Different Concentrations Of Thidiazuron Research Articles

Effects of TDZ and IBA on DNA methylation during callus induction in pear (<i>Pyrus ussuriensis</i> Maxim)

One of the essential epigenetic processes in higher eukaryotes, DNA methylation is essential for maintaining genome integrity and influencing gene expression. There aren't many research on how plant growth regulators (PGR) affect DNA methylation during the pear callus formation process. In this work, By using methylation-sensitive amplification polymorphism (MSAP), DNA methylation in pear callus caused by various quantities of thidiazuron (TDZ) and indole-3-butyric acid (IBA) was compared. The concentrations of TDZ and IBA could affect the callus induction rate and methylation level. It was discovered that induction rate and methylation were negatively correlated. The rate of pear callus induction was highest (54%) while the medium suppled with 3.0 mg·L^-1 TDZ and 4.0 mg·L^-1 IBA, although the matching DNA methylation level was lowest (27.96%). Additionally, there were significant difference in th level of genes' expression between different treatments and Both methylation and demethylation are regulated by these genes. As for genes related to auxin and cytokinin, gene expression analysis revealed that their levels of expression after different concentrations of TDZ combined with IBA present in differences significantly. Our findings provide light on DNA methylation mechanisms of plant tissue culture (PTC) dedifferentiation.

Study on exogenous application of thidiazuron on seed size of Brassica napus L.

Thidiazuron (TDZ) is a novel and efficient cytokinin commonly used in tissue culture, and numerous studies have demonstrated that TDZ can increase berry size. However, no study to date has explored the effect of TDZ on seed size of Brassica napus and the mechanism. To shed light on the effect of TDZ on the seed size of B. napus, four different concentrations of TDZ were applied to B. napus. Results indicated that TDZ treatment could increase the seed diameter and silique length of B. napus to varying degrees and 100 and 200 μmol/L TDZ treatments were the most effective with a 3.6 and 4.6% increase in seed diameter, respectively. In addition, the yield of B. napus was also substantially increased under TDZ treatment. On the other hand, confocal micrographs of embryos and cotyledon cells suggested that embryos and their cotyledon epidermal cells treated with 200 μmol/L TDZ were obviously larger in size than the control. Furthermore, TDZ promoted the upregulation of some key maternal tissue growth-related genes, including two G-protein signaling genes (AGG3 and RGA1) and two transcriptional regulators (ANT and GS2). The expression analysis of genes related to the auxin metabolic pathways, G-protein signaling, endosperm growth and transcriptional regulators confirmed that treatment with TDZ negatively regulated the key genes ABI5, AGB1, AP2, ARF2, and ARF18 during bud development stage and florescence. The results strongly suggested that TDZ might regulate the transcriptional levels of key genes involved in auxin metabolic pathways, G-protein signaling, endosperm growth and transcriptional regulators, which resulted in bigger cotyledon epidermal cells and seed size in B. napus. This study explored the mechanism of TDZ treatment on the seed size of B. napus and provided an important reference for improving rapeseed yield.

High-frequency shoot regeneration from flower bud derived callus of Gymnostachyum febrifugum Benth., an endemic medicinal plant to the Western Ghats

Gymnostachyum febrifugum Benth. is a small, scapigerous, rare and endemic medicinal herb indigenous to India, and belonging to the family Acanthaceae. This study reports an efficient protocol for high-frequency flower bud-derived callus induction and shoot organogenesis in G. febrifugum. Flower buds at 7 days before anthesis (dBA) were excised from the inflorescence and cultured on MS medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 2.275–9.1 µM) for callus induction. The optimum callus induction (78%) was obtained on MS medium supplemented with 6.825 µM 2,4-D. The calli when subcultured on MS medium supplemented with different concentrations of thidiazuron (TDZ; 2.275–11.375 µM) or 6-benzylaminopurine (BAP; 2.22–11.1 µM) alone or in combination with 1- naphthaleneacetic acid (NAA; 1.074–3.759 µM) induced shoots. The highest frequency (94%) and number of shoots (44.6 shoots/unit callus) were obtained on MS medium supplemented with 9.1 µM TDZ and 2.685 µM NAA. The optimum rooting frequency (95%) and number of roots (10.2) were observed on ½ MS medium supplemented with 14.76 µM indole-3-butyric acid (IBA). The rooted plantlets were acclimatized and transferred to soil with 94% success. A protocol for high-frequency shoot induction from flower bud derived calli has been standardized for G. febrifugum,an endemic medicinal herb to the Western Ghats.

English

The effect of different concentrations of thidiazuron (TDZ), benzyl amino purine (BAP), kinetin and 2,4-dichlorophenoxy acetic acid (2, 4-D) on callus induction in three elite cassava cultivars (agric-rouge, atinwewe and agblehoundo) was evaluated. Leaf explants harvested from greenhouse-grown cassava were sterilised using different concentrations of commercial bleach commonly called Jik (3.85% NaOCl) at different time intervals. The highest number (94%) of clean explants was obtained when 2% (v/v) Jik was used for 15 min. The explants were cultured in half MS media supplemented with different growth regulators TDZ, BAP, kinetin 2, 4-D, 100 mg/l myo inositol, 2% sucrose and gelled with 0.3% phytagel. Callus formation was observed from the cut edges of the leaves in all cultivars after 10 days in medium supplemented with TDZ, 12 days in BAP medium, and 15 days in kinetin medium. There were significant (p < 0.05) differences in callus formation among all cytokinins types and concentrations. However, there were no significant differences in callus formation in different 2,4-D concentrations. All 2,4-D concentrations produced 100% callus in all the cultivars. However, 2,4-D at 2 µM significantly produced the highest (2.48±0.30) callus weight in cultivar atinwewe. Furthermore, simple sequence repeats (SSR) and sequence-characterized amplified region of the induced calli on TDZ and 2,4-D media indicated the loss of CMD2 gene among induced calli compared to the mother plants. Key words: Cassava, simple sequence repeats (SSR) and sequence-characterized amplified region (SCAR), genetic stability, callus, cytokinins, auxins.

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36 h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0 μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0 μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5 μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15 μM TDZ for 24 h) explants was achieved on WPM augmented with 6.0 μM TDZ and 0.5 μM NAA after 8 weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (∼0.5 g) and were transferred onto WPM fortified with different concentrations (2.0–9.0 μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0 μM) and gibberellic acid (GA3, 0.5–3.0 μM). Maximum shoot number (10.4 ± 0.74) and average shoot length (4.75 ± 0.71 cm) were observed in WPM enriched with 2.0 μM BAP, 1.0 μM IAA and 1.5 μM GA3 after 8 weeks of incubation. The developed shoots (4 cm) were excised, pulse-treated for 24 h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0 μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60 days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.

Thidiazuron and Explant Type Effects on High-frequency In Vitro Mass Propagation of Cherry Tomato

Cherry Tomato is one of the most important vegetable crops cultivated for export in Egypt. In vitro culture response was assessed in tomato (Solanum lycopersicum L. var. cerasiforme) cv. (Summer Cherry) for optimum callus induction and plantlet regeneration. Callus induction was achieved within eight to 12 days directly on the cut surfaces of hypocotyl, cotyledon and leaf disc explants cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations of Thidiazuron (TDZ)] and benzyl adenine (BA) alone, but not in hormone free-medium. The highest callusing index (3.9 and 3.7) was obtained on hypocotyl explants cultured on MS medium supplemented with TDZ(1.0 and 2.0 mgl-1) followed by an index of 3.5 obtained from the same explant by using 0.5mgl-1 BA. However, for the leaf disc explants, the highest callusing index (3.1) was obtained on MS medium supplemented with BAat 2.0 mgl-1. After 8 weeks of culture, organogenesis was observed only on the explants cultured on medium containing different concentrations of TDZ and BA. The best shoot formation (93%) was obtained from leaf disc explant callus induced on MS medium containing TDZ. The highest number (13.4) of shootsexplant-1 was found when cotyledon explant callus was sub cultured on MS medium supplemented with 2.0 mgl-1 TDZ. Half strength of MS was found to be the best rooting medium, however, addition of IAA at 1.0 mgl-1 and IBA at 2.0 mgl-1 were found necessity to induce highest number of roots (22.5) and longer roots (11.0 cm), respectively. Acclimation of in vitro rooted plant is important for testing the post culture behavior of tissue culture regenerated plants. Cherry tomato derived from different explant sources under different concentration of TDZ and BA were not significantly different in their vegetative characters to those obtained from seed. However number of (raceme plant-1, flower raceme-1, fruit raceme-1, fruit plant-1) which produced by seed-derived plants was significantly less than those derived from in vitro propagated plants. This protocol would be valuable to create somaclonal variation and develop transgenic approaches for varietal improvement of cherry tomato.

In Vitro Techniques to the Conservation and Plant Regeneration of Malanga (Colocasia esculenta L. Schott)

Malanga (Colocasia esculenta) is a plant genetic resource that requires biotechnological strategies for conservation and propagation. One time-, labor-, and space-saving option is in vitro conservation and regeneration. The objective of this study was to develop a protocol for in vitro regeneration and conservation of germplasm of C. esculenta var. criolla. For conservation through minimal growth, we assessed several concentrations of Murashige and Skoog (MS) medium (one-third, one-half, and three-quarter strength), the growth retardant ancymidol (0, 1, 2, and 3 mg·L−1), and the osmoregulator polyethylene glycol (PEG-8000 mw) at different concentrations (0, 10, 20, and 30 g·L−1). For in vitro conservation, the percent survival, shoot number and length, and number of leaves and roots per explant were evaluated after 24 weeks. For in vitro regeneration, different concentrations of thidiazuron (TDZ: 0, 0.5, 1, 1.5, and 2 mg·L−1) and 6-benzylaminopurine (BAP; 0, 1, 2, 3, and 4 mg·L−1) were evaluated. After 4 weeks of cultivation, the percent response, shoot number, and number of leaves per explant were recorded. During in vitro conservation, it was noted that the treatment including 2 mg·L−1 ancymidol resulted in a retarded development, without affecting the survival of the C. esculenta germplasm. With regard to shoot regeneration, 7.60 shoots per explant were obtained using 2 mg·L−1 TDZ. Finally, 98% survival was achieved during the acclimatization process. This study will contribute to the establishment of genetic improvement programs through in vitro conservation and propagation of this valuable plant genetic resource.

English

The traditional way of grapevine (Vitis vinifera L.) propagation is time consuming and allows disease transmission from generation to generation. Moreover, it is difficult to improve this crop through conventional plant breeding methods. Therefore, the objective of this study was to develop efficient in vitro regeneration protocol for &lsquo;Canonannon&rsquo; and &lsquo;Chenin Blanc&rsquo; varieties of grapevine using leaf explants. MS medium supplemented with different concentrations of thidiazuron (TDZ) alone or in combination with &alpha;-naphthalene acetic acid (NAA), and 6-benzyl aminopurine (BAP) alone or in combination with indole-3-butyric acid (IBA) were used for regeneration of shoots from leaves. The regenerated shoots were transferred to shoot multiplication medium and subsequently to rooting medium and the plantlets were acclimatized after rooting. The rooting medium consisted of MS medium containing different concentrations of IBA or indole-3-acetic acid (IAA). The highest number of shoots per leaf explant was obtained from both &lsquo;Chenin Blanc&rsquo; (2.3 &plusmn; 0.3) and &lsquo;Canonannon&rsquo; (2.2 &plusmn; 0.2) on medium supplemented with 2.0 mg/L BAP. Among 16 different combinations of TDZ and NAA, the maximum number of shoots per explant (1.5 &plusmn; 0.2) was obtained from &lsquo;Canonannon&rsquo; on medium containing 1.0 mg/L TDZ and 0.1 mg/L NAA. However, when these shoots were transferred to shoot multiplication medium, 10 &plusmn; 0.51 shoots per explant were obtained from &lsquo;Chenin blanc&rsquo; on MS medium supplemented with 2.0 mg/L BAP. The highest number of roots per explant (8.3 &plusmn; 0.30) was obtained on medium containing 2.0 mg/L IBA. The survival rate of &lsquo;Chenin Blanc&rsquo; and &lsquo;Canonannon&rsquo; was 83.3 and 75 %, respectively after one month of acclimatization. &nbsp; Key words: &nbsp;Callus induction, growth regulators, hyperhydricity, organogenesis.

Thidiazuron (TDZ) induced organogenesis and clonal fidelity studies in Haloxylon persicum (Bunge ex Boiss & Buhse): an endangered desert tree species.

Haloxylon persicum (Bunge ex Boiss & Buhse), is one of the hardy woody desert shrubs, which is now endangered and/or nearing extinction. Urban landscape development and overgrazing are the major threats for the erosion of this important plant species. For conserving the species, it is critical to develop an efficient in vitro regeneration protocol for rapid multiplication of large number of regenerants. Leaf explants, cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) (0.5, 1, 2µM), showed significant difference in bud sprouting and adventitious shoot induction. The highest shoot bud formation was recorded on MS medium supplemented with 0.5µM TDZ. Shoot tip necrosis (STN), observed after first subculture of shoot buds in same medium, increased in severity with subculture time. Application of calcium (4mM) and boron (0.1mM) in combination with kinetin (10µM) in the subculture medium significantly reduced the intensity of STN. On an average eight shoots/explant were produced by alleviating this problem. ISSR marker analysis revealed monomorphic banding pattern between progenies and parents, indicating the true to type nature of the clones and its parents.

Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.)

The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro bud induction and proliferation of a potent medicinal woody shrub R. tetraphylla were investigated. Multiple shoots were induced from nodal explants on Murashige and Skoog medium augmented with different concentrations (0.2–1.0 µM) of TDZ. Continuous exposure beyond 5 weeks of singly supplemented TDZ resulted in hyperhydricity, fasciation and distortion of cultures. To avoid these deleterious effects, benzyl adenine (BA) was supplemented to the media. It not only mitigated the negative effects of prolonged TDZ exposure, but also enhanced shoot multiplication rate. About 30 shoots per explant with an average shoot length of 5.8 cm were obtained after 20 weeks of culture on MS medium fortified with 0.4 µM TDZ and 2.0 µM BA, regeneration frequency was also enhanced by 24% on this media as compared to singly supplemented TDZ. Microshoots (~ 5 cm) were successfully rooted on filter paper bridge in MS liquid medium augmented with different concentrations of indole-3-butyric acid (IBA) after 4 weeks of incubation. Best rooting response (80%) was achieved on MS medium supplemented with 0.3 µM IBA with (9.3 ± 1.45) mean number of roots and (3.3 ± 0.33 cm) root length per shootlet. The in vitro raised healthy plantlets with well developed root and shoot were successfully hardened off and acclimatized in thermocol cups containing sterilized planting substrate, Soilrite® for 8 weeks under culture room conditions prior to field transfer and successfully established in earthen pots with 90% survival rate. The genetic fidelity of in vitro regenerated plantlets was evaluated using random amplified polymorphic DNA markers. No polymorphism was observed in banding pattern among micropropagated and the donor plant, thus, exhibiting their genetic uniformity and clonal fidelity.

Quebra da dormência de macieiras ‘Daiane’ pelo uso do tidiazurom

The aim of this study was to evaluate the effect of different concentrations of thidiazuron (TDZ) combined with mineral oil (MO) or calcium nitrate on phenology, bud breaking and fruit production in ‘Daiane’ apple trees. The experiment was carried in Cacador, SC, during the 2013/2014 and 2014/2015 seasons. The treatments evaluated were control (without application); TDZ 250 mg L-1 + MO 3,5%; TDZ 125 mg L-1 + MO 3,5%; TDZ 250 mg L-1 + calcium nitrate 5%; hydrogen cyanamide (HC) 0,7% + MO 3,5%. A randomized block design was used, with six repetitions. The application of bud break promoters reduced the flowering period in 2013/2014 season, leading to a higher bud break of axillary buds than control in the two seasons. TDZ + MO was more effective for bud break in axillary buds than the other treatments. TDZ + MO and HC + MO had similar effect on bud break in terminal buds, with higher values than TDZ + calcium nitrate applications. In 2013/2014 season, the control and TDZ 250 mg L-1 + MO and HC + MO treatments showed similar fruit production, being higher when compared to the other treatments. For 2014/2015, the fruit weight was higher in plants submitted to applications of TDZ 250 mg L-1 + MO and HC + MO. The TDZ combined with MO is efficient in bud break induction of ‘Daiane’ apples, being a viable alternative to HC application.

Evaluation of genetic homogeneity of Jatropha curcas L. hybrid at an early stage of shoot bud formation from petioles using ISSR marker

ABSTRACTGenetic homogeneity is known to be the most important prerequisite in the micropropagation of Jatropha curcas L. to produce true-to-type plants. The detection of genetic homogeneity in clonal micropropagation for elite plants at an early stage is required, to avoid any increase in variation in the next stage of micropropagation. The genetic homogeneity was assessed during shoot bud formation from petiole explants of J.curcas (P1 × P3) hybrid with different concentrations of thidiazuron (TDZ) in a range of 0.5–4.0 mg/L using inter simple sequence repeat (ISSR) markers. Out of 23 ISSR primers, 16 primers produced clear, distinct and reproducible bands. A total of 96 bands, ranging in size from 100 to 1013 bp were generated. Based on the band data, a total of 94 bands were monomorphic (98%) and two bands were polymorphic (2%). All banding patterns from the shoot buds induced by 0.5, 1.0 and 2.0 mg/L TDZ were monomorphic, but 4.0 mg/L gave 2% polymorphism. These findings indicated that concentrations of 0.5, 1.0 and 2.0 mg/L TDZ did not trigger any somaclonal variation and could, therefore, be considered suitable for application in clonal micropropagation of J. curcas hybrid using petioles as explant material.

Micropropagation, berberine content and antitumor activity of Jeffersonia dubia (Maxim.) Benth et Hook

An efficient micropropagation protocol was developed for Jeffersonia dubia using sucker explants. High frequency of multiple shoot formation was induced when the sucker explants were cultured on Chu’s (N6) medium with different concentrations of thidiazuron (TDZ) plus 0.54 µM α-naphthaleneacetic acid (NAA). The maximum frequency of shoot formation (96.2 %) was obtained on N6 medium with 2.27 µM TDZ plus 0.54 µM NAA. The highest mean number of shoots per explant (13.6) was obtained in temporary immersion system using an immersion frequency of 30 s every 30 min. The highest frequency of rooting (100 %), number of roots per shoot (5.8), and root length (6.3) was observed in half-strength N6 medium supplemented with 2.69 µM NAA. The regenerated plantlets (30 days old) were successfully acclimatized in the greenhouse with 98 % survival rate. The berberine content and cytotoxicity were higher in in vitro-developed calli and shoots than in leaves of field-grown plants. The greatest content of berberine was found in shoots (1381 μg g−1) followed by calli (1092 μg g−1) and leaves of field-grown plants (92 μg g−1). At 1000 μg mL−1 concentration, growth inhibition rate of berberine, callus, shoot, and leaf (in vivo) extracts were 68.4, 57.1, 54.2, and 17.7 %, respectively.

Genetic modification of Rosa pinna (Clerodendrum philippinum Schauer) with gai mutant gene for improved plant architecture

Rosa pinna ( Clerodendrum philippinum Schaue r), is a shrub with attractive and fragrant flowers. However, the tall and spread architecture and high leaf to flower ratio hinder its use as an ornamental plant. Hence, changing the level of acceptance of C. philippinum by incorporating a dwarfing gene to the plant is considered useful. A study was conducted to find a suitable regeneration medium and ultimately to modify the plant architecture through transformation of the gibberellic acid inhibitor (gai) mutant dwarfing gene by particle bombardment. Effects of different concentrations of thidiazuron (TDZ:Nphenyl N’ 1,2,3-thidiazol-5-yl urea) on callus initiation and regeneration of shoots and the effects of micro career flying distance in gene transformation of C. philippinum were investigated. The regenerated shoots were first cultured on hormone free MS medium and transferred to a MS medium with 0.5 mg/L in dole butyric acid for rooting after four weeks. The shortest duration for callus initiation, the largest callus volume (28 days after establishment) and the highest number of shoots (75 days after establishment) were observed in 1.5 mg/L TDZ in the MS medium. Results of transferring the gai mutant gene to in vitro leaves of C. philippinum revealed that both the shortest time duration and the highest plant regeneration were at 9 cm micro-carrier flying distance. Leaf tissues of regenerated plants, which carry the gai mutant gene, positively responded for the beta-glucuronidase (GUS) assay. The regenerated shoots were rooted and successfully acclimatized. Tropical Agricultural Research Vol. 25 (1): 27-37 (2013)

Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis.

The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure.

Effects of Thidiazuron and Activated Charcoal on in vitro Shoot Proliferation and Rooting of Myrtle (Myrtus communis L.)

In this study, a micropropagation approach was developed for a commercially important myrtle clone ‘Asi Mersin’ in Turkey. The effects of different concentrations of thidiazuron (TDZ), 6-benzylaminopurine (BAP) and naphthalene acetic acid (NAA) on shoot formation and the effect of activated charcoal on rooting were studied. The most of the shoots were obtained on Murashige and Skoog (MS) medium containing 0.3 mg L-1 TDZ + 0.1 mg L-1 NAA in the 1st subculture (3.8 shoot), and from MS with 0.3 mg L-1 TDZ + 0.01 mg L-1 NAA in the 2nd subculture (4.0 shoot). The explants were rooted in ½MS containing indole-3-butyric acid (IBA), NAA and activated charcoal (AC). IBA applications induced more rooting than NAA. The medium supplemented with 1.0 mg L-1 IBA + 2.0 g L-1 AC resulted in the highest rooting ratio (80%). Addition of AC into the medium resulted in slight increase in the rooting ratio, significant increase in shoot length, and reduced darkening in the rooting area. Acclimatization was successful for 86% of the rooted plants

Indirect regeneration from in vitro grown leaves of three pear cultivars and determination of ploidy level in regenerated shoots by flow cytometry

The aim of the present investigation was to optimize a protocol for indirect regeneration in three commercially important pear cultivars: “Bartlett”, “Dargazi” and “Harrow delight”. Effects of different concentrations of thidiazuron (TDZ) and naphthaleneacetic acid (NAA) in modified Murashige and Skoog culture medium (1/2 strength micro elements, 1/4 strength macro elements and full strength vitamins and mio-inositol for induction phase & 1/2 strength macro and micro elements and full strength vitamins and mio-inositol for regeneration phase), explant types (upper and lower leaf sections) and cultivars were examined based on the quality and quantity of callus formation, indirect regeneration rate and mean number of shoots per explant. Three kinds of callus were detected during the induction phase. Only white and puffy callus (type I) proceeded to shoot regeneration while callus type II (similar to callus type I, becoming green and watery in some parts) and callus type III (green or creamy in color and watery) did not show regeneration potential. The highest percentage of explants producing callus type I (100%) and regeneration (64%) and the highest mean number of shoots per explant (10.6) was obtained from cv Dargazi on medium containing 16μM TDZ. The lower section of leaves was significantly more effective than the upper section regarding the parameters studied. Flow cytometry analysis indicated that regenerated shoots and donor shoots were identical with respect to the ploidy level. Shoot regeneration from organogenic callus was successfully achieved and regeneration capacity varied depending on genotype, explant type and plant growth regulator combinations.

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.

An efficient plant regeneration protocol from petiole explants of physic nut (Jatropha curcas L.)

Efficient and reproducible method for the in vitro regeneration of Jatropha curcas plants has been developed through direct shoot buds induction from petiole explants on Murashige and Skoog (MS) basal medium supplemented with different concentrations of thidiazuron (TDZ). The highest percentage of shoot buds induction (64.0%) was observed on MS medium supplemented with 0.52 mgL -1 TDZ with organic additives; adenine sulphate (50 mgL -1 ) + glutamine (100 mgL -1 ) + L-arginine (25 mgL -1 ) + citric acid (0.0025%) + ascorbic acid (0.005%). A maximum of six shoots per explant were elongated when explants with induced shoot buds were transferred on MS medium containing 3.0 mgL -1 6-benzyladenine (BA) and 1.0 mgL -1 indole-3-butyric acid (IBA). The regenerated shoots were rooted on half strength MS medium supplemented with 1.0 mgL -1 indole-3-acetic acid (IAA) and 0.2 mgL -1 IBA. The rooted plantlets were established in soil with more than 90% survival. Keywords: Jatropha curcas , thidiazuron, petiole, shoot buds, plantlets

Establishment of in vitro plant regeneration system for Chimonanthus praecox (L.)

An efficient protocol for plant regeneration of Chimonanthus praecox (L.) Link, was developed using leaves from seedlings of seeds. Shoots induction were influenced by cytokinins and nodal positions. The results show that the highest callus induction frequency was obtained on MS medium supplemented with 2.0 mg l −1 6-benzyladenine (6-BA) and 0.9 mg l −1 1-naphthalene-acetic acid (NAA) after 10 days incubation in darkness. The frequency of callus induction of different nodal leaves (from the first to the forth node) varied from 70.0 to 93.5%; the highest frequency of callus induction (93.5%) was observed in the second node group. Through many times subculture of callus, green and compact callus was selected and transferred to regeneration medium (MS, 1/2 MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ) (2.37, 4.74 and 9.48 mg l −1 ) and NAA (0.74, 1.48, and 2.96 mg l −1 ). It was shown that the optimal combination of the three factors was WPM + NAA + TDZ. The highest adventitious shoot regeneration frequency of leaf explants (71.33%) and the highest mean number of shoots per explant (2.23) were obtained on WPM supplemented with 2.37 mg l −1 TDZ and 1.48 mg l −1 NAA. Optimal rooting was seen on 1/2 MS medium with 1.5 mg l −1 IBA for eight days, followed by transfer to hormone-free MS medium. Rooted plantlets were easily acclimated in a glasshouse. Key words: Chimonanthus praecox, in vitro regeneration, adventitious buds.