Nickel hyperaccumulation in shoot cultures of Alyssum markgrafii

Shoot cultures of Alyssum markgrafii O.E. Shulz, endemic nickel hyperaccumulating species of central Balkan, were established and maintained on Murashige and Skoog medium supplemented with 0.2 mg dm^–3 benzyladenine (BA). Nickel in form of NiCl₂ . 6 H₂O was supplemented at 22 different concentrations ranging from 0.0001 to 15 mM but none of them was lethal to cultures. High Ni²⁺ concentrations (10 mM or more) arrested shoot growth which, upon transfer to Ni-free medium, commenced via axillary bud proliferation. Shoots that developed from axillary buds through the subculture manifested increased tolerance to Ni²⁺ expressed as shoot elongation. Shoot multiplication and dry biomass production decreased with increase of Ni²⁺ in medium. Only the accumulation of Ni²⁺ in tissues increased with Ni²⁺ content of the medium. Apart from shoot cultures, high Ni²⁺ accumulation was registered in undifferentiated callus cultured on medium with 0.5 mg dm^–3 BA and 0.5 mg dm^–3 naphthylacetic acid. Highest content of accumulated Ni was 2.37 g g^–1 (d.m.) in shoots and 2.65 g g^–1 (d.m.) in callus, both measured on medium with 15 mM Ni²⁺.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

Induced Activity of Superoxide Dismutase and Peroxidase of in vitro Plants by Low Concentrations of Ethanol

A rapid micropropagation protocol was established for Aloe vera L. var. chinensis (Haw.) Berger, Chinese Aloe. The effects of three factors, namely BA, NAA and sucrose, on bud initiation were evaluated by L₉ (3⁴) orthogonal design. The variance analysis of the experimental results showed that the actions of the three factors were all considerable. Among the three factors, sucrose was the most important for bud initiation followed by BA, and NAA had the weakest effect. The best medium for bud initiation was semi-solid MS supplemented with 2.0 mg l^–1 BA, 0.3 mg l^–1 NAA, 30 g l^–1 sucrose and 0.6 g l^–1 PVP (pH 5.8), on which Chinese aloe could multiply 15 times in 4 weeks. Some shoots rooted spontaneously on 1/2 strength MS medium, but the rooting percentage was improved in the presence of 0.2 mg l^–1 NAA. Rooted plantlets were acclimatized to greenhouse conditions. The young plantlets from tissue culture were transplanted successfully. In vitro propagation can be a useful tool in the conservation of this endangered medicinal species.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Micropropagation of juvenile and adult Annona squamosa

A micropropagation system for Annona squamosa L. (Sugar Apple) using hypocotyls of seedlings and nodal cuttings from 3-year-old plants was developed. Shoot proliferation was achieved with Woody Plant Medium supplemented with BA. Silver thiosulphate was added at 0.5 mg l^–1 to control leaf abscission. Rooting was obtained when subcultured shoots were preconditioned for 2 weeks in medium with 10 g l^–1 activated charcoal before treatment with 43 µm NAA or 39 µm IBA. Rooting was improved when galactose was used instead of sucrose in the rooting medium. The rooted plantlets were acclimatised successfully.Abbreviations NAA naphthaleneacetic acid - IBA indolebutyric acid - MS Murashige & Skoog Medium - WPM Woody Plant Medium - NN Nitsch Medium - Juv juvenile explant - Adu adult explant     

In vitro multiplication ofVanilla planifolia using axillary bud explants

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N⁶-benzyladenine (BA, 2 mg l^–1) and -naphthaleneacetic acid (NAA, 1 mg l^–1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l^–1 and NAA at 0.5 mg l^–1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.Abbreviations BA N⁶-benzyladenine - DMRT Duncan's multiple-range test - KC Knudson (1946) medium - KCB KC basal medium - Kn kinetin - MS Murashige and Skoog (1962) medium - MSB MS basal medium - 1/2 MSB half-strength MSB - MS-D double-phase MS medium - MS-L liquid MS medium - MS-S semi-solid MS medium - NAA -Naphthaleneacetic acid     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

Vegetative propagation of Begonia venosa Skan in vitro from inflorescence explants

Methods are described for the vegetative propagation of Begonia venosa Skan. Young flower buds are capable of producing callus which, contrasting to callus from leaves of adult plants, is very organogenic. For callus induction are required: BA and NAA at a conc. of 0.5 mgl^–1, 21 °C and low irradiance. Subculture of organogenic callus is optimal on a medium with 0.1 mgl^–1 BA and 2% glucose, whereas NAA is ommitted. Shoot development from preformed adventitious buds is enhanced by lowering the BA and glucose conc. Optimal rooting of excised shoots is obtained on a medium without regulators and a low glucose level. No visible mutations can be detected in the plant material, even not at the flowering stage.Abbreviations (BA) 6-Benzylaminopurine - (2iP) N⁶-(2-isopentenyl)adenine - (MS)(1) Murashige and Skoog - (NAA) 1-Naphthaleneacetic acid Publ. 535     

Plant Regeneration Through Somatic Embryogenesis in Leaf Derived Callus of Plumbago Rosea

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm^–3 kinetin and 2.0 mg dm^–3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm^–3 kinetic (Kn), 0.5 mg dm^–3 gibberellic acid (GA₃) and 0.1 mg dm^–3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm^–3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.     

Shoot Organogenesis from Immature Zygotic Embryo Cultures of Ginkgo biloba

Immature zygotic embryos were cultured on Murashige and Skoog's medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), benzyladenine (BA) and zeatin or with various concentrations of 2,4-D alone. The maximum number (8 per embryo) of adventitious buds formed from cotyledons of heart stage embryos cultured on MS medium with 1 mg dm⁻³ BA and 0.01 mg dm⁻³ NAA. The adventitious buds originated from procambial strands of immature embryo cotyledons and then developed into adventitious bud primordia within 20 d. Adventitious buds transferred to hormone free MS medium grew into shoots, but did not produce plantlets because the shoots failed to root. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

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.     

In vitro cormlet development in Crocus sativus

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm⁻³ benzyladenine (BA) and 80 g dm⁻³ sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm⁻³ BA, 3 mg dm⁻³ indolebutyric acid and 30 g dm⁻³ sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.