Proliferation Of Calli Research Articles

Microencapsulation of betacyanins accumulated calli, short-term cold storage, and retrieval of callus in Selenicereus undatus (Haw.) D.R. Hunt

The red color of dragon fruit pulp and peel is due to the accumulation of betacyanins. In the present study, callus cultures were established using in vitro germinated seedlings of the white dragon fruit plant (Selenicereus undatus). Murashige and Skoog (MS) medium with 6-benzylaminopurine (BAP) at 2.0 mg/L was used to induce red-colored friable callus (92.0 % response). The betacyanin accumulated calli were harvested and encapsulated using sodium alginate (NaC6H7O6) and calcium chloride (CaCl2). These synseeds were cold stored for 6 months at 4.0 °C temperature. The stored synseeds were germinated on 2.0 mg/L BAP in the medium. The highest germination rate (88 %) of synseeds was achieved with 3 % sodium alginate polymerized with 75 mM calcium chloride, resulting in firm, uniform, and round synseeds. The betacyanin-loaded synseeds could be stored, transported, and directly used as inoculums for the extensive proliferation of calli and cell cultures for the commercial production of betacyanin.

Tissue-specific and novel secondary metabolites identified by SPME/GC-MS in Artemisia annua calli cultured under different wavelength

The controlled environment in the in vitro conditions allow for a stable and high-quality production of metabolites. Also, the use of different tissues, under in vitro conditions, can produce a profile of secondary metabolites different from those produced under conventional systems. The present study aimed to develop a protocol for in vitro calli induction and proliferation of Artemisia annua, and to compare the secondary metabolite profile of in vitro calli, in vitro plantlets, and plants cultivated ex vitro. The effects of different light wavelengths on the production of secondary metabolites were also evaluated. The presence of phytoregulators 6-Benzylaminopurine (BAP) and Naphthalene Acetic Acid (NAA) promoted the induction and proliferation of calli, with an increase in diameter and fresh weight of the calli. SPME/GC-MS analysis showed differences in the profile of secondary metabolites produced by different A. annua cultivation systems and light wavelengths. White light sources (cold fluorescent) and red light (LEDs) resulted in a greater diversity of metabolites produced by the calli. In total, 25 metabolites were detected exclusively in calli of A. annua, four of which are reported for the first time in the species: menthyl acetate, geranyl acetate, 4-methoxy benzaldehyde, and alpha-santalene. Of these, six metabolites were uniquely identified in calli cultured under red LED, demonstrating calli response specificity. The results demonstrate the potential of in vitro culture of A. annua calli in the production of metabolites different from those obtained in different tissues, such as in vitro plantlets and plants grown in greenhouse.

Somatic embryogenesis and plant regeneration from radicles of olive (Olea europaea ‘Chemlal’) zygotic embryos

Olive improvement by biotechnological tools such as genetic transformation requires an efficient in vitro regeneration system. Somatic embryogenesis seems the most suitable process. Our work describes for the first time the regeneration of whole plants in the main olive cultivar in Algeria ‘Chemlal’ via somatic embryogenesis induced from radicles of mature zygotic embryos. The obtained results showed that the establishment of a competent embryogenic culture is highly influenced by the chemical composition of the calli induction and maintenance media as well as addition of growth regulators. More than 10 and 13 % of nodular calli were obtained after callogenesis respectively on MS and OMc solid media containing IBA and zeatin followed by transfer to the same media without zeatin and a reduced concentration of auxin, while embryogenesis rates of 3.3 and 6.7 % were obtained respectively with IAA on MS medium and NAA on both tested media. However, no embryogenesis was observed with 2, 4-D or control which induced less callogenesis. Subsequently, an ECO medium with IBA, zeatin and BA particularly in liquid culture, allows better calli proliferation and embryogenic expression compared to OM and MS media. Finally, matured somatic embryos germinate quickly on a solid OM basal medium and generate normal well-developed plantlets easily acclimatized to natural conditions.

Optimized rice transformation protocol for transformation of the blast susceptible Indica rice accession CO39

BackgroundMany rice transformation protocols have been reported, but optimization is still required to ensure efficient transformation of specific rice accessions. The modified rice transformation protocol presented here builds upon the original protocol: ‘An improved protocol for efficient transformation and regeneration of diverse Indica rice cultivars’ volume 7, Article number: 49 (2011), Plant Methods.ResultsFollowing the aforementioned transformation protocol, calli browning occurred and no Agrobacterium-mediated transformation could be achieved, but this could be remedied by increasing the concentration of l-Proline. Improved callus health lead to successful transformation and proliferation of calli on selection media, but a low frequency of plantlet regeneration occurred when calli were transferred to regeneration media. The efficiency of plantlet regeneration was greatly improved by removing antibiotics from regeneration media, with the presence of escapes selected against during subsequent transfer of plantlets to antibiotic containing rooting media. Transformation of CO39 callus was found to be possible 8 days after callus induction resulting in a time saving of 10 days compared to the original protocol.ConclusionsThis optimized transformation protocol allows for the generation and survival of healthy CO39 calli, efficient transformation of calli using Agrobacterium, and produces a high frequency of regenerated transgenic plants. These protocol modifications will be useful for optimizing the transformation and regeneration of other recalcitrant Indica rice cultivars, particularly those sensitive to antibiotics during plantlet regeneration.

Induction of somatic embryogenesis in Tecomella undulata (Sm.) Seem using a pistillate explant

Desert teak (Tecomella undulata (Sm.) Seem) a multipurpose ornamental tree native to the arid and semi-arid tropics has entered the endangered plant category mainly as a result of the species' ineffective seed reproduction system. The tree usually reproduces through a few root suckers in old stands. Conventional methods of plants multiplication could not offer a viable practice for its mass multiplication. Low adventitious rooting of the cuttings has been the principal cause of failure in its vegetative propagation. Hence, the present research was planned and conducted to evaluate the feasibility of somatic embryogenesis in this species, the pathway that bypasses the need for rooting stage by developing bipolar embryos. Ovary explant was cultured in modified Murashigue & Skoog (MS) medium supplemented with different auxins and cytokinins. The results showed α-naphthalene acetic acid (NAA) was superior in inducing embryogenic callus. NAA ranging 5.4-21.5 μM could induce the highest embryogenic calli which exhibited developing pro-embryogenic masses (PEM) and globular somatic embryos. The calli which were induced by the use of 2,4-dichlorophenoxyacetic acid (2,4-D) were poor in quality and showed no morphogenesis potency. Individual application of Thidiazuron (TDZ) and N6-benzyladenine (BA) induced good callogenesis at low concentrations but the calli were non-embryogenic with both. The proliferation of embryogenic calli was the best in a hormone-free medium. However, the media containing 40.5 and 54 μM NAA alone could induce somatic embryos along with callus proliferation. Low BA-contained medium (0.9-4.44 μM) led to recurrent somatic embryogenesis. Neither BA and GA3, nor the elevating sucrose concentration could cause further development and maturation of the somatic embryos induced during previous stages (callogenesis and callus proliferation). More research is required to optimize the maturation stage. The findings of the present study can be useful for future studies in the micropropagation of this recalcitrant specieslationships in Indian mustard under heat stress and the differential remobilization efficiencies in the advanced breeding lines.

Inisiasi, pertumbuhan, dan perkembangan kalus embriogenik tanaman stevia (Stevia rebaudiana)

The leaves of sweetener plant Stevia rebaudiana contain secondary metabolites of steviol glycosides which are very sweet, with no calorie and zero glycemic index. Propagation of stevia by seeds is ineffective due to its low germination rate and diverse progenies. The tissue culture of stevia can be used to mass propagate rapidly and is commonly conducted by shoot multiplication. Up to now, the technology of somatic embryogenesis (SE) in stevia has not been successful yet. SE is developed to increase the production scale, rejuvenate clonal-propagated plants, and plant genetic transformation. The research objective was to develop protocols for the initiation, proliferation, and development of embryogenic calli of stevia as potential materials for SE. The explants used were young leaves, nodes, and internodes of axenic plantlets of stevia BX clone. The explants were cultured on MS solid media containing different concentrations of auxin and cytokinin for callus initiation. Callus emerged after 2-3 weeks of culture. The calli obtained were proliferated by subculturing several times as material stocks for indirect SE. MS solid media added with 1 µM 3,4-D and 16 mM CaCl2 gave the highest callus multiplication rate (4.7 times in 3 weeks). The selection of embryogenic calli was made continuously to obtain a pure line of embryogenic calli. Three types of calli attained were friable, fast-growing, yellowish calli, shiny nodular calli, and greenish nodular calli. Histological studies revealed that cells of the nodular calli had been differentiated to potentially formed somatic embryos.

A novel micropropagation of Lycium ruthenicum and epigenetic fidelity assessment of three types of micropropagated plants in vitro and ex vitro.

Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.

In Vitro Calli Production Resulted in Different Profiles of Plant-Derived Medicinal Compounds in Phyllanthus amarus.

The efficient production of plant-derived medicinal compounds (PDMCs) from in vitro plants requires improvements in knowledge about control of plant or organ development and factors affecting the biosynthesis pathway of specific PDMCs under in vitro conditions, leading to a realistic large-scale tool for in vitro secondary metabolite production. Thus, this study aimed to develop an in vitro technique, through the induction and proliferation of calli, for production of plant fresh weight, and to compare the PDMC profile obtained from the plants versus in vitro calli of Phyllanthus amarus. It was successfully possible to obtain and proliferate two types of calli, one with a beige color and a friable appearance, obtained in the dark using Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and a second type with a green color, rigid consistency, and nonfriable appearance obtained under light conditions and MS medium plus 6-benzyladenine (6-BA). In vitro micropropagated plants that gave rise to calli were also acclimatized in a greenhouse and cultivated until obtaining the mass for PDMC analysis and used as a control. While the micropropagated-derived plants concentrated the lignans niranthin, nirtetralin, and phyllanthin, the Phyllanthus amarus calli proliferated in vitro concentrated a completely different biochemical profile and synthesis of compounds, such as betulone, squalene, stigmasterol, and β-sitosterol, in addition to others not identified by GC-MS database. These results demonstrate the possibility of applying the calli in vitro from Phyllanthus amarus for production of important PDMCs unlike those obtained in cultures of differentiated tissues from field plants.

Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile, Tripogon loliiformis

Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0mgL−1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL−1 6-benzylaminopurine (BAP) and 0.5mgL−1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL−1 BAP but with 0.25mgL−1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.

Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

Chinese chestnut (Castanea mollissima) is an important germplasm resource for the breeding of Castanea species worldwide with vital ecological and economic value. Biotechnology overcomes the limitations of traditional breeding and accelerates germplasm improvement. However, a genetic transformation system for Chinese chestnut has not yet been established. In this study, a stable and efficient Agrobacterium-mediated genetic transformation method for Chinese chestnut is described. Embryogenic calli of C. mollissima cv. ‘Yanshanhongli’ were used as the target material. The sensitivity of embryogenic calli to kanamycin was determined, whereby the proliferation of non-transformed calli was completely inhibited at 180 mg/L. Antibiotic inhibition results for Chinese chestnut embryogenic calli showed that 50 mg/L cefotaxime and 500 μM timentin completely inhibited the growth of Agrobacterium tumefaciens but did not affect the normal growth of Chinese chestnut embryogenic calli. When embryogenic calli were co-cultured for 2 days with Agrobacterium tumefaciens strain AGL1 harboring the PBI121-EGFP plasmid, an embryogenic callus transformation efficiency of 4.55% was obtained, and two transgenic chimera were acquired. This Agrobacterium-mediated transformation system for Chinese chestnut provides a fundamental platform for genetic improvement of core germplasm and for further verification of gene function.

Influence of Carbohydrates on Callus Proliferation During Somatic Embryogenesis in Pineapple [Ananas Comosus (L.) Merr. (Bromeliaceae) Var. Cayenne Smooth Cultivar CI 16

The improvement of pineapple (Ananas comosus var. Smooth Cayenne) by means of in vitro culture is less studied in Côte d'Ivoire despite the importance of this plant for this country’s economy. Our work consisted in highlighting nature and concentration effects of carbohydrates on the proliferation of calli in pineapple as a prelude to efficient embryogenesis. Callus proliferation was carried out from the base of pineapple vitroplants leaves. Thirty (30) explants were cultured on the tested culture medium. MS medium (micro- and macro elements of Murashige and Skoog) supplemented with vitamin Gamborg B5 was used as base medium to which were added 0.05 mg/L BAP, 3 mg/L picloram, 2 mg/L glycine, 1,000 mg/L glutamine, 100 mg/L casein hydrolyzate and 30 g/L carbohydrate. Sucrose was tested at different concentrations (20, 25, 30, 35 and 40 g/L). The results revealed that callus proliferation is strongly influenced (p ˂ 0.0001) by nature and concentration of carbohydrate. Sucrose with the highest dry matter content (61.34 mg) has a higher callogenic potential than the other studied carbohydrates. The concentration of 30 g/L sucrose significantly improved the calli proliferation in pineapple. Galactose and maltose were less favorable to proliferation.

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

Dioscorea rotundata, commonly known as white yam, is an important staple food crop widely cultivated in West Africa and provides food security to millions of people. Genetic improvements of this crop using the advanced biotechnology tools have been hampered hitherto by the recalcitrant nature of regeneration through somatic embryogenesis. Therefore, we have developed an efficient and reproducible system for plant regeneration via somatic embryogenesis. Explants of different types (immature leaf, node, stem internode, root segment, petiole, and axillary bud) of D. rotundata accession TDr 2436 were tested for their embryogenic potentials on Murashige and Skoog (MS) medium supplemented with various auxins (2,4-D, NAA, and picloram). Among all explants tested, axillary bud explants cultured on MS medium supplemented with picloram (0.5–12 mg/l) favored the production of calli. Maximum proliferation of calli (526 mg fresh weight/explant) was achieved on MS medium supplemented with picloram (0.5 mg/l), casein hydrolysate (600 mg/l), and proline (1 g/l). Histology analysis confirmed that the embryogenic calli produced on this medium were mixed with non-embryogenic calli. Transfer of calli on MS basal medium supplemented with activated charcoal (1 %) changed the color of calli to purple and promoted the production of somatic embryos (87 embryos/callus) as well as adventitious shoot buds. Furthermore, upon transfer to MS medium supplemented with BAP (0.4 mg/l), the embryos continued their differentiation and maturation and germinated into complete plantlets. The adventitious shoot buds produced multiple shoots on MS medium supplemented with BAP (0.4 mg/l). Well-developed germinated plantlets were acclimatized in the screen house with 90 % survivability. Histology studies confirmed that the regeneration of D. rotundata reported here followed dual regeneration pathways. The embryogenic calli regenerated through development of somatic embryos and germinated into complete plantlets, however non-embryogenic calli regenerated through organogenesis and developed multiple shoots. The developed protocol has potential for somatic hybridization, mass clonal propagation, and genetic transformation applications.

Morpho-anatomic of adventitious rhizogenesis in mini-cuttings of Eucalyptus grandis x Eucalyptus urophylla

The present work aimed to accomplish the histological analysis of the events involved in the adventitious rooting pattern of four Eucalyptus grandis x Eucalyptus urophylla clones. The mini-cuttings were collected from a mini-stump and the rooting experiments were set up under mini-hedge indoor semi-hydroponics by means of intermittent flooding fertirrigation. For histological analyses, the proximal ends of the mini-cuttings were collected after 0, 1, 2, 4, 6, 8, 10, and 12 days after planting them at greenhouse. After fixation with FAA70%, the samples were dehydrated in a graded ethanol series and infiltrated overnight in metracrilate resin, and finally embedded in resin. It was verified the endogenous origin of adventitious root primordia from vascular cambium, and in some cases, the formation and proliferation of calli at the proximal end of the mini-cuttings within 8 to 12 days.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

Efficient in vitro plant regeneration via indirect organogenesis for different common bean cultivars

An efficient protocol for the in vitro regeneration of Phaseolus vulgaris L. cv. CIAP7247F via indirect organogenesis was established. Cotyledonary nodes, cotyledonary nodes with one cotyledon and cotyledonary nodes with two cotyledons dissected from the embryonic axis of three-day-old germinated seeds, were used as primary explants. Seeds of different age were used for callus induction. Different concentrations of thidiazuron (TDZ) and 6-benzylaminopurine (BAP) were assessed for callus proliferation and shoot regeneration. Five cultivars were tested to determine the effect of genotype. Cotyledonary nodes with one and two cotyledons from fresh and four-month-old seeds were the most effective explants for callus formation. Callus proliferation medium containing 0.04mgl−1 of TDZ was optimum for proliferation of calli. A shoot regeneration frequency of approximately 3.0 shoots per callus was obtained on medium supplemented with 2.25 or 4.50mgl−1 of BAP. Efficient rooting of plantlets was achieved on shoot elongation and rooting medium. Regenerated in vitro plants grown in the greenhouse showed normal development and were fertile. Although different responses were observed depending on genotype, the protocol was efficiently applied for different commercial P. vulgaris cultivars (BAT93, BAT304, BAT482 and ICA Pijao), demonstrating the value of this procedure.

Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula

Leaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4–5 times every 2 weeks for the proliferation of calli. After 8–10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis.

English

Callus induction and somatic embryogenesis from hypocotyl explants of some Iranian cottons spp. (Hashem abad, Kerman, Termez and Sepid) were compared with Coker 312 through induction and formation of embryogenic calli on medium of Murashige and Skoog (MS) with Gamborg vitamins (B5) supplemented with the following compositions: MSB1 (0.5 mg/l zeatin), MSB2 (1 mg/l zeatin), MSB3 (0.5 mg/l 2,4-dichlorophenoxyacetic acid, 0.1 mg/l kinetin), MSB4 (1 mg/l 2,4-D, 0.5 mg/l kinetin, 0.5 mg/l zeatin) and MSB5 (2 mg/l α-naphtalene-3-acetic acid, 1 mg/l kinetin, 0.75 mg/l MgCl2). The optimum medium for the proliferation of embryogenic calli was MS medium containing B5 vitamins, 1 mg/l 2,4-D, 0.5 mg/l kinetin and 0.5 mg/l zeatin and the optimum medium for the development of somatic embryos was MS medium (NH4NO3 was removed and KNO3 amount doubled) containing B5vitamins, 40 g/l sucrose and without hormone. Media MSB1, MSB2 and MSB4 gave the highest percentage (100%) of calli induction in Coker 312 but the lowest induction (46.66%) was observed when Hashem abad explants were cultured in the MSB3 medium. Embryogenesis percentage of Termez (2.22 to 24.40%), Hashem abad (1.85 to 9.73%) and Sepid (9.06 to 22.28%) genotypes were significantly lower than that of Coker 312 (66.66 to 94.33%). The Kerman genotype did not show embryogenesis. In the histological studies, the different development stages of the embryos (globular, heart, torpedo and cotyledonary) together with callus cells were showed.    Key words: Hypocotyl explants, somatic embryo, in vitro regeneration, germination, somatic embryogenesis histology.

The CKH1/EER4 Gene Encoding a TAF12-Like Protein Negatively Regulates Cytokinin Sensitivity in Arabidopsis thaliana

The recessive ckh1 (cytokinin hypersensitive 1) mutant of Arabidopsis thaliana shows hypersensitivity to cytokinins, which promote proliferation and greening of calli. The CKH1 gene encodes a protein resembling TAF12 (TATA BOX BINDING PROTEIN ASSOCIATED FACTOR 12), which is a component of transcription factor IID (TFIID)- and histone acetyltransferase-containing complexes in yeast and animals. Microarray analyses revealed that a substantially greater number of genes responded to a low level of cytokinins in the ckh1 mutant than in the wild type. However, expression of cytokinin primary response genes was not significantly affected by the ckh1 mutation. These results suggest that the CKH1 protein regulates a set of genes involved in late signaling processes governing a range of cytokinin responses, including cell proliferation and differentiation.

Somatic embryogenesis in four tree legumes.

Somatic embryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.25–1.0 mg/l Kn (kinetin) and 2.0–3.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0 mg/l 2,4-D and 1.0–1.5 mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.0–2.0 mg/l 2,4-D or NAA and 0.25–1.5 mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.5–2.0 mg/l 2,4-D or NAA and 1.0–1.5 mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.0–2.0 mg/l 2,4-D, 1.0–1.5 mg/l kinetin, and 400–600 mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1 mg/l IAA and 0.25 mg/l BA; developed shoots and rooted in 1/2 strength MS medium supplemented with 0.1 mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber.