Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Direct somatic embryogenesis from pericycle cells of broccoli (Brassica oleracea L. var. italica) root explants

Cotyledon, hypocotyl or root explants of 7-day-old broccoli seedlings were cultured on Murashige and Skoog (MS) agar or liquid medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). The frequency of direct somatic embryo formation was 100% when root explants were cultured in liquid medium. Histological analysis indicated that somatic embryos were initiated directly from the pericycle cell layers of root explants as early as 1 day after liquid culture. Genotype did not affect the frequency of somatic embryo formation or the number of somatic embryos per explant. All broccoli genotypes examined had 100% somatic embryo induction efficiency, and the number of somatic embryos per 0.8 mm root segment ranged from 22.9 in ‘Luhui’ to 26.0 in ‘Haizi’. The number of normally developed somatic embryos in culture increased with increasing 2,4-D concentration. Plantlet regeneration frequency was the highest (73.3%) when germinated plantlets were transferred to 1/2 strength MS agar medium containing 1.0 mg l⁻¹ 6-benzyladenine (BA). When regenerated plantlets were transferred to a greenhouse, approximately 75% survived and there were no morphological differences between regenerated plants and seed-derived controls. The protocols established in this study will benefit large-scale vegetative propagation and transformation-based genetic improvement of broccoli.     

Regeneration of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch) cultivars and <Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">Prunus dulcis</Emphasis> rootstocks via organogenesis

Somatic peach plants were regenerated from callus derived from the base of stem explants of the scion cultivars ‘UFO-3’, ‘Maruja’, ‘Flariba’ and ‘Alice Bigi’, and the peach × almond rootstocks ‘Garnem’ and ‘GF677’. A protocol for organogenic plant regeneration was developed using three culture media containing different concentrations of 6-benzyladenine (BA) and indolebutyric acid to produce organogenic calli. Shoots were obtained from sliced calli after their transfer to a differentiation culture medium containing 2 mg l⁻¹ BA and 1 mg l⁻¹ α-naphthalene acetic acid. Using this procedure, up to 29 regenerated plants per callus were obtained. The highest regeneration rate was obtained with the peach × almond rootstocks. This work provides an effective protocol that could be utilized for peach transformation research.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Somatic embryogenesis and plant regeneration of <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> through suspension culture

A simple and reliable protocol for regeneration of okra through somatic embryogenesis from suspension cultures has been developed. Embryogenic callus was obtained from hypocotyl explants cultured on media with Murashige and Skoog (MS) salts, Gamborg (B5) vitamins, 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg dm⁻³ naphthaleneacetic acid (NAA), 25 mg dm⁻³ polyvinylpyrrolidone and 30 g dm⁻³ sucrose. More number and high frequency of healthy embryoids appeared individually in suspension culture containing MS salts, B5 vitamins, 2.0 mg dm⁻³ 2,4-D and 1.0 mg dm⁻³ kinetin. Formation of cell clusters from the single cells was clearly noticed during ontogeny. Matured embryos at the cotyledonary stage were transferred to agar solidified medium for germination. The best conversion of embrya into plantlets (67.3 %) was recorded on media with half strength MS salts, B5 vitamins, 0.2 mg dm⁻³ benzylaminopurine (BAP) and 0.2 mg dm⁻³ gibberellic acid (GA₃). The plantlets were transferred to soil and hardened in the plastic pots. After proper acclimatization, the plantlets regenerated through somatic embryogenesis were compared to seed grown plants to observe any variation.     

Efficient plant regeneration from immature embryo cultures of <Emphasis Type="Italic">Jatropha curcas</Emphasis>, a biodiesel plant

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1–1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog’s (MS) medium supplemented with IBA (0.5 mg l⁻¹) and BA (1.0 mg l⁻¹). The above medium when supplemented with growth adjuvants such as 100 mg l⁻¹ casein hydrolysate + 200 mg l⁻¹ l-glutamine + 8.0 mg l⁻¹ CuSO₄ resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 mg l⁻¹ polyvinyl pyrrolidone + 30 mg l⁻¹ citric acid + 1 mg l⁻¹ BA + 0.5 mg l⁻¹ Kn + 0.25 mg l⁻¹ IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-strength MS medium supplemented with 0.5 mg l⁻¹ IBA and 342 mg l⁻¹ trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (<Emphasis Type="Italic">Cinnamomum camphora</Emphasis> L.)

An efficient protocol for secondary somatic embryogenesis in camphor tree is reported. Secondary somatic embryos (SSEs), initially obtained from the primary embryos of a nascent embryogenic culture in 2002, were proliferated and maintained for more than 4 yr via cyclic secondary somatic embryogenesis. Throughout this period, the embryo populations retained a high level of competence for plant regeneration. SSEs were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos (MSEs). Histological observations of the various stages of secondary embryo development revealed four typical stages, namely, globular, heart-shaped, torpedo, and cotyledonary. The process of secondary embryogenesis continued in a cyclic way, with each newly formed embryo producing a subsequent generation of secondary embryos. In order to progress developmentally beyond proliferation cycles, cotyledonary embryos from one of embryogenic lines (L14) were cultured on Murashige and Skoog (MS) medium with 0.1–3.0 mg l⁻¹ abscisic acid (ABA) or 0.05–1.0 mg l⁻¹ thidiazuron (TDZ) in darkness for 2 mo to achieve maturation. Matured embryos were then transferred to MS-based germination medium containing either 0.1 mg l⁻¹ TDZ, 0.2 mg l⁻¹ indole-3-butyric acid (IBA), and 0.5 mg l⁻¹ 6-benzylaminopurine (BA) or 0.1 mg l⁻¹ TDZ and 0.2 mg l⁻¹ IBA and were cultured in light for germination. Over 50% of embryos matured in the presence of 0.5 mg l⁻¹ ABA were able to germinate with shoots and poor root system. Frequencies of embryos germinating normal shoots among different genotypes did not change significantly. A total of 93% of the shoots from the germinated embryos converted to plantlets on half strength MS medium with 0.5 mg l⁻¹ IBA by 3 wk. Plantlets acclimatized successfully to ex vitro conditions and developed as field-grown plants with normal appearance.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange

In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l⁻¹ was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹ kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l⁻¹ BA, 0.5 mg l⁻¹ KT, 0.1 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.     

Somatic embryogenesis and plant regeneration in date palm <Emphasis Type="Italic">Phœnix dactylifera</Emphasis> L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus

Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l⁻¹ 2,4-D and 0.3 mg l⁻¹ activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with 1 mg l⁻¹ ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and 306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l⁻¹ NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots. The growth of regenerated somatic plants was also monitored in the field.     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Plant regeneration and production of embelin from organogenic and embryogenic callus cultures of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm. f.—a vulnerable medicinal plant

Embelia ribes, an important vulnerable medicinal liana, was regenerated through organogenesis and embryogenesis using leaf explants. Leaf explants produced organogenic calluses on MS medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzylaminopurine. Shoot regeneration was obtained from organogenic calluses on MS medium containing different concentrations of thidiazuron (TDZ) and indole-3-acetic acid (IAA). The frequency of shoot bud organogenesis was highest (23.9 shoots/explant) in MS medium containing 0.5 mg l⁻¹ TDZ and 0.1 mg l⁻¹ IAA. The best result for induction of embryogenic callus was noticed in the combination of 2.0 mg l⁻¹ TDZ and 0.5 mg l⁻¹ 2,4-D. This callus, maintained in the same medium, showed the highest differentiation of embryos (56.5%) after 6 wk of culture. Embryos were transferred to MS medium supplemented with different concentrations of TDZ, and this facilitated conversion of embryos into plants. After 6 wk of subculture, MS medium with 0.05 mg l⁻¹ TDZ favored the highest percentage (52.2%) embryo conversion. As per the present protocol, 52.2% of the embryos underwent conversion, and a mean number of 29.5 shoots per culture was obtained. Shoots developed from both types of calluses were rooted on half-strength MS basal medium supplemented with 1.0 mg l⁻¹ indole-3-butyric acid. HPLC-UV assay demonstrated the highest embelin content (5.33% w/w) in the embryogenic callus cultures. Embelin was isolated from embryogenic callus and was identified using IR and ¹H NMR studies.     

High efficiency organogenesis and analysis of genetic stability of the regenerants in <Emphasis Type="Italic">Solanum melongena</Emphasis>

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ zeatin, 0.1 mg dm⁻³ indoleacetic acid and 10 g dm⁻³ sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0–2.0 mg dm⁻³ gibberellic acid and 4.0–8.0 mg dm⁻³ AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.     

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.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.