The effect of drying and submersion pretreatment on adventitious shoot regeneration from hypocotyl explants of flax (Linum usitatissimum L.)
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Hypocotyl explants of 3 flax cultivars were cultured for adventitious shoot regeneration in 3 different ways. Two pretreatment applications were compared with the routinely applied conventional regeneration protocol of culturing explants directly on Murashige and Skoog (MS) medium containing 1 mg L^{-1} 6-benzylaminopurine (BAP) and 0.02 mg L^{-1} naphthalene acetic acid (NAA). In the first pretreatment application, explants kept in a sterile cabin under an air flow for 30 min were immersed in MS solution containing 1 mg L^{-1} BAP and 0.02 mg L^{-1} NAA for 15 min; then pretreated explants were cultured on MS medium without any growth regulators (MS0). In the second pretreatment application, explants were kept in a sterile cabin under air flow for 30 min and then immersed in MS solution containing 1 mg L^{-1} BAP and 0.02 mg L^{-1} NAA for 15 min. The pretreated explants were then transferred to a culture medium enriched with 1 mg L^{-1} BAP and 0.02 mg L^{-1} NAA. Fresh and dry weights of hypocotyl explants, shoot regeneration percentage, shoot number per hypocotyl, shoot length, and total chlorophyll content were recorded. From the results, it could be seen that treating explants before culture initiation for regeneration with a liquid MS medium containing 1 mg L^{-1} BAP and 0.02 mg L^{-1} NAA for 15 min after keeping hypocotyls under an air flow in a sterile cabin for 30 min gave rise to the highest scores for tissue culture response.Keywords:
Linum
Kinetin
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Effects of different concentrations of IAA (0, 0.3, 0.6 mg/l) and 2ip (0, 0.3, 0.6, 0.9, 1.2 mg/l) and their combinations on callus induction and shoot regeneration of hypocotyl and its thin cell layer (TCL) explants in tomato was studied. Explants were prepared from hypocotyls of seedlings in the aseptic condition. Hypocotyl segments were more efficient than TCL explants for callus induction and it was occurred on the 96.0 percent of hypocotyl explants as compared to 76.5 percent of TCL explants. The mean diameter of formed calli on the hypocotyl explants were significantly more than TCL explants. The calli on hypocotyl explants were more regenerative than calli produced on TCL explants and 60.1 percent of calli produced on hypocotyl explants developed the shoots while the regenerated shoots from the calli of TCL explants were 21.45 percent. Percentage of shoot induction on TCL explants was maximum (47.44 percent) in the medium containing 0.3 mg/l 2ip and 0.6 mg/l of IAA. Explant typehad significant influence on the shoot number per callus. Calli developed on TCL explants regenerated more shoots significantly than hypocotyl explants. The recorded shoot number per callus of TCL and hypocotyl explants were 6.45 and 3.22, respectively. The mean length of developed shoots on the hypocotyl segments was significantly higher than that of TCL explants and maximum length of shoot was obtained on the medium containing only 0.3 mg/l IAA.
Callus
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Rotala rotundifolia (Buch-Ham. ex Roxb) Koehne, an aquatic plant belonging to the family Lythraceae, is used for the treatment of some diseases due to its medical and anti-microbial properties. This study presents multiple shoot regeneration from shoot tip and nodal explants of R. rotundifolia cultured on Murashige and Skoog (MS) nutrient medium containing 0.05-1.25 mg/L Kinetin (KIN) and 0.25 mg/L Gibberellic acid (GA 3 ) combinations for eight weeks. At the end of the second week, shoot formations began to be observed on the explants. High shoot regeneration frequencies were determined for both explants in the culture medium. The maximum number of shoots per explant was obtained from shoot tip (38.66) and nodal (30.77) explants cultured on MS medium containing 0.25 mg/L KIN + 0.25 mg/L GA 3 . Whereas the minimum number of shoots per explant was determined on MS medium containing 1.25 mg/L KIN + 0.25 mg/L GA 3 for both explant types. The highest shoot lengths for shoot tip (1.87 cm) and nodal (1.79 cm) explants were obtained on MS culture medium containing 0.75 mg/L KIN + 0.25 mg/L GA 3 and 0.50 mg/L KIN + 0.25 mg/L GA 3 , respectively. For in vitro rooting of the regenerated shoots, 2 cm long cut shoots were transferred to MS medium containing 0.25 mg/L indole-3-butyric acid (IBA). The rooted shoots were then successfully acclimatized to external conditions in the aquarium environment.
Kinetin
Gibberellic acid
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Enhance efficiency plant regeneration of three cactus species including Gymnocalycium damsii, Echinocereus rigidissimus and Echinocactus grusonii were studied. The objective was to investigate the effects of sucrose, explant types and BA on proliferation, growth and development of cactus. The shoot at size of 0.5 cm of three genotypes cactus were uncut halves and longitudinally cut pieces and cultured on MS (Murashige and Skoog) medium supplemented with 0, 15, 30 and 45 g/l sucrose. After 8 weeks of culture, the results found that each species gave various responses to different concentrations of sucrose. The explant uncut longingly of G. damsii cultured on MS medium supplemented with 15 g/L sucrose gave the highest shoot induction at 35.05%, number of shoot (1.28 shoots/explant) and number of root (1.60 roots/explant). For Echinocereus rigidissimus, the explant cut longingly and cultured on MS medium supplemented with 45 g/L sucrose gave the highest shoot induction at 60.25% and number of shoot (7.53 shoots/explant). The explants cut longingly of Echinocactus grusonii and cultured on MS medium supplemented with 45 g/l sucrose gave the highest shoot induction at 36.18% and number of shoot (3.69 shoots/explant). On the other hand, the explant uncut longingly of Echinocactus grusonii and cultured on MS medium supplemented with 15 g/L sucrose gave the highest shoot length (8.60 mm.), root induction (16.35%), number of root (11.50 roots/explant) and root length (2.40 mm). For effect of BA on growth and development of 3 genotypes cactus, the results showed that shoot of G. damsii and Echinocereus rigidissimus cultured on MS medium supplemented with 1 mg/l BA gave the highest shoot induction (60.25 and 75.25%) and number of shoot (10.40 and 3.53 shoots/explant), respectively, after 4 weeks of culture. On the other hand, shoot of E. grusonii cultured on MS medium supplemented with 2 mg/l BA gave the highest shoot induction at 67.25% and number of shoots at 2.27 shoots/explant.
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A rapid, simple and efficient protocol for in vitro multiple shoot induction and plantlet regeneration was achieved from three different explants of Cicer arietinum. The explants viz shoot tip, cotyledonary node and node were cultured on MS medium fortified with Benzyl Adenine (BA) (0.44-8.88μM) for multiple shoot induction. Multiple shoots proliferation was best observed at 4.44μM BA from all the three explants within two weeks of culture. Of the three different explants tested, cotyledonary nodes produced the maximum number of shoots. Shoot number per explant ranged between 7 and 15. Individual shoots were aseptically excised and subcultured in the same media for shoot elongation. The elongated shoots were transferred to Indole Butyric Acid (IBA) (2.46-12.30μM) for root induction. Rooting was observed within two weeks of culture. Rooted plantlets were successfully hardened under culture conditions and subsequently established in the field conditions. The recorded survival rate of the plants was 76.3%. Plants looked healthy with no visually detectable phenotypic variations.
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In vitro multiple shoot regeneration of Solanum nigrum L., an Indian medicinal plant was accomplished on MS medium utilizing shoot tip and nodal explants. Direct multiple shoots differentiated within 6 weeks when explants were cultured on MS medium containing BAP (1.0-5.0 mg/l) and KIN (1.0-5.0 mg/l) individually. Among various concentrations of cytokinins tested, maximum number of multiple shoots was obtained on MS medium supplemented with BAP (1.0 mg/l) from shoot tip (20.4±0.22) and MS medium supplemented with BAP (3.0 mg/l) from nodal explants (8.4±0.22). The in vitro regenerated shoots were rooted (8.4±0.16 roots per shoot) on MS medium supplemented with NAA (1.0 mg/l) within 2-3 weeks of culture and the regenerated plantlets could be successfully established in soil where they grow normally.
Solanum nigrum
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Rangpur lime (Citrus limonia Osbeck) in vitro organogenesis was studied based on explant type and cytokinin culture media supplementation. Four explants types collected from epicotyl or hypocotyl regions of in vitro germinated seedlings were evaluated. The epicotyl-derived explants consisted of epicotyl segments and the hypocotyl-derived explants consisted of the entire hypocotyl segment, the hypocotyl segment attached to a cotyledon fragment, and the hypocotyl segment divided longitudinally. The explants were cultured on EME culture medium supplemented with benzylaminopurine (0, 0.5, 1.0, or 1.5 mg L-1). The evaluation was performed after 6 weeks. Best results considering both the explant responsiveness and number of shoots developed per explants were obtained when epicotyl segments-derived explants were evaluated. Considering the explant responsiveness of hypocotyl segments-derived explants no difference was detected between the entire hypocotyl segment and the hypocotyl segment attached to a cotyledon fragment. Moreover, the percentage of responsive explants decreased when hypocotyl segments divided longitudinally were tested. No difference was detected for the number of shoots developed per explant considering the three types of hypocotyl-derived explants. Culture media supplementation with BAP was not essential for Rangpur lime in vitro organogenesis. However, adventitious shoot development was stimulated in concentrations between 0.5 - 1.0 mg L-1.
Epicotyl
Cotyledon
Organogenesis
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Abstract In this study, possible effects of 24-epibrassinolide (24-epiBL) pretreatment against NaCl stress were investigated using in vitro shoot organogenesis from cotyledon and hypocotyl explants in M-28 tomato hybrid cultivar. The cotyledon and hypocotyl explants of 10-day sterile seedlings were treated with 1 μM and 2 μM 24-epiBL solutions (prepared with 70% acetone) for 30 seconds and applied explants were cultured on MS medium supplemented with 2 mg/l 6-benzylaminopurine (BAP) + NaCl (0, 20, 40, 60, 80 and 100 mM). It was determined that the regeneration percentage as well as the shoot number/explant, the shoot length and the leaf number/shoot derived from both explants suffered NaCl stress after 30 days. 24-epiBL pretreatment against NaCl stress showed ameliorative effects and hypocotyl explants gave better results than cotyledon explants for these growth parameters. Different stages of shoot organogenesis from hypocotyl explants applied with 24-epiBL under salt stress were observed with scanning electron microscopy. As a result, it was shown that 24-epiBL treatment against NaCl stress may play an effective role in salt tolerance in M-28 tomato hybrid cultivar.
Cotyledon
Organogenesis
Lycopersicon
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