Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Adventitious shoot regeneration from cotyledonary explants of rapid-cycling fast plants of <Emphasis Type="Italic">Brassica rapa</Emphasis> L

Rapid-cycling fast plants (Brassica rapa; RCBr) is also known as Wisconsin Fast Plant and is widely used in K-12 and undergraduate studies. RCBr has a short generation time (seed-to-seed in 30–60 days), which allows for the completion of experiments in a semester. Previous studies have shown that cotyledonary explants with attached petioles are capable of generating shoots. However, there is no published adventitious shoot regeneration protocol to date. Sterile cotyledonary explants were excised; all edges and petioles were removed. Five-day-old cotyledonary explants produced shoots on a Murashige and Skoog medium containing 1.5 mg/L thiadiazuron (TDZ) and 0.5 mg/L 1-naphthaleneacetic acid (NAA) (FPM I) at a mean rate of 8.8%. This rate increased to 14.8% in explants placed on FPM I medium supplemented with 5.0 mg/L silver nitrate (AgNO₃) (SRM 2). The rate increased to 32.5% when 5-day-old explants, excised from the part of the cotyledon nearest to the petiole, were placed adaxial side up on SRM 2 medium. The shoot regeneration rate increased to 44.5% using 4-day-old cotyledonary explants. A shoot regeneration rate of 23% was observed among 9-day-old leaf explants. Shoots from cotyledonary explants were elongated on basal medium with 0.5 mg/L NAA, rooted on basal medium, and later acclimatized. This is the first report of shoot regeneration from cotyledonary explants of rapid-cycling Brassica rapa without pre-existing meristematic tissues.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.     

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.     

In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L.

Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to be highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminopurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation.Abbreviations MS Murashige and Skoog - IAA 3-indoleacetic acid - BA 6-benzylaminopurine     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important western-shipper cantaloupe and honeydew melons, ‘F39’ and ‘150’, respectively. Different media were tested to select a shoot regeneration system for each of three elite breeding lines ‘F39’, ‘141’ and ‘TMS’. Murashige &; Skoog (MS) basal medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.26 mg l^?1 abscisic acid (ABA) and 0.8 mg l^?1 indole-3-acetic acid (IAA) was used for shoot regeneration from cotyledonary explants in ‘F39’ and ‘150’. Kanamycin sensitivity as well as Timentin^? and Clavamox^® were evaluated using wild-type ‘F39’ and ‘150’ cotyledons. Kanamycin concentrations of 200 and 150 mg l^?1 were chosen as the threshold levels for ‘F39’ and ‘150’, respectively. No significant differences were found between Timentin^? and Clavamox^® in ‘F39’; however, Clavamox^® reduced the incidence of vitrification and increased the frequency of shoot elongation in ‘150’. A. tumefaciens strain EHA105, harboring pCNL56 carrying neomycin phosphotransferase II (nptII) and gusA reporter genes, was selected to establish a transformation protocol for ‘F39’ and ‘150’. Putative transformants were evaluated using β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) and Southern blot analyses. Based on these parameters, the transformation efficiency for cantaloupe ‘F39’ was 0.3% and that for honeydew ‘150’ was 0.5%.     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

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.     

Establishment of efficient and rapid regeneration system for some diploid wild species of <Emphasis Type="Italic">Arachis</Emphasis>

Though peanut tissue culture has advanced to a considerable extent using a number of explants with the subsequent production of transgenic plants, wild Arachis species appeared to be recalcitrant to using similar explants. In this study, the use of cotyledonary nodes as explants prepared from 7-day old seedlings resulted in the development of a simple and rapid regeneration protocol for five diploid wild species including A. diogoi, A. stenosperma, A. duranensis, A. cardenasii and A. correntina belonging to the genus Arachis for producing multiple shoots. Shoot bud initiation was observed 10 to 15 days after culture initiation. Responding cotyledonary nodes with shoot buds were subcultured to lower levels of cytokinin and finally to MS basal medium for further shoot development and elongation. Production of multiple shoots was observed in all the five diploid species with a maximum of 9 to 16 shoots were obtained per explant in the primary cultures. The number of shoot buds increased significantly with repeated explant subculturing with recovery up to 45 shoots from responding explants. These shoots were rooted efficiently on MS medium supplemented with 1 mg l⁻¹ naphthalene acetic acid and the time taken from explanting to the transfer of shoots to potting mixture was about 12 weeks. All rooted shoots were successfully established in soil in glass house and further transferred to field. These plants survived to maturity and set seed.     

Enhancement of Naphthaleneacetic Acid-Induced Rhizogenesis in T(L)-DNA-Transformed Brassica napus without Significant Modification of Auxin Levels and Auxin Sensitivity

Determination of the abscisic acid and indoleacetic acid (IAA) contents of floral stem segments of nontransformed and pRi A₄ T_L-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor) using a high performance liquid chromatography-enzyme-linked immunosorbent assay procedure and mass spectrometry controls showed that IAA levels were not modified. The regeneration abilities of the in vitro cultured explants were compared on media supplemented with several plant growth regulator combinations. No regeneration occurred on hormone-free media, and shoot production was similar in both genotypes when supplemented with benzyladenine. In the presence of naphthaleneacetic acid (NAA), transformed explants were characterized by faster root regeneration and reduced shoot organogenesis. The optimum for root formation was the same in nontransformed and transformed plants, but the sensitivity threshold was slightly lower in the latter. The NAA inductive period was shorter (14 versus 22 h) with transformed tissue. Root neoformation occurred about 72 h earlier on transformed explants. Our results suggest mainly that there is an acceleration of the auxinic signal transduction and/or that the events preliminary to the formation of roots occur faster in the transformed tissues than in the normal ones.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

A novel pathway for rapid shoot regeneration from the proximal zone of the inpocotyl of melon (Cucumis melo L.)

Summary Hypocotyl explants of melon (Cucumis melo L.) are capable of regenerating multiple shoots on Murashige and Skoog (1962) medium, augmented with 4.4 μM benzylademne. Regeneration from the hypocotyl is much more rapid than the more commonly reported regeneration from cotyledonary explants, producing shoots within 2 wk compared to more than a month required for cotyledon explants. The rapid regeneration response depends on the presence of a fragment of the cotyledon remaining attached to the hypocotyl. Controls were performed to ensure that the regeneration was not due to the presence of the shoot apical bud of the melon seedling after explant production. Scanning electron microscopy revealed that microsurgery to remove the apical bud left no excess bud material. Regeneration from the proximal part of the hypocotyl was due to production of a new shoot apical meristem, observed by histology. The apical meristem can be produced before leaf primordia in regeneration from the hypocotyl, in contrast to the regeneration process from the melon cotyledon.     

Production of transgenic diploid <Emphasis Type="Italic">Cucumis melo</Emphasis> plants

Melon (Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

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.     

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05–2.5 μM) was used as a supplement to the Murashige and Skoog’s (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.