In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

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

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

Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)

We describe here an improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens. The production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved. The system is based on the regeneration of shoots from hypocotyls extracted from mature seed. The shoot regeneration medium is Murashige and Skoog (MS) salts and vitamins supplemented with 7.5 M thidiazuron and 0.25 M indole-butyric acid. Transferring the explants after co-cultivation to shoot regeneration medium containing 80 mg l^-1 of kanamycin and 300 mg l^-1 of Timentin reduced the total number of regenerated shoots without affecting the transformation rate. Transformation rates using the described system averaged 1.2% of the hypocotyl slices producing transgenic plants, with a range of 0–4.2%. The transgenic shoots rooted at a rate of 90% on half-strength MS salts and vitamins supplemented with 5 M -naphthaleneacetic acid and 0.01 M kinetin. Plantlets were transferred to a greenhouse directly from culture tubes with a 90% average survival.Abbreviations ACO1 Prunus persica 1-aminocyclopropane-1-carboxylate oxidase - CP Coat protein - GUS -Glucuronidase - NPTII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PDV Prune dwarf virus - PNRSV Prunus necrotic ringspot virus - SGM Shoot growth medium - SRM Shoot regeneration medium - TIM Timentin (SmithKline Beecham, Philadelphia) - TomRSV Tomato ringspot virusCommunicated by K.K. Kamo     

High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.)

Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-D), 60 M 4amino-3, 5,6-picolinic acid (picloram), or 30 M 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 M dicamba plus 10 M 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 M dicamba, 20 M BA) resulted in regeneration of shoots from 45% of calli of the cultivar Baron. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,5,6-picolinic acid - BA 6-benzylaminopurine     

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 M N⁶-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 M indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 M IBA + 0.5% activated charcoal + 1 M BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Rapid in vitro Regeneration of Sesbania drummondii

This paper describes rapid propagation of Sesbania drummondii using nodal explants isolated from seedlings and young plants. The nodal segments proliferated into multiple shoots on Murashige and Skoog's (MS) medium supplemented with 22.2 M benzyladenine. MS medium containing 2.2 and 4.5 M thidiazuron induced 5 – 6 shoots per stem node from 3-month-old plants. Nodal explants when cultured on MS medium containing combinations of benzyladenine (8.8 and 11.1 M) and indole-3-butyric acid (0.24 – 2.46 M) or indole-3-acetic acid (0.28 – 2.85 M) gave lesser number of shoots. Callus induced on cotyledonary explants when subcultured on 2.2 M thidiazuron containing medium resulted in its mass proliferation having numerous embryoid-like structures. Indole-3-butyric acid (0.24 – 2.46 M) was found suitable for root induction. In vitro regenerated plants were acclimatized in greenhouse conditions.     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Thidiazuron-Induced High-Frequency Shoot Regeneration from Root Region of Robinia pseudoacacia L. Seedlings

High-frequency regeneration of shoots was achieved at root region of seedlings of Robinia pseudoacacia L. cultured from seeds on medium supplemented with thidiazuron (TDZ, 1.0 M). The roots of intact seedlings proliferated and formed a compact callus followed by differentiation of numerous shoots. Corresponding cultures on benzylaminopurine-containing medium exhibited much weaker response. Hypocotyl segments also formed shoots at a lower concentration of TDZ (0.1 M). The shoots formed on TDZ-containing medium were well-developed and readily rooted on hormone-free medium. The obtained plants after acclimation in culture room survived after transfer to soil.     

Rapid micropropagation of <Emphasis Type="Italic">Curcuma longa</Emphasis> using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Multiple shoots of Curcuma longa were induced by culture of bud explants for 1week in Murashige and Skoog (MS) liquid medium supplemented with 72.64M thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8weeks. The regeneration rate was up to 11.4±1.7shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

In vitro propagation of Syzygium travancoricum Gamble – an endangered tree species

Rapid multiplication in Syzygium travancoricum, an endangered tree species was achieved from nodal explants on Murashige and Skoog's (MS) medium supplemented with Woody Plant Medium (WPM) micronutrients and plant growth regulators. Multiple shoot induction from young 1–2-year-old seedlings was observed on basal medium supplemented with 17.7 M benzyladenine and 1.3 M -naphthalene acetic acid. A high number of multiple shoots (25 shoots/nodal explant) was observed by the third subculture on multiplication medium. The dark brown nodular callus obtained from in vitro grown shoots through axillary buds regenerated at a high frequency when transferred to 1/2 strength basal medium. The plants rooted on 1/2 strength basal medium supplemented with 1.1 M indole-3-acetic acid and were transferred to the field with 40% survival.     

In vitro shoot regeneration from olive cultivar tissues

Petioles, leaf discs and midribs of several olive (Olea europaea L.) cultivars, collected from potted greenhouse plants, field-grown and in vitro shoots, were used to test their morphogenic capacity. Adventitious shoots were induced only in petioles from in vitro-grown shoots of cultivars Moraiolo, Dolce Agogia and Halkidikis, grown on Olive Medium (OM) plus 18 M zeatin within 4 to 5 weeks. Regeneration was achieved, both on Murashige and Skoog (MS) and on modified OM, only in the dark. The highest regeneration was achieved directly from the proximal part of the petioles after 2 to 3 weeks in media containing 5 to 40 M thidiazuron, or with both 10 M 2-isopentenyladenine +2.2 M 6-benzyladenine with or without low auxin concentration (not more than 2.5 M). A few adventitious shoots were also regenerated from callus when it was shifted from auxin and cytokinin media to cytokinin only medium. The regeneration potential was higher in petioles collected from apical nodes than from basal ones. The adventitious shoots were transferred to solid half-strength MS medium supplemented with 4.5 M zeatin for further development. Several regenerated shoots were rooted and the plantlets hardened in the greenhouse. No apparent differences regarding morphological aspects were observed among the regenerated plantlets or with those obtained by stimulation of axillary buds.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea) - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - 1/2 MS half strength MS - OM Olive Medium - BN Bourgin & Nitsch     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid