Young Calli Research Articles

Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture (LBA4404: pSB1, pCAMBIA1301) and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6- and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants (T0) revealed the presence of 1–2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.

Desinfestação superficial, germinação e regeneração in vitro a partir de sementes de calêndula

Os objetivos deste trabalho foram: desenvolver um protocolo de desinfestação superficial de sementes, selecionar uma metodologia de germinação in vitro, avaliar o potencial de regeneração no cultivo in vitro e a influência do tempo de cultivo no tipo de calo formado e na regeneração a partir de calos de calêndula. Para a desinfestação superficial, foram testados diferentes tempos de imersão em solução de hipoclorito de sódio a 2,5%. Na germinação in vitro, foram testados: imersão em ácido sulfúrico absoluto; imersão em ácido clorídrico absoluto; retirada do tegumento e embebição das sementes; e embebição das sementes sem a retirada do tegumento. Para avaliar o potencial de regeneração in vitro, foram testadas diferentes concentrações de 6-benzilaminopurina (BAP) e ácido alfa-naftaleno acético (ANA) e tempos de cultivo. A imersão em solução de hipoclorito de sódio a 2,5% por 30 minutos aliada à remoção do tegumento promove a germinação in vitro de sementes de calêndula, efetuando uma desinfestação superficial satisfatória. Para a regeneração de partes aéreas e raízes a partir de sementes de calêndula não é necessária a suplementação com fitorreguladores. Na presença de BAP, independentemente da presença ou não de ANA, calos primários induzem à formação de calos esponjosos e friáveis e de calos verdes e rígidos, estes em menor número; na ausência de BAP é induzida a formação de calos pequenos. Calos jovens são mais eficientes em regenerar partes aéreas em calêndula.

Structural and chemical study of callus formation from leaves of Rubia tinctorum

Dedifferentiation was monitored in Rubia tinctorum L. leaves over a 14-d period after callus induction using transmission electron microscope (TEM), high performance liquid chromatography (HPLC), spectroscopy and thin layer chromatography (TLC). Photosynthetic pigment loss of leaves took 3–5 d coinciding with the first period of anthraquinone accumulation. Callus cells were discernible in the region of the vascular bundles and wounded edges of leaves after 10–14 d. Characteristic ultrastructural alterations were manifested in vacuolization, appearance of mitochondria, amount of smooth endoplasmatic reticulum and cytoplasm, caryolympha density of nuclei and cytoplasm content of cells. There were special events in the transfer cells: unequal divisions of dedifferentiated plastids and lytic activity in the cell wall. Our results show that mesophyll cells seem to be stopped at a particular level of dedifferentiation, while transfer cells embodied in veins of leaves pass through further alterations and lead to callus formation. Findings suggest that a sort of dedifferentiation drift manifests in the various cells of R. tinctorum leaves during callus induction and depending on their specialized status they achieve different levels of dedifferentiation. Approximately 4 weeks after callus induction, root growth has started from the young calli.

Production of haploids of neem (Azadirachta indica A. Juss.) by anther culture.

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.

Efficient callus formation and plant regeneration of goosegrass [Eleusine indica (L.) Gaertn.

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.

Adenylate Kinase in Tobacco Cell Cultures. II. Variability and Regulation of Isoform Activity Patterns in Different Cell Lines

Summary Specific activity and isoform patterns of adenylate kinase (AK) were investigated in tissue cultures of cloned tobacco cell lines that differed mainly in hormone requirement, proximate cause of hormoneautotrophy and neoplastic growth characteristics. Variations in the AK isoform pattern were related to growth and ageing processes, differentiation state of tissues, differences between culture cycles as well as the neoplastic characteristics of the cell lines. High specific AK activity and isoform patterns with dominating chloroplastic activities appeared as typical of green, photosynthetic and actively growing tissues (calli in growth phase, callus outer tissues). Lower specific AK activity and isoform patterns with dominating cytosolic activity were typical for senescent or non-photosynthetic tissue (calli in stationary phase, callus core tissues), but sometimes also occurred in entire young calli, apparently as a consequence of stress. This isoform pattern is therefore proposed as a stress marker in plant cell cultures. The isoform patterns of the different cell lines were analyzed in detail for the variations during different culture cycles and the differences appearing under optimal growth conditions. The observed characteristics were related to proximate cause of hormone-autotrophy and neoplastic state. The main differences appeared between normal and neoplastic, transformed tissues. Changes in compartmentation of AK activities under different metabolic situations are discussed with respect to the physiological characteristics of the cell lines, energy metabolism and ATP/ADP ratios of cellular compartments and the presumed role of AK in ageing and stress.

L-DOPA content, peroxidase activity, and response to H2O2 ofVicia faba L. andV. narbonensis L. in situ and in vitro

Comparative investigations onVicia faba L. andV. narbonensis L. demonstrate that externally applied H2O2 oxidizes L-DOPA to DOPAchrome in young calli and protoplasts isolated from precultured leaves ofV. faba, but not in those fromV. narbonensis. A specific staining method indicates that peroxidases and not polyphenol oxidases mediate the oxidation of L-DOPA in such tissues and cells. The change in peroxidase activity and L-DOPA content, measured during a long-term callus culture, and in mesophyll protoplasts of both species is described. An orange-red colour is only visible if sufficient amounts of active peroxidases and L-DOPA are present. Cells and tissues without L-DOPA, or with inactive peroxidases, show no reaction to H2O2. Wounding and cell division induce peroxidase activity. Using root cross sections it is demonstrated that cell divisions alone activate peroxidases, and that wounding is not an obligatory factor.

Growth and Cytology of Callus and Cell Suspension Cultures of Vicia faba L.

Callus cultures derived from root and shoot tissues of Vicia faba were initiated on a modified TORREY-FOSKET medium (L). Continuous subculturing of root-derived callus on SH-M medium led to two morphologically distinct strains (YSR and RWR), shoot-derived callus (S) did not change its morphology. The fast growing strain RWR (white color) with a 12 to 31 fold increase of fresh weight in a 30 days period has an average mitotic index of 4.3 %, the slowly growing strain YSR (8 to 9 fold increase of fresh weight, yellowish) has an average mitotic index of 4.2 %. Though this difference is not significant, there is a strong negative correlation (p<0.1 %) between the increase of fresh weight and the mitotic indices of the different cultures. The different degree of differentiation of both strains is also expressed by different cell sizes and nuclear diameters. The ploidy distributions within different strains are similar: 76 to 81 % ± diploid, 17 to 21 % ± tetraploid and 2 to 3 % >tetraploid (percentage of cells). The predominant chromosome numbers of 5, YSR and RWR are 14 to 16, including 3 or 4 M-chromosomes. Two distinct types of long-term cell suspension cultures of friable YSR- callus have been established in liquid MS or SH-M medium. They differ in their composition of single cells, cell clusters, and calli. The chromosome numbers are 11 and 13 to 14, respectively. Both suspensions are capable of regenerating calli from single cells. Though various culture media have been tested until now, chlorophyll synthesis has only been initiated in relatively young calli of both roots and shoots, but not in suspension-derived calli. At present root formation could hardly often be induced in shoot-derived calli.