Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro

We succeeded in cultivating onion plants in vitro with a high potential for shoot regeneration. The apex must be destroyed or injured to obtain axillary buds. This capacity was restricted to the abaxial base of the youngest sheaths. It was shown necessary to restore plant individuality before further proliferation; this process constituted one cycle. For successive regeneration each cycle was composed of three steps: shoot proliferation in the presence of a cytokinin, shoot individualization and plant development in the absence of growth regulators. Effect of growth regulators on the physiological status of onion plants cultured in vitro is discussed.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid     

Carbohydrate and nitrogen sources affect respectively in vitro germination of immature ovules and early seedling growth of Impatiens platypetala Lindl.

In vitro germination of 20-day old immature ovules of Impatiens platypetala Lindl. was inhibited at concentrations as low as 50 mM sucrose or mannitol and 100 mM glucose. Younger ovules (12, 14, and 16 days old) were similarly inhibited at 100 mM sucrose.Inorganic nitrogen concentration did not affect germination regardless of ovule age, but seedling fresh weight was significantly less and abnormal development of seedlings was significantly increased by total inorganic nitrogen concentrations higher or lower than 30 mM (at a ratio of 20: 10 mM NO₃ ^-: NH₄ ⁺) in the culture medium.     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

In vitro plant regeneration from leaf callus of Grindelia robusta Nutt

Abstract

A regeneration protocol from leaf explants of Grindelia robusta Nutt. was developed. The combination of 0.5 mg l^?1 IBA plus 0.5 mg l^?1 or 1 mg l^?1 BA added to Murashige-Skoog (MS) medium resulted in the best callus induction frequency; the combination of 0.4 or 0.9 mg l^?1 BA plus 1.2 mg l^?1 GA₃ resulted in the best shoot regeneration. Rooting was successful on MS medium supplemented with 0.5 mg l^?1 IBA. Hardening of G. robusta plants was accomplished in 30 days with 85% survival rate.     

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

Plant regeneration from somatic tissue of Rhododendron laetum x aurigeranum

The influence of the source of plant material (greenhouse-grown plants or in vitro shoot cultures), the type of tissue explant (shoot-tip, single-node stem segment, whole leaf, leaf strip or half-leaf section) and growth regulator concentration on shoot regeneration from somatic tissue of Rhododendron laetum × aurigeranum was evaluated. No regeneration response was obtained on explants from greenhouse-grown plants. Adventitious shoots were obtained from callus produced at the basal end of shoot-tip and single-node stem segment explants derived from in vitro-grown shoots cultured on Anderson's medium supplemented with 22.8 M IAA and 73.8 M 2iP. The greatest percentage of adventitious shoot regeneration (77%) was induced on leaf sections cultured in the presence of 22.8 M IAA and 147.6 M 2iP. Plant regeneration was accomplished with minimal callus formation. This technique represents a further step toward gene manipulation of Rhododendron.Abbreviations IAA 1-H-Indole-3-acetic acid - 2iP N-(3-methyl-2-Butenyl)-1H-purin-6 amine     

In vitro regeneration from excised leaves of Flaveria trinervia (Sprengel) C. Mohr

Flaveria trinervia (Compositae) leaves are used for the treatment of jaundice and fever. From the leaf callus cultures regeneration of plantlets has been achieved. The results showed that BAP greatly stimulated the bud formation in concentrations ranging from 2–5 mg l^–1 than at very low concentrations (0.2–1.0 mg l^–1). Roots developed on the regenerated shoots, over a range of treatments, but were most prolific in the medium containing 1 mg l^–1 IAA. Histological observations revealed that cultured spongy cells of the mesophyll were greatly enlarged and underwent repeated cell divisions leading to the formation of hard nodular callus from which shoot buds differentiated. The shoots obtained were readily rooted and transplanted into glass houses. Cytological studies of the callus showed abnormalities such as bridges, endomitosis and multinucleolate conditions. Root tip squashes of the regenerated plants showed no variations and were diploid in chromosome number.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA napthalene acetic acid - IAA indole acetic acid - BAP 6-benzyl aminopurine - Kn kinetin     

Adventitious shoot regeneration from immature embryos of sorghum

Eleven genotypes of sorghum were examined for their response in tissue culture, and the tissue culture system was optimized. The cultures were initiated from immature embryos taken approximately two weeks after flowering. The response of immature embryos varied with the genotype. `C. Kafir' and `PE932 025' showed the highest frequency of callus induction and regenerable callus formation under appropriate culture conditions. Regeneration occurred at high frequencies when cytokinins (kinetin or 6-benzyladenine) had been added in the callus induction medium, followed by regeneration medium devoid of growth regulators. The addition of proline and polyvinylpyrrolidone also enhanced shoot formation, but the addition of cytokinins to regeneration media did not improve shoot formation. On the revised culture medium, plants were regenerated from up to 100% of sorghum immature embryos.     

In vitro regeneration of plantlets from shoot callus of mature trees ofDalbergia latifolia

A successful procedure was established for in vitro mass multiplication of Indian rosewood (Dalbergia latifolia Roxb.). In vitro regeneration of plantlets was achieved from callus of shoot tips and shoot segments of over 50-year-old elite trees on Murashige & Skoog's medium containing naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). For rooting, regenerated shoots from the calli were excised and first treated with White's liquid medium or half-strength Murashige & Skoog's medium, supplemented with indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid for 48 h to 72 h. Following this treatment, plantlets were transferred to hormone-free half-strength MS medium. Rooted plantlets were then transferred to pots and grown in the greenhouse.Abbreviations BAP 6-benzylamino pruine - CH casein hydrolysate - CM coconut milk - 2, 4-D dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PVP-10 polyvinyl pyrrolidone - YE yeast extract     

Thidiazuron promotes adventitious shoot regeneration from pothos (Epipremnum aureum) leaf and petiole explants

Summary Regeneration of adventitious shoots of pothos (Epipremnum aureum Linden and Andre) ‘Jade’ was obtained using leaf and petiole explants preprated from shoot tips of 3-yr-old greenhouse-grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), 6-(4-hydroxy-3-methy-trans-2-butenyl-amino)purine (zeatin) or N-isopentenylaminopurine (2iP) individually with α-naphthaleneacetic acid (NAA) in 18 combinations. Callus was initiated from cut surface and along the midrib or major vein of leaf sections. Shoot regeneration from leaf and petoole explants occurred in 30d on medium containing 1, 5 or 10μM TDZ with 0.5 or 1.0μM NAA except petioles on medium with 10 μM TDZ and 1.0 μM NAA where regeneration failed. More time (50d) was needed for shoot regeneration when explants were cultured on medium containing either 2iP or zeatin with NAA. Regeneration frequencies were up to 20% and 50% for leaf and petiole explants, respectively. Shoot numbers per responding explant attained 30 for leaf and petiole explants on medium containing TDZ but only one to four on medium containing either 2iP or zeatin. These results indicate that TDZ is a more effective cytokinin for in vitro regeneration of pothos than either zeatin or 2iP.. Shoots elongated readily and rooted well on MS basal medium, without plant growth regulators. Plantlets acclimatized rapidly and grew vigorously in the greenhouse after transfer to pots containing a commerecial potting medium.     

In vitro adventitious shoot formation from embryonic and cotyledonary tissues of Pinus brutia Ten.

Adventitious buds were induced when isolated whole embryos, and excised cotyledons from treated seeds of Calabrian pine (Pinus brutia Ten.) were cultured on a cytokinin-supplemented medium. The adventitious buds formed directly from the cotyledons. The highest number of bud primordia were formed, with both embryos and excised cotyledons, after 6 weeks on a BAP — supplemented Schenk and Hildebrandt medium under a 16 h photoperiod. The buds, when separated and maintained individually on a full-strength medium without growth regulators, developed into well-formed shoots within 4 weeks. The average number of harvested shoots obtained (>1 cm in height) per seed over 24 weeks was 55; however, a maximum number of 152 shoots was obtained from one individual over the same period. The shoot forming capacity of the meristematic tissue was not lost after seven harvests.     

In vitro shoot proliferation and plant regeneration from kurrat (Allium ampeloprasum var. kurrat) seedlings

Shoots were produced from kurrat seedling and mature plant explants cultured in Murashige and Skoog medium (MS) alone or supplemented with 4.4 M benzyladenine (BA). Shoots were also produced from explants through a two-step procedure. Regenerated shoots were induced to form roots on MS medium with 5 g I^-1 activated charcoal. Plants were successfully established in soil.Abbreviations AC activated charcoal - BA benzyladenine - MS Murashige & Skoog's (1962) medium - 2,4-d 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

In vitro regeneration of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

Introduction and establishment of apricot in vitro through regeneration of shoots from meristem tips

Summary In this study different aspects of the in vitro introduction and establishment of apricot cultivars were investigated through meristem tip culture. The best time to introduce the meristems of ‘Canino’ was when buds were starting to swell. Various plant growth regulators were used at different concentrations on four distinct apricot cultivars to promote the development of the meristems to shoots which could then be micropropagated. Very diverse results were obtained depending on the genotype. In general, meristems did not survive without N⁶-benzyladenine. Concentrations of gibberellic acid from 2 to 4 mg 1⁻¹ (5.8–11.4 μM) promoted explant elongation. This step was critically important to obtain apricot shoots large enough to be transferred to proliferation medium.     

High frequency plant regeneration from stem explants of Brassica alboglabra Bailey in vitro

Culture conditions for shoot regeneration and proliferation, and rooting of Brassica alboglabra Bailey were optimized by a judicious selection of explants and manipulation of hormonal combinations in the culture medium. Both half and whole stem explants were more regenerative than cotyledons and hypocotyls. The highest shoot regeneration frequency (100%) accompanied by high number of shoots was obtained using half stem explants grown on Murashige & Skoog [14] medium supplemented with 2 mgl^-1 BA in combination with 1 mgl^-1 NAA, or 4 mgl^-1 2iP with 0.5 mgl^-1 NAA. For shoot proliferation, 4 mgl^-1 kinetin was most effective. The presence of auxin reduced shoot proliferation significantly. Maximum rooting (100%) of shoot cuttings was obtained either in the absence or in the presence of 0.5 mgl^-1 NAA, or IBA or IAA ranging from 0.1 to 8 mgl^-1.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin