Direct organogenesis and plantlet regeneration from mature zygotic embryos of masson pine (Pinus massoniana L.)

Mature zygotic embryos of masson pine were cultured as initial explants to investigate the process of direct organogenesis. Adventitious buds were initiated on DCR medium (Douglas-fir cotyledon revised medium) supplemented with 0.5 mg l⁻¹ N⁶-benzyladenine (BA) and 0.05 mg l⁻¹ indolebutyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest induction frequency of adventitious buds was 99.3%. Subsequent transfer of buds to medium with lower concentrations of plant growth regulators in time was necessary for differentation of high quality adventitious buds. After culturing on elongating medium, in which the proportion of cytokinins to auxins was reduced, shoots higher than 2 cm were transferred for root induction to GD medium with half of the concentration of macro-salts (½ GD) and with 2 mg l⁻¹ IBA and 0.05 mg l⁻¹ BA. The average root frequency was over 70%. After adventitious roots had appeared, the shoots were transferred to ½ GD medium with a lower concentration of IBA (0.2 mg l⁻¹) for further root development.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

Regeneration of <Emphasis Type="Italic">Pelargonium × hederaefolium</Emphasis> ‘Bonete’ from petiole explants

The adventitious shoot regeneration from petiole explants of Pelargonium × hederaefolium ‘Bonete’ was achieved via a mixed pathway i.e. organogenesis and somatic embryogenesis. The histological study of regenerated structures revealed the presence of both shoot primordia and embryo-like structures. The initial growth in petiole explants occurred on media with BAP + auxin or TDZ alone. However, the most effective regeneration (24 structures/explant) was noted in the presence of TDZ (2 mg l⁻¹) and IBA (0.1 or 0.2 mg l⁻¹). Moreover, the application of TDZ in the induction phase reduced the time needed for the formation of adventitious structures and positively influenced the further shoot development on the medium containing m-topolin and IBA.     

Rapid micropropagation of mature wild cherry

Explants taken from the mature vigorous tree of wild cherry (Prunus avium L.) were assayed for their organogenic capacity under various phytohormonal treatments. The highest rate of adventitious shoot multiplication was recorded at a combination of 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ thidiazuron (6.83 shoots per explant). No differences in multiplication rates were found among media supplemented with BAP, BAP + α-naphthaleneacetic acid (NAA) or BAP + indole-3-butyric acid (IBA). Shoot elongation was significantly affected by the concentration of BAP, regardless of auxin addition to medium. Up to 73 % of microshoots rooted after using 0.3 mg dm⁻³ IBA, otherwise the adventitious rooting occurred at reasonable frequencies in all auxin treatments. Regenerated plantlets were successfully hardened ex vitro and continued to grow after the transfer to soil. No morphological aberrations were observed in the regenerates.     

Plant regeneration from phyllode explants of Acacia crassicarpa via organogenesis

In this paper we report the establishment of Acacia crassicarpa regeneration through organogenesis. We used phyllode (leaf) explants excised from 60-day-old in vitro seedlings for green compact nodule induction and, tested Murashige and Skoog (MS) media supplemented with various concentrations of 1-phenyl-3-(thiadiazol-5-yl) urea (thidiazuron) (TDZ) and α-naphthaleneacetic acid (NAA). Under the optimized condition, green compact nodules and adventitious shoots were induced in 10 and 40 days, respectively, on the medium containing a combination of 0.5 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA. This medium also yielded the highest rate (56%) of adventitious shoots forming from the nodules. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoots to medium containing 0.1 mg l⁻¹ TDZ within 2 months. The elongated adventitious shoots were rooted at a rate of 96.5% on half-strength MS medium with 0.5 mg l⁻¹ 3-indolebutyric acid (IBA) in 1 month. Rooted plantlets were hardened and successfully established in soil with an 80% survival rate. To our knowledge, this is the first report describing a detailed protocol for regeneration through organogenesis using phyllodes as explants for A. crassicarpa.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Adventitious regeneration in blackberry (<Emphasis Type="Italic">Rubus fruticosus</Emphasis> L.) and assessment of genetic stability in regenerants

Adventitious shoot regeneration from leaves of blackberry cultivar Čačanska Bestrna was examined using 30 different combinations of treatment. Young, fully expanded leaves taken from in vitro proliferating shoots were cultured on Murashige and Skoog (MS) medium containing either N ⁶-benzylaminopurine (BAP) (2.0 mg l⁻¹) or thidiazuron (TDZ) (1.0 and 2.0 mg l⁻¹) alone, or either of them combined with indol-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.1, 1.0 and 2.0 mg l⁻¹). Indirect shoot formation was observed in 12 different treatments, though the efficacy varied greatly among types and concentrations of plant growth regulators. TDZ at 1.0 mg l⁻¹, applied either alone or combined with IBA, was significantly more effective than BAP in inducing shoot regeneration. The highest regeneration rate (41.66%) was obtained on medium containing TDZ alone. Cytological, flow cytometry and isozyme analyses were used for screening of genetic stability/instability in regenerants. Cytological study, based on chromosome counts in root tip meristems, and flow cytometry analysis indicated that adventitious shoots of Čačanska Bestrna are tetraploid with 2n = 4x = 28 as well as those derived from axillary buds. However, a study conducted on the peroxidase patterns of the different blackberry regenerating lines showed differences between some lines and control plants (in vivo plants and micropropagated plants). These differences were visible with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as hydrogenous donor for peroxidase detection.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

Expression of Flowering Repressor Gene CsSVP,Carbohydrates, and Antioxidants Affected by Plant Growth Regulators in Saffron

Improving flower yield through lengthening flowering duration is a primary breeding objective in saffron (Crocus sativus L.). Asexual reproduction in saffron limits biodiversity and conventional breeding. Hence, eliciting flowering-related gene expression by plant growth regulators is one way to achieve this aim. The phytohormones methyl jasmonate (MeJA) and 6-benzyl amino purine (BAP) signals are received by the MADs-box gene family. In this study, to elucidate the role of phytohormones on flower development, plant were treated with BAP (0 and 5 mg L^?1), and methyl jasmonate (MeJA) (0, 20, and 100 mM) at three developmental stages of the saffron life cycle. Then, the expression of the SHORT VEGETATIVE PHASE (CsSVP) gene as a MADS-box gene family was assessed in the saffron corm. The activities of antioxidant enzymes, soluble sugar, starch content, and soluble protein content were also measured in corm, leaf, and root tissues. The application of MeJA and BAP treatments resulted in down-regulation of CsSVP expression in the corm during dormancy. At the dormancy stage, catalase, peroxidase activity decreased, and ascorbate peroxidase activity increased following MeJA treatment. In contrast, an increment in catalase and peroxidase activity and reduction of ascorbate peroxidase activity were observed after treatment with MeJA during the flowering stage. This change in enzyme activity is most likely due to flowering, which demands the re-allocation of resources. As flowering is a process heavily influenced by the environment, plants treated with MeJA, which may mimic environmental stress, showed changes in antioxidant enzyme activity. Overall, these results suggested that MeJA and BAP treatments play a significant role in the vegetative-to-reproductive phase change in saffron.

     

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.     

Encapsulation of cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var <Emphasis Type="Italic">botrytis</Emphasis>) microshoots as artificial seeds and their conversion and growth in commercial substrates

An effective protocol for the mass production of cauliflower microshoots was refined using the meristematic layer of cauliflower curd. After the meristematic layer was surface sterilized and shaved off, a commercial blender was used for homogenization and several blending treatments were tested in the range 15–120 s and 30 s was found to be optimal in terms of the amount explants produced and their subsequent growth ability. Explants were cultivated in S23 liquid medium (4.4 g L⁻¹ MS (Murashige and Skoog) and 3% v/w sucrose) supplemented with several combinations of plant growth regulators (PGRs) including 1 and 2 mg L⁻¹ of Kinetin in combination with three types of auxins (indole butyric acid (IBA), Naphthaleneacetic acid (NAA) and Indole-3-acetic acid (IAA)), each at 1 and 2 mg L⁻¹ concentration. The use of 2 mg L⁻¹ Kinetin and 1 mg L⁻¹ IBA gave the best results in terms of its effects on explant induction. Microshoots of different sizes were encapsulated in a sodium alginate matrix and the optimal stage suitable for the production of artificial seeds was assessed in terms of both subsequent conversion and plantlet viability. The feasibility of cultivating cauliflower artificial seeds in commercial substrates (compost, vermiculite, perlite and sand) irrigated with different solution mixtures including sterilized distilled water (SDW), PGRs-free S23 medium and S23 medium supplemented with Kinetin (1 and 2 mg L⁻¹) and IBA or NAA at (1 and 2 mg L⁻¹) was investigated. The use of 2 mg L⁻¹ Kinetin and 2 mg L⁻¹ NAA applied with S23 gave the optimal response with both perlite and compost. This study showed high growth capacity of cauliflower artificial seeds in commercial substrates which is considered a promising step for their direct use in vivo.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

<Emphasis Type="Italic">Crambe tataria</Emphasis>: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l⁻¹ 2,4-D in combination with 2 mg l⁻¹ Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.     

A combined pathway of somatic embryogenesis and organogenesis to regenerate radiata pine plants

This study describes for the first time in Pinus genus a plant regeneration system via a combined pathway of somatic embryogenesis and organogenesis from immature seeds of radiata pine. Somatic embryos were obtained from embryogenic line 2162 of Pinus radiata D. Don on EDM basal medium containing 60 μM ABA and 6% sucrose. The explants used for organogenesis experiments were either freshly collected somatic embryos or somatic embryos germinated for 1 week. Germination medium was half-strength LP medium, supplemented with 0.2% activated charcoal. Different induction periods and BA concentrations were assayed for shoot induction. After induction treatments, explants were elongated on the same medium used for germination stage. Rooting medium was quarter-strength LP medium supplemented with three different auxin treatments: 1.5 mg L⁻¹ 1-naphthalene acetic acid (NAA), 1.5 mg L⁻¹ indole-3-butyric acid (IBA) and 1 mg L⁻¹ IBA with 0.5 mg L⁻¹ NAA (MIX). The effect of the photon flux (120 mmol m⁻² s⁻¹ and darkness) in the first week of the explants in the rooting media was also tested. This methodology could offer an alternative to overcome some problems associated with somatic embryogenesis such as the seasonality of embryogenic tissue (ET) initiation or a low embryo production from the ET, a particularly important issue in the case of genetically transformed ETs.     

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.