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1.
High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l−1 BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l−1 α-naphthaleneacetic acid and 0.5 mg l−1 BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l−1 BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.  相似文献   

2.
An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L−1 6-Benzylaminopurine (BAP) + 0.1 mg L−1 Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L−1 BAP + 0.1 mg L−1 IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L−1 BAP + 0.1 mg L−1 IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L−1 Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.  相似文献   

3.
The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l?1 6‐benzylaminopurine and 0.15 mg l?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l?1 AgNO3 was very beneficial to shoot regeneration in B. napus and Ag2S2O3 (10 mg l?1) was even superior to AgNO3 (2.5 mg l?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.  相似文献   

4.
Data presented herein provides a rapid and efficient method for Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima for hairy root cultures as well as for enhancing Shikonin production. Etiolated explants viz. shoot tip, nodal, leaf and internodal segments were co-cultivated with Agrobacterium rhizogenes for induction of hairy root. Among the various explants employed, leaf explant showed maximum 70.7% response followed by shoot tip 48.3%, nodal segment 38.7% and internodal segment 9.3%. Integration of Ri plasmid rolB gene in the transformed hairy root cultures was confirmed by PCR analysis using forward (FrolB) and reverse (RrolB) primers of rolB gene resulting in the amplification of 0 ∼ 0.8 kb fragments. Medium compositions have been optimized for in vitro induction of Shikonin in hairy root cultures of Arnebia hispidissima. Hairy roots on hormonefree MS medium showed red spots in the older part of the tissues which turned white after a second subculture. Whereas hairy roots cultured on RC medium showed faster growth and produced large amount of Shikonin. The Shikonin content in transformed hairy root culture was estimated by recording absorbance at 620 nm and quantified against authentic sample of Shikonin. Shikonin content was estimated to be 0.85 mg g−1 fresh weight of tissue at the end of the 50 days of culture. The results presented herein will help to design strategies for bridging the gap between ever increasing demand and supply of raw products necessary for obtaining Shikonin for cosmetic, dyeing, food, medicinal, and pharmaceutical industries.  相似文献   

5.
Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA3, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.  相似文献   

6.
Summary Multiple shoots buds were obtained successfully from shoot tips of Acacia saligna by placing explants into solidified Murashighe & Skoog (1962) medium (MS medium) supplemented with 5.0 to 9.0 mg/L BAP. Sequential culture treatment was highly effective for shoot elongation using MS medium containing 0.3 mg/L BAP and 0.2 mg/L IAA. The shoots rooted best on MS medium supplemented with 2.0 mg/L IBA. Plantlet survival after transfer to soil was more than 90%. The shoot proliferation method described could be used for the mass clonal propagation of selected genotypes.  相似文献   

7.
Protocols of plant regeneration have been developed for Brassica carinata for creating somaclonal variation for plant type and adaptability, so that this species can fit into cropping systems in Indian agriculture. The response of cotyledonary and stem explants was assessed for callus induction and shoot regeneration on MS and B5 basal media containing different combinations of auxin and cytokinin concentrations. MS medium supplemented with BA and NAA favoured callus induction. Supplementing MS with combinations of BA and IAA, as also with BA alone, regenerated shoots from the ex pi ants with a high frequency. The frequency of shoot regeneration and the mean number of shoots per explant were higher in cotyledons than in stem explants on identical growth regulator combinations. On B5 medium, supplemented with BA (2 mg/l) and IBA (0.4 mg/l), compact callus was produced which regenerated shoots on transfer to medium containing BA (0.8 mg/l). Genotypic differences among carinata accessions for regeneration were also observed.  相似文献   

8.
The importance of fast-trackt generation advancement in developing superior germplasm has been recognized in breeding of many crop species. To address this issue in tomato, immature seeds were excised from fruit at different maturity stages and transferred to culture medium. The best culture medium was modified full strength Moorashige–Skoog (MS) salts supplemented with 0.1 mg l−1 IAA, 0.5 mg l−1 IBA, 0.5 mg l−1 GA3 and 2% sucrose. If the excised seeds were able to grow, most showed shoot formation after a week. Seeds extracted as early as 10 days after pollination were successfully cultured provided they were transferred aseptically and without injury. No morphological or physiological changes in regenerated plants and their fruit relative to the parent were detected. Germination from immature seeds of tomato is a simpler alternative to in vitro culture of immature embryos or callus, as it can be undertaken in comparatively less stringent laboratory conditions. Using this approach, five generations can be produced in a year in contrast to a maximum of three generations with conventional methods. This offers an opportunity for rapid generation advancement aimed towards population development when coupled with marker assisted selection in tomato breeding for biotic and abiotic stress tolerance.  相似文献   

9.
The genus Crocus comprises plants with a potential to be developed as a new ornamental crop but to date, there are not many reports on in vitro propagation of many members of this genus. The present study involves in vitro propagation of Crocus cancellatus with ornamental and horticultural value. Two different types of corm explants (apical and basal halves of corms) were cultivated onto Murashige and Skoog’s (MS) medium supplemented with different levels of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). One to five cormlets emerged from every responding explant through direct organogenesis. Apical halves of corms were more highly responsive than basal halves and produced a maximum multiplication rate with 3.45 ± 0.06 cormlets per explant in 95.33 ± 2.33% of the explants in MS medium supplemented with 3% sucrose and 2 mg L−1 NAA and 1 mg L−1 BAP. The effect of cold storage temperature on in vitro cormlets sprouting was studied. Cormlets stored at 4°C for 8 weeks had more statistically significant positive effects on cormlets sprouting from the controls. In vitro rooting of cormlets was induced on MS medium without plant hormones.  相似文献   

10.
In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

11.
Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l–1 BA+0.05 mg l–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l–1 BA+0.1 mg l–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.  相似文献   

12.
Summary To improve the efficiency of papaya anther culture, we investigated (1) hormonal medium conditions for inducing haploids or dihaploids; (2) identified the sex of established plantlets using a sex-specific DNA molecular marker and (3) estimated their ploidy by flow cytometry analysis of DNA content. Anthers with a mixture of uninucleate, mitotic, and binucleate microspores were collected from a male plant, and cultured on MS agar medium with different concentrations of CPPU and NAA. An embryo induction rate of 13.8% was attained on MS agar medium with 0.01 mg l−1 CPPU and 0.1 mg l−1 NAA. The induced embryos were subcultured on medium with 0.0025 mg l−1 CPPU. Rooting of the developed shoots was promoted by treating their basal parts with 1500 mg l−1 IBA in a 50% ethanol solution for about 10 seconds. All the embryo-derived plantlets (27 plants) were identified as female, implying that they were derived from microspores. In addition, 26 plants were determined to be triploids and one to be tetraploids. We also observed a wide range of morphological variation (e.g., in tree height and fruit size) among the established plants. Based on the results, we discussed a potential value of anther culture techniques for the breeding of papaya.  相似文献   

13.
Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L−1 kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L−1 sucrose and 0.5 mg L−1 indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.  相似文献   

14.
Acorus calamus is an important medicinal plant which has been used in Indian traditional medicine since time immemorial. Various bioactive molecules, viz., acorin, α- and β-asarone, asaryldehyde, caryophylene, isoasarone, methylisoeugenol, and safrol have been isolated from this plant. However, the use of this plant for medicinal purpose has been recently banned due to the high toxic property of β-asarone. The triploid Acorus calamus is reported to be low in β-asarone content and thus found to be the ideal raw material for medicinal use. The present investigation represents our finding for successful in vitro clonal propagation of the elite triploid accessions of Acorus calamus for mass propagation. In the dual-phase culture system consisting of agar-solidified Murashige and Skoog medium overlaid by liquid fraction of the same medium, maximum multiple shoot induction was favored by supplementation of α-naphthaleneacetic acid (0.5 mg L−1) and 6-benzylaminopurine (2.0 mg L−1). In vitro rooting of the microshoots was maximum in the medium supplemented with indolebutyric acid at 2.0 mg L−1. The well-rooted microshoots could be successfully hardened and transplanted in the field. This result can be reproduced and is a viable protocol for successful clonal propagation of the seedless triploid Acorus calamus for conservation and sustainable development.  相似文献   

15.
Although several studies have been made on the micropropagation of Jatropha curcas using agar base mediums, none of them have been by using liquid medium systems. The effects of explant type and temporary immersion system (test tube, jar with filter paper boat, and growtek bioreactor) on the micropropagation of J. curcas were studied. The explant type influenced shoot quality, multiplication coefficient (MC), and rooting. Leaf explant produced more and longer shoots than nodal explant. Use of filter paper (FB) boat prevented hyperhydricity and allowed proliferation of nodal explants cultured in liquid MS (Murashige and Skoog) medium supplemented 6-benzylaminopurine (BAP) and Kinetin (KN). The best shoot bud induction (92.1±3.1%) was achieved in liquid MS medium supplemented with 2.0 mg/L KN. Leaf regeneration efficiency was compared in growtek bioreactor and in jar containing liquid MS medium supplemented with 0.5 mg/L Thidiazuron (TDZ). The best shoot bud regeneration (78.7±2.1%) was obtained in growtek bioreactor. Shoot buds achieved from nodal segment and leaf were subcultured on filter paper boats in jar and bioreactor containing liquid MS medium supplemented with BAP, Indole butyric acid (IBA), Indole-3-acetic acid (IAA), and KN. Best shoot proliferation and elongation was obtained in filter paper boats containing liquid MS medium supplemented with 1.5 mg/L BAP, 0.5 mg/L IAA, and 0.2 mg/L KN. The number of multiple shoot buds was higher in leaf explants as compared to nodal explants and the highest number of multiple shoot buds was recorded from leaf explants. Up to 76.4% rooting efficiency was obtained when the shoots were ex vitro rooted. The generated plants well established in the nursery and grew normally in outdoor conditions. The protocol has good potential for application in large-scale propagation of J. curcas using liquid medium.  相似文献   

16.
Multiple shoots were efficiently regenerated from cotyledonary node and shoot tip explants of Pisum sativum within 15 days on MS medium containing B5 vitamins and supplelmented with 2.0 mgl-1 6-benzylaminopurine. The elongated shoots produced on the same medium were excised and transferred to MS medium containing half strength ammonium nitrate (8.25 gml-1) and supplemented with auxins (indole-3-butyric acid or naphthalene acetic acid) either alone or in combinations with gibberellic acid. Rooting and flowering were observed on the 7th and 15th day after their transfer to rooting medium. The flowers self-fertilised in vitro and produced mature pods within 25 days of rooting. These seeds were germinable both in vitro and in vivo. In vitro seeds sown in pots under field conditions developed into flowering plants, and subsequently produced pods with viable seeds. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

17.
The present study describes the procedure for micropropagation of Dracocephalum kotschyi L. using shoot tips from in vitro-germinated plants. The best response was observed for shoot tips on MS medium containing 5 mg 6-benzylaminopurine L?1 and 0.2 mg 1-naphthaleneacetic L?1 acid. Regeneration for other types of the explant hypocotyls and cotyledons did not show satisfactory results so that the explants did not develop into normal shoots and in turn developed into the calli after 12 days of culture. Histochemical analysis showed that only the shoot tip revealed a direct induction of more teratological protuberances that arise around the cut end of the explants. Elongation of shoot buds was obtained on MS medium containing 1 mg BAP L?1 + 0.5 mg IBA L?1. Regenerated shoots rooted best on the same medium of elongation. After hardening, the rooted plants were transferred to the greenhouse where they grew, matured, and flowered normally with a survival rate of 95%. We concluded that the present protocol can be efficiently used for mass propagation of Dracocephalum kotschyi.  相似文献   

18.
An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog’s, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation.  相似文献   

19.
We established an advanced protocol for in vitro propagation of Aloe vera via comparison of basal media, sucrose contents, growth hormone combinations, and additional supplementation with various polyamines. The maximal number and growth of shoots after 5 weeks was obtained using MS media including 30 g L?1 sucrose supplemented with 1.0 mg L?1 BA and 0.1 mg L?1 NAA. To improve shoot production, various concentrations of putrescine, spermidine, and spermine were added under optimal growth hormone conditions (MS media supplemented with 30 g L?1 sucrose, 1.0 mg L?1 BA, and 0.1 mg L?1 NAA). Maximal shoot number and growth after 5 weeks were achieved with supplementation of 50 mg L?1 spermidine. Regenerated plants were successfully acclimatized in soil with 100% efficiency. Cytogenetic inspection revealed that the regenerated plants maintained intact chromosomes identical to those of plants grown in field conditions. This protocol provides a valuable alternative for mass production of elite Aloe vera.  相似文献   

20.
H. Paul    B. Henken    Th. S. M. de  Bock W. Lange 《Plant Breeding》1992,109(4):265-273
Plant regeneration from callus cultures of Allium trifoliatum subsp. hirsutum fertile accession F-370, was studied as a means for clonal multiplication and germplasm storage of Allium spp. Callus was induced on in votro-cultured basal leaf explants. Best proliferation was obtained on modified BDS medium supplemented with (mg/1): 0.75 picloram, 2.0 benzyl adenine, and 900 casein hydrolysate. Shoot and root organogenesis were obtained in 3 to 5 month old subcultured calli, on BDS or MS medium supplemented with (mg/1): either 0.03 picloram or no auxin, 2 BA or 2-isopentenyladenine, and 900 casein hydrolysate. Direct bulb formation, without shoot elongation, occurred on BDS medium with 10 mg/1 IBA. Under these conditions, callus formation and organogenesis were not obtained with A. trifoliatum subsp. hirsutum var. sterile, a male-sterile genotype. Most regenerants were phenotypically normal, but some abnormal shoots were also observed, i.e. shoots with vitrified or extremely broad leaves. Isozyme polymorphism analysis of seven proteins in the latter regenerants, and in several callus cultures, revealed significant deviation from the original pattern in esterase, 6-phosphogluconate dehydrogenase and superoxide dismutase. No such deviations were detected in normal regenerated plants.  相似文献   

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