Preliminary observations on organogenesis inTaraxacum officinale tissue cultures

Summary Tissue cultures ofTaraxacum officinale have been isolated from the secondary thickened root. Callus development and leaf and root formation occur on a basal medium supplemented with coconut milk and IAA or NAA, and the addition of kinetin to these media enhances callus growth and organogenesis. Cultures grown on the basal medium with coconut milk and 2,4-D show only callus growth, but organogenesis is induced by the substitution of IAA for 2,4-D. In the 2,4-D grown callus a layer of secondary meristematic tissue is present and organogenesis apparently occurs from localized regions of this tissue which have undergone de-differentiation to the primary meristematic condition.     

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     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

THE EFFECTS OF GIBBERELLIC ACID AND A GROWTH RETARDANT ON OVULE FORMATION AND GROWTH OF EXCISED PISTILS OF NIGELLA RANUNCULACEAE

The effects of gibberellic acid (GA₃) and the growth retardant AMO-1618 on ovule formation in excised pistils of Nigella sativa L. were studied by sterile culture techniques. Gibberellic acid promoted pistil growth and inhibited ovule formation. The role of endogenous gibberellins in ovule formation and pistil growth was investigated by adding AMO to the basal medium. Both pistil lengths and ovule formation were reduced significantly with increasing concentrations of AMO. The addition of low concentrations of GA₃ to the medium restored pistil growth but did not reverse the inhibitory effect of AMO on ovule formation. The addition of kinetin or indoleacetic acid (IAA) to the medium containing AMO had no effect on pistil lengths. However, with the addition of 10⁻⁷ m kinetin, the number of ovules in pistils was increased but not to the levels found in pistils grown in the absence of AMO.     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

THE EFFECTS OF HORMONES UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on defined medium containing kinetin, indoleacetic acid (IAA), or gibberellic acid (GA₃). Only when 10⁻⁶ or 10⁻⁷ m kinetin was added to the basal medium was there a significant increase in the number of initiated whorls of primordia. Buds on the basal medium or on medium with IAA or GA₃ failed to initiate carpels. On medium with 10⁻⁶ or 10⁻⁷ m kinetin, buds successfully initiated a normal whorl of five carpels. A high level of inorganic nitrogen was also required for the initiation of carpels. With 10⁻⁵ m kinetin, individual buds initiated from 6–18 carpels. Staminodial primordia of these buds were replaced with carpels, or the floral apex enlarged to accommodate a single whorl of many carpels. Kinetin did not support the further differentiation of the floral organs. Sepals, petals, and carpels did differentiate on medium with GA₃, but stamens aborted. However, on medium with GA₃ and kinetin, stamen primordia differentiated into short filaments and anthers. Further unknown growth factors appear to be required for the complete differentiation of floral primordia into mature organs.     

Morphogenesis in Tissue Cultures of Different Organs of Nigella sativa

Calli were isolated from root, stem and leaf segments of Nigella sativa (Fam. Ranunculaceae) on White's medium containing napthaleneacetic acid and coconut milk. From all the three types of calli, roots were differentiated. Shoot development occurred in stem and leaf calli only after the omission of first coconut milk and then of auxin from the basal medium and also in IAA (2.0 mg/ml) and coconut milk (15 % v/v) in the medium. Frequency of organ formation and the maintenance of this capacity depend upon the nature as well as the age of the callus tissue.     

<Emphasis Type="Italic">In vitro</Emphasis> stem elongation of stemless carline thistle

In vitro culture of stemless carline thistle was established using immature zygotic embryos. A satisfactory bud multiplication was achieved on MS medium supplemented with BAP (1 mg L^–1) and IAA (0.2 mg L^–1). Maximum rooting of buds was induced upon short cultivation (4 or 8 days) on an auxin-supplemented medium. Highest number of roots was obtained with NAA in the medium while the longest roots developed on the IAA-supplemented medium. Plantlets that subsequently developed were in rosette form if grown in light (16/8 h light to darkness photoperiod) and with elongated stems if raised in darkness. Light grown plantlets treated with GA₃ showed dose dependent stem increase in length, reaching maximum at the concentration of 10^–4 M. This was correlated with the length and the average number of internodes. If cultivated in the presence of ancymidol, dark grown plantlets showed reduced stem length. However, the inhibitory effect of the growth retardant on stem elongation was completely overcome by the addition of GA₃.     

In vitro development of plantlets from axillary buds of Acacia auriculiformis — a leguminous tree

Multiple shoots have developed from axillary buds excised from in vitro grown seedlings of Acacia auriculiformis on Gamborg's (B5) basal medium supplemented with coconut milk (5 or 10%) and benzylaminopurine (10^-6M). These shoots, if transferred individually to indole-3-acetic acid (10^-7M) or naphthaleneacetic acid (10^-6 or 10^-7M) augmented B5 medium, produced roots at their base.Abbreviations B5 Gamborg's basal medium (BM) - CM coconut milk - BA 6-benzylaminopurine - Kn kinetin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Evaluation of the effect of three growth regulators in the germination of <Emphasis Type="Italic">comparettia falcata</Emphasis> seeds under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Summary The effect of 3-indoleacetic acid (IAA), 6-furfurylaminopurine (kinetin), and gibberellic acid (GA₃) on germination of the orchid Comparettia falcata was evaluated in a factorial experiment (4×4×4) with Murashige and Skoog (1962) basal medium. It was established that seeds of this orchid could be maintained under aseptic conditions as long as the necessary nutrients and appropriate concentrations of growth regulators were provided. Of the three growth regulators used, IAA significantly decreased seed germination of Comparettia falcata. There was a synergistic effect in the kinetin:GA₃ combination that produced a positive response in both percentage seed germination and development of seedlings. This study describes a single medium-based protocol able to achieve more than 160000 seedlings within 21 wk, starting from a single capsule, sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.     

In vitro plant regeneration of Hedychium roxburghii blume through rhizome-meristem culture

Full grown plants have been raised successfully in vitro by culturing meristems excised from rhizome of Hedychium roxburghii Blume on Murashige and Skoog's medium supplemented with NAA and kinetin. Zeatin associated with IAA stimulated the development of buds on the basal region of the stem. A low concentration of NAA (0.2 mg.1^-1) enhanced the root formation on young excised buds.     

In vitro propagation of Populus x wilsocarpa — a hybrid of ornamental value

Populus x wilsocarpa, a hybrid of important ornamental value, cannot be seed-propagated, nor grafted, since a compatible rootstock has not been identified. A micropropagation protocol consisting of a series of steps was therefore developed to facilitate the commercial production of this species. The technique involved the transfer of swelling buds to a growth initiation medium with the following composition: N6 macronutrients, MS micronutrients and vitamins supplemented with 0.5 mg l^-1 BAP. The best buds were from dormant twigs, stored at 0–2°C and then forced to burst prior to culture initiation. Shoot multiplication was on a basal WPM medium including 0.1 mg l^-1 BAP and 0.001 mg l^-1 NAA. Shoot elongation and rooting was also on a basal WPM medium supplemented with 1.0 mg l^-1 GA₃ followed by a transfer to a peat-perlite mix in the greenhouse.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid (GA₃) - MS Murashige and Skoog [17] - NAA naphthaleneacetic acid - N6 medium [Chu et al., 7] - WPM woody plant medium [16]     

Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var ‘capitata’)

Summary Protoplasts were enzymatically isolated from the first leaves of cabbage (Brassica oleracea var capitata, F1 hybrid Baochun). Sustained cell division and somatic embryogenesis were obtained after culturing the protoplasts in modified liquid DPD medium supplemented with CaCl₂ · 2H₂O 800 mg/l, 2,4-D 0.5 mg/l, kinetin 1 mg/l, 0.3 M mannitol and sucrose 20 g/l. Upon transferring cell colonies onto a modified Murashige and Skoog (MS) agar medium, small calli were gradually formed. Callus proliferated on MS medium supplemented with hormone combinations of 2,4-D 0.1–0.5 mg/l and kinetin 3–4 mg/l. Multiple shoots were induced on differentiation medium supplemented with 3 mg/l of kinetin and 0.1 mg/l of gibberellic acid GA₃. After transferring differentiated shoots onto MS medium supplemented with indoleacetic acid (IAA), kinetin, GA₃ at 0.1 mg/l each and 500 mg/l of N.Z. amine, intact plants were eventually produced.     

Regulation of seed germination and polarity in seedling development inOrobanche aegyptiaca by growth substances

InOrobanche aegyptiaca PEES. (Orobanchaceae) the mature seed is tiny and contains a subglobose embryo which is not differentiated into radicle, hypocotyl, plumule, and cotyledons. In aseptic seed cultures on medium TB supplemented with yeast extract or coconut milk, both roots and shoot originated from the morphological radicular pole of the embryo (monopolar pattern). The bipolar mode of seedling formation, that is a shoot originating from the plumular pole and roots from the radicular pole, ensued on the basal medium THS and on TB supplemented with certain concentrations of IAA, kinetin, GA3, or strigol.     

Der Einfluß von Wachstumsregulatoren auf Protein- und Enzym-Spektren kultivierter Explantate aus Rüben von Cichorium intybus L.

Summary On a simple nutrient medium in explants from roots of Cichorium intybus form shoots visible after about 14 days. Gibberellic acid (GA₃) does not influence the spontaneous development of the chicory explants. GA₃ in combination with kinetin inhibits shoot formation whereas kinetin alone promotes the process. On the other hand high concentrations of IAA inhibit the regeneration of shoots.The soluble proteins of chicory cultures treated with growth regulators were examined by disc-electrophoresis. It was shown that the proteins detected by staining with Amido black, phosphatases, esterases and glutamate dehydrogenase (GDH) present in the original root tissue remained constant under the different culture conditions during a period of 12 days. The quantitative changes of some of the proteins, phosphatases and esterases observed during the culture period were identical for all the different cultures in spite of the different morphogenetic behaviour. Only the activities of GDH and peroxidase changed after treatment with different growth regulators; however, in these cases, there was also no direct connection with the morphogenetic responses of the cultures.The specific activity of the GDH-band was promoted by IAA and at the same time the formation of peroxidases was inhibited. Kinetin delayed the formation of peroxidases during the first days of the culture period but promoted it later on. There was a repression by IAA of a specific kationic peroxidase. In the tissues treated with GA₃ the activity of peroxidases was always higher than in the control tissue. This effect of GA₃ can be partly explained by the fact that GA₃ inhibits the release of peroxidases of the explants into the nutrient medium.     

Plant regeneration via somatic embryogenesis from protoplasts of Uganda cherry orange (Citropsis schweinfurthii)

Protoplasts isolated from embryogenic callus of Citropsis schweinfurthii (Engl.) Swing. & M. Kell were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA, 0, 300, 600 or 900 mg l^–1 malt extract and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg l^–1 BA and 600 mg l^–1 malt extract. MT basal medium containing 5% sucrose and supplemented with 0.01 mg l^–1 kinetin was found to be a medium suitable for the development of globular somatic embryos derived from protoplasts into heart-shaped somatic embryos with cotyledon-like structures. The highest percentage of shoot formation was obtained using 0.1 mg l^–1 GA₃. A complete protoplast to-plant system was developed for C. schweinfurthii, which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃ - ME malt extract     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.