Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Summary Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - CA casamino acids - B5CA B5 with 0.2% casamino acids - IBA indolebutryric acid; Picloram (4-amino-3,5,6-tricnloro-2-pyridinecarboxylic acid) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - BA 6-benzylaminopurine     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

In vitro organogenesis and somatic embryogenesis from leaf explants of Leucosceptrum canum sm.

Plantlets were obtained from leaf explants of a Labiatae tree — Leucosceptrum canum Sm. using plant tissue culture techniques. Two types of calli proliferated from the leaf explants when grown on different media, one of which was amenable to somatic embryogenesis. Differentiation of the embryoids started from the fourth passage of culture and continued up to the seventh passage. The number of embryoids decreased with the age of the callus. The capacity of such embryoids to form entire plantlets was studied using different nutrient mileux. Embryoids formed plantlets on Murashige and Skoog's (MS) medium fortified with benzylaminopurine plus indolebutyric acid. Organogenesis was observed in shoot-buds derived from explants of in vitro regenerated plantlets on MS basal medium supplemented with benzylaminopurine. Culture regenerated plantlets were transferred to MS medium without sucrose and growth hormones; finally transferred to pots containing sterile vermiculite where they are growing.Abbreviations MS Murashige and Skoog's medium - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn kinetin - BAP benzylaminopurine - CW coconut water     

Application of the disk method: responses to growth regulators

The paper disk method of screening several plant growth regulators was evaluated. Leaf explants ofVigna unguiculata (L) Walp. were placed on solidified Murashige and Skoog's minimal organics medium containing 0.5 mg/l nicotinic acid. Hormones were tested, singly and in combinations, on paper disks in large Petri plates (150×20 mm). Hormones tested were 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), picloram (4-amino-3,5,6-trichloropicolinic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), BA (6-benzyladenine), 2iP (2-isopentenyl adenine), and kinetin [6-(furfurylamino)-purine]. Root formation was stimulated by IAA and IBA; dicamba, picloram, 2,4-D, and 2,4,5-T stimulated callus formation. All cytokinins tested suppressed root formation. Dicamba in combination with either 2iP or kinetin induced the greatest callus formation. Root formation was optimal with kinetin and either IAA or IBA. The disk method provided a rapid, nonquantitative evaluation of callus and root formation from leaf disks.     

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     

Somatic embryogenesis and plant regeneration of squash Cucurbita pepo L cv. YC 60

Plant regeneration from tissue cultures of summer squash, Cucurbita pepo L., cv. YC60, has been observed. Somatic embryos organized from shoot apex derived callus cultured on Murashige and Skoog (MS) medium supplemented with 1.2 mg/l 2,4,5-trichlorophenoxyacetic acid, 0.8 mg/l benzylaminopurine, and 0.1 mg/l kinetin. Embryos developed into plantlets by transfer of immature somatic embryos to MS medium with 0.05 mg/l NAA and 0.05 mg/l kinetin. Regenerated plants appeared morphologically normal and set fruits with seeds which could germinate normally.Abbreviation BAP 6-benzylaminopurine - 2,4-D 2, 4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - KN kinetin - NAA -naphthyleneacetic acid - MS Murashige and Skoog - 2,4,5-T 2, 4,5-trichlorophenoxyacetic acid     

Plant regeneration from mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

A three media transfer system for direct somatic embryogenesis from leaves ofSenecio x hybridus Hyl. (Asteraceae)

Bisected leaves were cultured on semi-solid Murashige & Skoog medium amended with 13.5 M 2,4-D and 4.5 M BA for 7–14 days, transferred to 0.5% activated charcoal medium (without growth regulators) for three days, then transferred to MS basal medium. Control explants remained on initiation medium for comparison to transferred explants. Twenty-eight days after initiation of cultures, explants exposed to 11–14 days on induction medium yielded the largest number and most developmentally advanced embryos. Mature, white somatic embryos from transferred explants were visible after 35 days. Conversely, somatic embryos on control explants were not morphologically mature until 2–4 months. Mature embryos from transferred explants germinated without a resting stage, whereas embryos from control explants appeared quiescent. Histological examination confirmed embryo anatomy, however embryos, regardless of treatment, had abnormal cotyledons and regenerated plants had multiple stems.Abbreviations AC activated charcoal - BA 6-benzylaminopurine - CrAF III chromium trioxide, acetic acid and formaldehyde - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1-naphthalenacetic acid     

Regulation of organogenesis and somatic embryogenesis by auxin in melon,Cucumis melo L.

Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA 3indoleacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Influence of auxin on the establishment of bilateral symmetry in monocots

To study the influence of auxin on the shift from radial to bilateral symmetry during monocot embryogenesis, the fate of young wheat (Triticum aestivum L.) zygotic embryos has been manipulated in vitro by adding auxins, an auxin transport inhibitor and an auxin antagonist to the culture medium. The two synthetic auxins used, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), induced identical phenotypes. In the most severe cases, the shift from radial to bilateral symmetry was blocked resulting in continuous uniform radial growth. The natural auxin indole-3-acetic acid (IAA) induced the same phenotype. The effect of 2,4,5-T and 2,4D depended on their concentrations and on the developmental stage of the isolated embryos. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin transport inhibitor, the overall embryo symmetry was abnormal. The relative position of the shoot apical meristem in comparison with the scutellum was anomalous. The quality of shoot apical meristem and the scutellum differentiation was altered compared with normal developed embryos. No root meristem was differentiated. The effect of TIBA depends on its concentration and on the developmental stage of the isolated embryos. By contrast, 2-(pchlorophenoxy)-2-methylpropionic acid (PCIB) which is described as an auxin antagonist, has no visible direct effect on the embryonic symmetry. These observations indicate that auxin influences the change from radial symmetry to embryonic polarity during monocot embryogenesis. A model of auxin action during early wheat embryo development is proposed.     

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     

Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.)

Whole plant regeneration via somatic embryogenesis was obtained in pea (Pisum sativum L.) using explants from immature embryos or shoot apex segments. The induction of somatic embryos required picloram or 2,4-D. Germination of fully-developed embryos was accomplished by subculture on medium with only cytokinin and then on medium supplemented with cytokinins in combination with a reduced auxin concentration. Plantlets obtained from both zygotic embryos and shoot apices were transferred to soil and were grown to maturity. Nine plants were examined cytologically, revealing three tetraploids (2n=4x=28) and six diploids (2n=2x=14).Abbreviations Picloram 4-amino-3,5,6-trichloropicolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - IBA indole-3-butyric acid KAES Journal Article No. 87-3-4     

Influence of photoperiod and medium formulation on peanut somatic embryogenesis

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of -naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) - B5 Gamborg et al. (1968) - picloram 4-amino-3,5,6-trichloropicolinic acid - FN Finer & Nagasawa (1988) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Origin of somatic embryos from cultured shoot tips ofSorghum bicolor (L.) moench

Summary Histologic examination of shoot-tip explants, 1 wk after culture initiation on Murashige and Skoog medium with 2.5 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/liter kinetin, reveals active meristematic centers inside cultured tissue. Clusters of cells in these meristematic centers exhibit remarkable resemblance to the initial three divisions in the zygotic embryo. Several such meristematic groups of cells are observed in the cultured explant at this stage. Embryogenesis is obviously initiated very early in this tissue in the presence of 2,4-D. A well-defined, white globular embryogenic callus develops in culture in about 4 wk, and it consists of clusters of embryoids with large cells characterized by thick cell walls, numerous lipoidal vesicles, and localized areas of carbohydrate storage. These cells resemble the scutellar tissue of the embryo. However, there are cells within this tissue that themselves appear embryogenic. They undergo cell division giving rise to small clusters of cells. As long as 2,4-D is present in the medium, the cells apparently retain the capacity to proliferate and to produce more cells capable of embryogenesis. Embryogenesis seems to occur via two processes, initiation of somatic embryos early in culture and secondary embryogensis from the scutellar tissue that forms in vitro.     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.