Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

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.     

Effects of 3,4-dihydroxybenzoic acid on tobacco (Nicotiana tabacum L.) cultured in vitro. Growth regulation in callus and organ cultures

ABSTRACT

The influence of 3,4-dihydroxybenzoic acid (protocatechuic acid), a naturally occurring benzoic acid derivative, on tobacco (Nicotiana tabacum L.) cell and tissue cultures was examined. The response to 0.1, 10 and 1000 µM 3,4-dihydroxybenzoic acid was tested with regards to cell proliferation in leaf explants, callus growth and shoot formation. Effects on shoot and root growth in micropropagated plants were also analysed. The highest concentration of 3,4-dihydroxybenzoic acid strongly inhibited the proliferation of leaf tissues, callus growth, shoot regeneration and root growth in micropropagated plants. On the contrary, the lowest concentration (0.1 µM) showed auxin-like activity by stimulating cell dedifferentiation, callus induction and rooting of leaf tissues. The presence of auxins and cytokinins in the media contrasted 3,4-dihydroxybenzoic acid inhibition of callus growth at all tested concentrations.     

Shoot regeneration from in vitro-derived leaf and root explants of <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>

In vitro culture is currently used to produce plant material for ex situ conservation of endangered species. In this study, an efficient protocol for shoot regeneration from leaves and roots was developed for Centaurea ultreiae, a critically endangered species. Organogenesis from leaf and root explants was promoted by incubating these explants on half-strength Murashige and Skoog (MS) medium in the presence of one of four different cytokinins [6-benzyladenine (BA), zeatin, kinetin or N⁶-(2-isopentenyl) adenine (2iP)], each provided at five different levels. Shoot organogenesis was induced in both explants. The best response, 90% of leaf explants producing a mean of 2.48 shoots per explants and 94.3% of root explants producing a mean of 5.60 viable shoots per explants, was observed when explants were incubated on a medium containing 0.55 μM BA. Histological studies revealed connectivity between vascular tissues of regenerated shoots and cambial cells of leaf explants. Moreover, adventitious shoots were derived from pericycle cells of root explants and parenchymatic cells of callus tissues.     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

Microwave treatment induced mutations and altered gene expression in <Emphasis Type="Italic">Vigna aconitifolia</Emphasis>

Primary leaf explants of aseptically grown seedlings of moth bean [Vigna aconitifolia (Jacq.) Marechal] immersed in water or not were treated in microwave oven (2450 MHz, 800 W cm⁻²) for 1, 3, 5 and 7 s before culturing. Callusing and shoot emergence from these explants were enhanced up to microwave exposure lasted 5 s while longer treatment of water-immersed explants delayed callusing. One polypeptide (26.6 kD) was up regulated in the callus derived from microwave treatment in water-immersed explants. RAPD analysis detected alteration in DNA sequences due to microwave treatment in water-immersed explants for 7 s. The frequency of mutation was 1.6 % (4 bands out of 248) over all the cultures analyzed and the same was 13 % (4 bands out of 31), if amplicons generated at 7 s treatment alone were considered.     

Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants

Abstract

Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for their anticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenols and ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petiole and root explants are reported. Callus induction and proliferation depended on the genotype and the experimental conditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on medium supplemented with 2,4-D (0.1 mg l^?1) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l^?1) plus 6-benzil-ammino-purine (BAP) (0.22 mg l^?1) for A. sedifolius. The content of total polyphenol and ascorbic acid was estimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lower than in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reduced ascorbic acid pool increased. These findings demonstrate that Aster callus cultures produce antioxidant compounds and as such might be a model system to investigate the regulation and production of these important metabolites.     

Effect of plant growth regulators on morphogenesis and forskolin production in Plectranthus barbatus Andrews

Plectranthus barbatus (syn. Coleus forskohlii) is the only known source of forskolin, a compound with a wide range of pharmacological activities. Here, an efficient protocol for adventitious root regeneration from leaf explants of P. barbatus was developed. Different concentrations of plant growth regulators individually and in combination were used to induce roots in vitro. Morphogenic responses and forskolin production varied depending on the concentrations of plant growth regulators added to the medium. Lower concentrations of auxins trigger callus proliferation while higher concentrations induced adventitious root regeneration. Of all the auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2 (2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP), and 4-amino-3,5,6-trichloropicolinic acid (picloram) induced callus, whereas α-naphthaleneacetic acid (NAA), indole-3-acetic acid, and indole-3-butyric acid induced rhizogenesis. Use of picloram at 1.0 and 0.5 mg l⁻¹ resulted in the formation of friable callus, and when combined with 0.5 mg l⁻¹ 6-benzylamino purine (BA), rhizogenic callus was produced. The cytokinins BA and kinetin produced a mixed response of multiple shoot regeneration, callus proliferation, and rhizogenesis. The maximum forskolin content of 1,178 mg kg⁻¹ dry weight was found in root cultures initiated on Gamborg’s B₅ medium supplemented with 0.5 mg l⁻¹ NAA. The biosynthesis of forskolin was differentiation dependent, and rhizogenic cultures exhibited the maximum biosynthetic potential for forskolin.     

Indirect shoot organogenesis from leaves of Dieffenbachia cv. Camouflage

A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N ⁶-(Δ²-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and morphology have been observed in regenerated plants.     

In vitro morphogenesis from excised leaf explants of Digitalis obscura L.

The morphogenic capacity of Digitalis obscura leaf explants cultured in vitro has been studied, noting factors promoting the differentiation of roots, buds and shoots as well as those promoting callus proliferation. Complete plant regeneration was obtained only by first culturing the leaf explants in a medium with NAA and BA to induce formation of buds, and subsequently transferring them to a medium without growth regulators to achieve the further development of shoots.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid     

The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

As a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil-grown plants and in vitro-grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10^?6 M-1 ×10^?4 M) of the auxins 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and mdole-3-acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil-grown plants. The cytokinins examined 6-benzyl-aminopurine (BAP) and 6-4-hydroxy-3-methyl-2-enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10^?6 M in combination with BAP at a concentration of 1× 10^?6 or 1 × 10^?4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10^?6 M) combined with BAP (1 × 10^?6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil-grown plants, and NAA (1 × 10^?5 M) combined with BAP (1 × 10^?6 or 1 × 10^?5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4-D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4-D and BAP at concentrations of 1 × 10^?6 M allowed differentiation of the resistant and susceptible cultivars. These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.     

Leaf explant response in in vitro culture of St. John’s wort (Hypericum perforatum L.)

For centuries Hypericum perforatum has been used in natural medicine. In the last decades, it has also attracted the attention of pharmaceutical industry due to its promising anti-depressant properties. The important factor in pharmaceutical application of plant material is its stable content of active compounds. Such stability requires standardized conditions of growth, e.g. an in vitro culture. Our aim was to establish a medium allowing for an effective regeneration of shoots from the standardized leaf explants in in vitro conditions. Cultures of the leaf explants carried out in darkness, on Murashige and Skoog agar medium, supplemented with auxins (2,4-dichlorophenoxyacetic acid, 2-metoxy-3,6-dichlorobenzoic acid, α-naphtaleneacetic acid, indole-3-acetic acid) and cytokinins (kinetin, N⁶-(benzyl)adenine, thidiazuron) resulted in callus formation. The callus produced roots on media containing indole-3-acetic acid or α-naphtaleneacetic acid alone. On media supplemented with auxins and cytokinins, indirect shoot organogenesis was also observed. The most efficient shoot formation was observed with 2.85 μM of indole-3-acetic acid and 4.44 μM of benzyladenine. Regenerated shoots were rooted on Murashige and Skoog without plant growth regulators medium or on a medium supplemented with indole-3-acetic acid. From a single leaf explant (one fifth of the leaf) after a month of the culture, 35 regenerated shoots were obtained (allowing for the formation of about 180 vegetative shoots per leaf). Successful multiplication of shoots from a standardized explant makes it possible to obtain a great quantity of uniform plant material for biotechnological purposes.     

Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum ait

Callus and suspension cultures, established from seedling and leaf explants of Solanum laciniatum Ait were analysed for solasodine using a spectrophotometric assay. Solasodine concentration in both types of culture ranged from 0.5 – 1.0 mg/g dry wt., with a small number of callus explants containing higher concentrations. There was no overall fall in concentration as a result of serial subculture, and in suspension cultures the level remained constant throughout a single passage. Solasodine concentration was enhanced by the induction of organogenesis in both primary leaf explants and callus. ABA, added at 0.04 mg 1^?1, increased solasodine yield in callus cultures whilst CEPA, at concentrations of 10 mg 1^?1 and higher, inhibited production. Dark grown callus contained significantly more solasodine than light grown.     

Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

In vitro Regeneration of Gerbera jamesonii Bolus from Leaf and Petiole Explants

Regeneration of adventitious shoots from leaf and petiole pieces of Gerbera jamesonii has been obtained on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins and cytokinins. About 75’77 per cent of the calli from both types of the explants produced 12’15 shoots per callus with 3 mg l^?1 SAP. Auxins and kinetin, separately failed to produce shoots. The shoots regenerated on the callus induction medium (elM). The regenerated shoots multiplied with 1 mg l^?1 SAP, were rooted on MS medium containing 1mg l^-1 BAP + 0.1 mg l^-1 IAA. The plants obtained were transferred to pots and acclimatized with 60’70 per cent success.     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

Biosynthesis and accumulation of a medicinal compound,Picroside-I,in cultures of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.     

Benzyladenine-induced inhibition of flowering in Chenopodium rubrum in vitro is not related to the levels of isoprenoid cytokinins

The effect of 6-benzylaminopurine (BAP) on floweringand on endogenous levels of isoprenoid cytokinins wasinvestigated in explanted terminal shoots of Chenopodium rubrum cultivated in vitro. Themother plants were grown under continuous light andexplants were cut off when the 6th leaf primordiumoriginated at the shoot apex. The explants wereexposed to one dark period of 13 hours inductive forflowering or to continuous light on medium with orwithout BAP (0.05;0.2;0.4 mg.l^-1). Undernon-inductive conditions no flowering was observedeither in the control or after BAP treatment. Afterreceiving one inductive dark period, the controlexplants flowered. However, BAP application either atthe beginning of the inductive dark period and/orduring the following light cultivation inhibitedflowering and stimulated initiation and growth of leafprimordia. In the case of the most efficient BAPconcentration (0.05 mg.l^-1) flowering wasinhibited by 80% and the number of leaf primordia wasincreased by 3. Explantation caused a significantincrease in the total amount of endogenous cytokininsin the explants within first 13 h, provided they werekept in light. When explants were kept in darkness,only a slight increase in cytokinin levels wasobserved. BAP treatment had no influence on the levelsof endogenous cytokinins either in light or indarkness. We may thus conclude, that BAP applicationinhibited flowering of photoperiodically inducedterminal shoot explants and stimulated leaf primordiaformation with no significant effect on changes inlevels of endogenous isoprenoid cytokinins. This maysuggest the direct ability of BAP to regulate morphogenesis.