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     

In vitro propagation of Anacardium occidentale L.

Multiple buds have been induced from cotyledonary nodes of cashew, Anacardium occidentale L. Maximum number of buds were initiated on Murashige & Skoog's (MS) medium supplemented with 117 mM sucrose, 14.6 mM maltose and 22.2 M BA. The buds were harvested at each subculture after elongation on MS medium supplemented with 100 ml l^-1 coconut water, 14.6 mM maltose, increased sucrose (146 mM) and decreased BA (4.4 M). Excised microshoots were rooted in vitro on MS medium in the presence of 117 mM sucrose, 2.9 M IAA and 4.9 M IBA. Plantlets have been successfully transferred to soil and have been established in the field.     

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.     

Shoot organogenesis and plant regeneration in mulberry (Morus bombycis Koidz): Factors influencing morphogenetic potential in callus cultures

Regeneration of multiple shoots via callus induction and organogenesis was achieved in mulberry (Morus bombycis). Pre-soaked internodal explants in 4.4–8.9 M benzyladenine (BA) formed callus on Linsmaier and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (9.05 M), -naphthaleneacetic acid (2.85 M) and BA (2.2 M). Explants soaked for 48 to 72 h in low levels of BA produced loose and nodular callus that showed regeneration ability. Calluses developed adventitious shoot buds within 3–4 weeks on medium containing BA (8.9 M). Fifteen-week-old calluses developed fewer shoot buds than five-week-old calluses, indicating a decrease in morphogenetic potential with increasing duration of callus cultures. Semi-thin section microscopy was used to evaluate incapability of sustained regeneration. Development of normal shoot bud primordia, due to sub-surface reorganisation, was high in young calluses. The decline in the frequency of shoot bud primordia formation with callus ageing is due to reduced cell division activity in epidermal as well as sub-epidermal layers.     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

Effects of Activation of NMDA and AMPA Glutamate Receptors on the Extracellular Concentrations of Dopamine, Acetylcholine, and GABA in Striatum of the Awake Rat: A Microdialysis Study

The effects of activation of the AMPA and NMDA ionotropic glutamate receptors on the extracellular concentration of dopamine, acetylcholine, (ACh) and GABA in striatum of the awake rat was investigated. Also the levels of DOPAC, HVA, and choline (Ch) were included in this study. Seven to eight days after stereotaxical implantation of a guide-cannulae assembly, microdialysis experiments were performed. The dopamine and ACh content of samples were measured by HPLC coupled to electrochemical detection. GABA was measured using fluorometric detection. Perfusion of AMPA (1, 20, 100 mM) produced a dose-related increase of dopamine and a dose-related decrease of DOPAC and HVA. AMPA 100 M decreased extracellular concentrations of ACh and increased the extracellular concentration of Ch and GABA. Perfusion of NMDA 500 M increased the concentration of dopamine and decreased DOPAC and HVA. Also, NMDA 100 M decreased DOPAC. NMDA 500 M decreased the extracellular concentrations of ACh and increased the concentrations of Ch and GABA. Perfusion of the AMPA/kainate-antagonist DNQX (100 M) blocked the effects of AMPA (100 M) on dopamine, DOPAC, HVA, ACh, and GABA concentrations. Perfusion of the NMDA-antagonist CPP (100 M) blocked the effects of NMDA 500 M on dopamine, DOPAC, HVA, ACh, Ch, and GABA concentrations. These results suggest an interaction between glutamate-dopamine-ACh-GABA in striatum of the awake rat.     

Micropropagation ofPinus sylvestris

Plantlet regeneration from axillary buds, induced on seedling apices ofPinus sylvestris, is described. The influence of medium containing 1, 5, 10 or 50 M BA on the initiation of buds on 3, 6 and 9-week-old seedlings was investigated. With higher BA concentrations the number of apices that formed buds and the total number of buds increased, wheres their length decreased. Soaking the explants for 2 h in 111 M BA and for 5 h in 44 M BA gave better results, best expressed in longer buds. After 30 days, axillary buds were separated and transferred to a medium with 2% sucrose. Separated buds exposed to far-red light during the first week elongated better in comparison with the control. 64% of shoots rooted after a 24 h period of treatment with 53.8 M NAA incorporated in 0.6% water agar and transferred to 1/16-strength Murashige & Skoog medium as modified by Cheng supplemented with 1% sucrose.     

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     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

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.     

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

Summary The effects of high-concentration, 2-h liquid pulses of N⁶-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 M BA, and 100 and 200 M TD. Shoots developed best from buds induced with 300, 400, and 800 M BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 M BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.Abbreviations BA N⁶-benzylaminopurine - TD thidiazuron - SH Schenk and Hildebrandt (1972) - NAA 1-naphthaleneacetic acid Paper 2691 of the Forest Research Laboratory, Oregon State University, Corvallis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the authors or Oregon State University.     

Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Organogenesis in vitro of Nothofagus alpina (P. et E.) Oerst, Fagaceae

In vitro shoot and root regeneration of 2-year-old Nothofagus alpina plants was achieved from several types of expiants cultured in vitro on a modified Woody Plant Medium formulation. Multiple shoot formation was obtained from leaf expiants using 0.45–2.27 M thidiazuron and 0.0049–0.098 M indolebutyric acid. Excised axillary buds formed shoots and roots in the presence of 0.0049 M benzyladenine and 2.46 M indolebutyric acid, or in the absence of plant growth regulators. Nodal sections rooted when 2.46 M indolebutyric acid at was supplied in the medium. Subcultured shoots originating from nodal sections showed a high regeneration rate through multiple shoot and root formation.Abbreviations BA benzyladenine - GA₃ giberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron - WPM McCown's Woody Plant Medium - Z zeatin     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

In vitro micropropagation of Piper longum – an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 M benzyladenine (BA) and 4.64–13.9 M kinetin (K). Maximum number of shoots were induced with 8.9 M BA and 4.64 M K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 M BA along with 3.31–12.4 M picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 M BA and 8.28 M P. Elongated shoots were separated and rooted in MS supplemented with 2.46 M indole butyric acid (IBA). Plantlets, thus developed were established in soil.     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.