Maternal Effect on Embryogenesis in Tobacco Overexpressing Rice Phytochrome A*

When analyzing tobacco lines overexpressing various types of rice phytochrome A, we observed seedlings with fused cotyledons. Phytochrome A is a member of the phytochrome family of plant red/far-red absorbing photoreceptors. Reciprocal crossings with wild-type tobacco indicated that this abnormal phenotype was maternally inherited. Mother plants that were expected to produce abnormal seedlings, were raised under different conditions and seeds collected separately from individual capsules. The frequency of abnormal seedlings increased in seeds from later flowers but decreased when mother plants were raised under continuous white light. The interaction of the overexpressed phytochrome A proteins with endogenous plant hormones might be responsible for cotyledon fusion. The abnormal phenotype could partially be recovered by application of gibberellic acid to intact flowers at the time of pollination. In contrast, the synthetic auxin NAA slightly enhanced the degree of cotyledon fusion. Even wild-type tobacco seedlings exhibited partial fusion of cotyledons if flowers were treated with 2,4-D and occasionally the shoot apex was replaced by a second root. Application of GA₃ to the flowers, in contrast, impaired the development of the radicule. These observations are discussed with respect to maternal effects in plant embryogenesis.     

Efficient and repetitive production of leaf-derived somatic embryos of Oncidium

Oncidium cultivars gave different embryogenic responses of leaf explants when affected by auxins (2,4-D, IAA, IBA and NAA), cytokinins (2iP, BA, kinetin, TDZ and zeatin), sucrose, NaH₂PO₄, casein hydrolysate, peptone, and glutamine. The best embryogenic responses of cv. Sweet Sugar were at 20 g dm⁻³ sucrose, 85 mg dm⁻³ NaH₂PO₄ and 3 mg dm⁻³ kinetin, respectively. The development of somatic embryos on leaf explants of cv. Sweet Sugar was delayed for about 10 – 20 d in comparison with cv. Gower Ramsey. On growth regulator-free medium, about 40 % of leaf derived embryos of cv. Gower Ramsey were fused together in their basal parts and so called multiple-state embryos. However, under the same condition, the embryos of cv. Sweet Sugar were all in multiple-state form.     

Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method

Factors influencing in vitro growth rates of soybean embryos were investigated using embryos isolated in the cotyledon stage. The influence of these factors on final plant recovery from the embryos cultured was tested. Sucrose and glucose could serve as carbon sources with final plant yields being higher with sucrose than with glucose. A culture medium containing only KNO₃ (25 mM) as the nitrogen source supported embryo growth. Adding glutamine (10 mM) to the medium containing KNO₃ increased final plant recovery to 25%. Of several vitamin supplements tested a combination of pyridoxine-HCl, nicotinic acid and pantothenic acid (0.5; 1.0; 0.5 mg l^-1) provided the best growth and plant yield. Of the plant growth regulators tested IAA, BAP and GA₃ stimulated embryo growth and plant development when added to the medium at a low concentration (0.1 M). The optimal temperature for in vitro growth of cotyledon stage embryos was 27°C. Temperatures above 30°C caused growth retardation and reduced plant yield. A protocol for culturing soybean cotyledon stage embryos under conditions ensuring high plant recovery is proposed.Abbreviations IBA indole-3-butyric acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside     

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     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

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     

Embryo-lethal mutants of Arabidopsis thaliana: analysis of mutants with a wide range of lethal phases

Summary Embryo-lethal mutants of Arabidopsis thaliana were isolated by treating mature seeds with an aqueous solution of ethyl methanesulfonate (EMS), screening the resulting M-1 plants for siliques containing 25% aborted seeds following self-pollination, and verifying the presence of induced mutations in subsequent generations. Thirty-two recessive lethals with a Mendelian pattern of inheritance were examined in detail. Developmental arrest of mutant embryos ranged from the zygotic stage of embryogenesis in mutant 53D-4A to the linear and curled cotyledon stages of development in mutants 112A-2A and 130B-A-2. These lethal phases did not change significantly when plants were grown at 18 °C rather than at 24 °C. Differences between mutant lines were found in the color of arrested embryos and aborted seeds, the percentage and distribution of aborted seeds in heterozygous siliques, the size of arrested embryos, and the extent of abnormal development. Unusual mutant phenotypes included the presence of unusually large suspensors, distorted and fused cotyledons, reduced hypocotyls, and arrested embryos without distinct cotyledons or hypocotyl tissue. The isolation of eight new mutants with a non-random distribution of aborted seeds in heterozygous siliques provides further evidence that many of the genes that control early stages of embryogenesis in plants are also expressed prior to fertilization.     

Roles of arabinogalactan proteins in cotyledon formation and cell wall deposition during embryo development of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Arabinogalactan proteins (AGPs) are a class of highly glycosylated, widely distributed proteins in higher plants. In the previous study, we found that the green fluorescence from JIM13-labeled AGPs was mainly distributed in embryo proper and the basal part of suspensor but gradually disappeared after the torpedo-stage embryos in Arabidopsis. And (β-d-Glc)₃ Yariv phenylglycoside (βGlcY), a synthetic reagent that specifically binds to AGPs, could inhibit embryo development. In this study, as a continuous work, we investigated the AGP functions in embryo germination, cotyledon formation, and cell wall deposition in Arabidopsis embryos by using immunofluorescent, immunoenzyme, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) techniques. The results showed that 50 μM βGlcY caused inhibition of embryo germination, formation of abnormal cotyledon embryos, and disorder of cotyledon vasculature. Compared with the normal embryos in vitro and in vivo, the AGPs and pectin signals were quite weaker in the whole abnormal embryos, whereas the cellulose signal was stronger in the shoot apical meristem (SAM) of abnormal embryo by calcofluor white staining. The FTIR assay demonstrated that the cell wall of abnormal embryos was relatively poorer in pectins and richer in cellulose than those of normal embryos. By TEM observation, the SAM cells of the abnormal embryos had less cytoplasm, more plastid and starch grains, and larger vacuole than that of normal embryos. These results indicated that AGPs may play roles in embryo germination, cotyledon formation, cell wall cellulose and pectin deposition, and cell division potentiality during embryo development of Arabidopsis.     

Plant regeneration from cotyledon protoplasts of Xinjiang muskmelon

Cotyledon protoplasts were isolated from seedlings of Xinjiang muskelon (Cucumis melo var.saccharinus) grown under sterile conditions and cultured in modified Miller medium. High frequency division of the protoplast-derived cells was observed. Agarose bead culture with B₆S₃ tobacco crown gall nurse cells was found most suitable for the cotyledon protoplasts when compared with other culture methods. Intact plants were regenerated from the protocalli by a two-step culture procedure with liquid and then solid media.Abbreviations BAP 6-benzylaminopurine - B₆S₃ crown gall tumor cells of tobacco - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2(Nmorpholino) ethanesulfonic acid - MS Murashige and Skoog medium(1962) - NAA -naphthaleneacetic acid - N6 Zhu et al. medium (1975)     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

fist: an Arabidopsis mutant with altered cell division planes and radial pattern disruption during embryogenesis

A T-DNA-tagged, embryo-defective Arabidopsis thaliana mutant, fist, was identified and shown to exhibit defects in nuclear positioning and cell division orientation beginning at the four-cell stage of the embryo proper. Cell division orientation was randomised, with each embryo exhibiting a different pattern. Periclinal divisions did not occur after the eight-cell embryo proper stage and fist embryos lacked a histologically distinct protoderm layer. Terminal embryos resembled globular-stage embryos, but were a disorganised mass containing 30–100 cells. Some terminal embryos (5%) developed xylem-like elements in outer surface cells, indicating that the fist mutation affects radial pattern. A soybean β-conglycinin seed storage protein gene promoter, active in wild-type embryos from heart stage to maturity, was also active in terminal fist embryos despite their disorganised globular state. This indicated that some pathways of cellular differentiation in fist embryos proceed independently of both organised division plane orientation and normal morphogenesis. Endosperm morphogenesis in seeds containing terminal fist embryos was arrested at one of three distinct developmental stages and appeared unlinked to fist embryo morphogenesis. The β-conglycinin seed storage protein gene promoter, normally active in cellularised wild-type endosperm, was inactive in fist endosperm, indicating abnormal development of fist endosperm at the biochemical level. These data indicate that the fist mutation, either directly or indirectly, results in defects in cell division orientation during the early stages of Arabidopsis embryo development. Other aspects of the fist phenotype, such as defects in endosperm development and radial pattern formation, may be related to abnormal cell division orientation or may occur as pleiotropic effects of the fist mutation. Received: 15 July 1997 / Accepted: 9 September 1997     

Morphogenetic routes of long-term embryogenic callus culture of <Emphasis Type="Italic">Areca catechu</Emphasis>

Early morphogenetic events and repetitive embryogenesis from callus culture of betel nut palm (Areca catechu L.) were studied using scanning electron microscopy. On Murashige and Skoog (MS) medium supplemented with 2 mg dm⁻³ dicamba, callus culture has capacity to form plantlets via somatic embryogenesis and to form secondary embryos for about 4 years. However, various abnormal embryos without differentiation of the leaf sheath and shoot apical meristem were observed, which showed bell-shaped and then cup-shaped or mushroom-shaped structures. These abnormal embryos contained distinctive structures, including a disk-shape interior region, surfaces with grooves and a stalk-like posterior region. During subculture, these abnormal embryos enlarged, became deformed and gradually lose their shape and then converted into nodular, compact embryogenic callus. It was also found that secondary embryos originated from interior surfaces or posterior regions of abnormal embryos, and gave rise to the next cycle of normal and abnormal embryos.     

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tsatsai</Emphasis>)

Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.     

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.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Somatic embryogenesis and plant regeneration inCoronilla varia L. (crownvetch) long-term tissue cultures

Spontaneous recovery of regeneration abilities was observed in a long-term (about two-year-old) crownvetch (Coronilla varia L.) tissue culture permanently grown on MS medium containing 1 mg 1^-1 IAA. Somatic embryos and later complete plants differentiated from initially regenerating roots. The formation and development of embryos was accompanied by a 10- to 20-fold increase in the content of cardioactive glycosides hyrcanoside and deglucohyrcanoside in the culture biomass. The effect of auxins (IAA, NAA, 2,4-D) and cytokinins (6-BAP) on calogenesis and somatic embryogenesis was examined; further development of somatic embryos was enhanced by light. Primary explants which were newly isolated from sterile R₁ plantlets showed differential, organ-specific ability of somatic embryogenesis which was highest in root cuttings. Regenerated plants were transferred to field culture; two-year-old cultures of regenerated plants showed in most cases phenotypic deviations from the original material, especially dwarfism.     

The influence of flask sealing on <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

The influence of flask sealing on eggplant morphogenic responses and morpho-anatomical characteristics was evaluated. Eggplant seeds from the cultivar Embu were disinfected and inoculated in MS medium supplemented with B₅ vitamins, 0.55 mM myo-inositol, 2% (w/v) sucrose, and 0.65% (w/v) agar. NAA (53.7 μM) and IAA (0.57 μM) were added to the medium to elicit morphogenic responses from cotyledon and hypocotyl explants via somatic embryogenesis and organogenesis, respectively. The plates were sealed with Micropore® 3M, Parafilm®, or polyvinyl chloride (PVC) film. The effect of glass vessel capping on morphogenesis was also evaluated for shoot apexes inoculated on medium containing half-strength MS where the capping consisted of polypropylene lids with or without two vents (0.45-μm MilliSeal® air vent) and PVC film. Leaf histological analysis and leaf bleaching from each treatment were performed. No significant differences were observed in the number of embryos and root primordia in media containing either 53.7 μM NAA or 0.57 μM IAA. However, embryogenic calli fresh weight was higher for PVC and Parafilm®. Morphogenesis from the shoot apex was influenced, except the plant height. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures compared to plants under other treatments. This treatment resulted in more expanded leaves, wider stems, and higher dry and fresh weights. In all treatments, the number of stomata was higher in the abaxial surfaces of leaves. Our results indicate that the flasks with vents provided air exchange beneficial for plant morphogenesis.     

In vitro regeneration of the tropical multipurpose leguminous tree Sesbania grandiflora from cotyledon explants

Summary A system using cotyledon pieces as explants and a BAP/NAA containing medium was developed for in vitro mass propagation of Sesbania grandiflora, a tropical nitrogen-fixing leguminous tree. The age and the lighting conditions of seedlings providing the explants were shown to be critical factors for both bud induction and bud elongation. Optimal choice for an efficient and reproducible bud induction process consisted of dark-grown seedlings, 24/36 h-old-post-imbibition, that yielded up to 96% of explants producing more than 30 buds each, after one week in culture. Bud development occurred throughout a direct organogenesis pathway, from the proximal and adaxial cut surface of the explants as proved by histological studies. Additional sites of regeneration were also obtained after wounding on the epidermal surface of explants, suggesting a large distribution of regenerative cells all along the explants. Bud elongation, i.e. stem differentiation and leaf growth, was improved by bud isolation from cotyledon explants and their further subculture in liquid bud elongation media for one week. Rooting was obtained on an auxin medium after 3 weeks and plants were established in soil with 92% success.Abbreviations BAP 6-BenzylAminoPurine - NAA a-Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - IAA Indole-3-Acetic Acid - GA3 Gibberellic Acid - MS Murashige and Skoog (1962) medium