<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Somatic embryogenesis in clonal neem, <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. and analysis for <Emphasis Type="Italic">in vitro</Emphasis> azadirachtin production

Summary Efficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately 15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic embryos.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Saponins in tissue culture of <Emphasis Type="Italic">Primula veris</Emphasis> L.

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

Summary Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl⁻¹ agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Morphactin influences guggulsterone production in callus cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the efficacy of different plant growth regulators was evaluated for optimizing its production. Morphactin was found to be effective in enhancing the accumulation of guggulsterones in callus cultures. Maximum callus growth was recorded on medium containing morphactin (0.1 mg l⁻¹) and 2iP (2.5 mg l⁻¹), whereas maximum guggulsterone production occurred when the calluses were cultured on medium containing 0.1 mg l⁻¹ morphactin and 1.0 mg l⁻¹ 2iP. Morphactin and 2iP interacted significantly to enhance the callus growth and guggulsterone production by about 8-folds in one-year-old cultures. However, the effect of morphactin on callus growth and guggulsterone production was not uniform over the levels of 2iP tested. Such an effect of morphactin has never been reported on the production of secondary metabolites.     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Shoot regeneration from in vitro-derived leaf and root explants of <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>

In vitro culture is currently used to produce plant material for ex situ conservation of endangered species. In this study, an efficient protocol for shoot regeneration from leaves and roots was developed for Centaurea ultreiae, a critically endangered species. Organogenesis from leaf and root explants was promoted by incubating these explants on half-strength Murashige and Skoog (MS) medium in the presence of one of four different cytokinins [6-benzyladenine (BA), zeatin, kinetin or N⁶-(2-isopentenyl) adenine (2iP)], each provided at five different levels. Shoot organogenesis was induced in both explants. The best response, 90% of leaf explants producing a mean of 2.48 shoots per explants and 94.3% of root explants producing a mean of 5.60 viable shoots per explants, was observed when explants were incubated on a medium containing 0.55 μM BA. Histological studies revealed connectivity between vascular tissues of regenerated shoots and cambial cells of leaf explants. Moreover, adventitious shoots were derived from pericycle cells of root explants and parenchymatic cells of callus tissues.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Pluchea lanceolata</Emphasis>, a medicinal plant,and effect of heavy metals and different aminopurines on quercetin content

Summary An effcient regeneration protocol has been developed from leaf explants of Pluchea lanceolata, a medicinally important plant of the family Asteracea. Nodular callus was initiated from young leaf segments cultured on Wood and Braun medium containing 2.0% sucrose (WB) supplemented with kinetin. On WB+5.0 mg l⁻¹ kinetin, 100% plant regeneration with 14±0.5 plantlets was obtained. Regenerated plantlets with well-developed root systems were transferred to pots and subsequently to the field. With respect to the effect of metals on morphogenic events and quercetin content of the cultures, lower concentrations of CuSO₄ proved to be beneficial but higher concentrations were detrimental. The presence of 200 μM ZnSO₄ or 150 μM CuSO₄ also resulted in the highest level of quercetin in regenerants. The yield of quercetin in culture varied with stages of differentiation.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Regeneration of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) plants from anther culture and somatic tissue with increased resistance to<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis>

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz     

Tissue culture and plant regeneration of the salt marsh monocots <Emphasis Type="Italic">Juncus roemerianus</Emphasis> and <Emphasis Type="Italic">Juncus gerardi</Emphasis>

Summary Tissue culture and plant regeneration protocols for the salt marsh plants Juncus roemerianus Scheele and Juncus gerardi Loisel, were developed. J. roemerianus callus was induced from mature seeds cultured on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BA), 5.37 μM α-naphthaleneacetic acid (NAA), 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 ml l⁻¹ coconut water (callus induction medium). The callus was subcultured on MS medium containing 2.22 μM BA, 5.37 μM NAA, and 9.05 μM 2,4-D for callus maintenance. Shoot regeneration occurred 2 wk after transferring the callus onto shoot regeneration medium, which consisted of MS medium containing BA or thidiazuron. A high frequency of shoot regeneration was obtained when the medium contained 13.3 μM BA. Regenerated shoots were transferred to MS medium supplemented with 10.7 μM NAA for root production. Rooting did not occur in the shoots regenerated on the thidiazuron-containing media. The callus induction medium for J. roemerianus was also effective in inducing callus of J. gerardi from young inflorescences. The same medium was also used for callus maintenance. Shoot regeneration occurred 10 d after transferring the callus onto MS medium supplemented with 0.44 μM BA and 0.57 μM indole-3-acetic acid. Root regeneration occurred after transferring the shoots onto MS medium plus 0.44 μM BA and 14.8 μM indole-3-butyric acid. The regenerated plants of both J. roemerianus and J. gerardi grew vigorously in potting soil in the greenhouse. J. roemerianus regenerants also grew well in a saltwater-irrigated field plot. Tissue culture-produced plants of J. roemerianus and J. gerardi can be used for planting in created or restored wetlands.