Peroxidase and catalase changes during in vitro adventitious shoot organogenesis from hypocotyls of Albizia odoratissima L.f. (Benth)

Hypocotyls of Albizia odoratissima cultured on shoot induction medium (MS medium with 7.5 μM BAP and 0.5 μM NAA) showed adventitious shoot organogenesis under light with 16 h photoperiod. Similar cultures under total darkness produced non-morphogenic calli. The changes in the specific peroxidase and catalase activity, total protein content and acidic isoperoxidase pattern were compared between the culture showing shoot organogenesis and culture producing non-morphogenic calli. It was found that in vitro shoot bud differentiation is accompanied by an increase of the specific activities of peroxidase and catalase in culture kept under light. In parallel with the above changes the total protein content reached to the maximum level and also a new isoperoxidase (P10) expressed on the 21st day in cultures kept under light. Conversely, culture producing non-morphogenic calli underwent a reverse change in specific peroxidase activity. This change in antioxidant enzyme activities corresponds to the histological observation of shoot bud differentiation in cultures kept under light.     

Shoot regeneration from organogenic callus of sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

A complete method to regenerate adventitious shoots and to produce field-ready trees from three commercial cultivars of sweet cherry (Prunus avium L.) is described. The effects of explant types, pre-treatments, basal media, and phloroglucinol on cultivars Bing, Sweetheart, and Lapins were investigated. Callus developed on four explant types: apical shoot tips isolated from orchard trees; and punctured shoot tips, stem sections, and shoot bases of in vitro shoot cultures. Callus formed on Bing (5%), Sweetheart (8%), and Lapins (20%) shoot tips from orchard trees after 4 months on Murashige and Skoog medium (MS) at half-strength with 3 μM benzylaminopurine (BA). In vitro-derived explants formed callus after 3 months on Woody Plant Medium with 3 μM BA (W3B): punctured shoot tips (Sweetheart and Lapins 67%), stem sections (Sweetheart 31%, Lapins 27%), and shoot bases (Sweetheart 10%, Lapins 17%). Pre-treatment of shoot cultures on MS with 3 μM BA and 1 mM phloroglucinol increased callus formation three-fold on shoot base explants. Callus was separated from parental explants and maintained on MS with 3 μM BA. Shooting was induced by transferring callus to W3B. At 2 weeks, shoot development approached 100%. By 4 weeks, 7–17 shoots had formed on each explant. Callus was maintained for 1.5 years with no decrease in shoot production. Shoots were grafted onto Mazzard (P. avium) rootstocks with 54% (Sweetheart), 57% (Lapins), and 21% (Bing) success after 5 weeks.     

Effect ofin vitro storage at 4°C on survival and proliferation of two apple rootstocks

One cm long shoot explants of dwarf apple rootstocks P 2 and M.9 taken from 2 year-old cultures were stored at 4°C in the dark in three media differing in concentration of growth regulators. Every 6 weeks, some explants were transferred into proliferation medium and multiplication rate was observed during three or four consecutive passages. In a second experiment, the influence of explant type (1 cm long shoot tips, 1 cm long middle part of shoots or three-shoot tufts smaller than 1 cm) and transfer time to the cold room (immediately, 10 days, or 20 days after subculture) on explant survival and proliferation were analysed.Survival of explants was influenced by composition of the storage media. On medium without 6-benzylaminopurine, 70% of P 2 and 17% of M.9 explants became necrotic during 18 weeks of storage. P 2 rootstock proliferated better in three passages after storage than did unstored controls. Storage of M.9 rootstock reduced proliferation in the first and second passages if stored in media containing 6-benzylaminopurine in comparison with unstored controls. Explants stored as tufts and transferred to the cold room directly after subculture produced more shoots during two passages than cultures stored as single shoots.     

Plant regeneration from the root-derived embryonic tissues of Rosa hybrida L. cv. Charming via a combined pathway of somatic embryogenesis and organogenesis

This study describes culture conditions for a plant regeneration system via a combined pathway of somatic embryogenesis and organogenesis in root explant cultures of the commercial rose cultivar 'Charming'. Root explants formed white calluses at a frequency of 30% after 6 weeks of culture on Schenk and Hildebrandt (SH) medium supplemented with 11 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. After 6 weeks of transfer to SH medium without growth regulators, initial white calluses gave rise to globular somatic embryos at a frequency of 2.8%, which were subsequently dedifferentiated to embryonic tissues. Somatic embryos or embryonic tissues initially derived from root explants did not undergo development beyond cotyledonary stage. To produce adventitious shoots, embryonic tissues were sliced and cultured on SH medium with 0.5 mg l⁻¹ 6-benzyladenine. After 4 weeks of culture, 28% of embryonic tissue explants formed adventitious shoots. Regenerated shoots were rooted on half strength SH medium with 0.1 mg l⁻¹ α-naphthalaneacetic acid and subsequently grown to maturity. Root-derived embryonic tissues were proliferated by subculture, while retaining the capacity for shoot production for a few years.     

Direct Adventitious Shoot Formation from Apical Shoot Explants of Euphorbia tirucalli

The induction of adventitious buds from apical shoot explants of Euphorbia tirucalli was studied. On average, 10.5 adventitious buds were efficiently induced in a ring on the segment from one apical explant on MS (Murashige and Skoog) medium supplemented with 0.5 mg l⁻¹ thidiazuron and 0.5 mg l⁻¹ benzylaminopurine. The adventitious buds could develop into adventitious shoots during subsequent cultures on hormone-free MS medium. For rooting, shoot clumps were cultured on half-strength MS medium containing 0.2 mg l⁻¹ α-naphthaleneacetic acid or indole-3-butyric acid. All the rooted plants survived establishment in soil within 2 months.     

Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees

Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.     

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.)

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, -naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 M indole-3-butyric acid (IBA) and 3–12 M N₆-benzylaminopurine, thidiazuron (TDZ), or 6-(,-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4°C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 M TDZ and 2 M IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Micropropagation of mature <Emphasis Type="Italic">Cornus mas</Emphasis> ‘Macrocarpa’

Sprouting axillary buds sampled from a mature 27-year-old shrub of Cornus mas ‘Macrocarpa’ were used as starting material for in vitro culture establishment. Multiple shoot cultures, grown on basal woody plant medium with the pH adjusted to 5.6–5.7 and supplemented with 6-benzylaminopurine in combination with 1-naphthaleneacetic acid, were capable of continuous axillary and adventitious shoot proliferation up to 1 year. Later on, growth ceased, shoot tip necrosis appeared and shoot cultures died. Transfer of living shoots onto modified woody plant medium with the pH adjusted to 6.8–7.0 led to vigorous growth of multiple shoot cultures without any loss of multiplication rates or decreased vitality for several years. The use of 6-benzylaminopurine in combination with 1-naphthaleneacetic acid proved superior to the application of thidiazuron which induced a frequent formation of short and fasciated shoots. 1-naphthaleneacetic acid promoted in vitro adventitious rooting frequency up to 73.3%, whereas indole-3-butyric acid was not effective. Ex vitro acclimatized plants did not show any visually detectable morphological variation.     

α-Galactosidase from sweet chestnut seeds

The major sugars of fresh seeds of Castanea sativa were shown to be raffinose, stachyose and sucrose. Drying seeds at 25° for 14 weeks increased the ratio raffinose: stachyose from 1.1 to 3.5, reduced sucrose content by ca 50 % and decreased total extractable α-galactosidase. The enzyme activity was resolved into two peaks, a high MW form I (apparent MW215 000) and a low MW form II (apparent MW 53 000). The latter form was predominant in the extract of fresh seeds whereas the former was the main form in the 14-week dried seeds. An increase in the amount of enzyme I was also observed when a buffered extract (pH 5.5) of fresh seeds was stored at 4°. Enzymes I and II had pH optima of 4.5 and 6, respectively. Both enzymes hydrolysed p-nitrophenyl α-d-galactoside at a much greater rate than the natural substrates raffinose, stachyose, locust bean gum and carob gum. However, enzyme I showed preference for stachyose as compared to raffinose; the opposite order was observed for enzyme II.     

In vitro regeneration of brahmi shoots using semisolid and liquid cultures and quantitative analysis of bacoside A

The major objectives of this study were to investigate an efficient rapid protocol for mass propagation of adventitious shoots of brahmi using semisolid and liquid cultures; and to assess the amount of bacoside A accumulated in the regenerated shoots. Leaf explants were grown in vitro on Murashige and Skoog semisolid medium supplemented with 0.5, 1.0, 1.5, 2.0 and 2.5 mg l⁻¹ 6-benzyladenine or kinetin (KN) or thidiazuron (TDZ) for 4 weeks. Adventitious shoots developed from leaf explants on all cytokinin supplemented media. After 4 weeks of incubation, leaf explants were split into two batches and one set was subcultured on semisolid medium and another set in liquid medium containing same concentration of cytokinins where they have come from. Highest rate of shoot regeneration was observed for explants cultured on medium with 2 mg l⁻¹ KN. The fresh and dry weight of shoots was also highest with this treatment. Liquid cultures were found suitable for proliferation of shoots (155.6 shoots per explant) and they also favored highest biomass accumulation (8.60 g fresh and 0.35 g dry biomass). The bacoside A contents were determined in shoots using HPLC. Analysis revealed that, the contents were highest with shoots regenerated on medium supplemented with 2 mg l⁻¹ KN. The amount of bacoside A was highest in the shoots regenerated in liquid medium (11.92 mg g⁻¹ DW) and it was 2.2-fold higher compared to shoots grown on semisolid cultures.     

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

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

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Direct adventitious shoot induction and plant regeneration of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm F.

An efficient micropropagation system via direct shoot organogenesis from hypocotyl segments of Embelia ribes Burm F. was developed. A high frequency (84%) of adventitious shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with additives (283.85 μM ascorbic acid [AA], 118.96 μM citric acid [CA], 142.33 μM cysteine, and 684.22 μM glutamine) and 1.13 μM of thidiazuron (TDZ) after 4 weeks following culture. Further development of shoot primordia into well-grown shoots of 4–5 cm in length was achieved by sub-culturing explants along with shoot primordia on MS medium supplemented with 0.44 μM benzyl adenine (BA) and 0.49 μM indole butyric acid (IBA) for three sub-culture periods with an interval of 15 days between them. The highest shoot multiplication was obtained when explants were incubated on MS medium supplemented with 2.2 μM BA and 0.49 μM IBA in 4 weeks. All in vitro developed shoots, 3–4 cm in length, rooted when grown on half-strength MS basal medium along with 2.47 μM IBA within 4 weeks. Moreover, 100% of shoots developed roots when these were treated with 4.93 μM IBA for 20 min and then transferred to pots containing soilrite mix and grown in the greenhouse. In vitro and ex vitro rooted plants showed a survival of 85 and 95% respectively, during hardening in the greenhouse for a 6-week period.     

Selection and optimization of a high-producing tissue culture of <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Biomass growth, ginsenoside and polysaccharide production in different ginseng tissue cultures, including callus culture, adventitious root culture and hairy root culture, were studied, and the active component contents were compared with that of native ginseng roots. The adventitious root culture was confirmed to be a very nice system, which grew fast and contained a rather high content of ginsenosides. Then, the culture conditions of adventitious root culture were optimized. The results showed that salt strength, various sucrose concentrations, ammonia/nitrate ratios and phosphate concentrations had significant influences on adventitious roots growth, secondary metabolite and polysaccharide synthesis in ginseng. The best culture conditions for ginsenoside production seemed to be 0.75 salt strength Murashige and Skoog medium, 4% sucrose, 9 mM ammonia to 36 mM nitrate, and 1.25 mM phosphate, while the optimization for polysaccharide accumulation seemed to be 0.75 salt strength, 6% sucrose, 9 mM ammonia to 36 mM nitrate and 3.75 mM phosphate source. Appropriate conditions allowed for a maximum ginsenoside yield of up to 132.90 mg/L and polysaccharide yield of 407.63 mg/L to be obtained after 4 weeks of culture.     

Direct shoot regeneration from nodal,internodal and petiolar segments of <Emphasis Type="Italic">Piper longum</Emphasis> L. and in vitro conservation of indexed plantlets

Three explants namely, nodal, internodal and petiolar segments were used to establish in vitro cultures of Piper longum. Multiple shoots were induced on semi-solid Murashige and Skoog (MS) medium supplemented with 1 mg/l 6-benzyladenine (BA). Addition of ascorbic acid (40 mg/l) considerably reduced browning of tissue and medium. Best shoot regeneration was observed from petiolar explants and was, therefore, used for all further studies. An indexing method was introduced for checking bacterial contamination in well established shoot multiplication cultures. It was found that bacterial infection was quite high in shoots derived from nodal and internodal explants while it was least in those obtained from petiolar segments. Only shoots that indexed negative for endogenous bacteria were used for proliferation and in vitro conservation studies. At the end of 4 weeks in proliferation medium which consisted of MS supplemented with 0.5 mg/l BA and 40 mg/l ascorbic acid as many as 22 shoot buds of 41 mm length could be obtained. Shoot buds developed into clusters for ease of further proliferation. A step of shoot elongation for 2 weeks in liquid MS basal medium was found to be beneficial for getting long and healthy shoots for rooting. Single shoots were rooted in 0.25 mg/l indole butyric acid that could be successfully acclimatized under nethouse conditions. A conservation strategy was also developed. The shoot cultures could be maintained without subculturing for as long as 8 weeks in MS medium supplemented with 1 mg/l paclobutrazol (PBZ) and 40 mg/l ascorbic acid.     

Improved clonal propagation of <Emphasis Type="Italic">Spilanthes</Emphasis><Emphasis Type="Italic">acmella</Emphasis> Murr. for production of scopoletin

A reproducible protocol for clonal propagation of Spilanthes acmella has been established. Routinely, the cultures were established in spring (January–April) season because of the highest aseptic culture establishment and high frequency shoot proliferation. Incorporation of 5 μM N⁶-benzyladenine (BA) to Murashige and Skoog (MS) basal medium showed 100% bud-break and promoted multiple shoot proliferation in cultures. Interestingly, a higher concentration of BA (7–15 μM) promoted stunted shoots with pale leaves while a lower concentration (1–3 μM) resulted in shoots with long internodes and excessive adventitious root proliferation from all over their surface. For recurrent shoot multiplication, single node segments from in vitro-developed shoots were excised and cultured on MS + BA (5 μM) medium where 20.3-fold shoot multiplication was achieved every 5 weeks. Finally, these shoots were successfully rooted on half-strength MS medium (major salts reduced to half-strength) with 50 g l⁻¹ sucrose, with a frequency of 100%. Transplantation survival of micropropagated plants was 88.9%. Additionally, accumulation of scopoletin, a phytoalexin, was revealed for the first time in the uninfected leaves of Spilanthes. Further, the quantitative estimation by HPLC with a fluorescence detector showed that the amounts of scopoletin content (0.10 μg g⁻¹ DW) in the leaves of micropropagated plants are comparable to those of field-grown mother plants. The study thus signifies the effectiveness of in vitro methodology for true-to-type plant regeneration of Spilanthes and their later utility for biosynthesis and constant production of scopoletin throughout the year.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

Establishment of adventitious root culture of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7 μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6 months with regular subcultures after 4 weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7 μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25 ml), culture period (4 weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2 g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.     

Adventitious shoot regeneration fromFagus sylvatica leaf explantsin vitro

Summary Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.