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1.
A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.  相似文献   

2.
An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L−1 activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L−1 activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.  相似文献   

3.
The effect of various concentrations of CuSO4 on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO4. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO4 was significantly higher.  相似文献   

4.
Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.  相似文献   

5.
A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.  相似文献   

6.
Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.  相似文献   

7.
Various explants of Minthostachys andina (Brett.) were evaluated for their morphogenic potential under in vitro culture conditions. Axillary buds derived from 2 year-old plants grown in MS-medium supplemented with 4.4 or 8.8 μM BA and 0.054 μM NAA, initiated shoot growth and new shoot formation. Under subculture in NN medium, shoots were rooted in the presence of NAA (1.6, 2.7 or 5.3 μM) alone or in combination with IBA (9.8 μM), and the regenerated plantlets were later acclimatised in the greenhouse. Also, polynodal segments from seedlings initiated multiple shoots and plantlets when initially cultured in presence of NN-liquid salt medium supplemented with 2.2-17.7 μM BA or 4.5-13.6 M TDZ in combination with different auxin-like growth regulators and after a final transfer for root initiation. The same types of responses were found in hypocotyl and leaf explants, which produced adventitious shoots in the presence of TDZ. The use of antioxidants helped to prevent browning and favoured organogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

8.
Zygotic embryos of Karwinskia parvifolia, isolated from seeds obtained from different regions of Mexico, were cultured on Woody Plant Medium (WPM) supplemented with 0.06 μM indole-3-acetic acid, 0.03 μM gibberellic acid, and 2 μM 6-benzylaminopurine. The growth of embryos and multiplication of shoots from stem segments were achieved. Rooting of excised shoots could be initiated on basal WPM medium with prolonged subculture period to 2 months, or on WPM medium supplemented with 10 μM 1-naphthaleneacetic acid. Multiplication capacity of shoots and rooting of K. parvifolia differed in dependence on the origin of explant material. The shoot multiplication was much lower than that of Karwinskia humboldtiana. The rooting depended on the origin of K. parvifolia seeds. The regenerated plants were successfully transferred to glasshouse. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

9.
A successful system of direct organogenesis is described for the wild citrus tree, Citrus halimii Stone which used in vitro seedling explants cultured on Murashige and Skoog medium supplemented with 0.4–11.1 μM 6-benzyladenine. Hypocotyl was the best explant for multiple shoots regeneration. Maximum number of shoots was obtained on medium with 2.2–11.1 μM 6-benzyladenine. Rooting of regenerated shoots was best on Murashige and Skoog medium supplemented with 2.7 μM α-naphthalenacetic acid. In vitro and ex vitro rooted plantlets survived (on average 83.3%) after being transferred to the soil mixture consisting of soil, sand and organic material (1: 1: 1) and kept in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

10.
Multiple shoots were regenerated from leaf explants obtained from in vitro grown shoot cultures of pineapple. Each leaf was horizontally cut into three pieces (~ 0.5 cm, basal, middle and tip) and cultured onto MS basal medium supplemented with 2% sucrose and various growth regulators.The explant containing the basal part of the leaf gave rise to tiny protuberances which grew into shoots.The highest number of shoots were obtained on MS basal medium supplemented with 2,4-D (0.90 µM) and 2iP (0.98 pM).These shoots were subcultured ontowhite’s basal medium supplemented with 1% sucrose, NAA (0.54 µM) and IBA (1.97 µM). Plantlets produced in vitro were transferred to paper cups containing autoclaved soil or Soilrite, hardened in the greenhouse and established in soil.The protocol provides an easy propagation system for pineapple, an otherwise vegetatively propagated fruit crop.  相似文献   

11.
Plant regeneration from cultured immature inflorescence segments (3–5 mm) of sugarcane (Saccharum sp) var. CP 5243 was obtained via somatic embryogenesis. Embryogenic callus culture was initiated on MS medium supplemented with 2,4-D (13.5 μM) over 30 days. The callus was subcultured every 15–20 days on MS medium supplemented with 2,4-D (4.5 μM), arginine (50 mg l-1) and proline (500 mg l-1). The callus was subjected to five treatments: 2,4-D (4.5 μM), Picloram (8.2 μM) and Dicamba (22.6 μM). SPC was determined at the beginning, after 20 days in culture, and every 24 hours thereafter up to 72 hours. SDS-PAGE electrophoresis was performed based on soluble protein content. Some differences were found between SPC and bands (intensity and number) for all treatments associated with shoot formation. The results point out the association of soluble protein content and callus regenerative ability of sugarcane cv. CP5243 and suggest the presence of a marker protein (between 55–70 kDa) for embryogenic callus regeneration ability in this cultivar. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

12.
Thidiazuron (TDZ), a substituted urea with cytokinin-like activity stimulated shoot proliferation in chickpea (Cicer arietinum L). Embryonic axis with half portion of both cotyledons was more responsive explant, producing ~22 shootslexplant at 1.0 µM TDZ concentration but higher concentration of TDZ (5.0 µM) reduced both the shoot proliferation and growth. An incubation time of 12 h at 1.0 µM TDZ was sufficient for induction of multiple shoot formation.TD2 induced high frequency of shoot formation as compared to BAP and also minor salts of MS medium played an important role in increasing the number of shoots. Roots could be induced in these shoots in MS medium supplemented with 0.5 µM IBA.  相似文献   

13.
Plant regeneration ability of ginseng (t Panax ginseng C.A. Meyer) via organogenesis was studied. Compact callus was induced from four different types of explants-leaf, petiole, flower stalk, and root of t in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.5 μM) and kinetin (0.46 μM) were conditioned for two weeks on the same medium. These calli differentiated into adventitious shoots when cultured on 1/2MS basal medium plus kinetin 4.7 μM and silver thiosulphate 10 μM. An addition of GA3 (2.9 μM) and BA (4.4 μM) to MS basal medium, however, induced high frequency t in vitro flowering (86.1%) and multiple shoot budding which affected the normal complete development of plantlets. Plantlets with a well-developed root system were obtained six weeks after regenerated shoots had been transplanted to 1/2 MS20 medium containing IBA 1.2 μM. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all the regenerants were typically diploids as the mother plants were, indicating that nuclear DNA content remained stable during cell differentiation. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

14.
The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N1) from the basal cut end similar to cytokinin–auxin combination (N2). Between two types of nodular tissues, N2 was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA3). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.  相似文献   

15.
A rapid clonal propagation method for Fibigia triquetra (DC.) Boiss. (Brassicaceae), a rare Croatian stenoendemic species, has been developed. Shoots originated from aseptically germinated seeds were used for culture initiation. The highest multiplication rate of 9.2 shoots per explant was achieved in a 4-weeks-culture period in the third subculture on half strength Murashige and Skoog medium supplemented with 0.5 μM 6-benzylaminopurine and 2.9 μM gibberellic acid. Excised shoots were rooted best with addition of 8.6 μM indole-3-butyric acid on the same basal medium. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

16.
An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA3). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.  相似文献   

17.
Multiple shoot formation from cotyledonary node segments of Eastern redbud   总被引:4,自引:0,他引:4  
A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 μM BA and 0.5 or 1.0 μM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 μM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.  相似文献   

18.
Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L?1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1–3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L?1) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4 °C for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21 to 66.7% of the SE generated for both genotypes.  相似文献   

19.
Somatic embryogenesis was obtained from transverse thin cell layers (tTCLs) of Digitaria sanguinalis. tTCLs (0.2 - 0.4mm thick, 1mm in diameter) were excised from 4-week-old seedlings and placed onto Murashige and Skoog media supplemented with a varying concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (from 1 μM to 100 μM) and sucrose (from 3% to 24%). Somatic embryos were obtained in the dark 7-10 days after inoculation from tTCLs excised at specific levels on the seedling and cultured in the presence of 2,4-D (5 μM to 10 μM) and sucrose (3 to 6%). The exposure of the tTCLs to light decreased the percentage of tTCLs forming somatic embryos. Viable plantlets were obtained 2 weeks after transfer onto a cytokinin-containing medium. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

20.
Efficient and simple, organogenesis (direct and indirect) and somatic embryogenesis (cell suspension) systems were developed for in vitro propagation of Cyrtanthus mackenii, a valuable economic plant from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of sucrose, plant growth regulators (PGRs), glutamine, phloroglucinol (PG) and 6-(2-hydroxy-3-methylbenzylamino) purine (PI55). MS medium solidified with 8 g L?1 agar (MSS) containing 40 g L?1 sucrose, 10 µM picloram, 2.5 µM benzyladenine (BA) and 20 µM glutamine produced a higher number of shoots from white nodular callus. This was however, not significantly different to direct shoot regeneration on media containing 10 µM picloram, 2.5 µM BA and a reduced concentration of sucrose and glutamine. The regenerated shoots were rooted best with MSS medium incorporating 10 µM PG. The number of somatic embryos (SEs) were significantly higher using liquid MS medium containing 30 g L?1 sucrose, 0.5 µM picloram, 1 µM thidiazuron or BA and 3 µM glutamine or gibberellic acid. The embryos were germinated in PGR-free MSS medium. All plantlets were successfully acclimatized in the greenhouse. Histological studies confirmed the different developmental stages and bipolar structure of SE. The organogenesis and somatic embryogenesis protocols provides a system for large scale propagation and germplasm conservation. Developed protocols can be used for clonal production and pharmacological and genetic transformation studies.  相似文献   

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