Cotyledon Segments Research Articles

Simultaneous expression of serine acetyltransferase and cysteine synthase results in enhanced sulfate uptake and increased biomass in Ipomaea aquatica

The long range goal of the present study is to practically utilize Ipomoea aquatica (water spinach) for phytoremediation of polluted water with sulfuric compounds. In higher plants, the sulfate assimilation pathway consists of 5 key enzymes, among which serine acetyltransferase (SAT) and cysteine synthase (CS) constitute one of the rate limiting steps. Subsequently we have attempted to improve the sulfur assimilation capacity of I. aquatica using genes encoding these two enzymes. Cotyledon segments of seedlings were transformed with Arabidopsis SAT and rice CS genes under the control of the cauliflower mosaic virus 35S promoter. Among 3,245 cotyledon explants, 325 regenerated shoots, and two showed a high tolerance to hygromycin, designated as SR3 and SR10. In transgenic lines, the SAT activity was over 2-fold, and the CS was 3-fold higher than those in the wild type control. The cysteine and glutathione contents were also 6- and 2-fold higher than the control, respectively. When cultured in the presence of 1 g l � 1 (7 mM) sulfate, they accumulated sulfate as much as 20 mg g � 1 fresh weight, being 5-fold higher than the control. Under standard culture conditions, transgenic lines grew faster than the control, showing a 20% increase in fresh weight within 5 weeks cultivation. These results suggested that strengthening of SAT and CS resulted in increase not only in sulfate uptake, but also in total biomass.

In vitro organogenesis in watermelon cotyledons

The objective of this work was to study the in vitro organogenesis of Citrullus lanatus, by the induction of adventitious buds in cotyledon segments cultured in medium supplemented with cytokinin. Explants were collected from one, three and five-day-old in vitro germinated seedlings, considering the distal and proximal cotyledon regions. The data obtained showed that in vitro organogenesis of watermelon occurred with higher efficiency, when cotyledon segments from the proximal region collected from three-day-old seedlings were cultivated in medium MS, supplemented with BAP (1 mg L-1) and coconut water (10%). The histological study showed that the organogenesis occurs directly, without callus formation, on epidermal and subepidermal layers of the explants. Adventitious shoots were characterized by the development of shoot apical meristem and leaf primordia. The formation of protuberances, that do not develop into adventitious buds, was also observed.

In vitro induction of callus from cotyledon and hypocotyl explants of Glycine wightii (Wight & Arn.) Verdc.

With the objective to promote in vitro callus induction, cotyledon and hypocotyl segments of "perennial soybean" (Glycine wightii (Wight & Arn.) Verdc.) were inoculated in basal medium MS supplemented with sucrose (1.5 e 3%) and 0.8% agar and different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). The explants were maintained in a dark growth room at 28ºC. The best callus induction was observed in explants (cotyledon and hypocotyl) maintained in medium containing the combination of 2,4-D (1 mg.L-1), kinetin (0.1 mg.L-1) and 3% sucrose. To promote callus subculture, the MS medium was supplemented with different combinations of 2,4-D (0.5 to 4.0 mg.L-1), with or without kinetin (0.1 mg.L-1) and sucrose (1.5 e 3%). The calli were maintained 35 days in a dark growth room at 28ºC. The results indicated that the use of 2,4-D 1.0 mg.L-1 + kinetin 0.1 mg.L-1 + sucrose 3% provided the highest average weight of cotyledons calli fresh matter, whereas the use of 2,4-D 2.0 mg.L-1 + kinetin 0.1 mg.L-1 + sucrose 3% provided the highest average weight of hypocotyl calli fresh matter. High concentrations of 2,4-D, independent of kinetin and sucrose concentrations, promoted oxidation and reduction in fresh weight from calli of cotyledon and hypocotyls.

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.

Production of Abrus pulchellus ribosome-inactivating protein from seeds callus culture

Ribosome inactivating proteins (RIPs) were isolated from callus culture that were established from seed explants of Abrus pulchellus. Cotyledon segments of immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin (2,4-D), citokinin (kinetin and BA) and sucrose in order to determine the best callus induction. A. pulchellus type 2 RIP (pulchellin) expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained after 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins (0.1 M Tris–HCl pH 7.6 buffer, containing 0.15 M NaCl, 3 h at room temperature). A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the presence of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, peak released by 0.1 M galactose, appeared to be composed of two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to that obtained with seeds.

In vitro conservation of Cedrela fissilis Vellozo (Meliaceae),a native tree of the Brazilian Atlantic Forest

Two in vitro conservation methods have beendeveloped for the ex situ conservation of germplasm fromCedrela fissilis, an economically important tree of theBrazilian Atlantic Forest. The first method involves the medium-term storage, at 25°C, of artificial seeds comprising alginate encapsulatedvegetative propagules (shoot tips, cotyledonary and epicotyl nodal segments).Maximum post-storage (3 months) viabilities of 96–100% wereachieved for encapsulated shoot tips and cotyledonary nodal segments stored onwater-solidified agar (at 0.4–0.7% w/v). Encapsulated shoot tips storedfor 6 months on 0.4% (w/v) agar showed the highest survival rates(44%). Seeds of C. fissilis were successfully cryopreserved(100%) after direct immersion in liquid nitrogen. Ex situstorage procedures are now available for the medium- to long-term conservationof C. fissilis. These approaches offer new opportunitiesfor the conservation, sustainable management and utilization of this valuablefast growing timber tree.

Protective Role of Thidiazuron Treatment on Cucumber Seedlings Exposed to Heavy Metals and Chilling

The action of thidiazuron, a synthetic growth regulator, was studied on 7-day-old cucumber seedlings (Cucumis sativus L., cv. Monastyrskii) exposed to chilling and sublethal concentrations of lead and copper ions. The extent of injury was assessed from the electrolyte leakage from cotyledonary leaves into distilled water. Separate application of each stress factor induced an increase in membrane permeability; however, their combined application caused a weaker response. A preliminary treatment of seedlings with thidiazuron fully or partly prevented the stress-induced stimulation of electrolyte leakage from cotyledon segments. It is concluded that thidiazuron elevates plant resistance to adverse environments.

Somatic Embryogenesis of Cucumber (Cucumis sativus L) Using Seed Cuttings Obtained from Pre-Mature Fruit

Seed cuttings including zygotic embryo axis with its two cotyledon parts (2-5 mm2) were extracted from green-pre-mature cucumber fruit and used as seed explants, or they were used after dividing them (after 3 days culture) into embryo axis and cotyledon parts. Their embryogenic potential increased with the increase of fruit maturity, the maximum was obtained when 3 weeks old fruits were used. The most efficient explant type was the cotyledon segments. Efficient cucumber embryogenesis was obtained using a two steps procedure: 1-somatic embryos were initiated and multiplied on Murashige and Skoog (MS) medium supplemented with 4.5μM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 90 mM sucrose and 0.8% agar (NMM medium), 2-embryo maturation and germination were accomplished using the same medium without 2, 4-D (MGM medium). The most important sign for successful transfer from the first to the second step was the appearance of slight greenish patches on the embryogenic mass (EMS), which appeared in 10 weeks.

Explantes de cupuaçuzeiro submetidos a diferentes condições de cultura in vitro

Este trabalho objetivou avaliar as respostas morfogenéticas de diferentes explantes de cupuaçuzeiro (Theobroma grandiflorum Schum.) submetidos a várias condições de cultura in vitro. A utilização de 2,4-D nas concentrações de 4,52; 6,78 e 9,05 µM promoveu a formação de calos friáveis em eixos embrionários e o TDZ nas concentrações de 2,0 e 3,0 µM induziu uma alta frequência de calos friáveis e menor oxidação em segmentos de folhas jovens. O 2,4-D combinado com KIN, promoveu a iniciação de calos friáveis e o ANA combinado com KIN, a rizogênese em segmentos cotiledonares. Na presença de 2,26 µM de 2,4-D e 13,94 µM de KIN, houve a formação de calos friáveis em segmentos caulinares. A adição de água de coco, 2iP e ANA estimulou o rápido crescimento e a progressão de culturas de calo em eixo embrionário, segmentos caulinares e segmentos de folhas jovens clorofiladas.

Efficient Agrobacterium-mediated transformation of Lotus japonicus with reliable antibiotic selection

We have developed a new procedure for transforming a model legume, Lotus japonicus, that yields transformed plants from transverse cotyledon segments without contamination from the presence of non-transformants that survived the antibiotic selection. L. japonicus was transformed with the HPT gene as a selectable marker and the GUS reporter gene, both of which were driven by cauliflower mosaic virus 35S promoter. The efficacy of selection with hygromycin was tested using the assay of GUS activity in putative transformants. The integration of the GUS gene was also confirmed by polymerase chain reaction analysis of the genomic DNA. The integrated T-DNA was stable and inherited as a dominant trait. This procedure may have potential effectiveness and application in large-scale transformation for insertional mutagenesis or gene tagging.

Callus induction and plantlet regeneration from mature cotyledonary segments of groundnut (Arachis hypogaea L.)

We evaluated the efficiency of callus induction and plantlet regeneration from mature cotyledonary segments of groundnut cultivars VRI-2 and VRI-3. Callus cultures were induced from mature tissues using NAA and IAA in combination with KIN or BAP. Maximum induction was recorded with 3.0 mg/L IAA and 1.0 mg/L BAP. However, green, compact, and nodular calli were obtained in 2.5 mg/L of IAA or NAA combined with 1.0 mg/L of either BAP or KIN. Fresh and dry weights were highly influenced by auxin concentration. Compact and nodular calli were then transferred to shoot induction media. The highest mean number of shoots was observed in 3.0 mg/L BAP plus 0.5 mg/L IAA. Finally, the resulting plantlets were rooted with IBA and NAA.

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 μM N6-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 μM thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 - 14.43 μM gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 μM abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 μM BAP and the plantlets were hardened and transferred to soil.

Agrobacterium tumefaciens -mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l−1 α-naphthaleneacetic acid (NAA) and 1.0 mg l−1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 μg ml−1 Km, 200 μg ml−1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T1 seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.

Somatic Embryogenesis and Plantlet Regeneration of Melia azedarach L (Ghora Neem) from Cotyledonary Segments

Embryogenic calli were obtained within 3–4 weeks on MS medium supplemented with 3% sucrose, 200 mg I-1 caseinhydrolysate, and 3 mg l−1 each of 2,4-D and NAA from 3–4 days imbibed seeds. Heart-shaped embryos differentiated when growth regulators were withdrawn gradually. Quantification of somatic embryo formation showed a direct relationship between initial response, somatic embryo formation and its subsequent germination. The somatic embryos germinated into emblings with 90% conversion frequency on MS medium containing 2% sucrose.

Efficient Plant Regeneration of Ipomoea aquatica by Direct Shoot Formation from Cotyledon Segments.

An efficient method for regeneration of Ipomoea aquatica was established. Cotyledons with petioles produced adventitious shoots near the excised margins situated only on the distal side, which rooted within one week in a 1/2 MS solid medium without phytohormones. The frequency of adventitious shoot formation depended on the developmental stage of the seedlings. Upon transfer to hydroponics in a greenhouse, all plantlets grew to maturation.

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l−1) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l−1) + thidiazuron (1 mg l−1). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem.

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l−1) and indole-3-acetic acid (IAA) (0.5 mg l−1). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l−1) and subsequent subculture in IBA-free medium.

In vitro regeneration from cotyledons of watermelon

Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four weeks, then the shoots were transferred to hormone free medium for root induction.
 This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.

Somatic embryogenesis, plant regeneration and cyanogenesis in Manihot glaziovii Muell. Arg. (ceara rubber).

The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3-6 mm long) adjacent to the apex in Murashige and Skoog medium containing 18 μM 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μM 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10-4 M kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos. Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of linamarase activity were almost the same in leaves and stem tissues and very low in roots.

Comparative Study Of Anthraquinones From Embryogenic Callus Tissue And Zygotic Embryos Of Frangula Alnus And Rhamnus Catharticus

Organogenesis and somatic embryogenesis were achieved in cotyledon cultures of Frangula alnus Mill. (alder buckthorn) and Rhamnus catharticus L. (common buckthorn), and in zygotic embryo cultures of F. alnus. Histological examination of cotyledonary segment cultures revealed that adventitious buds and somatic embryos arose indirectly through an intervening callus phase. In comparison to zygotic embryos, embriogenic callus of Frangula alnus contained significantly more anthraquinones in the form of free aglycones and different participation of chrysophanol and physcion within synthesized complex of metabolites. Also, in comparison to shoots, root cultures and in particular undifferentiated callus of Frangula alnus, embryogenic callus contained more chysophanol and aloe emodin and less physcion derivatives. Production of physcion derivatives was significantly increased in embryogenic calluses of R. catharticus compared to zygotic embryos. On the other hand, the emodin content was lower than in zygotic embryos. In comparison to shoot, root cultures and in particular undifferentiated callus of common buckthorn, embryogenic callus contained significantly more derivatives of physcion and emodin. The content of chrysophanol and its derivatives was decreased proportionally.