Effects of boron on somatic embryogenesis of Daucus carota

Although the micronutrient boron (B) apparently exerts no influences on the induction of somatic embryogenesis of cultured carrot petiole explants, strong influences on the development of somatic embryos were observed (0–8 mg l⁻¹ B). At lower B concentrations the development of roots is promoted with simultaneous retardation of shoot development and at higher B concentrations shoot development is favored at the expenses of the development of the root system. Parallel to this, the ratios of concentrations of endogenous indole-3-acetic acid to total cytokinins (with one exception) changes continuously from 4 (zero B) to 0.22 (8 mg l⁻¹ B). Analogies to morphogenetic reactions following the application of various ratios of auxins/cytokinins to the nutrient medium of cultured tissue (Skoog and Miller, 1957 and others) are suggested. An increase in the B concentration in the nutrient medium results generally in a reduced concentration of endogenous abscisic acid.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Metabriggsia ovalifolia</Emphasis>

An efficient propagation and regeneration system via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia, was established. High activity cytokinins [6-benzyladeneine (BA) and thidiazuron (TDZ)] and low activity auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA)] could directly induce adventitious shoots from leaf or petiole explants within 5 weeks. Cytokinins (TDZ or BA) combined with auxin (NAA) in the induction media induced more adventitious shoots than when auxins or cytokinins were used alone. Adventitious shoots could be induced and also mass-propagated on media containing 2.5–5.0 μM TDZ (or BA) and 0.25–0.5 μM NAA. Adventitious roots differentiated at the proximal end of shoots on rooting media containing half-strength MS salts and 0.5 μM IBA, 0.5 μM NAA, 0.1% activated charcoal or no plant growth regulators. Over 90% of plantlets survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite) in basins.     

Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize

Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0–100 μM) and five auxins (0–5 μM). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 μM) and a higher concentration of picloram (5 μM) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation.     

Influence of growth regulators on somatic embryogenesis in sesame

Somatic embryos were induced from hypocotyl-derived callus of sesame (Sesamum indicum Var. TMV 6). The influence of different auxins and cytokinins on somatic embryogenesis was investigated. Among the different auxins tested, 2,4-dichlorophenoxy acetic acid was the most effective and resulted in the highest frequency of responding cultures and highest average number of somatic embryo per responding cultures napthaleneacetic acid, indoleacetic acid, indolebutyric acid and 2,4,5-trichlorophenoxypropionic acid were also effective for embryogenesis, but 2,4,5-trichorophenoxyacetic acid and napthoxyacetic acid were not beneficial. The combined effect of cytokinins with 2,4-d was also studied. Among the four cytokinins tested, 2.2 chμM benzyladenine with 13.6 chμM 2,4-d slightly enhanced embryogenic efficiency; while kinetin, zeatin, 6-γ-γ-dimethylallylaminopurine enhanced the frequency of responding cultures. There was a decrease in the number of somatic embryos per culture in the presence of all cytokinins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Direct somatic embryogenesis and shoot organogenesis from leaf explants of Primulina tabacum

An efficient propagation system via somatic embryogenesis and shoot organogenesis and plant regeneration system for endangered species Primulina tabacum Hance was established. Thidiazuron (TDZ) was the key plant growth regulator for inducing somatic embryogenesis and kinetin (KIN) and 6-benzylaminopurine (BAP) were the key cytokinins for inducing shoot organogenesis from leaf explants. TDZ combined with BAP or KIN in the induction Murashige and Skoog medium induced both somatic embryos and adventitious shoots. Leaf explants with abaxial site in contact with the medium induced less somatic embryos or adventitious shoots compared to inversely placed leaf explants and the optimum pH was 6.5–7.0. Secondary somatic embryos or adventitious shoot could be induced from primary somatic embryos using TDZ and BAP. Shoots developed adventitious roots on rooting medium containing 0.5 μM indole-3-butyric acid and 0.2 % activated carbon. Over 90 % of plantlets survived following acclimatization and transfer to potting mixture (sand:Vermiculite:limestone; 1:2:1).     

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.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity

An efficient in vitro regeneration protocol was developed for medicinally important aromatic plant Anethum graveolens. Nodal segments were cultured onto Murashige and Skoog (MS) basal medium supplemented with different auxins and cytokinins singly as well as in combinations. The optimum callus induction (93.33 %) was obtained on medium fortified with 2.2 μM N⁶-benzyladenine (BA) and 0.21 μM α-naphthaleneacetic acid. The best shoot regeneration (85.7 %) with 12.86 shoots per explant was achieved in two weeks when callus was subcultured on MS medium amended with 2.2 μM BA and 1.85 μM kinetin. The average length of regenerated shoots varied from 3.15 to 4.8 cm. The rooting of regenerated shoots was nearly 100 % on ? MS augmented with 4.9 μM indolebutyric acid with a maximum root length of 5.1 cm. Plantlets were successfully acclimatized with 60 % survival rate. During organogenesis, catalase and ascorbate peroxidase activity increased while superoxid dismutase activity decreased. Clonal fidelity of in vitro raised plants has been checked by random amplified polymorphic DNA using 10 selected decamer primers. It has been found that regenerated plants are true to type plants.     

Induction of somatic embryogenesis and organogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L., a dye-yielding medicinal plant

Oldenlandia umbellata L., commonly known as “Indian madder”, is an ancient Indian herb valued as a source of red color dye and various medicinal products. In this study, successful protocols have been developed for induction of somatic embryogenesis and organogenesis in O. umbellata. Emerging young leaves, shoot apices, and stems were used as explants, grown on Murashige and Skoog (MS) media supplemented with various auxins, including indole acetic acid, indole butyric acid, napthaleneacetic acid (NAA), and 2,4-Dichlorophenoxyacetic acid, each at levels ranging between 0.1 and 0.5 mg/l, cytokinins, including benzyladenine (BA) and kinetin, each at concentration ranging between 0.5 and 5 mg/l, with and without coconut milk (CM) at levels of 0.5–5%. For callus induction, NAA at 2.5 mg/l was optimal; while, for rapid embryogenic callus induction, 0.2 mg/l NAA, 0.5 mg/l BA, and 0.1% CM induced the highest frequency (95.86%). Shoots developed upon transfer of embryogenic calli to MS medium containing 1.5 mg/l BA, 0.3 mg/l NAA and 1% CM. For root induction, 0.3 mg/l NAA and 1.0% CM promoted highest and earliest rooting. C. Rajasekaran contributed equally to this work.     

Mixture screening and mixture-amount designs to determine plant growth regulator effects on shoot regeneration from grapefruit (<Emphasis Type="Italic">Citrus paradisi</Emphasis> macf.) epicotyls

The objective of this study was to improve shoot regeneration from grapefruit. Because many commercially grown citrus types are apomictic, important in vitro applications such as Agrobacterium-mediated transformation commonly use epicotyl explants from in vitro seedlings; thus, adequate adventitious shoot production is an important prerequisite for efficient use of these applications. Eight plant growth regulators were studied—six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-[γ,γ-dimethylallylamino] purine, zeatin riboside trans-isomer and meta-topolin) and two auxins (α-naphthalene acetic acid and indole-3-acetic acid). An iterative design strategy was followed that included mixture and mixture-amount experimental designs suitable for resolving proportional and concentration effects; in vitro effects of cytokinins and auxins are affected by both proportion and concentration. One-centimeter-long explants were excised from the epicotyl of etiolated, in vitro-grown seedlings. Explants were placed onto experimental formulations and cultured in growth cabinets at 27°C over 6 wk, which included 2 wk in the dark followed by 4 wk in the light. The results indicated that (1) 6-benzylaminopurine or zeatin riboside were the most effective cytokinins for inducing shoot regeneration in citrus; (2) zeatin riboside singly or in combination with indole-3-acetic acid resulted in the highest quality, the greatest number of explants with buds/shoots, and the greatest shoot number; and (3) 6-benzylaminopurine and indole-3-acetic acid improved shoot regeneration vs. 6-benzylaminopurine at a considerably lesser cost than zeatin riboside and indole-3-acetic acid.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot bud differentiation and plantlet regeneration in <Emphasis Type="Italic">Feronia limonia</Emphasis> L. (Swingle)

Summary In vitro adventitious shoot bud regeneration systems are considered most suitable for Agrobacterium-and biolisticsmediated genetic transformation to obtain transgenic plants. In the present investigation, multiple adventitious shoot buds could be induced directly from Feronia limonia hypocotyl explants inoculated on Murashige and Skoog (MS) medium containing different growth regulators. During the initial phase, the hypocotyl segments nearer to the cotyledons responded quickly compared to those closer to the root. The response, however, was comparable in both the segments in subsequent subculture. Of the various cytokinins, 2.22 μM 6-benzylaminopurine (BA) proved to be more effective compared to kinetin (Kn). The two-way interaction of BA and Kn significantly influenced shoot regeneration and contributed the most among the interactions studied. The best response, however, was obtained when 2.22 μM BA and 2.32 μM Kn were combined. Although the effect of auxins like α-naphthaleneactic acid (NAA) combined with cytokinins evoked a significant responsein terns of number of shoot buds, this response did not supersede the effect of combined cytokinins. Vone of the polyamines tested induced shoot buds on hypocotyl segments. Adventitious shoots were multiplied on MS medium containing 2.22 μM BA, 6.96 μM Kn, and 0.05 μM NAA. More than 60% of the shoots produced roots when cultured on medium containing one-quarter strength MS salts, 10% suerose, 0.6% agar, and 7.36μM indole-3-butyric acid. The adventious origin of shoot buds showing continuous vascular connections was confirmed through histological investigations.     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

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.     

In vitro shoot culture and microtuber induction in the steroid yam Dioscorea composita Hemsl

The individual effects of sucrose, plant growth regulators and basal salt media formulations were investigated on microtuber induction and development in shoot cultures of the steroid yam Dioscorea composita. Sucrose at 8% (w/v) was the single most significant medium constituent for microtuber induction. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5 μM showed strong inhibitory effects on microtuber induction. By contrast, the auxins α-naphthaleneacetic acid and indole-3-butyric acid at 5.0 μM showed striking promotive effects on microtuber induction and growth. In the presence of either one of these auxins at 5.0 μM shoot cultures produced microtubers weighing 300–400 mg fresh weight whilst kinetin, 6-(γ,γ-dimethylallylamino)-purine, 6-benzyladenine and abscisic acid failed to promote microtuber growth (microtubers weighed generally <200 mg). Media formulations Lloyd and MacCown and White supported the lowest frequencies of microtuber induction when kinetin was present at 2.5 μM. Anderson Rhododendron was as effective as Murashige and Skoog overall in promoting both microtuber induction and growth. When removed from cultures and planted in sterilized moist sand, microtubers sprouted readily (60–87% within 2 weeks) and produced vigorous shoot growth and after 5–7 months minitubers of sizes (30–80 g) suitable for direct field planting. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oligopeptides as plant growth regulators

It is generally accepted that plant growth and development are regulated by the known plant hormones. Some objections to the functions of auxins and cytokinins in the induction of shoot and root primordia are reported. Instead of them oligopeptides of special amino acid sequences could be the endogenous signals. There exist structure relationships between auxins and parts of the α-helical oligopeptides of defined amino acid sequences. The same is true for cytokinins. The most difficult part of this hypothesis is its verification. Using protonemata ofFunaria hygrometrica bud induction by various oligopeptides was investigated. The most active peptide tested is leucine-tryptophan. On the other hand endogenous oligopeptides containing [¹⁴C]-leucine in the moss protonemata during endogenous bud initiation were looked for. Three to four different oligopeptide spots seem to be related to bud induction.     

Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

The molecular path to in vitro shoot regeneration

Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.