Shoot Apex Culture Research Articles

A novel technique for Musa acuminata Colla ‘Grand Naine’ (AAA) micropropagation through transverse sectioning of the shoot apex

A new approach is described for in vitro shoot proliferation using transverse sections of individual shoot apices as explants, and its efficiency was compared to conventional shoot apex culture of banana ‘Grand Naine’. Individual shoot apex explants were inoculated on Murashige and Skoog (MS) media supplemented with 0, 10, 20, and 30 μM of 6-benzylaminopurine (BAP) in combination with 1.0 μM α-naphthaleneacetic acid (NAA) and maintained for 6 mo with monthly subculture. Shoot proliferation increased at each subculture to a maximum of 23.65 shoots on 20 μM BAP-supplemented medium. To increase the proliferation rate, individual shoot apices cultured on MS media containing 10 μM BAP were split into three transverse sections, and the middle section produced the highest number of shoots (7.22). These shoots, when cultured on MS medium supplemented with 20 μM BAP, produced 8.26 shoots per explant vs. 6.18 and 7.94, respectively, for media supplemented with BAP at 10 or 30 μM. From just one transverse section, 32.77 shoots could be produced after 6 mo vs. 11.79 shoots via direct regeneration from individual shoot apex explants. MS medium supplemented with 6 μM IBA induced vigorous rooting within 4 wk. Acclimatization was achieved under greenhouse conditions, and the highest survival rate was 88.63%. Randomly amplified polymorphic DNA (RAPD) analysis showed that plants from shoot apex cultures had 23.46% polymorphism, while those from transverse sections showed no genetic variation when compared to the parent plants.

In vitro culture of shoot apices from juvenile and adult yellow passion fruit plants

The aim of the present study was to evaluate in vitro plant regeneration from the shoot apices of juvenile and adult passion fruit plants and provide a basis for future clonal cleanup of superior genotypes through the use of tissue culture. The studies were divided into three experiments to evaluate the effect of age and origin of the donor plant, the culture medium methodology and the size of the explants. The shoot apices were inoculated in Murashige & Skoog (MS) medium with 3% sucrose and three 6-benzylaminopurine (BAP) concentrations: 0, 1 or 2 mg L-1. Next, the samples were transferred to a half-strength mineral salt medium (½ MS) and supplemented with 1 mg L-1 indolebutyric acid (IBA). Two cultivation methods were used: one with variations in the cultivation period in the MS medium (26 or 60 days) and the other with or without transfer to half-strength mineral salt medium (½ MS). The methodology by which explants are maintained in MS medium supplemented with 1 mg L-1 BAP for 26 days, followed by transfer to ½ MS medium supplemented with 1 mg L-1 IBA for 21 days of cultivation and then transfer to ½ MS medium for 14 days is more appropriate for regenerating plants from the shoot apices of adult Passiflora edulis (AGBP accession 38).

In vitro propagation of Gentiana scabra Bunge - an important medicinal plant in the Chinese system of medicines.

BackgroundGentiana scabra Bunge commonly known as ‘Long dan cao’ in China has been used in traditional Chinese medicines for more than 2000 years. Dry roots and rhizome of the herb have been used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are the main bioactive compounds which attribute to the pharmacological properties of this plant. The species is difficult to mass propagate by seed due to the low percentage of germination and limited dormancy period. Wild populations in some locations are considered to be in the endangered category due to over exploitation.ResultsIn the present study, we report an efficient micropropagation system. Shoot apices of six weeks old in vitro grown G. scabra plants were used as explants for the in vitro propagation. Induction of multiple shoots (9.1/explant) was achieved on the culture of shoot apices on half strength Murashige and Skoog’s basal medium (MSBM) containing 2.0 mg/L−1 6-benzylaminopurine (BA), 3% sucrose and 0.9% Difco agar. In vitro shoots induced profuse rooting on half strength of MSBM supplemented with 0.1 mg/L−1 1-naphthaleneacetic acid (NAA), 3% sucrose and 0.3% gelrite. A two-stage ventilation closure procedure during the in vitro culture, and transparent sachet technique enhanced the survival rate of G. scabra plantlets to 96% in the greenhouse. Tissue culture plants flowered after 5 months of transfer to pots.ConclusionsA simple and an efficient in vitro propagation protocol of Gentiana scabra Bunge by optimizing the medium composition and ventilation closure treatments has been developed. The protocol can be very useful in germplasm conservation and commercial cultivation of G. scabra plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0056-4) contains supplementary material, which is available to authorized users.

Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas)

Aims: Sweet potato (Ipomoea batatas) cv. “Miyazakibeni” was used as material for shoot apex culture, reverse transcription-polymerase chain reaction (RT-PCR) and clonal propagation to establish an efficient and practical virus-free seedling supply system in production of vegetatively reproductive plants. Study Design: At first, efficient plant regeneration was achieved from shoot apex culture of sweet potato. Secondly, RT-PCR method was used to detect the sweet potato feathery Original Research Article British Biotechnology Journal, 4(1): 51-63, 2014 52 mottle virus (SPFMV) viral infection of tuber surface of edible sweet potato using the RNAs from the plants obtained from shoot apex culture. Finally, the virus-free plants verified by RT-PCR were propagated clonally by culture of suckers cut from stems of the virus-free plants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2008 and December 2012. Methodology: The best efficiency for material sterilization was tested using different concentrations (0.1% 1.5%) of sodium hypochlorite solution (SHS) and the treated times (5 min – 20 min). Theshoot apexes less than 0.3mm in size were cultured on Komamine and Nomura (1998) (KN) medium and Murashige and Skoog (1962) (MS) medium. The regenerated plants were used for RNA extraction and then, used for RT-PCR for detection of SPFMV. Based on the result of RT-PCR, the suckers cut from stems of virus-free plants were cultured and propagated clonally and routinely within a short period. Results: The combination of 0.3% of SHS and 10 and/or 20 min gave the best result (100%) of surviving rate for material sterilization. The culture of shoot apexes less than 0.3 mm in size gave plant regenerating rates of 82% and 65% on KN and MS medium, respectively. The results of RT-PCR of RNAs from plants obtained from shoot apex culture and plants of SPFMV infection showed that SPFMV virus was clearly removed by shoot apex culture conducted in this study. For clonal propagation, 80-100% of suckers cut from the stems of the virus-free plants detected grew into complete plants after 6 weeks of culture, indicating that the virus-free plants could be routinely propagated 5 times in number each time and repeatable by the short circle. The sweet potato produced in field showed no symptom called as russet crack-like symptom (RC-LS) even after cultivation two seasons. Conclusion: Overall, an efficient and practical virus-free seedling supply system was established in sweet potato by the three steps of 1) virus-free plant regeneration from shoot apex culture, 2) quick detection of SPFMV using RNA of the regenerated plants by RT-PCR, and 3) the verified virus-free plants were propagated clonally and routinely within a short period using culture of suckers cut from the stems of virus-free plants.

Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

Optimisation ofin vitroregeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants - ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined effects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l−1) dramatically decreased necrotic response of explants. However, citric acid, used together with ascorbic acid completely inhibited shoot growth in shoot apex cultures. Glutathion evoked hyperhydricity of explants. Activated charcoal induced rooting on media which are commonly used for shoot proliferation. Combination of acetosyringone with antioxidants influenced shoot proliferation, except of variant with ascorbic acid. Citric acid was the best and universal antioxidant in faba beanin vitrocultures and its use is recommended for faba bean genetic transformation experiments.

SHOOT APEX CULTURE OF DORMANT BUDS IN CHESTNUT

Chestnuts (Castanea spp.) can be propagated by tissue culture methods such as meristem tip culture, shoot tip culture and bud culture. However, some limiting factors prevent using these methods extensively. For example, the explant collection periods of these methods are short, and the collection time is critical. In addition, shoot formation ratios of explants on the initial media and survival of these shootlets thereafter are generally low due to factors such as infection, browning, and vitrification. In this study the shoot apex of the dormant buds were used as the explants source for the first time. By use of this method the explant collection period was much longer and the mass multiplication probability increased. Three chestnut cultivars native to Turkey - 'Sanaslama', 'Osmanoglu' and 'Haciomer' Castanea sativa Mill. - were used in the study in two years of experiments 2007 and 2008. Two main media were used as initial to obtain shootlets: MS (1/2×NH 4 NO 3 and KNO 3 ) and DKW (Driver-Kuniyuki walnut medium). In multiplication step MS (1/2×NH 4 NO 3 medium was used with 0.5, 1.0, 1.5 mg/L BAP concentrations and in rooting MS (1/2×NH 4 NO 3 with 1.0 and 2.0 mg/L IBA concentrations were used. Shootlet formation from the explants in 2008 was higher (66.66-95.45%) than in 2007 (42.00-75.00%). MS (1/2×NH 4 NO 3 and KN0 3 ) medium has given successful results in this respect. Therefore it can be recommended as a growing medium. The multiplication coefficient varied between 3.12 to 3.57 in 2007 and 3.95 to 4.76 in 2008 according to the cultivars. Rooting of the shootlets was difficult, and no rooted shootlet was obtained. However the experiment is now being continued, and new rooting media will be tested. The results obtained from this study showed that this method can be efficiently used in the mass multiplication of chestnuts.

Efficient in Vitro Regeneration and Micropropagation of Medicinal Plant Momordica tuberosa Roxb

Attempts have been made to establish protocol for in vitro propagation of Momordica tuberosa (Cogn) Roxb. using nodal segments and shoot apices obtained from field-grown mature plants. In vitro regeneration was achieved from nodal explants on Murashige and Skoog's (MS) medium supplemented with 6-benzyladenine at 2.22, 4.40, 6.62, and 8.90 μM and kinetin at 2.32, 4.60, 6.92, and 9.30 μM either alone or in combination (BA + Kn). Within the ranges evaluated, the regeneration medium containing 4.40 μM BA combined with 4.60 μM Kn showed highest regeneration efficiency, with 9.0 ± 0.49 shoots per explant. Such in vitro regenerated shoots attained a maximum length of 10.0 ± 0.47 cm. The regeneration medium containing BA + Kn was used to subculture regenerated shoots at 4-week intervals. BA at 13.30 μM induced regeneration from shoot apex cultures, and 75% of explants showed regeneration efficiency with 7.8 ± 0.66 cm as the mean length of shoot. Such shoots could be subcultured on medium containing BA. Microshoots were rooted onto MS medium supplemented with 4.90 μM indole-3-butyric acid. Regenerated plants were established in the greenhouse with 90% survival rate. The protocol is simple, rapid, and efficient for in vitro propagation of nodal explants and shoot apices of M. tuberosa.

Effects of Type of Explant and Dark Preconditioning on Bud Formation in Habenaria radiata (Thunb.) in Vitro

Habenaria radiata is a terrestrial orchid with beautiful bird-shaped petals. The wild H. radiata population has been severely affected by environmental disruption and overexploitation. In micropropagation of H. radiata, although aseptic germination has been studied, tissue culture methods have not yet been established. Shoot apexes and leaf explants from vegetative plants and flower stalks, stolons, and floret explants from reproductive plants were chosen for this study. Explants were cultured on half-strength inorganic salts and full-strength vitamins of Murashige and Skoog (1/2 MS) medium containing 30 g·L−1 sucrose, 8 g·L−1 agar (pH 5.6) supplemented with 4.44 μM N6-benzyladenine, and 0.54 μM α-naphthaleneacetic acid. After 8 weeks of culture, the highest survival rate was obtained with floret explants excised from plants at the reproductive phase. In floret culture, the number of adventitious bud formation per explant was 5.4 per upper floret and 4.0 per lower floret. Dark preconditioning, which inhibited browning and contamination, of explants before shoot apex culture increased survival rates of explants (53%) and bud formation (83%). Consequently, a tissue culture method using florets and shoot apexes as explant material was established for H. radiata.

Screening of topical sterilants for shoot apex culture of Acacia mearnsii

The surface sterilisation procedure is of integral importance to any micropropagation technique. This process should do the least amount of plant damage, whilst reducing microbial contamination to an acceptable level. The objective of this research was to investigate alternative sterilisation agents to the dangerous chemical mercuric chloride (HgCl2), and to determine the effect of sterilant exposure time on shoot apex culture of Acacia mearnsii. Explants were cultured on MS medium supplemented with 2.0 mg l–1 BA and monitored for 21 d for signs of contamination and shooting. Household bleach (NaOCl), diluted 1:3 in water, resulted in decontaminated explant levels not significantly different to the 0.2% HgCl2 treatment and resulted in shoot development levels significantly higher than the 0.2% HgCl2 treatment. There was no significant effect of sterilant exposure time on decontaminated explant levels, whilst the shortest exposure time resulted in significantly higher shooting levels than the other two time periods tested.

Evaluation of different approaches to buckwheat (Fagopyrum esculentum Moench.) micropropagation

Plant regeneration by different techniques was evaluated in three buckwheat cultivars: indirect regeneration from cotyledons and hypocotyls, direct regeneration by nodal segment cultivation and induction of multiple shoots from seedling apices. Regenerated shoots were obtained in all procedures. The effects of BA (6-benzylaminopurine), media composition and gelling agent were tested. The regeneration efficiency of shoot apex culture was 2.65–3.33 nodal segments/explant. Cotyledon and hypocotyl segments produced 1.25–2.44 shoots per explant plated. Nodal segment cultivation yielded 4.1–4.8 new nodal segments/explant in 4 weeks. Eighty percent of shoots rooted on the basal medium. Rooting was improved (up to 95.6%) by IBA (3-indolebutyric acid) addition to the culture medium. Regenerated plantlets were transferred to the soil. The most efficient and simple micropropagation of buckwheat was nodal segment cultivation on MS medium solidified by agar with the addition of 1 mg/l BA. This method is advisable for rapid multiplication, in vitro conservation or rescue of genetic resources.

Evaluation of orange-fleshed sweet potato (Ipomoea batatas L.) genotypes for salt tolerance through shoot apex culture under in vitro NaCl mediated salinity stress conditions

Fifteen genotypes of sweet potato were evaluated for salinity stress tolerance under in vitro NaCl mediated salinity stress conditions (MS, MS + 0.5% and MS + 1.0% NaCl). The growth parameters such as number of leaves, number of shoots, number of roots, length of plantlets and length of roots decreased significantly among the genotypes with increase in level of salinity. Of the 15 genotypes tested, six genotypes (108X1, 90/606, 90/696, CIP 8, S-30X15 and SP-61) were unable to sprout even at 0.5% NaCl and were characterized as susceptible to salt stress, three genotypes (CIP 6, 90/774 and CIP 3) which could tolerate 0.5% NaCl as moderately tolerant and six genotypes (CIP 12, CIP 13, JO 14, JP 13, SB-198/115 and Gouri) as tolerant to salinity at 1.0% NaCl. Amongst the six genotypes showing tolerance to 1.0% NaCl, the exotic genotypes––JP 13, CIP 12 and indigenous one SB-198/115 continued to exhibit significant higher values for growth parameters over the susceptible one. Based on the performance under NaCl mediated salinity stress (1.0%), the pattern of salinity tolerance in the genotypes through shoot apex culture was JP 13 > SB-198/115 > JO 14 > Gouri > CIP 12 > CIP 13. The effect of salt stress on the activity of antioxidative enzymes was studied in leaves of 8-week-old plantlets of those six genotypes, which responded at higher NaCl stress along with a susceptible genotype 90/606. In leaves of salt stressed plants, superoxide dismutase (SOD), guaiacol peroxidase (GPX) and catalase (CAT) activities increased when compared with the stress free control. The increase was more pronounced in the tolerant genotypes than that in the susceptible one. These results indicate that oxidative stress may play an important role in salt stressed sweet potato plants and that the greater protection of tolerant plants from salt induced oxidative damage results, at least in part, through the increase in the activity of antioxidant enzymes.

Rejuvenation by Shoot Apex Culture Recapitulates the Developmental Increase of Methylation at the Maize Gene Pl-Blotched

Cytosine methylation provides an attractive epigenetic modification for the global maintenance of phases in plant development; however, there are few known examples of specific genes whose methylation status changes in a developmentally regulated manner. Pl-Blotched, an allele of purple plant1 (pl1), which encodes a myb-like transcription factor that regulates anthocyanin production in maize, is one such gene: certain cytosines at the 3' end of this allele are hypomethylated in seedlings, become hypermethylated in organs formed in the adult phase, and are hypomethylated again in the next generation. We tested whether this developmental pattern of low juvenile cytosine methylation followed by higher methylation in adult tissues could also be observed in plants "rejuvenated" via shoot apex culture. We found that cytosine methylation patterns at Pl-Blotched were indeed recapitulated in culture-rejuvenated plants, showing hypomethylation in leaves with juvenile patterns of differentiation (even though they were made by an old meristem) followed by hypermethylation in later-formed leaves. Our results show that methylation status at that locus is determined by the developmental phase of the shoot, rather than by the age of the meristem forming it. These results support the hypothesis that DNA methylation is employed by the plant to maintain an epigenetic state.

Gentian Tumorous Symptoms are Transmitted by Grafting and are not Observed by Shoot Apex Culture

Gentian Tumorous Symptoms are Transmitted by Grafting and are not Observed by Shoot Apex Culture

Effects of Sampling Season of Explants and Growth Stage of Mother Plants on the Regenerative Capacity in Cultured Shoot Apices of Chrysanthemum (Dendranthema*grandiflorum (Ramat.) Kitam.)

The effects of the sampling season of explants and the growth stage of mother plants on the regenerative capacity and growth characteristics of regenerants in the shoot apex culture of chrysanthemums were investigated. Shoot apices from two edible cultivars of the autumn-flowering type, 'Abokyu' and 'Enmeiraku', were cultured for three months on a modified Shizuoka Agricultural Experiment Station medium supplemented with NAA (1 mg·liter-1). Every two or three weeks from April to September 1991, shoot apices were collected from the terminal and axillary buds of non-pinching plants and cultured. The regeneration rate varied with the cultivars used and the sampling season. When the terminal buds of primary lateral shoots that developed after pinching were cultured, high percentages of regenerants were obtained from vigorously growing mother plants in both cultivars. Even in the early reproductive growth phase (involucre- formation stage), the explants maintained some regenerative capacity. The growth characteristics of regenerants differed with the cultivars used and the growth stages of the mother plants.

茎頂培養によるナシ粗皮病の枝病徴の消失

茎頂培養によるナシ粗皮病の枝病徴の消失

Possible consequences of fungal contamination on the RAPD fingerprinting in Miscanthus (Poaceae)

Fungal contamination has been frequently reported in higher plants. In Miscanthus species, a wide range of fungal flora has also been recorded previously, including an investigation based on nrITS amplification. In order to understand the effects of the fungal genomes on the random amplified polymorphic DNA (RAPD) fingerprinting, callus specimens were obtained from the tissue culture of shoot apices of Miscanthus. RAPD fingerprinting with 60 oligoprimers was conducted with genomic DNA extracted from leaf tissue collected in the field and from the greenhouse, as well as callus derived from the same individuals. Extra bands were detected in the RAPD fingerprints amplified with 44 primers (84.6%) from the genomic DNA of both the field and greenhouse leaf tissue of most Miscanthus taxa examined, except for M. sinensis var. condensatus. Positive PCR amplification of organelle DNA non-coding spacers with both leaf and callus DNA ruled out the possibility that such DNA fingerprinting discrepancies were due to loss of organelles in the callus after consecutive subcultures. Among the 44 primers, one yielded no amplified fragments from the callus DNA, indicating that the amplified DNA fragments from leaf-tissue DNA were likely to be derived from fungi. The contaminating fungal DNA not only caused the overestimation of genetic diversity in the host plants, but also interfered with the phylogenetic inference. Systematic inconsistency occurred between the UPGMA dendrograms of leaf and callus DNA fingerprints. The detection of contaminating fungal DNA suggested that precautions are required for PCR-based fingerprinting when field materials are used for DNA resources. A method for quick screening of the contaminating fungal DNA with universal primers for the nrITS (internal transcribed spacer) region is suggested.

Germplasm Preservation of Wild Arachis Species through Culture of Shoot Apices and Axillary Buds from In Vitro Plants

A study was conducted to evaluate in vitro techniques for germplasm preservation of wild species of Arachis. Nodal segments excised from in vitro-grown plants of A. retusa, A. macedoi and A. burchellii were used to examine the effects of explant position and age of the donor plant. Explants were excised from plants maintained in culture for 30, 60, 90 or 180 d, numbered I – V from top to bottom and cultured on MS medium supplemented with 2.7 µM NAA or different BAP concentrations (0, 4.4, 13.2 and 22 µM). The age of the donor plant has not influenced the responses of the four genotypes studied. In contrast, shoot regeneration ability was significantly affected by the original explant position, decreasing from top to bottom. In media supplemented with different BAP concentrations, multishoot formation was induced from apical segments at low frequencies (10 – 20%) and segments of all positions originated calluses at the explant basis after 30 d of culture. The culture of nodal segments in the presence of 2.7 µM NAA as the sole growth regulator is recommended for the multiplication of in vitro collections of wild groundnut species in order to avoid callusing and adventitious shoot formation.

MICROPROPAGATION OF Helianthus maximiliani (Schrader) BY SHOOT APEX CULTURE / MICROPROPAGACION DE Helianthus maximiliani (Schrader) POR EL CULTIVO DE VAINAS DE LOS VASTAGOS / MICROPROPAGATION DE L’Helianthus maximiliani (Schrader) AU MOYEN DE LA CULTURE DE L’APEX DE LA POUSSE

SUMMARYH.maximiliani was micropropagated using culture of shoot apices on modified Murashige and Skoog medium (DV). Further propagation of in vitro grown plants was done by culture of their nodal segments and shoot tips on the same medium supplemented with phloridzin, silver nitrate and casein hydrolysate (DV'). Rooting was induced by dipping the explants into IBA solution prior culture. Viable protoplasts (90%) were isolated from leaf mesophyll. These protoplasts divided (18%) in culture in agarose droplets.

Effects of Using Virus Free Plants in the Chinese Yam (Dioscorea opposita Thunb.) Cultivation and Practical Method to Distinguish a Japanese Yam Mosaic Virus Plant.

To improve productivity in the cultivation of Chinese yam 'Tanba-yamanoimo' (Dioscorea opposita Thunb.), we investigated the effect of virus free plants on the yield of a high yielding variety 'Shintanmaru' at a commercial field in 1998, 1999 and 2000. The weights of rhizophore of the virus free clone obtained from shoot apex culture were significantly heavier than infected plants with Japanese yam mosaic virus. The rates of yield increase were 27% in 1998, 17% in 1999 and 23% in 2000. The infected plants with Japanese yam mosaic virus showed the disease symptoms as mosaic in leaf, leaf transformation, leaf miniaturizing, and a delay in sprouting and growth. They could be easily distinguished from the virus free plants by their appearance. The results of distinction by symptoms in appearance and by serum test were nearly the same, with few exceptions. Because the former method was simple and not costly, it was a more effective and practical way to obtain virus free plants in seed propagation.

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system.

In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l-1) and thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l-1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l-1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l-1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex.