High frequency plant regeneration from cotyledon and hypocotyl explants of ornamental kale

A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg dm⁻³ 6-benzylaminopurine (BA) and 0.1 mg dm⁻³ naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response. The addition of 3.0 mg dm⁻³ AgNO₃ was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium.     

Organogenic regeneration of soybean from hypocotyl explants

Summary Thirteen soybean genotypes representing maturity groups IV−VI were compared for organogenic responses on three media cultured under two lighting conditions with hypocotyl sections excised from 7-d-old seedlings. All soybean lines responsed by producing adventitious shoots on the acropetal end of the hypocotyl explants, confirming genotype-independence of shoot initiation. Media containing 6-benzyladenine (BA; 5.0–10 μM) induced the greatest numbers of shoots. Histological studies confirmed the adventitious nature of arising shoots by indicative formation of meristematic zones and shoot primordia from parenchymatous tissues of central pith and plumular trace regions of the hypocotyl. Incompletely excised cotyledonary buds also contributed to shoot initiation. Degrees of responses were media-dependent and varied with regard to genotype. Centennial, Epps, and Lyon gave the greatest individual responses. Between cultivars (across all treatments), the regeneration potential (percentage of explants producing meristem-like structures or shoot primordia) 4 wk after initiation ranged from 47 to 75%. Four wk later, regenerative ability (number of shoots produced per responding explant) and regeneration efficiency (number of shoots produced per explant plated) yielded 1.4–7.1 and 1.0–5.0 shoots, respectively. The optimized protocol included initiation on a medium containing 5.0 μM BA for 4 wk, then transfer onto a shoot elongation medium (0.36 μM BA) for 4 wk. For 11 genotypes tested, 66–100% of excised shoots produced roots after 4 wk on media containing 12.5–29.2 μM indole-3-butyric acid. Of 109 regenerants transplanted to soil, 94% survived and no sterility has been observed on those mature enough to flower.     

In vitro plant regeneration from seedling-derived explants of ramie [Boehmeria nivea (L.) Gaud]

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B₅ vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Phytochrome A and phytochrome B1 control the acquisition of competence for shoot regeneration in tomato hypocotyl

 We analysed the light-dependent acquisition of competence for adventitious shoot formation in hypocotyls of phytochrome A (fri) and phytochrome B1 (tri) mutants of tomato and their wild type by pre-growing the seedlings under different light quality. The regenerative response in vitro of explants from etiolated seedlings was reduced in comparison to that displayed by light-grown ones. Our results indicate that the light-dependent acquisition of competence for shoot regeneration in the tomato hypocotyl is regulated by phytochrome and antagonistically by a blue-light receptor. By using phytochrome mutants and narrow wave band light we showed that it is mediated at least by two distinct phytochrome species: phytochrome B1 and phytochrome A. The action of phytochrome B1 during seedling growth was sufficient to induce the full capacity of the subsequent regenerative response in vitro in explants from all positions along the hypocotyls. In contrast far-red light acting through phytochrome A did not induce the full capability of shoot regeneration from middle and basal segments of the hypocotyl when phytochrome B1 was absent (tri mutant). A few middle and basal hypocotyl explants pre-grown in blue light regenerated shoots. Received: 12 April 1999 / Revision received: 5 July 1999 · Accepted: 6 August 1999     

Cytokinins,donor plants and time in culture affect shoot regenerative capacity of American elm leaves

Cytokinins, donor plants and their time in vitro as well as basal media were investigated for their influence on shoot regenerative capacity of American elm (Ulmus americana L.) leaves. Leaves excised from six 2-year-old seedlings formed adventitious shoots when placed on Driver and Kuniyuki Walnut (DKW) medium supplemented with 7.5, 15 or 22.5 M of benzyladenine (BA) or thidiazuron (TDZ). Thidiazuron induced significantly higher regeneration percentages on elm leaves than BA, regardless of concentration used. Donor plant also affected the efficiency of shoot regeneration, with certain seedlings having 1.5 to 7 times more explants forming shoots as compared to other seedlings tested. By subculture 15, the average number of shoots per regenerating explant increased at least 3-fold for leaves on media with BA or TDZ for the one donor plant that survived continued subculturing. Leaf explants from donor plants with the highest regenerative capacity had a higher percentage of shoot formation on DKW than MS medium. Explants from productive donor plants should be placed on DKW medium supplemented with TDZ to improve shoot regeneration efficiency from American elm leaves.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Rapid shoot regeneration from thin cell layer explants excised from petioles and hypocotyls in four cultivars of <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The present work describes a procedure that allows for the easy and rapid induction of caulogenesis in four cultivars of Brassica napus L. from transversal Thin Cell Layers (tTCLs). In order to investigate the regeneration ability of this crop, the effects of genotype, explant source and culture medium were examined on shoot regeneration. The tTCL explants were excised from hypocotyl and petiole of 2-week-old seedlings and cultured on a solid basal MS medium supplemented with α-naphthaleneacetic acid (NAA: 0.1–0.4 mg l⁻¹), 6-benzylamino-purine (BAP: 1–4 mg l⁻¹) and sucrose (20–40 g l⁻¹). A significant genotypic effect was observed between the four cvs; Jumbo and Drakkar displayed higher capacities to produce shoots than Pactol and Cossair. Regeneration commenced earlier and the percentage of shoot-producing explants as well as the number of shoots per regenerating explant was greater. The comparison between the regeneration ability of different explants showed that the hypocotyls exhibited a high rate of shoot organogenesis when they were cultured on MS medium supplemented with 3 mg l⁻¹ BAP, 0.3 mg l⁻¹ NAA and 30 g l⁻¹ sucrose. Adventitious shoot buds developed from 46% of the tTCLs, with a mean of 7.5 buds. Furthermore, the method was fast with shoot formation occurring by 7 days culture. Plantlets regenerated from all shoots and developed normally. The regenerated plants were fertile and identical to source plants.     

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Beta vulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

High regenerative nature ofMentha arvensis internodes

Media and incubation conditions have been defined for highly efficient regeneration of shoots from internode explants of slow and fast growing cultivars ofMentha arvensis. Internodal segments excised from thein vitro raised shoots were inoculated on the MS medium supplemented with combinations of 5 concentrations of l-napthalene acetic acid (NAA) and 3 concentrations of 6-benzyl amino purine (BAP). The media containing 2 μg ml⁻¹ NAA, 10 Μg ml⁻¹ BAP and 1 μg ml⁻¹ NAA, 5 μg ml⁻¹ BAP proved best for shoot regeneration and growth responses on cv Himalaya and cv Kalka explants, respectively. In 12 weeks time, on average one explant of cv Himalaya produced about 200 shoots and that of cv Kalka produced about 180 shoots. The Himalaya explants required higher concentrations of NAA and BAP for high efficiency proliferation as compared to the Kalka explants. The experiments demonstrated that internodal tissue inMentha arvensis can be induced to obtain direct shoot regenerants with high efficiency. The analysis of the RAPD profiles of 100 regenerated plantlets each of cv Himalaya and Kalka showed more than 99.9% homogeneity in bands with respect to the parents.     

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 mol m-² s-^-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation ( 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L^–1 for 3 h and then subculturing on MS medium without growth regulators.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

In vitro propagation of Cleome spinosa (Capparaceae) using explants from nursery-grown seedlings and axenic plants

Summary Two independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.