Influence of genotype and explant source on the in vitro regeneration ability of different melon varieties

Nine genotypes of melon (Cucumis melo L.) were selected for the investigation of regeneration. Most of the tested varieties showed regeneration ability on medium containing 0.5 mg l?1 or 1 mg l?1 BA, but following the appearance of shoot buds, only six varieties produced leafy shoots. The effect of combinations of BA with different auxins (IAA, NA, 2,4-D) and ABA in the culture medium on shoot regeneration was tested on cotyledon explants of 'Hógolyó' and 'Hale's Best'. To establish optimal conditions for the adventitious shoot induction six types of seedling-derived explants were prepared from seedlings of four different ages. The best results for shoot forming capacity were achieved with cotyledons followed by decapitated seedlings and hypocotyls derived from 4-day-old seedlings. Cotyledon segments of 'Hógolyó' and 'Hale's Best' were also cultivated on media with different concentrations of IAA and BA supplemented with 0.26 mg l?1 ABA. The highest number of well-formed plantlets was counted for 'Hógolyó' on the medium supplemented with 0.9 mg l?1 BA+ 0.6 mg l?1 IAA+ 0.26 mg l?1 ABA. This is the first report on the in vitro regeneration of 'Hógolyó' from decapitated seedling and hypocotyl explants and of 'Javított Zentai', 'Muskotály', 'Hógolyó', 'Tétényi csereshéjú' and 'Magyar Kincs' from cotyledon explants.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

In vitro regeneration of Minthostachys andina (brett) Epling - a Bolivian native species with aromatic and medicinal properties

Various explants of Minthostachys andina (Brett.) were evaluated for their morphogenic potential under in vitro culture conditions. Axillary buds derived from 2 year-old plants grown in MS-medium supplemented with 4.4 or 8.8 μM BA and 0.054 μM NAA, initiated shoot growth and new shoot formation. Under subculture in NN medium, shoots were rooted in the presence of NAA (1.6, 2.7 or 5.3 μM) alone or in combination with IBA (9.8 μM), and the regenerated plantlets were later acclimatised in the greenhouse. Also, polynodal segments from seedlings initiated multiple shoots and plantlets when initially cultured in presence of NN-liquid salt medium supplemented with 2.2-17.7 μM BA or 4.5-13.6 M TDZ in combination with different auxin-like growth regulators and after a final transfer for root initiation. The same types of responses were found in hypocotyl and leaf explants, which produced adventitious shoots in the presence of TDZ. The use of antioxidants helped to prevent browning and favoured organogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N ⁶ –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA₃), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA₃ (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg∙L ^-16-BA与0.5 mg∙L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg?L ^-16-BA与0.5 mg?L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium

A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N ⁶-benzyladenine (BA) concentrations or with 2 mg?l^?1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg?l^?1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.     

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10^–7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10^–7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.Abbreviations ABA Abscisic acid - BA 6-Benzylaminopurine - GD Gresshoff and Doy (1972) nutrient medium - NAA -Naphthaleneacetic acid.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media

 The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10^–2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed. Received: 24 January 2000 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

Regeneration and production of transgenic Lilium longiflorum via Agrobacterium tumefaciens

Efficient transformation of lilies is required for their genetic improvement in ornamental and marketable qualities. Although Lilium longiflorum can be transformed by particle bombardment and Agrobacterium, the transformation frequency is low. In this study, we tested new Agrobacterium-mediated transformation methods using shoot segments combined with two different regeneration systems. Shoot segments were co-cultivated for 2?d with Agrobacterium tumefaciens strain AGL1/pCAS04 harboring a binary vector carrying the neomycin phosphotransferase II driven by a promoter from the maize ubiquitin gene. The effect of different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on regeneration was investigated. The results indicated that Murashige and Skoog (MS) medium with 4.4???M BA and 0.5???M ??-naphthalene acetic acid was optimal for shoot formation, and the nodal stem was the best explant for shoot induction. MS medium with 9.0???M 2,4-D and 0.4???M BA was optimal for callus induction. The direct shoot formation method regenerated 187 plantlets per 100 explants, and 74.4% of the regenerants were positive in transgene PCR. The callus regeneration method regenerated 20 plantlets per 100 explants, and 31.5% of them were PCR positive. Southern blotting confirmed the insertion of transgene in the plant host genome. The direct shoot formation method is more than 20-fold more efficient than previously reported transformation method in this species.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees

Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.     

Effect of vessel type and growth regulators on micropropagation of Capsicum annuum

Leaves from 14-d-old Capsicum annuum L. cv. Anaheim seedlings were cultured on Murashige and Skoog (MS) medium containing different combinations of indole-3-acetic acid (IAA) and 6-benzyladenine (BA). After 3 months, cultures were transferred to new medium where BA was replaced with 9 μM isopentenyladenine (2iP) to enhance the growth of shoot buds. Developing shoots were elongated and rooted on MS medium enriched with 9 μM indole-3-butyric acid (IBA). All cultures were maintained in 250 cm³ baby jars covered with a clear polypropylene lid with or without microporous polypropylene membrane. Vessel type and plant growth regulators significantly affected callus morphogenic appearance, organogenesis and in vitro plantlet growth. Ventilated vessels supported photomixotrophic culture and improved regeneration and growth of plantlets. Higher plantlet dry mass and content of photosynthetic pigments, and lower stomatal density of plantlets grown in ventilated than in non-ventilated vessels facilitated ex vitro acclimation and growth.     

Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO₃). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.