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.     

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.     

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.     

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     

Regeneration of pea (Pisum sativum L. cv. Century) plants by in vitro culture of immature leaflets

In vitro regeneration of plants from immature leaflets of 3 day-old pea (Pisum sativum L. cv. Century) seedlings was studied under defined nutritional, hormonal and environmental conditions. Immature leaflets isolated from the second and third apical leaves of aseptically germinated seeds were cultured on MS medium containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and supplemented with 0.1, 1, and 10 M concentrations of naphthaleneacetic acid (NAA) and 1 and 10 M levels of benzyladenine (BA) in various combinations. Shoot regeneration from the primary callus occurred within 45 to 90 days of culture in most of the hormone combinations. Although the number of calli producing shoots was maximal at 10 M levels of NAA and BA, multiple shoot regeneration was predominant at a combination of 0.1 M NAA and 10 M BA. Indoleacetic acid (IAA) and kinetin (K), both at 10 M, also induced shoot regeneration. No shoots were regenerated when 10 day-old leaflets were used as explants. Root production generally occurred on non-shoot regenerating calli. Roots were induced to differentiate by transferring the regenerated shoots onto half-strength B5 medium supplemented with 1 M NAA.NRCC # 19712Visiting Scientist, supported by a research fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (Rep. Argentina). Permanent address: Facultad de Ciencias Agrarias, IBONE, Casilla de Correos 209, Corrientes (3400), Argentina.     

Restoration of regeneration potentiality in prolonged culture of Digitalis purpurea

Regeneration potential was restored in 2-year-old cultures of proliferating shoots of Digitalis purpurea L. under the influence of 14.43 M gibberellic acid, while cytokinins were ineffective. The retrieved shoots grew normally in a modified Murashige and Skoog's medium supplemented with 0.98 M indole-3-butyric acid (IBA) and 27.1 M adenine sulphate and rooted 100% in the presence of 0.49 M IBA alone.NBRI Research Publication No. 415 (N.S.)     

Regeneration of peach plants from callus derived from immature embryos

Summary Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.44 M benzyladenine (BA) to media containing 0.27 M -naphthaleneacetic acid (NAA) and 2.2 M BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 M indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

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 meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

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.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid     

Plant regeneration from cotyledons of Prunus persica,Prunus domestica,and Prunus cerasus

Shoots were regenerated from the proximal region of immature cotyledons (with the embryonic axis removed) of Prunus persica (peach) and from the same area in mature cotyledons of P. domestica (plum) and P. cerasus (sour cherry) on MS medium containing (in mgl^-1) thiamine-HCl, 0.4; nicotinic acid, 0.5; pyridoxine-HCl, 0.5; sucrose, 25 000; and 0.7% agar. The medium was supplemented with 0.0–2.5 M indole-butyric acid and 5–12.5 M thidiazuron. Cultures were incubated at 24 °C under 16h photoperiod. Shoots regenerated adventitiously over a broad range of thidiazuron concentrations and 2.5 M indole-butyric acid in 35 days. The presence of the embryonic axis inhibited the development of shoots. Regenerated shoots of peach and plum were rooted on half-strength MS inorganic semi-solid medium with 2.5–5.0 M indole-butyric acid. Rooted plants were acclimatized and transferred to the greenhouse.Abbreviations BAP 6-benzylaminopurine - IBA indole-butyric acid - TDZ N-phenyl-N-1,2,3-thidiazol-5-ylurea - IAA indole-acetic acid - NAA -naphthalene-acetic acid     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

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.     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 M NAA and 10 M BAP, and embryo maturation was promoted after transfer to a medium containing 1 M NAA and 10 M BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 M NAA and 1 M BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 M NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.Abbreviations BAP 6-benzylaminopurine - B5 medium of Gamborg et al. 1968 - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium of Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

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.