<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Embryogenic tissue initiation and somatic embryogenesis in Fraser fir (<Emphasis Type="Italic">Abies</Emphasis> fraseir [Pursh] Poir.)

Embryogenic suspensor mass (ESM) was established from immature seeds of Fraser fir. The initiation frequency of ESM was dependent on genotype, collection time, medium, and plant growth regulators (PGR) used. The ESM initiation potential was higher with seeds collected in late June (clone 16-273, 4.7%) or early July (clone 16-45, 2.2%) and decreased as the zygotic embryos matured. Excised proembryo stage of zygotic embryos was most appropriate to initiation of ESM. Most of the ESM arose from the seeds that were at the proembryo stage. From the four different culture media we compared, seven ESM lines were obtained: two lines from Murashige and Skoog (MS) medium with 4.4 μM benzyladenine (BA), one from Schenk and Hildebrandt (SH) medium with 4.5 μM thidiazuron (TDZ), and four from SH with 4.4 μM 6-benzyladenine. However, only one ESM line from clone 16-273 (June 24, SH+TDZ) could be proliferated in subsequent culture. Different concentrations of l-glutamine and casein hydrolysate (CH) in the medium were also compared for their effect on ESM proliferation. The highest proliferation rate (1.16-fold) was obtained from SH medium supplemented with 250 mg/L CH and 3.42 mM l-glutamine. In contrast, the lowest rate was noted when 1,000 mg/L CH plus 3.42 mM l-glutamine (0.17-fold) was added to the medium. As for somatic embryo maturation, the highest number of mature precotyledonary (100.1/g⁻¹ FW ESM) or cotyledonary (64.3/g⁻¹ FW ESM) somatic embryos was obtained on a medium containing 20 or 80 μM abscisic acid, 10% polyethyleneglycol, 4% maltose, and 0.3% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium containing 0.3% gellan gum. The somatic plantlets were recovered from the germination medium and transferred to soils.     

<Emphasis Type="Italic">In vitro</Emphasis> fruiting and seed set of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. CV. Sweet banana

Summary Plantlets of Capsicum annuum L. ev. Sweet Banana regenerated via somatic embryogenesis from immature zygotic embryos were capable of producing flower, fruit, and seed when cultured in small tissue culture containers. In vitro floral buds were first formed on plantlets that grew on plantlet development medium [agar-gelled Murashige and Skoog (MS) basal medium containing 1 mgl⁻¹ (5.3 μM) α-naphthaleneacetic acid (NAA)] in a growth room at 22°C and continuous illumination. However, floral buds rarely developed further into mature flowers. This problem was overcome using the vented autoclavable plant tissue culture containers. In vitro fruit formation and ripening was observed when liquid half-strength MS basal medium supplemented with 5 μg ml⁻¹ silver thiosulfate, 1 mg l⁻¹ (5.3 μM) NAA, and 3% sucrose was added to the surface of the plantlet development medium. Hand-pollination improved fruit set. Further research in needed to determine why the pepper seeds formed in vitro failed to germinate.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Induction of somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of <Emphasis Type="Italic">eruca sativa</Emphasis> mill

Summary A protocol was developed for high frequency somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of Eruca sativa. Explants grown on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-D formed embryogenic callus after 4 wk of culture. Secondary somatic embryos were also produced from primary somatic embryos on MS medium containing 0.56 μM 2,4-D. Somatic embryos developed into mature embryos on MS medium in the presence of 45 gl⁻¹ polyethylene glycol. After desiccation, somatic embryos developed into plantlets by culturing the mature somatic embryos on 1/2 x MS medium containing 0.24 μM indole-3-butyric acid.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

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.     

Plant regeneration <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis in <Emphasis Type="Italic">Pinus kesiya</Emphasis> (Rolye ex. Gord.) influenced by triacontanol

Embryogenic cultures were initiated and established for the first time in 3 different genotypes of Pinus kesiya using mature zygotic embryos and triacontanol. Mature zygotic embryos produced white-mucilaginous embryogenic callus when cultured on half strength MSG (Becwar et al. 1990) basal medium supplemented with 90 mM maltose, 2.0 g l⁻¹ Gellan gum, 9.0 M 2, 4-D and 10 g l⁻¹triacontanol. On subculture of such embryogenic callus on the maintenance medium (II) containing 2.0 M 2,4-D and 2.0 g l⁻¹ triacontanol induced cleavage polyembryogenesis with proembryos. The percentage of somatic embryogenesis was not similar in all the three genotypes. The highest percentage of somatic embryogenesis (88.5 %) was recorded in PK04 genotype. Somatic embryos were successfully germinated on half strength MSG basal medium without growth regulators. Somatic seedlings showed fast growth and a survival rate of 95%. This work for the first time reveals that triacontanol can be used as an effective growth regulator for inducing somatic embryogenesis in conifers.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

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.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

<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.     

Organogenesis and somatic embryogenesis in <Emphasis Type="Italic">Ariocarpus kotschoubeyanus</Emphasis> (Lem.) K. Schum. (Cactaceae), an endemic and endangered Mexican species

Summary Shoots by direct and indirect organogenesis and somatic embryos were induced from tubercles excised from Ariocarpus kotschoubeyanus seeds germinated in vitro. Shoot formation was greatest (6.3 per explant) when explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA; 13.3 μM) and α-naphthaleneacetic acid (NAA; 5.4 μM). Individualized shoots were rooted in half-strength MS; the addition of activated charcoal (1 g l⁻¹) and the use of sun cap closures to seal containers improved the rooting of shoots. Nearly 20% of the explants produced somatic embryos on media containing combinations of BA (8.9–22.2 μM) and NAA (0.5–5.4 μM). The establishment of plantlets in soil presented no significant problems. In vitro culture is a useful option for mass propagation of A. kotschoubeyanus and contributes to its conservation.     

Influence of auxins in direct <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis of <Emphasis Type="Italic">Euphorbia nivulia</Emphasis>, a lectinaceous medicinal plant

Summary Somatic embryo (bipolar) or shoot (monopolar) morphogenesis in mesophyll cells of Euphorbia nivulia Buch.-Ham in vitro was dependent on the type of auxin supplementing Murashige and Skoog (MS) medium containing benzyladenine. Direct in vitro morphogenesis, i.e., organogenesis, and somatic embryogenesis were significantly influenced by seasonal growth of the donor plant, explant position (proximal, mid, and distal), and light. Explants collected in march/April were superior to July/August material. Proximal explants underwent morphogenesis more readily than mid- and tip-derived explants. Incubation in the light favored morphogenesis while darkness was inhibitory. Kinetin (Kn) was also inhibitory to morphogenesis. MS medium enriched with different levels of N⁶-benzyladenine (BA) alone, or in combination with α-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), induced adventitious shoots directly. Explants collected in March/April cultured on medium with 13.3 μM BA and 2.69 μM NAA developed the highest number of shoots, a mean of 15.2 shoots per proximal explant. Developed shoots rooted the best on half-strength MS medium with 2.46 μM indole-3-butyric acid, which developed a mean of 5.2 roots per shoot. Rooted healthy shoots could be transplanted to small pots, with an 80% survival rate. Addition of 2,4-dichlorophenoxyacetic acid (2.4-D) to BA-supplemented medium was obligatory to develop somatic embryos. MS medium containing 2.26 μM 2,4-D and 4.44 μM BA induced a mean of 44.8 somatic embryos per proximal explant. The embryos passed through distinct stages of embryogenesis, namely globular, heart, torpedo, and early cotyledonary. The embryos (88%) underwent maturation on half-strength MS medium with 2.89 μM gibberellic acid (GA₃), and its subsequent transfer on half-strength MS basal medium in light conditions facilitated 80% conversion of embryos to plantlets. Direct shoots or embryos were originated from the mesophyll cells. Somatic embryo development was concurrent with the independent origin of vasculature in the bulbous basal portion. The survival rate of embryo-derived plants was 90%.     

Effect of auxins on indirect <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis and expression of <Emphasis Type="Italic">gusA</Emphasis> transgene in a lectinaceous medicinal plant, <Emphasis Type="Italic">Euphorbia nivulia</Emphasis> Buch.-Ham.

Summary The types of auxin in Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.