Formation Of Shoot Primordia Research Articles

Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium

A study on the effect of various phytohormonal combinations on in vitro propagation of Cocoyam [Xanthosoma sagittifolium (L.) Schott] was conducted to develop an improved and efficient in vitro regeneration protocol for its mass multiplication. Histological analysis to understand the in vitro regeneration pattern and genetic fidelity assessment of regenerated plants were also carried out. Single shoots excised from in vitro established cultures of X. sagittifolium were used as explants. Among the 32 different phytohormonal combinations tested, indirect organogenesis with intervening callus phase was observed on majority of the media combinations. Meristematic clump formation was optimally achieved on all the tested media combinations with maximum 43.54 ± 0.51 shoot primordia on MS medium containing 0.2 mg/L BAP + 0.1 mg/L NAA followed by 36.44 ± 0.76 shoot primordia on MS medium having 2.5 mg/L TDZ. Micro-morphological analysis of different morphogenetic structures revealed that the regeneration of cocoyam is well executed via meristematic nodules, shoot primordia formation that may evolve in to proper shoots. Adventitious shoots (> 2 cm) were successfully (100.00 ± 0.00%) rooted on the half-strength MS medium containing IBA (0.05–1.0 mg/L) and IAA (0.05–0.5 mg/L). The number of roots ranged from 0.78 ± 0.31 on the control half-strength MS medium to 13.94 ± 0.46 on half-strength MS supplemented with 1.0 mg/L IBA. Considering somaclonal variations as a potential restriction to in vitro multiplication of plants, genetic stability was assessed using 40 ISSR primers. The PCR amplification profiles obtained from all the tested propagules (calli, meristematic clumps, regenerated plantlets) were similar to the mother plants indicating the homogeneity of the individuals raised through the regeneration protocol reported here.

Enhancement of In Vitro Production of Volatile Organic Compounds by Shoot Differentiation in Artemisia spicigera.

Callus initiation, shoot formation and plant regeneration were established for Artemisia spicigera, a traditional medicinal plant growing in Armenia, Middle-Anatolia and Iran, and producing valuable volatile organic compounds (VOCs) that are mostly represented by monoterpenoids. Optimal callus initiation and shoot production were obtained by culture of hypocotyl and cotyledon explants on MS medium comprising 0.5 mg L−1 naphthalene acetic acid (NAA) and 0.5 mg L−1 6-benzyladenine (BA). Consequently, the shoots were transferred onto the MS media supplemented with 1 mg L−1 of indole-3-butyric acid (IBA) or 1 mg L−1 of NAA. Both types of auxin induced root formation on the shoots and the resulting plantlets were successfully grown in pots. The production of VOCs in callus tissues and regenerated plantlets was studied by gas chromatography–mass spectrometry (GC-MS) analysis. Although the potential of undifferentiated callus to produce VOCs was very low, an increased content of bioactive volatile components was observed at the beginning of shoot primordia differentiation. Intriguingly, the volatiles obtained from in vitro plantlets showed quantitative and qualitative variation depending on the type of auxins used for the rooting process. The acquired quantities based on total ion current (TIC) showed that the regenerated plantlets using 1 mg L−1 NAA produced higher amounts of oxygenated monoterpenes such as camphor (30.29%), cis-thujone (7.07%), and 1,8-cineole (6.71%) and sesquiterpene derivatives, namely germacrene D (8.75%), bicyclogermacrene (4.0%) and spathulenol (1.49%) compared with the intact plant. According to these findings, in vitro generation of volatile organic compounds in A. spicigera depends on the developmental stages of tissues and may enhance with the formation of shoot primordia and regeneration of plantlets.

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics. The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.

STUDY ON SHOOT MORPHOGENESIS IN-VITRO OF MORINDA CITRIFOLIA L.

Morinda citrifolia L. is a valuable medicinal plant, used to treat many diseases, such as sleeplessness, backache, high blood pressure... To study the shoot morphogenesis in Morinda citrifolia L. for propagation in the future, we examined the effects of BA, Zeatin and NAA on adventitious shoot formation of hypocotyl. The results showed that the regeneration of adventitious shoot comprised three steps of morphogenesis. In this process, Zeatin 1mg/l stimulated the formation of shoot primordia in dark (after a week), earlier than BA at the same concentration. Shoot generation was slow (after five weeks) on MS medium supplemented with 0,1mg/l and Zeatin 1mg/l. Roles of respiration rate and endogenous hormones were discussed to understand the physiological changes in the adventitious root formation.

Plant regeneration with maintenance of the endosperm ploidy level by endosperm culture in Lonicera caerulea var. emphyllocalyx

An endosperm culture of Haskap (Lonicera caerulea var. emphyllocalyx) was established to develop polyploid plants and investigate the regeneration ability of the endosperm. Based on histological analysis of embryo and endosperm development, endosperms at the globular to early torpedo-stages of developing embryos were used to initiate an endosperm culture. Formation of shoot primordia was observed on Murashige and Skoog (MS) medium (Physiol Plant 15:473–497, 1962) containing benzyladenine and indole-3-butyric acid. Shoot primordium formation was confirmed in some genotypes with regeneration frequencies ranging between 1.9 and 10.0%. These proliferated on ½ MS medium containing 2.89 μM gibberellic acid (GA3), and then elongated and rooted on MS medium containing 0.44 μM BA and 2.89 μM GA3. These shoots developed into plantlets on ½ MS medium. Plantlets maintained ploidy of the endosperm following flow cytometric analysis, thus confirming that these were derived from the endosperm. These results indicated that endosperms were capable of regeneration.

Light quality influences the polyamine content of lettuce (Lactuca sativa L.) cotyledon explants during shoot production in vitro

Light quality has previously been shown to influence morphogenesis in lettuce cotyledon explants, with white or red light promoting adventitious shoot production, and blue light inhibiting it. Endogenous polyamine (PA) concentrations were compared between explants cultured under different light qualities. Explants cultured under white or red light accumulated PAs during shoot primordia production, with a 5.6-fold increase compared to initial concentrations under white light, and 6.7-fold increase under red light. These results suggest polyamines are involved in the formation of shoot primordia. After 18 days in culture PA concentrations decreased under white light, and to a lesser extent under red light, signaling a shift in polyamine metabolism that correlates with shoot expansion, which occurs more readily under white light. Explants cultured under blue light accumulated polyamines for the first 7 days, to a level 1.3 times greater than initial values, followed by a gradual decline during the remainder of the culture period. Explants cultured under blue light also contained a greater proportion of PCA-insoluble conjugated PAs, compared to explants under white or red light, which contained greater proportions of free or PCA-soluble conjugated polyamines. The ratio of putrescine to spermidine was also different with a lower Put:Spd ratio being associated with shoot production under white or red light, and higher Put:Spd ratio being associated with culture under blue light.

Jasmonic acid inhibition of in vitro shoot organogenesis in Pinus radiata cotyledons

Summary The effect of jasmonic acid (JA) on de novo shoot formation in excised cotyledons of radiata pine ( Pinus radiata D. Don), was examined. JA had no effect on shoot-forming (SF) tissues at concentrations up to 10 −6 mol · L −1 . At concentrations greater than that, JA caused a reduction in the number of shoots formed, as well as the lengths of the shoots and fresh and dry masses of the tissues. Reciprocal transfer of excised radiata pine cotyledons from a SF medium with 10 −5 mol · L −1 JA to a SF medium without JA and vice versa showed that any exposure of the cotyledons to JA either during the shoot induction phase (days 0-21) or the shoot development phase (beyond day 21) led to a reduction in shoot formation. However, the JA effect was significantly less if the cotyledons were not exposed to JA during the initial 10 days in culture; indicating that the JA effect was mainly during shoot primordia formation and the subsequent development into shoots.

Shoot Bud Histogenesis from Mature Embryos and Shoots of Eastern White Cedar (Thuja occidentalis L.) Cultured In vitro

The histological events associated with shoot primordia formation from mature embryos and in vitro cultured shoots of eastern white cedar, Thuja occidentalis, were examined. Axillary meristems were not present in the axils of the cotyledons. Embryos cultured on N⁶-benzyladenine medium developed two shoot buds from the apical position. The two shoot primordia arose from the peripheral region of the main shoot apex. Concomitant with the shoot bud initiation, the cells at the summit of the shoot apex began to accumulate phenolic substances. This region did not grow further and the apex was divided into two domes giving rise to two shoot buds. In the original shoot explants, small groups of cells with dense cytoplasm and large nuclei could be found in the axils of the leaves. These axillary meristems were initiated during leaf primordia differentiation. Little further development occurred in these meristems in the controls; as they remained as a small group of meristematic cells separated by vacuolated cells, they became "detached meristems." In the presence of zeatin, cell division in these detached meristems was initiated after 2-3 d of culture. Axillary buds became visible after about 25 d of culture.

Histology of Shoot Bud Ontogeny from Seedling Root Segments of Brassica napus L.

Root explants of Brassica napus cultured in vitro form adventitious shoots. The root buds originated at the base of the newly initiated lateral root. Cells in association with the differentiating phloem of the developing lateral roots were the sites for root bud formation. A nodular mass of cytoplasmic cells developed by day 7 at the base of the lateral root. This group of cells continued to divide an enlarge. The cells in the peripheral region of the nodular cell mass differentiated further into a meristematic zone. The meristematic cells grew towards the periphery of the cortex by crushing the outer layer of cortical cells. Further development of the meristematic layer resulted in the formation of shoot primordia with organized shoot apical meristems and leaf primordia.

In Vitro Differentiation of a Wild Peanut, Arachis villosulicarpa Hoehne1,,2

Abstract Immature leaflets 3 to 5 mm in length from Arachis villosulicarpa Hoehne seedlings were cultured in vitro on a medium consisting of the major and minor salts of Murashige and Skoog (MS), Gamborg's B5 vitamins, 20 g/L sucrose, 0.8% Difco agar, 4.44 μM N-6 benzyladenine (BA) and 5.37 μM napthaleneacetic acid (NAA). Hard callus with many shoot primordia formed on all leaflets within 4 to 6 weeks. Calli with primordia were further cultured for plant formation on 150 other media (MS major salts at 1/2 or 1/4 strength, full strength minor salts and vitamins, sucrose at 5 or 10 g/L, BA and NAA at 0 to 8 μM). Reduced concentrations of major salts with a high ratio of BA to NAA enhanced the formation of shoot primordia. Roots differentiated on media with 1/4 strength major salts, no BA, and 6 to 8 μM NAA and 5 g/L sucrose. Shoots differentiated from the branch points of roots after 2 to 4 months of culture. Plants were obtained via shoot primordia from callus and from the adventitious shoots from roots.