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     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss)

Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l^–1), kinetin (0.8 mg l^–1) and adenine sulphate (6 mg l^–1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l^–1), kinetin (0.08 g l^–l) and adenine sulphate (0.6 mg l^–1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l^–1) and all of these grew on transfer to soil. Received: 5 May 1996 / Revision received: 23 August 1996 / Accepted: 5 October 1996     

Micropropagation of Ulmus pumila L. from mature trees

Summary Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l^–1 BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l^–1 naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.     

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     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

In vitro micropropagation of a Cucurbita interspecific hybrid cultivar – a root stock plant

An efficient in vitro micropropagation protocol was developed for direct shoot growth of interspecific Cucurbita hybrid variety using shoot–tips of 5-day-old explants. The excised shoot–tips were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and naphthaleneacetic acid) with various combinations and concentrations for the study of shoot induction. The best condition for shoot growth was with 3 mg l^–1 6-benzyladenine (BA) in MS medium. The shooting frequency was 84% and five shoots were obtained from each explant after 30 days of culture. Shoots (11.5 cm length) were rooted most effectively in 1 mg l^–1 indole-3 butyric acid (IBA)-supplemented MS medium. The highest root formation rate was 93% and all rooted shoots were transplanted into soil.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^&#x2212;1 2,4-dichlorophenoxyacetic acid and 0.5&#x2013;2.0 mg l^&#x2212;1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0&#x2013;2.0 mg l^&#x2212;1 &#x03B1;-naphthaleneacetic acid (NAA) and 0.5&#x2013;2.0 mg l^&#x2212;1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^&#x2212;1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^&#x2212;1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

<Emphasis Type="Italic">Aloe vera</Emphasis> transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration

A system for genetic transformation and subsequent plant regeneration via indirect organogenesis from callus was developed for Aloe vera. Young seedlings served as primary explants. Callus cultures were established on Murashige and Skoog (1962) medium supplemented with 3 mg l⁻¹ benzylaminopurine and 2 mg l⁻¹ indole acetic acid. A protocol was developed to switch from the differentiated stage, using in vitro shoots or young regenerated plants, back to the de-differentiated stage of the callus and vice versa. Long-term maintenance of this callus paved the way for genetic manipulation of Aloe vera. Calluses were bombarded with a plasmid containing uidA and hpt genes, both under the control of the 35S promoter. Dithiothreitol and gibberellic acid were found to play a major role in reducing tissue necrosis following bombardment. Transformed shoots were regenerated under stepwise selection in hygromycin-containing liquid medium supplemented with different antioxidants. Amberlite XAD-4 resin was embedded into alginate beads and added to the selection medium. Amberlite was best for adsorbing different phenolic compounds and blocking explant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg l⁻¹ thidiazuron and 0.1 mg l⁻¹ indole butyric acid. Murashige and Skoog medium supplemented with 1 mg l⁻¹ zeatin riboside promoted shoot elongation. Rooting and plant development were obtained on Murashige and Skoog basal medium supplemented with 15 mg l⁻¹ hygromycin lacking growth regulators. The transgenic nature of the regenerated plants was verified by histochemical GUS assay and Southern blot hybridization.     

Efficient plant regeneration in vitro in buckwheat

An in vitro highly efficient plant regeneration system was established from hypocotyl segments in buckwheat (Fagopyrum esculentum Moench.). Calli were induced on Murashige–Skoog (MS) medium containing 1.0 mg l^–1 to 2.0 mg l^–1 2,4-dichlorophenoxyacetic acid and 1.5 mg l^–1 6-benzylaminopurine. Shoot buds were formed on subcultured pieces of callus. A high frequency (over 80%) of shoot differentiation was obtained on MS medium supplemented with 2.0 mg l^–1 6-benzylaminopurine and 1.0 mg l^–1 6-furfurylaminopurine. The regenerated shoots rooted readily on MS medium plus 0.2 mg l^–1naphthaleneacetic acid and 0.2 mg l^–1 indole butyric acid. The regenerated plantlets were acclimatized and successfully transferred to pots. Chromosome examination showed that the regenerated plants had normal chromosome number (2n=16).     

High frequency production of haploid embryos in asparagus anther culture

Summary A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l ^–1 casein hydrolysate, 800 mg l^–1 glutamine, 2 mg l ^–1 NAA, 1 mg l ^–1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l ^–1 NAA, 0.1 mg l ^–1 kinetin and 0.65 mg l ^–1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l ^–1 GA₃. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962)     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

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.     

In vitro callus induction and regeneration studies in Withania somnifera

Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-D (2 mg l⁻¹) and KN (0.2 mg l⁻¹). Shoots differentiated best from axillary shoot base callus on MS medium containing BA (2 mg l⁻¹). Regenerated shoots rooted best on MS medium containing IBA (2 mg l⁻¹) alone, and IBA (2 mg l⁻¹) with IAA (2 mg l⁻¹). Plantlets were transferred to pots containing sand and soil mixture, acclimatized in a culture room and afterwards transferred to the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l^–1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l^–1 ancymidol (A-Rest^TM), 0.1 mg l^–1 NAA and 0.1 mg l^–1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.     

In vitro establishment of a cycloclonal chain from nodal segments and apical buds of adult hazel (Corylus avellana L.)

Apical buds (0.5 cm) and nodal shoot segments (1.5 cm) excised from: A) field-grown branches, B) newly developed shoots from the forced outgrowth of axillary buds on A branches, C) newly developed shoots from the forced outgrowth of axillary buds on A branches submitted to cold storage were used as primary explants. Results indicate that three months cold storage greatly increases morphogenic capacity and reduces contamination and oxidation of tissues. Consequently, a multiplying chain could be easily established by culturing the tissues on a modified Murashige & Skoog (1962) medium plus 6-benzyl-aminopurine 5 mg l^-1, indole-3-acetic acid 0.01 mg l^-1 and gibberellic acid 0.1 mg l^-1. During the initiation and proliferation phases, both the proliferation and the elongation rate were significantly increased when a double-phase culture system (Viseur 1987) was used, giving rise to a higher microplant production than the one obtained using previously described methods. Plant regeneration was achieved by immersing the single microshoot's basal end in an IBA (0.1–1 mg ml^-1) solution for 10 s followed by a 20-day culture on a 1/2 MS2 medium.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - MS1 and MS2 modified Murashige & Skoog media - NAA 2-maphtaleneacetic acid     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10⁶ g fwt^–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10⁴ ml^–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10⁴ ml^–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl^–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l^–1 sucrose, NAA (0.2–0.5 mg l^–1), zeatin riboside (0.5–2.0 mg l^–1) and GA₃ (0.5–1.0 mg l^–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l^–1 agar-solidified B5 medium containing 30g l^–1 sucrose, IBA (0.01 mg l^–1) and BAP (1.0 mg l^–1). Elongated shoots developed roots after transfer to 8.0g l^–1 agar-solidified, hormone-free MS medium with 30 g l^–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA³ gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid