ‘TxTOOO’ sorghum [Sorghum bicolor (L.) Moench] cultivars was analyzed for cytokinins to determine whether the delayed leaf senescence of nonsenescent sorghums may be associated with transport of greater quantities of cytokinins from roots. Cytokinins were assayed by high-performance liquid chromatography (ultraviolet absorption) with ˡ⁴C-labeled zeatin (Z) as an internal standard. For field-grown plants, the amount of Z + zeatin riboside (ZR) in xylem sap per gram shoot dry weight was 1.51 times higher for the nonsenescent sorghum. Vegetative plants were grown in solution culture for ⊄ 1 mo and then irradiance and N supply were altered for 7 or 8 d prior to collection of sap for 24 h. With high N and high light, Tx7000 and Tx2817 had nearly identical Z + ZR in the sap per gram shoot fresh weight. Treatments of 10% N supply or 50% light reduced growth only slightly. With 10% N, however, Z + ZR averaged 38% of the control for Tx7000 and 59% for Tx2817; with 50% light, Z + ZR averaged 55% of the control for Tx7000 and 136% for Tx2817. Thus, Z and ZR transport from roots to shoots of nonsenescent Tx2817 was less susceptible to reduction by low N and was actually increased by low light. High xylem sap cytokinin contents for Tx2817 with 50% light suggests that its delayed senescence and enhanced resistance to adverse conditions may be due to high cytokinin production by roots when the carbohydrate supply to the roots is reduced. Contribution of the Texas Agric. Exp. Stn. Journal Article no. TA27045.

Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Abstract: The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.

Adventitious Bud Formation from Callus Cultures of Japanese Persimmon

Abstract: Callus cultures were initiated in the dark from leaf primordia, stem internodes, and young leaves of adult Japanese persimmon (Diospyros kaki L.) to induce adventitious buds. A high frequency of regeneration occurred on Murashige and Skoog medium (MS) with half the normal NH 4 NO 3 and KNO 3 concentration (1/2N) and containing 10 μM zeatin or 1 μM 4PU-30 in combination with 0.1 μM IAA, or MS(1/2N) medium containing 0.03 to 0.1 μM IAA or 0.01 to 0.03 μM NAA combined with 10 μM zeatin (...)

Physiology and application of phenylurea cytokinins

Abstract: Certain phenylurea derivatives: N1-(2-chloro-4-pyridyl)-NZ phenylurea (4-PU30, Fulmet, Forchlorfenuron) (Takahashi et al. 1978), N1-(1,2,3-thiadiazol-5y1)-N521-phenylurea (Thidiazuron, Dropp-50) (Baskakov et al., 1981; Kulaeva, 1982; Mok et al., 1982), N1-(pyrrolidino-3-sulfonyl-phenyl)-N521-phenylurea (PSPPU) (Karanov et al., 1985) and N1-(4-methoxy-6-methyl-sim.triazine-2yl)-N5- (2-chlorophenyl-sulfonyl) urea (Glean-75, DPX-4189, Chlorsulfuron) (Levit, 1983) exhibit cytokinin activity exceeding that of adenine derivatives (e.g. kinetin, BA, zeatin).

Micropropagation and Simultaneous Rooting of Actinidia deliciosa var. deliciosa 'Hayward'

Abstract: Nodal segments and shoot tips of axenic shoot cultures of 'Hayward' kiwi- fruit were inoculated on modified Murashige and Skoog (MS) medium supplemented with zeatin at 1 mg·liter -1 and IAA at 0.05 mg·liter -1 (H1) or on MS medium without growth regulators (H2). Inocula cultured on H2 medium all developed into normal plantlets, while those cultured on H1 medium developed into shoots, 18% of them abnormal. Rooting of H1 shoots was induced by a 24-h immersion in a solution of IRA at 20 mg·liter -1 . H2 plantlets were directly transferred to soil. Statistical treatment of the results revealed no significant differences, in terms of plant development, between the two micropropagation methods used. However, the presence of a functional root system on 5-week-old H2 plantlets resulted in 100% plant survival, but only 70% of in vivo-rooted shoots from H1 survived. Nevertheless, H1 still allowed for an important reduction of costs and manipulation. Chemical names used: indole3-acetic acid (IAA).

Shoot Regeneration from Cotyledonary Protoplasts of Melon(Cucumis melo L. cv. Charentais)

Abstract: Protoplasts, isolated from cotyledons of aseptically germinated seeds of the melon 'Charen-tais', were used to investigate the effects of hormones and osmotic conditions on cell divi-sion and plantlet regeneration.1. Division of protoplasts occurred at a high frequency during the initial incubation peri-od on the modified MS medium (200 mg•liter-1 NH4NO3 and 1% sucrose) containing a wide range of 2, 4-D concentrations. Protoplast division was continuous in a medium containing 0.05 mg•liter-1 2, 4-D and 0.5 mg•liter-1 BA even as the protoplasts mature and formed cell walls.2. Three osmotic conditions; mannitol only and an equimolor conc. of mannitol and sucrose and that of mannitol and glucose were used to clarify the role of the osmoticum on the shoot regeneration from calli. No obvious difference among the three osmotic conditions was ob-served in the frequency of cell division at initial culture and in callus formation. The callus grown in a medium containing mannitol and glucose differentiated shoots at the highest fre-quency among the media tested.3. More shoots were regenerated in a MS medium containing 0.5 mg•liter-1 GA3 and 2 mg•liter-1 BA than were formed with a combination of GA3 and zeatin. The GA3 com-bined with cytokinins promoted more shoots formation from calli than did the IAA-cytokinin combinations.The following protocol for shoot regeneration of melon was quite efficient: a) Culture the protoplasts in the modified MS medium supplemented with 0.05 mg•liter-1 2, 4-D, 0.5 mg•liter-1 BA, 0.2 M mannitol and 0.2 M glucose. b) Transfer the callus grown in the above medium to regeneration medium supplemented with 2 mg•liter-1 BA and 0.5 mg•liter-1GA3. On the average, three shoots per callus were regenerated.Many regenerated shoots may appear abnormal and will not elongate normally, in which case, transplant the explant intact onto a MS medium containing 0.1 mg•liter-1 BA and 0.2 mg-liter-1 GA3. Subculture the explant several times to promote elongation of the shoots. Finally, subculture the elongated shoot on MS agar medium with 1 mg•liter-1 NAA for 24 hr and then transfer it to one without any hormone. The treated shoot will regener-ate roots and grow into a normal plant.

I. Spatial distribution of endogenous cytokinins in relation to leaf growth

Abstract: Immunoaffinity purification of zeatin, dihydro zeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adeno sine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydro zeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spa tial distribution of endogenous cytokinins in rapidly ex panding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly ex panding leaves was found to be correlated with the rate of leaf expansion which is high (~40%/24 h) in the basal leaf tissue and low (~ 10%/24 h) near the leaf tip. Ap plied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.