Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture

The effect of genotype, growth regulators and preconditioning of donor plants on callus induction in anther culture of flax was investigated. Anthers were cultured on modified MS medium supplemented with five different combinations of plant growth regulators. The results suggested that specific combinations of growth regulators must be designed for each genotype. Major differences between the present results and previous reports are discussed. The influence of sucrose concentration was also investigated. For flax cultivar, 'Mikael', callus induction was higher in medium supplemented with 1 mg l(-1) BAP and 2 mg l(-1) 2,4D containing 6% sucrose, while this combination of growth regulators significantly increased callogenesis in cultivars 'Lirina', 'Barbara' and 'Szaphir' when supplemented with 9% or 12% sucrose. The preconditioning of donor plants influenced callogenesis in subsequently isolated anthers. Anthers from donor plants grown at a lower temperature (18/14 degrees C) significantly increased callus induction over those from plants grown at a higher temperature (22/18 degrees C), although each genotype still required optimization of growth regulator combinations in the induction medium. Only 'Mikael' regenerated shoots when the callus was from induction medium supplemented with 2 mg I(-1) BAP and 1 mg l(-1) NAA.     

Cultivation of Iris ensata Thunb. callus tissue

A continuous callus culture was obtained from zygotic embryos of Japanese iris (Iris ensata Thunb.) on the Murashige-Skoog medium supplemented with 2 mg/l alpha-naphthylacetic acid and 0.5 mg/l 6-benzylaminopurine (BAP). It was found that a successful callusogenesis required isolated embryos at the wax stage of endosperm development. The optimal combination of phytohormones for the growth of callus tissue was 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l BAP. The pigment composition of I. ensata callus tissue was studied. It was demonstrated that subcultivated callus tissue contained red pigments of flavonoid nature. Under stress cultivation conditions, yellow pigments were formed and the content of red pigments increased.     

Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Biosynthesis of precious metabolites in callus cultures of <Emphasis Type="Italic">Eclipta alba</Emphasis>

Eclipta alba (False daisy) is an important medicinal plant with well-known antihepatotoxic activity. However, no previous in vitro studies are available for its callus culture for increased production of antioxidant secondary metabolites. Herein, we maintained a competent protocol for callus culture of E. alba using stem and leaf explants grown on MS medium containing various concentrations of thidiazuron, 6-benzylaminopurine (BAP) either alone or in association with α-naphthalene acetic acid (NAA). Among all the applied plant growth regulators, BAP along with NAA resulted in maximal dry biomass of 18.0 and 13.8 g/l for stem and leaf explants, respectively. Furthermore, the highest production of phenolics (375.7 mg/l for stem-associated callus and 298 mg/l for leaf-associated callus) and flavonoids (62.0 and 52.3 mg/l for stem- and leaf-associated callus, respectively) were found to be present in optimized callus culture. Antioxidant activity was also elucidated for both stem and leaf derived calli. The highest antioxidant activities (~?93.5%) were witnessed for stem and leaf associated calli at set concentrations of 3.0 mg/l BAP?+?1.0 mg/l NAA and 4.0 mg/l BAP, respectively. High-performance liquid chromatography analyses revealed optimum accumulation of coumarin (1.98 mg/g DW) and wedelolactone (49.63 mg/g DW) in leaf associated callus and desmethylwedelolactone (69.96 mg/g DW), β-amyrin (0.8179 mg/g DW) and eclalbatin (0.3202 mg/g DW) in stem associated callus at optimized concentration.     

A simple culture method for Brassica hypototyl protoplasts

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2–4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4–6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3–4 weeks.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4 — dichlorophenoxyacetic acid     

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc], an under-utilised grain legume

The morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

Induction of direct somatic organogenesis in onion (Allium cepa L.) using a two-step flower or ovary culture

A novel method for direct organogenesis in onion (Allium cepa L.) resulting in the formation of multiple shoot structures induced on mature flower buds or ovaries in a two-step culture procedure is described. Flowers were cultured on an induction medium containing 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l 6-benzylaminopurine (BAP). After 6 days (superior to 3 or 12 days), flowers or extracted ovaries were transferred to a differentiation medium containing 2 mg/l thidiazuron (TDZ). Medium solidification with gellan gum was superior to agar or agar/gellan gum mixture. A similar regeneration frequency was achieved at high (100 g/l) and lower (50 g/l) sucrose content. Regeneration was obtained from all 12 cultivars or inbred lines examined, although the efficiency and the occurrence of hyperhydricity varied depending on genotype and procedure used. Studies of plant growth regulators revealed that in the induction medium, the auxin 2,4-D was superior to 5 mg/l naphthaleneacetic acid or picloram, which partially or completely inhibited regeneration. Omitting cytokinin in the induction medium or substitution of BAP with 2 mg/l 2iP lowered regeneration, while substitution with 1 mg/l TDZ was equally effective. In the differentiation medium, lower concentrations of TDZ (1 and 0.5 mg/l) or substitution of TDZ with 5 mg/l BAP were equally or less effective. Received: 14 October 1998 / Revision received: 19 November 1998 / Accepted: 30 November 1998     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Specific Features of Cultivation of Iris ensata Thunb. Callus Tissue

A continuous callus culture was obtained from zygotic embryos of Japanese iris (Iris ensata Thunb.) on Murashige–Skoog medium supplemented with 2 mg/l -naphthylacetic acid and 0.5 mg/l 6-benzylaminopurine (BAP). It was found that successful callusogenesis required isolated embryos at the wax stage of endosperm development. The optimal combination of phytohormones for the growth of callus tissue was 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l BAP. The pigment composition of I. ensata callus tissue was studied. It was demonstrated that subcultivated callus tissue contained red pigments of flavonoid nature. Under stress cultivation conditions, yellow pigments were formed and the content of red pigments increased.     

Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.)

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.     

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).