Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Somatic embryogenesis and plant regeneration from embryogenic callus of soybean,Glycine max L.

During induction of somatic embryogenesis from immature embryos of soybean, a smooth-shiny and a rough callus were obtained. The smooth-shiny type developed from callus derived from cotyledonary tissue and cultured on media containing 10 mg/l 2,4-D. The rough type was derived from immature embryos, cotyledons, hypocotyls and hypocotyl segments from germinated seedlings on a medium containing various growth regulators. Plants were obtained from the smooth-shiny type but rough callus did not differentiate into plants. The conditions for formation of both callus types and regeneration of mature soybean plants from the smooth-shiny type were defined.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - ABA abscicic acid - GA3 gibberellic acid - BA benzyladenine - B5 Gamborg et al. - LS Linsmaier & Skoog - MS Murashige & Skoog - P proline     

Growth Hormones and Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on solid or liquid medium with macro-nutrients according to Wimber (van Raalte 1967) and iron, micro-nutrients and vitamins according to Nitsch (1968) the medium also contained 2% sucrose. The effects of 1) the auxins; indol-3yl-acetic acid (IAA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D); 2) the cytokinins; 6-furfurylaminopurine (kinetin) and benzyladenine (BA) and 3) the gibberellin; gibberellic acid (GA) were examined alone or in combinations. IAA had no effect alone. NAA resulted in optimal fresh weight at 10 μM and the protocorms were vigorous, but lighter green than usual. 2,4-D caused a high weight increase at 1 μM, but the protocorms were abnormal. Higher concentrations of NAA and 2,4-D inhibited chlorophyll synthesis. On solid medium kinetin (100 μM) induced a growth of many small shoots, but had no effect on the fresh weight. In liquid medium, kinetin promoted a callus formation and fresh weight increase. BA had effects similar to kinetin, but at lower concentrations. GA alone promoted shoot and leaf growth. Combinations of kinetin and NAA resulted in a maximal fresh weight increase at kinetin concentrations one tenth of the NAA concentrations. The optimal growth and the best development occurred at 10 μM NAA and 1 μM kinetin. NAA and kinetin together could limit the shoot and leaf growth induced by GA.     

Plant regeneration from mesophyll protoplasts of the tree legume Pithecellobium dulce benth

Conditions were developed for the isolation, culture and regeneration of mesophyll protoplasts of the tree legume, Pithecellobium dulce Benth. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) was essential to induce initial cell divisions and addition of naphthaleneacetic acid (NAA) improved the response. Sustained division and cell colony formation were achieved from the protoplasts cultured in a modified KM8P medium containing 2,4-D (2.3 μM), NAA (3 μM) and benzyladenine (BA) (2.3 μM). Dilution of the osmotica included in the protoplast culture medium was necessary to induce sustained proliferation of the protoplast-derived cells. Differentiation of shoots from the protoplast-derived calli occurred on Murashige and Skoog (MS) medium supplemented with BA (5 μM) and indole-3-acetic acid (1 μM). Omission of 2,4-D from the culture medium, after the initial 2 weeks of protoplast culture, was obligatory to induce shoot morphogenesis.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

Amyloplast formation in cultured tobacco cells; effects of plant hormones on multiplication, size, and starch content

In BY-2 cultured tobacco cells (Nicotiana tabacum L.), depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) and addition of benzyladenine (BA) caused amyloplast formation, a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and facilitated by the addition of BA. An increase in the starch content of BY-2 cells was always accompanied by a reduction in cell multiplication. However, when hormonal conditions were unsuitable for amyloplast formation, the starch content of the cells did not increase, even if cell multiplication was forcibly terminated by the addition of aphidicolin. This result indicates that the hormonal conditions themselves, and not the decrease in cell multiplication, induce amyloplast formation in BY-2 cultured tobacco cells.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - NAA 1-naphthylacetic acid - PMSF phenyl methyl sulfonyl fluoride     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

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     

新疆雪莲体细胞胚胎发生

通过体细胞胚胎发生途径实现了新疆雪莲（Saussurea involucrata Kar．et Kir．）的植株再生。选用新疆雪莲子叶为外植体,接种于MS＋0．5mg·L^-12,4-D＋0．05—1mg·L^-1BA的固体培养基上,进行愈伤组织的诱导。从第1次继代培养的愈伤组织中挑选出黄绿色、颗粒状、质地致密的腔陛愈伤组织,转移到含0．05—0．1mg·L^-1 2,4-D的MS液体培养基中进行悬浮培养,20天后可分化产生大量球形胚。继代过程中相继加入PEG和GA3,可以促进体细胞胚的分化和生长。体细胞胚在含有5mg·L^-1 GA3的MS固体培养基上,可发育成完整的植株。     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

不同植物生长物质对牛角藓愈伤组织诱导的影响

相同剂量的GA3和2,4-D对牛角藓愈伤组织诱导效果明显,而6-BA、KT和NAA则抑制。光照对愈伤组织的形成有一定的促进作用。     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

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

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

Sapogeninbildung in suspensionskulturen von Digitalis purpurea

The effect of various growth conditions on the saponin level in suspension cultures of Digitalis purpurea has been investigated. The effect of time cultivation, 2,4-D concentration and 2,4-D combined with NAA with/or without casein hydrolysate, on saponin concentrations is described.     

Neo-formation of flower buds and other morphogenetic responses in tissue cultures of Melia azedarach

Melia azedarach has great interest because of its insecticidal properties. Recently, the occurrence of precocious flowering in tissue cultures of this species was reported. This paper describes some in vitro morphogenetic responses using hypocotyl segments as explants and MS basal medium. Amongst the results we report are: (a) in basal medium, 5% of the explants neo-formed floral buds and flowers, and 80% formed vegetative shoots; (b) flower neo-formation could not be controlled or increased by addition of benzyladenine, or lowering the nitrogen level; (c) benzyladenine increased the regeneration of vegetative shoots; (d) compact green calluses were eventually formed in basal medium, and vigorous friable calluses can be easily induced with 0.5 mM 2,4-D; (e) green calluses could be subcultured and regenerated into plants, and, from friable calluses, cell suspensions were started; (f) histological studies showed that neo-formations originate in the wound tissue or from the inner tissue of the hypocotyl.     

Effect of GA3 and 2,4-D foliar application on the anatomy of date palm (Phoenix dactylifera L.) seedling leaf

Two concentrations (10-5M and 10-3M) of both GA3 and 2,4-D were used as foliar spray to evaluate the response of date palm (Phoenix dactylifera L.) cv. Khedri seedlings. They affected some of the anatomical characteristics of the first leaf emerging after the beginning of the spray. The high concentration of GA3 increased the size of the midrib and its vascular bundle numbers. Both low and high concentrations of 2,4-D inhibited the formation of the midrib. 2,4-D in both low and high concentrations decreased the number of vessels in both protoxylem and metaxylem and also decreased their diameters, where as GA3 in low and high concentrations have less effect on the number of vessels and its diameters. GA3 in high concentration increased the number of vascular bundles in 1mm long of the leaf blade, while 2,4-D in low and high concentrations decreased their numbers. 10-3M of 2,4-D increased the size and layers of special hypodermal cells.