Adventitious shoot formation and plant regeneration from Pinus pinaster Sol. ex Aiton

Summary Pinus pinaster plants were regenerated from cotyledons excised from in vitro germinated seeds and axenically cultured on induction medium (GMD). 6-Benzyladenine (2.2 μM) induced the highest frequency of direct bud formation from cotyledons. An average of 13.1 ± 2.1 elongated shoots per cotyledon was obtained. Germination time influenced shoot induction, and the organogenic potential decreased with explant age. Cotyledons remained for 21 d on induction medium, and in order to promote adventitious shoot elongation, they were transferred to Gupta and Durzan’s DCR medium without growth regulators, containing 0.5% (wt/vol) activated charcoal and 3% (wt/vol) sucrose. Rooting was achieved by application of an indole-3-butyric acid, (396.6 μM) pulse (24 h at 4° C), followed by transfer to a sterile mixture of peat plus perlite (1:1 vol/vol). Ninety-eight to 100% of the regenerated plants were successfully acclimatized. All plants have survived after transfer to the field.     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast.

Summary Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing 2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be transferred to soil after being acclimatized.     

Evidence for the existence of a universal crown-gall tumor initiation enhancer

Tumor initiation of different dicotyledonous plant species inoculated with Agrobacterium tumefaciens B₆ has been studied in vivo and in vitro. The tumor formation in weakly susceptible plants can be strongly enhanced by exogenously applied active extract fractions derived from highly susceptible Helianthus cotyledons. It is found that highly susceptible plants (Kalanchoë, Lycopersicon and Pinto beans) contain an active tumor initiation enhancer which is clearly similar to the compound(s) found in Helianthus cotyledons. No activity can be detected in extracts derived from weakly susceptible plants (Coleus, Phaseolus) or in those obtained from crown-gall tumor tissues.Abbreviations HS high susceptibility for tumor initiation - LS low susceptibility for tumor initiation - PEF purified active extract fraction     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

In vitro induction of multiple shoots and plant regeneration inVigna

Summary Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B₅ vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.     

Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc.

The efficiency of any plant regeneration system lies in part in its wide applicability to diverse genotypes. In Asiatic Vigna, cotyledon and cotyledonary node explants from 4-day-old seedlings of 27 genotypes were cultured in a medium consisting of MS salts, B5 vitamins, 3.0% sucrose and 1.0 mg l^-1 BA. Direct and efficient multiple shoot regeneration (80–100%) from the cotyledonary nodes was obtained in all epigeal species namely radiata, mungo, aconitifolia, subspecies radiata var. sublobata, mungo var. silvestris and in the hypogeal but allotetraploid glabrescens. In contrast, two other hypogeal species V. angularis and V. umbellata failed to initiate shoots from the nodes. However, adventititious shoots developed at the basipetal cut (hypocotyl) in 35–67% of V. angularis explants. These results provide evidence in support of the existing genomic grouping within subgenus Ceratotropis, which designates AA, A₁A₁ and A₁A₁/- to epigeal, hypogeal and the allotetraploid species, respectively. Mean shoot production ranged from 3.3 to 10.4 shoots per explant during the first subculture and varied significantly among the responsive genotypes within 4 species. Additional shoots were obtained in all genotypes after subsequent subculture. However, cotyledons were not as regenerable as cotyledonary node explants. Although significant differences in rooting were observed among the shoots of the 15 genotypes, the response was generally higher in MS basal medium (MSO) than in MS with 1.0 mg l^-1 IAA. Regenerated plants were successfully transferred to soil (50–100% survival rate) and all surviving plants were reproductively fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from immature cotyledon explant cultures of bean (P. coccineus L.)

A method for plant regeneration, via organogenesis and somatic embryogenesis, from P. coccineus is described. Immature cotyledons from plants regenerated and cloned in previous experiments were used. The highest percentage of regeneration (37.5%) was observed from the cotyledons of clone C7 on a modified Murashige & Skoog basal medium to which (2-isopentenyl)adenine (2iP, 10 mg l^-1) and 2-naphthoxyacetic acid (NOA, 0.05 mg l^-1) were added. On average, the same medium gave the highest percentage of regeneration also from the cotyledons of the 7 clones of P. coccineus used in the experiment. Newly regenerated plants were normally fertile. The procedure described may serve for induction of somaclonal variation for in vitro selection of valuable genotypes and for genetic transformation by A. tumefaciens.Abbreviations 6BA 6-benzylaminopurine - CCC chlorocholine chloride - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - NOA 2-naphthoxyacetic acid     

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na⁺/H⁺ antiporter) and Arabidopsis AVP1 (vacuolar H⁺-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K⁺/Na⁺ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H₂O₂ generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.Feng-Yun Zhao and Xue-Jie Zhang contributed equally to this work     

Partial purification and characterization of the soluble DNA polymerase (polymerase-α) from seedlings of Pisum sativum L.

Radish seedlings (Raphanus sativus L. Saxa Treib) were grown in the dark with or without added kinetin (2 mg/l=9.29 M). Low-temperature (77°K) fluorescence emission and absorption spectra of etiolated cotyledons were registered at increasing seedling age before and immediately, 30 s and 30 min after one 1-ms flash. Kinetin was found to induce a higher accumulation of the phototransformable protochlorophyll(ide) P_657–650 in the etiolated cotyledons, especially from day 6 to day 10 after germination. The amount of the P_657–650 protochlorophyll(ide) resynthesized during a 30-min dark period after a 1-ms flash decreased with seedling age. It was smaller in cotyledons from kinetin-treated seedlings at day 6 after germination and at that age only. The ability to perform the Shibata shift decreased with increasing seedling age. In cotyledons from 10- and 13-day-old seedlings, the shift was accomplished to a greater extent when the plants were grown in the presence of kinetin.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants overexpressing <Emphasis Type="Italic">Ramosa1</Emphasis> maize gene show an increase in organ size due to cell expansion

The structure of the plant inflorescence and flower is an important agronomic and ornamental trait studied for its potential economic applications. In particular, the capacity to modify flower size has always been a breeder’s goal. Genetic and molecular studies have shown that the Zea mays gene Ramosa1 (Ra1) is involved in inflorescence branching regulation. In fact the ra1 loss of function mutation causes extra branching of the inflorescence. In this work we suggest a possible utilization of the Ramosa1 maize gene as a tool to modify inflorescence architecture and flower size in transgenic plants. In fact overexpression of this gene in Arabidopsis plants promotes an increase in reproductive organ size. Pollen, seeds, cotyledons, leaves and roots are also larger than those of the wild type. Analysis of organs from transformants showed that cell expansion was increased without apparently affecting cell division. These results suggest that the RA1 protein is able to up-regulate cell expansion in all organs of Arabidopsis plants.     

The effects of herbicides and mycoparasites at different moisture levels on carpogenic germination in Sclerotinia sclerotiorum

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant upon colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis, P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57 M_r protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38 M_r protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24 h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.     

Evidence for a relationship between malate metabolism and activity of 1-sinapoylglucose: L-malate sinapoyltransferase in radish (Raphanus sativus L.) cotyledons

The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl--D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO ₃ ^- as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO ₃ ^- also NH ₄ ⁺ . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO ₃ ^- nitrogen were subsequently supplied with NH ₄ ⁺ nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO ₃ ^- /NH ₄ ⁺ -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO ₃ ^- /NH ₄ ⁺ -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid.Abbreviation MSO L-methionine sulfoximine - SinGlc 1-O-sinapoyl--D-glucose - SinMal O-sinapoyl-L-malate - SMT 1-O-sinapoyl--D-glucose:L-malate sinapolytransferase     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

In vitro organogenesis in two dioecious species,Rumex acetosella L. and R. acetosa L. (Polygonaceae)

Callus cultures were established from dioecious plant species Rumex acetosella and R. acetosa, using cotyledons, hypocotyls and stem tips of aseptically germinated seedlings as primary explants. Cultures were also established from male and female R. acetosella adult plants, starting from vegetative lateral buds. Cell division was induced using a high 2,4-D concentration, while bud induction and multiplication were stimulated on a medium with high BAP/IAA ratio. Cotyledon fragments of both species produced only rhizogenic calli. Hypocotyl-derived calli of R. acetosella produced buds, while those of R. acetosa showed no bud forming response under these conditions. Bud multiplication occurred in stem tip cultures of both species and in lateral bud cultures of R. acetosella. Calli derived from male plants produced more buds than those from female. Shoots were easily rooted using IBA, and plantlets were effectively transferred to soil. Flowering was not induced in culture. The sex of regenerated male and female plants was not altered by the culture conditions.     

Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.