One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

Summary The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl₃ or ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.Abbreviations BA 6-benzylaminopurine - 2 4-D 2,4-dichlorophenoxyacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid - FW fresh weight - MS Murashige and Skoog (1962)     

Plant regeneration from cultured leaf explants of eight wild tomato species and two relatedSolanum species

Summary Optimum conditions for plant regeneration from cultured leaf explants were ascertained for eight different wild tomato (Lycopersicon) species and two closely relatedSolanum species. Of the eight media tested, basal MS medium supplemented with 5 μM of the cytokinin 6-benzyladenine proved to be the best overall regeneration medium. Regeneration frequency varied significantly between species with maximum frequency of regeneration observed forL. chilense, L. peruvianum, andS. lycopersicoides.     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Effect of calcium restriction on cardenolide accumulation in two cell lines of Digitalis thapsi grown under different light regimes

The effect of calcium-deprivation on growth and the production of cardenolides in two undifferentiated cell lines of Digitalis thapsi maintained under three different light regimes (16 h photoperiod, darkness, or continuous light) was investigated. Growth was stimulated by continuous light in both cell lines cultured in complete medium. The light regime did not affect cardenolide accumulation in the cells of the hypocotyl-derived line; by contrast, continuous light or darkness increased the production in the leaf-derived line. The elimination of calcium favoured cardenolide production independently of the origin of the suspensions and the light regime, this beneficial effect being predominantly manifested under continuous light.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Regeneration of plants from cultured root explants of mothbean (Vigna aconitifolia L. Jacq. Marechal)

Summary Decapitated seedling root explants of seven cultivars of mothbean (Vigna aconitifolia) cultured on Murashige and Skoog's basal medium without any phytohormone gave rise to plantlets at the end of 4–5 weeks. Addition of cytokinins such as BA, Z, Kn and 2,i-P enhanced the frequency of plant regeneration and also the average number of shoot buds/culture. The buds originated directly from cortical cells or through callussing and subsequent differentiation from the surface. The plantlets obtained were successfully transferred to the field.Abbreviations BA benzyladenine - 2,i-P 6---dimethylallylaminopurine - Kn kinetin - TIBA 2,3,5-Triiodobenzoic acid - Z zeatin     

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.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

Morphogenesis from cultured leaf tissue ofSorghum bicolor-culture initiation

Summary In this paper we present further studies on the generation of tissue cultures from leaves of the cerealSorghum bicolor (L.) Moench. It could be shown that during differentiation the leaf tissue rapidly loses the ability to respond to conventional tissue culture techniques. This was probably related to a loss of sensitivity towards 2,4-D, an otherwise most potent growth regulator in tissue culture. The immature tissue which proved to be sensitive proliferated over a wide range of concentration with a broad optimum of about 0.6–6 mg 1^–1 2,4-D. This concentration range appears to be only slightly higher than that described for many dicotyledonous tissue cultures. The relevance of these findings is discussed with reference to the well known dual function of 2,4-D, namely as a selective herbicide and a potent artificial auxin. The implications of these attributes to the practical application of cereal tissue culture is stressed.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - 4-CPA 4-Chlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA 3-Indoleacetic acid - Kinetin 6-Furfurylaminopurine - 6-BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - ABA Abscisic acid - MS Murashige and Skoog     

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^−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l^−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l^−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l^−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^−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.     

Root and shoot initiation by leaf,stem, and storage root explants of sweet potato

Explants from stem, leaf, and storage root tissue of sweet potato (Ipomoea batatas L.) cv. Jewel, were placed on media conaining 0.1, 1.0, and 10 mg/1NAA with 0.1, 1.0, or 10 mg/1BA in a factorial experiment. Some callus formed in every treatment, but the best callus growth was on media containing 1.0 mg/1NAA and 10 mg/1BA. Roots formed over a range of treatments but were most prolific on the medium containing 1.0 mg/1NAA and 0.1 mg/1BA. Some de novo formed roots subsequently produced shoot buds in culture. Shoot formation increased the longer the original explants remained in culture without subculture. Roots could be subcultured indefinitely on agar solidified medium, but shoot regeneration did not occur after two subcultures. Shoot formation was greatest when the roots were subcultured on medium containing 1.0 mg/NAA and 0.1 mg/1BA. The cultivar Caromex followed the same regeneration pathway, but the number of shoots formed was considerably less. Regeneration in both Jewel and Caromex explants was enhanced by light.Paper No. 8292 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.This work was done as a partial requirement for the M.S. degree at North Carolina State University.     

Carpology,germination and seedling performance in Digitalis thapsi L. (Scrophulariaceae)

Summary

We analyse the fruiting phase of the life cycleof Digitalis thapsi L. We studied the production and size of seeds and capsules, and the interpopulational variability and inter-relationships of these parameters. The capsule volume, number of seeds per capsule, of seeds per individual, and of capsules per individual were inter-related and fairly affected by the medium, whereas seed weight was found to bea more independent character. A germination study showed that the species has agreat germinative capacity and has positive photoblasty. It is non-dormant especially if incubated under experimental conditions close to the climatic conditions of the site of provenance of the samples. An increase in temperature speeds up the process without increasing germinability. The morphological and biometric characteristics of the first stages of development of the species following germination were analysed. It was found that seed weight is not positively correlated with cotyledon length nor with any of the other parameters con-sidered, and that the length of the fifth leaf is a useful parameter to express inter-populational genetic variability. Seedling production in spring seems to be influenced more by physical than by chemical factors.     

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 propagation of Albizia odoratissima L.F. (Benth.) from cotyledonary node and leaf nodal explants

Summary Cotyledonary node and leaf nodal explants excised from 14-d-old in vitro-grown seedlings of Albizia odoratissima were cultured on a Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), N ⁶-(2-isopentenyl) adenine (2-iP) and kinetin, used either solely or in combinations. The highest frequency for shoot regeneration (82.5%), the maximum number of shoots per explant (6.9), and the maximum shoot length (2.55 cm) were obtained from cotyledonary node explants cultured on a MS medium containing 10 μM BAP and 10 μM 2-iP with 30 gl^−1 sucrose. Successful rooting was achieved by placing the microshoots on MS medium with 25 μM indole-3-butyric acid (IBA) for 24h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was best for plant acclimatization, in which 75% of plants survived.     

In vitro adventitious root induction in Antirrhinum majus L.

A broadly applicable method for the successful induction of root systems in a number of cultivars of A. majus has been determined. This involves a double filter-paper bridge with a liquid medium for root induction and allows the transfer of culture-grown plantlets to a glasshouse environment with minimal disturbance to the plant as a whole. 100% survival of transferred plantlets has been achieved with the inclusion of a few simple precautions upon shoot transfer and during the initial stages of plant establishment in vivo.     

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.