Transformation of <Emphasis Type="Italic">PttKN1</Emphasis> gene to cockscomb

We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5 mg l⁻¹ 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l⁻¹ 6-BA, 0.5  mg l⁻¹ NAA and 0.5  mg l⁻¹ IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.     

Changes in biomass allocation and phenolic compounds accumulation due to the effect of light and nitrate supply in <Emphasis Type="Italic">Cecropia peltata</Emphasis> plants

Cecropia peltata is popularly known as “guarumbo” in Mexico and is used in traditional medicine for treatment of diabetes mellitus. C. peltata plants were cultivated in a hydroponic system under controlled conditions. Gradients of light (20, 30 and 100 μmol m⁻² s⁻¹) and nitrate concentrations (13, 2 and 0.2 mM) were applied to estimate their effect on biomass allocation and accumulation of bioactive (chlorogenic acid and isoorientin) phenolic compounds over a 28-day period. According to carbon nutrient balance (CNB) hypothesis predictions, biomass accumulation in foliage was stimulated by the highest irradiance (100 μmol m⁻² s⁻¹); similarly, at highest irradiance in combination with lowest nitrate concentration (0.2 mM), root growth was stimulated (root-to-shoot ratio increased twofold with respect to the control). In these conditions, total phenolics (TP) and chlorogenic acid (CGA) contents were higher in aerial parts than in roots, with a 3.8-fold increase in TP and a 7.7-fold increase in CGA in foliage with respect to the control plants. Isoorientin was accumulated at very low levels. Antioxidant activity and total phenolic content showed a strong positive correlation. Phenylalanine ammonia-lyase activity (PAL) in aerial parts exhibited significant changes (>twofold) by highest irradiance. C. peltata plants allocate biomass and/or phenolic compounds to compensate the oxidative damage (increase in MDA levels) due to changes in light and nitrate restriction. The results are the basis for the establishment of a system of C. peltata culture in view of the potential use of C. peltata in therapeutic preparations for the treatment of diabetes mellitus.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

Micropropagation and production of arbutin in oriental strawberry tree (<Emphasis Type="Italic">Arbutus andrachne</Emphasis> L.)

A protocol for micropropagation of Arbutus andrachne from seeds was developed. Results indicated that none of the seeds cultured on Murashige and Skoog (MS) medium, with or without plant growth regulators (PGRs), germinated. Seeds soaked in 250 mg l⁻¹ gibberellic acid (GA₃) at 4°C for 3 days, then cultured on water-agar medium containing 2.0 mg l⁻¹ GA₃ exhibited 80–100% germination and developed into usable seedlings. Shoot proliferation was tested on MS or B5 medium containing different concentrations of cytokinin. No shoot proliferation was observed on PGR-free medium. Proliferation was more successful on MS than on B5 medium. On both media, the most successful proliferation was obtained using zeatin as a cytokinin type. Rooting was tested on MS medium containing different concentrations of auxin. Rooting failed on PGR-free medium and on medium containing indole-3-acetic acid (IAA), 0.25 or 0.5 mg l⁻¹ indole-3-butyric acid (IBA), or 0.25, 0.5 or 2.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooting (40%) was most successful with 1.0 mg l⁻¹ NAA. Rooted plantlets exhibited 80% survival in all mixtures of peatmoss and perlite, and acclimatized plants were successfully grown in the greenhouse. Quantitative analysis of arbutin performed on in vivo and in vitro leaves using high-performance liquid chromatography (HPLC) revealed that in vivo leaves contained higher arbutin content (0.3–0.81% w/w) than in vitro leaves (0.09% w/w). The highest yield of arbutin in vivo was detected in leaves collected in August, and the lowest yield in leaves collected in December.     

Effects of donor plants and plant growth regulators on naphthoquinone production in root cultures of <Emphasis Type="Italic">Impatiens balsamina</Emphasis>

The effects of type of explant (leaves and roots), donor plants, and plant growth regulators on naphthoquinone (NQ) production of Impatiens balsamina L. root cultures were evaluated. The root cultures were initiated in liquid Gamborg’s B5 medium supplemented with 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ kinetin (Kn) and 1.0 mg l⁻¹ 6-benzyladenine (BA). The present investigation indicated that the root cultures established from the leaf explants produced higher total NQ content [1.01 ± 0.046 mg/g dry weight (DW)] than those established from the root explants (0.62 ± 0.023 mg/g DW). The leaf explants of four I. balsamina strains including white flower plant (IbW), pink flower plant (IbP), violet flower plant (IbV) and red flower plant (IbR) were used to establish the root cultures. Based on HPLC analysis, IbP strain produced the highest total NQ content (3.39 ± 0.072 mg/g DW), while IbR strain produced the lowest one (1.45 ± 0.055 mg/g DW). The root cultures established from the IbP explant were capable of producing higher content of total NQs (2.76 ± 0.093 mg/g DW) than those established from the other strains. The results suggest that the tissue cultures initiated from the high-yielding donor plants should be capable of producing higher content of secondary compounds than those initiated from low-yielding donor plants. In addition, plant growth regulator manipulation exhibited that a combination of 0.1 mg l⁻¹ NAA, 1.0 mg l⁻¹ Kn and 2.0 mg l⁻¹ BA is capable of increasing NQ production (2.97 ± 0.072 mg/g DW) in I. balsamina root cultures.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

<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.     

An elite protocol for accelerated quality-cloning in <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

A novel, efficient, and simple protocol was developed on in vitro mass propagation and acclimatization of Gerbera jamesonii Bolus cv. Sciella, an ornamental plant with attractive flowers. Shoot tip was used as the primary explant for in vitro establishment in which Murashige and Skoog (MS) medium supplemented with a low level of NAA (0.5 mg l⁻¹) and BAP (1.5 mg l⁻¹) promoted earliest axillary bud initiation within 5 d in 91.6% of the inoculants. Five axillary buds were initiated from a single explant within 13 d after inoculation. A very high rate of shoot multiplication (14 shoots per inoculated axillary bud) and proliferation was achieved when MS medium was fortified with a relatively higher level of BAP (2 mg l⁻¹) and 60 mg l⁻¹ ADS within 27 d of multiple shoot culture. A maximum number of well-developed roots per plant was observed in MS medium with 0.5 mg l⁻¹ IAA in the next 26 d. In the easy low-cost acclimatization process of 20 d, a combination of sand, soil, cow urine, and tea leaves extract (1:1:1:1; v/v) ensured 95% survival rate. Sixty-one well-acclimatized plants were obtained from a single shoot tip within 86 d. The sustained multiple shoot culture for 15 mo paved the way toward the conservation of genetic resources as well as beneficial economics. The clonal fidelity study of micropropagated and sustained cultured clones using ISSR primers ensured the continuous supply of quality propagules retaining genetic uniformity. The in vitro-generated plants performed better over conventionally propagated plants in the field condition.     

In vitro root culture of <Emphasis Type="Italic">Ocimum sanctum</Emphasis> L. and evaluation of its free radical scavenging activity

Young leaf explants of Ocimum sanctum L. incubated on solidified Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 1-naphthaleneacetic acid (NAA) and 0.2 mg l⁻¹ kinetin (Kn) developed rhizogenic callus. When these were subcultured onto MS medium supplemented with 1.5 mg l⁻¹ 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg l⁻¹ NAA, friable rhizogenic callus was observed. Upon transfer of this friable callus onto liquid MS medium containing 4 mg l⁻¹ NAA and 1.3 mg l⁻¹ 6-benzyladnine (BA) under continuous agitation at 90 rpm and 16 h photoperiod, roots with an optimum dry weight of 1,460 mg l⁻¹ were obtained. An ethyl acetate extract of these roots exhibited 1, 1–diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity.     

Effects of plant growth regulators and <Emphasis Type="SmallCaps">l</Emphasis>-glutamic acid on shoot organogenesis in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed in China, Mongolia and Siberia, where it is produced as a forage product. In this report, we establish a highly reproducible plant regeneration system through somatic embryogenesis. Two explants, mature seeds and leaf base segments were used; these parts displayed different responses to combinations of growth factors that affect embryogenic callus induction, callus type optimization and plant regeneration. The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of 5.0 mg l⁻¹ l-glutamic acid. The inclusion of 5.0 mg l⁻¹ l-glutamic acid was found to significantly promote primary callus induction, embryogenic callus formation and callus status improvement. Subculturing on maintenance medium for 1–2 months before plant regeneration was found to be essential for the optimization of callus type and the maturation of embryogenic callus. Callus relative water content and growth rate were simultaneously investigated during callus maintenance, and found to possibly be related to callus type. Shoots were differentiated from the embryogenic callus on the optimal medium with MS salts containing 0.2–0.5 mg l⁻¹ α-naphthalene acetic acid (NAA), 2.0 mg l⁻¹ kinetin (Kn) and 2.0 g l⁻¹ casamino acids in 71.0 and 69.2% of wild-type (WT) and Jisheng No.1 (JS) plants, respectively. Plant regeneration was variable depending on NAA levels, and the addition of casamino acids stimulated the maturation of embryogenic callus and plant regeneration. Transferring callus with shoots onto half-strength MS medium resulted in rooting within 1 week. The growth of regenerated plants was also surveyed in the field. This is the first report of plant regeneration through somatic embryogenesis from mature seeds and leaf base segments of L. chinensis.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

Fast versatile regeneration of <Emphasis Type="Italic">Trifolium alexandrinum</Emphasis> L.

Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly, Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies were obtained from intact cotyledons on NAA/BA (0.05 mg l⁻¹ NAA combined with 2.0 mg l⁻¹ BA) and NAA/TDZ (0.05 mg l⁻¹ NAA combined with 1.0 mg l⁻¹ TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively. 21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l⁻¹ NAA combined with 0.5 mg l⁻¹ BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric acid (IBA: 0.24 mg l⁻¹) or NAA (2.0 mg l⁻¹) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L⁻¹ Kinetin (Kn), 0.5 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.5 mg L⁻¹ 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl₂ solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS₂ solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m⁻² s⁻¹ provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Functional analysis of an <Emphasis Type="Italic">iaaM</Emphasis> gene in parthenocarpic fruit development in transgenic <Emphasis Type="Italic">Physalis pubescens</Emphasis> L. plants

An efficient somatic embryogenesis system for Physalis pubescens L. (husk tomato) was developed prior to transformation. Subsequently, cotyledonary explants of P. pubescens were transformed with a chimeric construct containing an iaaM gene from driven by the fruit-specific promoter 2A12 to develop parthenocarpic fruits. Following selection of explants on Murashige and Skoog (MS) medium containing containing 75 mg l⁻¹ kanamycin (Km), 36 km-resistant callus clusters were recovered, and these were regenerated into whole plants. Expression of the iaaM gene was detected in confirmed transgenic fruits. The 0.9-kb 2A12 promoter was capable of directing expression of the introduced iaaM gene in transgenic P. pubescens fruits, but iaaM expression was absent from both leaves and flowers. Quantitative measurements of indole-3-acetic acid (IAA) content during fruit development indicated that the IAA levels in transgenic lines increased from anthesis through young fruits and peaked at fruit maturity. On average, IAA contents in transgenic fruits were two-fold higher than those in control fruits. Under greenhouse condition, vegetative growth, morphology, and the flowering of transgenic plants were comparable to those of control plants. However, the fruits of transgenic lines ripened earlier and had fewer seeds per fruit than did control plants.