ABA content and sensitivity during the development of dormancy in lily bulblets regenerated in vitro

We measured ABA content and sensitivity in bulblels of Lilium speciosum Thunb , regenerating from scale explants in vitro at temperatures (15, 20 or 25°C) that allowed the development of various levels of dormancy (very low, intermediate or high, respectively). The one-step purification and the accuracy of the immunoassay were confirmed by HPLC and by liquid chromatography/mass spectrometry. ABA content was not correlated with dormancy development. Sensitivity to ABA was determined as the difference in sprouting performance of excised bulblets on medium with and without ABA. In bulblets regenerating at 20 or 25°C. ABA sensitivity was high during the period of dormancy establishment and decreased thereafter. Dormant hulblets were almost completely insensitive to ABA. The changes in sensitivity to ABA were confirmed by measuring the level of ABA in bulblets at the time of sprouting. This level was, as expected, highest in bulhlels with low ABA-sensitivity. Briefly cold-treated bulblets, in which dormancy may he re-established by culture at 20°C, again became sensitive to ABA. ABA sensitivity decreased with increasing temperature bulblets that regenerated at I5°C and hardly developed any dormancy, were very sensitive to ABA. It was concluded that in addition to ABA sensitivity another, still unknown, factor played a key role in dormancy development.     

The development of dormancy in bulblets of Lilium speciosum generated in vitro. II. The effect of temperature

Upon harvest, lily ( Lilium speciosum Thunb. cv. Rubrum) bulblets generated in vitro under standard conditions (11 weeks at 20°C) were dormant and needed a cold treatment prior to planting. During culture in vitro at 20°C, the bulblets proceeded through three phases: (1) at first they were non–viable and non-dormant (up to 5 weeks), (2) then viable and non-dormant (5–9 weeks) and (3) finally viable and dormant (from 9 weeks onwards). At 15°C, the bulblets became viable but did not develop dormancy, even after protracted culture. The results suggest that the development of dormancy depends upon an accumulation of'heat units'occurring at temperatures higher than 15°0. At 25°C, the succession of the three phases occurred more rapidly than at 20°C and heat units were accumulated more rapidly. During the third period, the chilling requirement increased showing that heat units continued to be accumulated during this period.
Dormancy connotes an arrest of growth. In lily bulblets, however, the number of scales continued to increase after the induction of dormancy at 20 or 25°C. Many of the scales initiated before the onset of dormancy were formed by swelling of a petiole, whereas, after the onset of dormancy, all scales were formed directly from a primordium. We conclude that the development of dormancy corresponds to a switch in the development of the primordium. Thus, after the induction of dormancy the primordium lost the ability to become a leaf and always developed into a scale.     

低温对东方百合试管鳞茎解除休眠及生长的影响

以东方百合‘索邦’和‘西伯利亚’鳞片为外植体得到的一代试管小鳞茎为试材,研究0℃冷藏不同时间对试管鳞茎生理指标变化,以及试管鳞茎解除休眠和移栽后生长发育的影响。结果表明,冷藏0--28dN,随着冷藏时间的延长,试管鳞茎出苗率逐渐增加,冷藏处理28d达到最高,之后逐渐降低。冷藏期间,试管鳞茎中淀粉含量持续降低,而可溶性总糖和还原性糖含量表现出先升高后下降的趋势,冷藏处理28d的含量最高。同时,随着冷藏时间的延长,IAA和ZR含量逐渐升高,ABA含量逐渐下降,而GA。含量表现出先上升后下降的趋势,并且也在冷藏处理28dN-含量达到峰值。另外,采用隶属函数值对低温处理的试管苗栽培1年后所收获种球的数量、质量等指标进行评价,结果显示,均以低温处理28d时达到最大值。由此得出,0℃低温处理试管小鳞茎28d为解除休眠及促进生长发育的最适处理时间。     

Disrupting Abscisic Acid Homeostasis in Western White Pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) Seeds Induces Dormancy Termination and Changes in Abscisic Acid Catabolites

To investigate the role of abscisic acid (ABA) biosynthesis and catabolism in dormant imbibed seeds of western white pine (Pinus monticola), ABA and selected catabolites were measured during a combined treatment of the ABA biosynthesis inhibitor fluridone, and gibberellic acid (GA). Fluridone in combination with GA effectively disrupted ABA homeostasis and replaced the approximately 90-day moist chilling period normally required to break dormancy in this species. Individually, both fluridone and GA treatments decreased ABA levels in the embryos and megagametophytes of white pine seeds compared to a water control; however, combined fluridone/GA treatment, the only treatment to terminate dormancy effectively, led to the greatest decline in ABA content. Fluridone treatments revealed that a high degree of ABA turnover/transport occurred in western white pine seeds during the initial stages of dormancy maintenance; at this time, ABA levels decreased by approximately two-thirds in both embryo and megagametophyte tissues. Gibberellic acid treatments, both alone and in combination with fluridone, suggested that GA acted transiently to disrupt ABA homeostasis by shifting the ratio between biosynthesis and catabolism to favor ABA catabolism or transport. Increases in phaseic acid (PA) and dihydrophaseic acid (DPA) were observed during fluridone/GA treatments; however, increases in ABA metabolites did not account for the reduction in ABA observed; additional catabolism and/or transport of ABA and selected metabolites in all probability accounts for this discrepancy. Finally, levels of 7′ hydroxy-ABA (7′OH-ABA) were higher in dormant-imbibed seeds, suggesting that metabolism through this pathway is increased in seeds that maintain higher levels of ABA, perhaps as a means to further regulate ABA homeostasis.     

In vitro bulblet production of Lachenalia

In vitro bulblet formation and subsequent transplanting of bulblets to soil were studied in order to develop a cost-effective method for the mass production of three Lachenalia varieties. Clumps of adventitious shoots regenerated from leaf explants were used. Bulblet formation was initiated after 2 weeks when shoots were subjected to low temperature (4–15 °C). The size (age) of the adventitious shoot affected the bulblet size, and shoots shorter than 4 mm did not form bulblets. Larger bulblets formed on medium containing 6% sucrose compared to 3% sucrose. Following bulblet initiation, illumination was not necessary for the completion of bulblet formation. Bulblets went into dormancy 3–4 months after they had been initiated or when the culture medium dried out, and they were released from dormancy when the natural night temperatures started to decrease in the late summer. The survival rate of the bulblets after transplanting was directly correlated to the size of the bulblets.The most important factors influencing in vitro bulblet formation of Lachenalia were sucrose concentration, temperature and length of explant shoots. Received: 12 June 1998 / Revision received: 8 September 1998 / Accepted: 23 September 1998     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and bulblet growth from lily bulbscale explants as affected by retardants,sucrose and irradiance

Bulbscales of oriental lily hybrid Star Gazer were used as the explants. Bulblets were formed on the basal portion of the excised bulbscales on MS medium supplemented with growth retardants, different sucrose concentrations and exposed to continuous light or dark. ^{Alar, Cycocel} and Paclobutrazol in concentration 1 mg dm⁻³ produced higher number of bulblets as compared to the control. The number of bulblets, however, decreased with the increase in concentration of the growth retardants. The number of bulblets was higher at 90 than at 60 g dm⁻³ sucrose and when the bulbscales were exposed to continuous light than to darkness. The growth retardants, higher sucrose concentration and continuous dark stimulated fresh mass of bulblets. The number of bulblets having roots and leaves decreased in medium with Alar, Cycocel and Paclobutrazol as compared to the control. A few bulblets produced roots and leaves in medium with 90 g dm⁻³ sucrose and none of the regenerated bulblets produced leaves under continuous dark.     

Role of Endogenous Abscisic Acid in Potato Microtuber Dormancy

Potato (Solanum tuberosum L. cv Russet Burbank) microtubers generated in vitro from single-node explants contained substantial amounts (approximately 250 pmol/g fresh weight) of free abscisic acid (ABA) and were completely dormant for a minimum of 12 weeks. Microtubers that developed in the presence of 10 [mu]M fluridone (FLD) contained considerably reduced amounts (approximately 5-25 pmol/g fresh weight) of free ABA and exhibited a precocious loss of dormancy. Inclusion of exogenous racemic ABA in the FLD-containing medium suppressed the premature sprouting of these microtubers in a dose-dependent manner. At a concentration of 50 [mu]M, exogenous ABA restored internal ABA levels to control values and completely inhibited FLD-induced precocious sprouting. Exogenous jasmonic acid was ineffective in suppressing FLD-induced sprouting. Application of FLD to preformed, fully dormant microtubers also resulted in a reduction in internal ABA content and precocious sprouting. These results indicate that endogenous ABA is essential for the induction and maintenance of potato microtuber dormancy.     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

Effect of Methyl Jasmonate on Morphology and Dormancy Development in Lily Bulblets Regenerated In Vitro

Scales of lily bulbs are swollen petioles. Lily scale fragments cultured in vitro regenerate bulblets consisting of scales that may or may not carry a leaf blade. The bulblets are dormant and require a cold treatment to sprout. We added the gaseous plant growth regulator methyl jasmonic acid (MeJA) in the headspace of the tissue-culture container and studied the effect on plantlet morphology (scale/leaf-blade formation) and dormancy development in three lilies, Lilium speciosum “Rubrum No. 10,” L. longiflorum “Snow Queen,” and the Asiatic hybrid “Connecticut King.” Methyl jasmonic acid strongly reduced leaf-blade formation in Lilium longiflorum and Connecticut King. This was a specific effect as scale formation was affected much less. The specific inhibition of leaf-blade formation was not observed in Lilium speciosum. In this lily, high concentrations of methyl jasmonic acid (MeJA) inhibited leaf-blade and scale formation to similar extents. Methyl jasmonic acid reduced dormancy development in all three lilies, with the largest effect observed in Connecticut King. In this Asiatic hybrid, almost all bulblets that had regenerated at 300 or 1000 μl l⁻¹ MeJA in the headspace, did not require a dormancy-breaking treatment to achieve sprouting after planting in soil. Previously, it has been found in lily that treatments that reduce leaf-blade formation promote dormancy development. The present findings with MeJA do not agree with this. In the three lilies, the various parameters that were studied—regeneration, scale weight, leaf-blade weight, and dormancy development—were very differently affected by MeJA.     

Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum

Production of bulblets of Muscari armeniacum through tissue culture is enhanced when 1 g/l activated charcoal is added to a modified Murashige and Skoog (MS) medium. Bulblet regeneration is direct from bulb scale explants with no intermediate callus growth. Bulblets can be transferred successfully to a greenhouse environment directly from aseptic culture.     

Phase change in lily bulblets regenerated in vitro

During the development of the lily ( Lilium ), three phases can be distinguished: the juvenile, the vegetative adult and the flowering phase. Juvenile bulblets sprout with one or a few leaves whereas vegetative adult bulblets sprout with a stem with elongated internodes. The transition to the vegetative adult phase was studied in lily ( Lilium  × cv. Star Gazer) bulblets regenerating on bulb scale segments in vitro. The phase change was marked by the development of a tunica-corpus structure in the apical meristem which leads to the formation of an actively growing stem primordium. This structure is absent in juvenile bulblets. Juvenile bulblets first developed competence for phase change during a culture period of at least 6 weeks at 25°C. Subsequent induction of the phase change occurred during a period of 2 weeks at lower temperature (15°C). A major factor influencing phase transition was bulblet weight. Small bulblets never formed a stem whereas large bulblets always formed a stem under inducing conditions. Large bulblets more often formed a stem than small ones but the relation between bulb growth and phase transition was not absolute. A high sucrose concentration, a large explant and a prolonged period for competence development stimulated bulb growth but also phase transition independently of growth. Lowering the concentration of MS-minerals reduced bulb growth but did not affect phase transition. Under these conditions, phase change was correlated with a low phosphorus content.     

Control of seed dormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesis imposes dormancy maintenance

The physiological characteristics of seed dormancy in Nicotiana plumbaginifolia Viv. are described. The level of seed dormancy is defined by the delay in seed germination (i.e the time required prior to germination) under favourable environmental conditions. A wild-type line shows a clear primary dormancy, which is suppressed by afterripening, whereas an abscisic acid (ABA)-deficient mutant shows a non-dormant phenotype. We have investigated the role of ABA and gibberellic acid (GA₃) in the control of dormancy maintenance or breakage during imbibition in suitable conditions. It was found that fluridone, a carotenoid biosynthesis inhibitor, is almost as efficient as GA₃ in breaking dormancy. Dry dormant seeds contained more ABA than dry afterripened seeds and, during early imbibition, there was an accumulation of ABA in dormant seeds, but not in afterripened seeds. In addition, fluridone and exogenous GA₃ inhibited the accumulation of ABA in imbibed dormant seeds. This reveals an important role for ABA synthesis in dormancy maintenance in imbibed seeds. Received: 31 December 1998 / Accepted: 9 July 1999     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Involvement of Endogenous Abscisic Acid in Onset and Release of Helianthus annuus Embryo Dormancy

Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed.     

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l^–1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l^–1 ancymidol (A-Rest^TM), 0.1 mg l^–1 NAA and 0.1 mg l^–1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.     

Contribution of explant carbohydrate reserves and sucrose in the medium to bulb growth of lily regenerated on scale segments in vitro

Bulb size is an important factor determining phase change in Lilium : phase change only occurs in bulblets over a certain threshold weight. After phase change has occurred, bulblets sprout with a stem with many leaves. Juvenile bulblets sprout with only a few leaves. The factors contributing to bulb size were studied during in vitro regeneration of bulblets on scale segments. The larger the explants, the larger the regenerated bulblets. Explant size influenced bulb growth during the complete culture period. Bulb growth was stimulated by a high sucrose concentration. The contribution of the medium and the explant reserves to bulb growth were studied in large and small explants using labelled sucrose. Sucrose was mainly taken up through the cut surfaces. In freshly cut explants, the rate of uptake was correlated with the size of the contact area, but at later stages, when regenerating organs were present, the difference in uptake rate of small and large explants almost disappeared. Small explants had a larger sink activity than large ones. Explants with regenerating organs took up more sucrose than freshly cut explants. Sucrose uptake and bulb growth were rather constant in the later phases and doubled when the sucrose concentration was doubled. Partitioning of label from the sucrose over the various organs, was also rather constant in time: approximately 25% was accumulated in the bulblets, 35–45% in the explant and 30–35% was converted to CO₂. About half of the label in the explant was recovered at the proximal side where regeneration takes place. Sucrose, once incorporated in the storage pools in the explant, either remained in the explant or was converted to CO₂. Redistribution to the growing bulblets hardly occurred. The percentage of bulb growth that could be attributed to uptake of medium components was constant over the regeneration period: 45–50% for large and 65–75% for small explants.     

Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorum Thunb.)

Bulb scale propagation makes it difficult to obtain a large number of bulblets from disease-free stocks in a short time. The establishment of improved micropropagation procedures by in vitro culture is therefore desirable. Easter lily (Lilium longiflorum Thunb.) filaments with and without anther were excised and cultured in vitro with different media and culture conditions. In cultures of filaments with anther, callus developed and led to bulb, shoot, and root formation, whereas in cultures of filaments lacking anther, callus development did not occur. Among the various media tested, the B5 medium combined with darkness and the N6 medium combined with darkness or light, both supplemented with 9% sucrose, proved to be superior. A total of 1260 plants were regenerated from callus, acclimatized under a mist, and transferred to the greenhouse with a 100% success rate. No morphological abnormalities were observed among plants regenerated from filament-derived callus and all plants displayed isozyme banding patterns identical to the original cultivar. Chromosome observations revealed that all callus-regenerated plantlets tested were diploid (2n=24). The results suggest that in vitro culture of filaments with anther can be cultured for mass propagation. Received: 5 February 1997 / Revision received: 12 May 1997 / Accepted: 2 June 1997     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.