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1.
The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l−1 KIN, 0.25 mg l−1 BAP, 0.1 mg l−1 IAA and 100 mg l−1 CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l−1 IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g−1 fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.  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 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   

4.
R. K. Jain  Ulrike Brune  W. Friedt 《Euphytica》1989,43(1-2):153-163
Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.  相似文献   

5.
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   

6.
Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium  相似文献   

7.
As a medicinal plant, the importance of evening primrose (Oenothera biennis L.) is due to its unsaturated fatty acids in the seeds and roots, and also oenotherine and comfarol in the leaves. Low germination and difficulties in seed production are the main problems encountered with growing this plant in the field. As an alternative approach, an in vitro experiment was set up for the evaluation of evening primrose production via direct and indirect regeneration of the cultivars NC-1 and VNK. For callogenesis and direct regeneration, the explants from the apical bud and petiole were cultured on MS medium supplemented with 0.25, 0.75, and 1.25 mg L?1 of both BAP and Kinetin (KIN). Indirect regeneration was performed by placing apical buds, petioles, and leaf explants on MS medium supplemented with 0.5 and 1 mg L?1 2,4-D and 0.5, 1, and 1.25 mg L?1 of both BAP and KIN. The highest shoot induction from direct regeneration was obtained with apical bud explants of VNK treated with 0.75 mg L?1 BAP. The highest callus weight (3.17 g) obtained from indirect regeneration was with petiole explants treated with 1 mg L?1 2, 4-D and 1 mg L?1 BAP in VNK cultivars. The highest number of torpedo embryogenic clusters (23.8) was obtained from the VNK petiole explants treated with 0.5 mg L?1 2, 4-D and 1.25 mg L?1 BAP. BAP had higher positive effects on in vitro production of evening primrose than KIN in both direct and indirect regeneration. In general, results indicated that VNK was more potent for regeneration than NC-1 and concentrations of 0.75 mg L?1 BAP for direct and 0.5 mg L?1 2, 4-D and 1.25 mg L?1of BAP for indirect regeneration had a higher efficiency for increasing in vitro production of evening primrose.  相似文献   

8.
The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.  相似文献   

9.
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   

10.
Jatropha curcas, the energy plant has attained great attention in recent years because of its biodiesel production potential; however, oil and deoiled cakes are toxic. A non-toxic variety of J. curcas is reported from Mexico. A simple and efficient protocol has been developed for plant regeneration using cotyledonary petiole explants of non-toxic variety of J. curcas. The percentage of induction of shoot buds (59.11%), and the number of shoot buds (5.01) per explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). These induced shoot buds multiplied when subcultured on MS medium supplemented with 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BAP), and 5.5 μM α-naphthaleneacetic acid (NAA) for 4 weeks and subsequent elongation achieved on MS medium supplemented with 2.25 μM BAP and 8.5 μM indole-3-acetic acid (IAA). Shoots more than 2 cm long were harvested and cultured on MS medium containing different concentrations and combinations of IBA, IAA, NAA, and 0.25 mg L?1 activated charcoal, and 19.91% rooting was achieved in 15 μM IBA, 5.7 μM IAA, and 16.5 μM NAA after 4 weeks with more than 90% survival rate.  相似文献   

11.
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   

12.
Factors affecting in vitro propagation and microtuberization were evaluated for Gloriosa superba L., an endangered ornamental cum medicinal plant having limited reproductive capacity. Surface sterilization of tuber explants with 0.1% mercuric chloride (HgCl2) for 5 min eliminated the contamination effectively with highest survival rate. Among the various combinations used, Murashige and Skoog (MS) medium with 2.0 mg L?1 6-benzylaminopurine (BAP) + 0.5 mg L?1 α-naphthalene acetic acid (NAA) containing 3% sucrose with 16-h photoperiod exhibited the greatest in vitro tuberization (3.2) with the highest shoot regeneration frequency (90%). The longest tuber regeneration occurred on MS media containing 4% sucrose. Transfer of in vitro-regenerated shoots to half-strength MS medium with 1.0 mg L?1 indole-3-butyric acid (IBA) + 0.5 mg L?1 NAA showed maximum root induction (66.6%). The in vitro-grown plantlets were successfully acclimatized and transplanted to sterilized soil and sand mixture (3:1) in the glasshouse with 70% survival. The colchicine content was determined in the tubers of ex vitro plants by HPLC using the same retention time (1.5 min) as that of the standard colchicine. This revealed that the micropropagation protocol developed by us for rapid mass production could be used as raw material for colchicine extraction and provides a basis for germplasm conservation and genetic improvement of G. superba.  相似文献   

13.
A protocol was developed for plant regeneration from leaf explants (laminaand petiole) of Aster cordifolius cultivar `White Elegans', which ischimeric for chromosome number and the ligulate flower colour. Explantswere cultured on MS or Gamborg's B5 medium in presence of IAA, 2,4-Dand BAP, alone or in combination. The highest frequency of shootregeneration was obtained on B5 medium supplemented with 2,4-D(0.09 mg l-1) and BAP (1.8 mg l-1). After the invitro rooting stage and acclimatization, 90% of plantlets survived in thegreenhouse. The regeneration process occurred by indirect organogenesisas shown by cyto-histology. The regenerants maintained the originalchimerism for chromosome number. Pink flowers instead of theexpected white ones were obtained in all regenerants. Ahypothesis for the uniform colour change is discussed. Variations weredetected in the regenerant generation (R0) for quantitative characterssuch as capitulum and disc diameters, and number of ligulate flowers. SinceAster can be vegetatively propagated, selection of variants in R0can provide interesting material for breeding purposes.  相似文献   

14.
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   

15.
Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin  相似文献   

16.
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   

17.
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   

18.
A simple and efficient protocol for direct in vitro shoot multiplication and plant regeneration was established for an important aromatic medicinal plant, Alpinia calcarata. Preinduction of rhizome segments in medium containing 8.8 μM 6-benzylamino purine (BAP) rescued the buds from dormancy in 60% of the cultures. An average of 6.2 shoots were produced from rhizomatous bud explants on Murashige and Skoog (MS) medium supplemented with 5 μM BAP, 10 μM kinetin, and 2.5 μM α-Naphthalene acetic acid (NAA). The mother cultures retained their morphogenetic potential upto four subcultures and a maximum of 1.77-fold increase in shoot multiplication was recorded after the 3rd subculture. Rooting was simultaneously induced during subculture on shoot multiplication medium eliminating an additional step for rooting induction. Rooted plantlets were successfully acclimatized in pots in the greenhouse and subsequently established in the experimental garden without any visible symptoms of wilting and necrosis. The genetic fidelity of regenerated plants was evaluated by adapting to two PCR-based DNA marker techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) which detected no variability in the in vitro multiplied plantlets of A. calcarata. This efficient method of clonal multiplication may be useful for commercial scale multiplication, and in situ and ex situ conservation of elite germplasm of A. calcarata.  相似文献   

19.
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   

20.
Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l?1 naphthylacetic acid (NAA) + 1.5 mg l?1 6‐benzylaminopurine (BAP) and with 0.3 mg l?1 NAA + 2.0 mg l?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.  相似文献   

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