<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation

A reliable plant regeneration system is described for the production of adventitious shoots from root explants of spinach. Explants from roots of axenic shoots and roots induced on cultured hypocotyl explants were used for adventitious shoot induction. Explants from apical, middle and basal root regions were incubated on Nitsch and Nitsch medium supplemented with α-naphthaleneacetic acid, gibberellic acid and kinetin. Optimum shoot regeneration was from explants of apical and middle root regions on medium with 20 μm α-naphthaleneacetic acid and 5.0 μm gibberellic acid. Shoots originated directly from root tissues without an intervening callus phase. Adventitious shoots were rooted and were grown to maturity in the glasshouse. This plant regeneration procedure has been exploited in preliminary studies of Agrobacterium-mediated transformation. Received: 27 February 1996 / Revision received: 22 August 1996 / Accepted: 30 September 1996     

Efficient Regeneration of Tetraploid Isatis indigotica Plants via Adventitious Organogenesis from Hypocotyl Explants

An efficient in vitro plant regeneration system via hypocotyl segments of tetraploid Isatis indigotica Fort. was established. Murashige and Skoog's (MS) and Gamborg's (GB₅) media were found to be superior to White medium for promoting shoot regeneration. The highest shoot regeneration (92 %) was achieved from hypocotyls cultured on MS medium containing 8.9 M benzyladenine (BA) and 2.7 M naphthaleneacetic acid (NAA), with an average of 4.2 shoots developed per explant. Plant regeneration was also improved when the explants were cultured in MS basal medium containing 3 % (m/v) sucrose and grown under a 12-h photoperiod. The developed shoots were well rooted in a half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA) and were morphologically normal after transfer to soil.     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis> Sm. using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

An efficient in vitro method for mass propagation of Potentilia potanii wolf

Summary This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and &#x03B1;-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl^&#x2212;1 BA and 1.0 mgl^&#x2212;1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl^&#x2212;1 NAA and 0.5 mgl^&#x2212;1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

Thidiazuron induced adventitious shoot regeneration in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

A high frequency adventitious shoot regeneration protocol was developed for henbane (Hyoscyamus niger L.) using thidiazuron (TDZ). Hypocotyl, cotyledon and stem explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of N⁶-benzylaminopurine and TDZ. MS medium supplemented with 16 μM TDZ was the most effective for providing 100 % regeneration frequency associated with a 19.53 shoots per hypocotyl explant. Plantlets were rooted on MS medium supplemented with different concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid. High rooting and survival was achieved using half strength MS medium supplemented with 8 μM IBA.This study was supported by The State Planning Commission of Turkey (DPT) and University of Ankara (Project Nos.: 98K120640 and 2001K120240).     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro

High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 M indole-3-acetic acid and 50 M benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.     

High frequency plant regeneration from cotyledon and hypocotyl explants of ornamental kale

A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg dm⁻³ 6-benzylaminopurine (BA) and 0.1 mg dm⁻³ naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response. The addition of 3.0 mg dm⁻³ AgNO₃ was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium.     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.