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1.
Summary

We have developed a two-step procedure for rooting of tea microshoots in vitro. The effectiveness of different auxin treatments for root formation was found to differ. Among the auxins tested, 25 μM -naphthalene acetic acid (NAA) gave the best results, with 100% rooting, compared to 25 μM indole-3-butyric acid (IBA) or 25 μM indole-3-acetic acid (IAA), which induced 17% and 58% rooting, respectively. Incubation of tea microshoots on 0.33 Murashige and Skoog (MS) medium, supplemented with 25.0 μM NAA or 175.0 μM IBA for 10 d, followed by transfer to auxin-free 0.33 MS medium resulted in 100% rooting, whereas 50.0 μM IAA induced 91.7% rooting. Besides the different auxin treatments, the strength of the MS medium, the duration of incubation of microshoots in auxin-containing medium, the sucrose concentration, the gelling agent, the pH of the medium, the incubation temperature, the light intensity, and the quality of the shoots also played a significant role during in vitro rooting of micropropagated tea shoots. Among the combinations tested, the most effective results were obtained when green microshoots were incubated on 0.33 MS medium supplemented with 25.0 μM NAA, 50.0 mM sucrose, pH 5.5, gelled with 0.2% (w/v) PhytagelTM for 10 d at 25° – 30°C at a light intensity of 40 μmol m–2 s–1, followed by transfer of shoots to auxin-free 0.3 MS medium. This resulted in 100% rooting and, on average, 11 long roots were formed per shoot. Anatomical changes during adventitious rooting of micropropagated tea shoots in vitro were also studied to understand the process of rooting.  相似文献   

2.
Summary

The influence of different growth regulators and additives on shoot multiplication from nodal explants of Psoralea corylifolia was investigated. Prolific shoot multiplication was achieved within 4 weeks of culture on Murashige and Skoog (MS) medium supplemented with 5 μM benzyladenine (BA), 5 μM ascorbic acid (AA), 100 mg l–1 casein hydrolysate (CH) and 5% (v/v) coconut water (CW). Shoots elongated on half-strength MS basal medium devoid of inositol, but containing 5 μM 2-isopentenyladenine (2iP), 10 g l–1 sucrose and 8 g l–1 agar. Elongated shoots rooted on half-strength MS basal medium supplemented with 3 μM indole-3-butyric acid (IBA), 10 g l–1 sucrose and 7 g l–1 agar within 5 d of culture. The in vitro-raised plants were established successfully in 2:1:1 (v/v/v) garden soil:farmyard soil:sand, and maintained in a growth chamber with 100% survival. Acclimatised plants were transferred to a glasshouse and established successfully in the field. Flowers and fruits appeared after 4 months and resembled those on source plants. This system could be used for rapid commercial propagation of P. corylifolia for conservation strategies and to produce phytomedicines.  相似文献   

3.
Summary

Dormant axillary buds excised from crowns of pineapple (Ananas comosus L., Merr.) cultured on growth regulator free Nitsch medium sprouted after 8–10 d. Sprouted buds produced multiple shoots (7–10 shoots per bud) upon transfer to solidified Murashige and Skoog medium supplemented with 9.67 μM NAA, 9.84 μM IBA and 9.29 μM KIN. Each isolated shoot upon subculture to liquid medium of the same composition further proliferated to form more multiple shoots (60–65 shoots) and were maintained on a gyratory shaker (90–100 rpm). In vitro grown shoots were rooted on White medium supplemented with 0.54 μM NAA and 1.97 μM IBA. In vitro plantlets were established in cups with soilrite and hardened for four weeks. Phenotypic variants such as albinos, white streaked shoots and shoots with elongated internodes were observed in in vitro cultures. Approximately 520 in vitro produced plantlets were established in the field and these plants exhibit somaclonal variation. Thirty-eight plants were found to be yellowish, spineless with anthocyanin streaks and three were anthocyanin rich, spined plants.  相似文献   

4.
Summary

A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA3. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.  相似文献   

5.
The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L−1 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L−1 BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L−1 BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L−1 BA and 0.5 mg L−1 indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L−1 BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.  相似文献   

6.
Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.  相似文献   

7.
Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.  相似文献   

8.
Summary

Efficient in vitro procedures for mass propagation of four commercially important Echinacea species have been deveoped. Plants of E. angustifolia, E. pallida, E. paradoxa and E. purpurea were regenerated by three methods, namely axillary bud proliferation, adventitious shoot formation and somatic embyrogenesis. Shoot tips obtained from in vitro germinated seedlings, adventitious shoots or somatic embryo-derived plantlets, when cultured on Murashige and Skoog medium enriched with 1 μM 6-benzylaminopurine, 2 μM kinetin, 0.5 μM indole-3-butyric acid and 4 mg–1 paclobutrazol multiplied three-fold within 3–4 weeks in culture. Incorporation of paclobutrazol in the shoot multiplication medium was necessary to recover healthy and robust shoots suitable for rooting. Direct, high-frequency shoot formation on intact leaves of shoots grown on 6-benzylaminopurine and kinetin-supplemented media, an unusual and novel observation made in this study, occurred in all the species studied. Rooting of in vitro developed shoots was achieved relatively easily with Murashige and Skoog basal medium rather than with auxin-enriched media. Culturing of hypocotyl explants on medium containing 3,6-dichloro-o-anisic acid (commonly known as dicamba), or 2,4-dichlorophenoxyacetic acid, resulted in direct somatic embryogenesis in all the species examined. The presence of cytokinin was required for somatic embryo germination, but further development of germinated somatic embryos into normal plantlets occurred in Murashige and Skoog medium. We conclude that the procedures described here could be used for rapid propagation as well as genetic transformation of commerically cultivated Echinacea species.  相似文献   

9.
Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.  相似文献   

10.
Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N6-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.  相似文献   

11.
Summary

An efficient, reproducible protocol has been developed for in vitro multiplication of Sida cordifolia L. High-frequency, multiple shoots (90%) were obtained indirectly from nodal explants. Callus was induced when nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg l–1 Kinetin (Kn). These nodal calli were then cultured in order to differentiate multiple shoots on MS medium supplemented with 0.5 mg l–1 Kn plus 0.5 mg l–1 naphthaleneacetic acid (NAA). Roots were induced from these multiple shoots following culture on MS medium supplemented with 0.8 mg l–1 NAA for 4 weeks. Finally, these in vitro plantlets were hardened, acclimatised, and successfully transferred to the field.  相似文献   

12.
Summary

To improve the yield and quality of essential oils, chromosome-doubling in mint cultivars was induced by treating shoots with colchicine in vitro. Shoot tips of three mint cultivars (‘68-7’, ‘73-8’, and ‘HU 39’) were cultured in vitro and treated at 2 months using either of two methods to induce chromosome-doubling. Explants were immersed separately in each of three concentrations of colchicine [0.1, 0.2, or 0.3% (w/v)] for 24 h or 48 h. Alternatively, shoots were cultured on solid 1.0× MS (Murashige and Skoog) medium supplemented with one of five concentrations of colchicine (10, 20, 30, 40,, or 50 mg l–1) for 30 d. After each treatment, ploidy levels were measured by flow cytometry. The results showed that high yields of 4n plants were induced by the immersion of shoots in 0.2% (w/v) colchicine for 24 h (13.3%), or by culturing shoots on MS medium containing 20 mg l–1 colchicine for 30 d (17.3%). The immersion method, which gave a survival rate of 93.3%, was more convenient and less phytotoxic to produce 4n mint plants. Compared with untreated plants, we observed fewer but larger stomata in chromosome-doubled plants. We also observed significant differences in the size, internode length, leaf length, leaf width, and stem width in chromosome-doubled mint plants.  相似文献   

13.
Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.  相似文献   

14.
Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (1?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on 1?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.  相似文献   

15.
To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l−1 6-benzylaminopurine (BA) and 1.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l−1 BA and 1.0 mg l−1 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l−1 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l−1 BA and 1.0 mg l−1 a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.  相似文献   

16.
Factors affecting the establishment, proliferation and adventitious root initiation of ‘Northern Spy’ are described. Shoot-tips established best in Linsmaier and Skoog medium with 5 μM BAP and 1 μM IBA supplemented with 1 mM phloroglucinol, but after the fourth sub-culture, proliferation was similar in media without phloroglucinol. Shoot proliferation was influenced by cytokinin type and concentration, light level and ex-plant type. Of the cytokinins tested, BAP was superior to zeatin, kinetin and 2iP. Proliferation was better with 10 μM BAP than with 5 or 1 μM BAP, but at 10 μM shoots were dwarfed. Single nodes and basal mass expiants produced significantly more shoots than did shoot-tips. Shoot proliferation was maximum at photon flux densities of 75 μmolm?2s?1. Roots can be readily induced with IBA, but salt concentration and the physical support on which shoot-tips are grown influence both root initiation and growth. Half-strength salts and 1 μM IBA gave best root initiation. With agar, coarse sand, perlite and rotated liquid medium, 90–100% root initiation occurs, but root growth was poor in agar.  相似文献   

17.
《Scientia Horticulturae》2002,95(3):213-221
Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA3; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l−1 activated charcoal.  相似文献   

18.
Summary

Giant reed (Arundo donax L.), a promising energy crop, is vegetatively-propagated from fragments of stems and rhizomes. This may limit large-scale cultivation, since it is time-consuming and involves considerable cost and effort. Tissue culture is an alternative to conventional methods of vegetative propagation and may represent a useful tool for large-scale propagation of plants for biomass production programmes. This report describes a protocol for the large-scale in vitro propagation of giant reed by adventitious bud formation. Stem nodes with dormant buds proved to be the most effective to initiate in vitro cultures giving the highest percentage of differentiated shoots (77%) compared to the other plant fractions tested. A sterilisation procedure using 5 g l–1 HgCl2 enabled the production of sterile explants. Moreover, early results indicated that late Autumn excision dates not only gave a higher percentage of well-developed shoots ( > 80%), but also a lower level of bacterial contamination (15 – 20%). 6-Benzylaminopurine (BAP) at 3.0 mg l–1 was most effective in promoting shoot multiplication when added to a basal medium containing Murashige and Skoog (MS) macro- and micro-nutrients, Morel’s vitamins, 30 g l–1 sucrose, and 7.0 g l–1 bacteriological agar supplemented with 1.0 mg l–1 indole-3-acetic acid (IAA) and 0.05 mg l–1 gibberellic acid (GA3). Rooting was successfully induced on the same basal medium used for proliferation, modified by halving the MS macro-nutrients and replacing BAP and the other growth regulators with 2.0 mg l–1 1-naphthaleneacetic acid (NAA). Successful acclimatisation (> 95% survival) of plantlets was carried out, even in late Winter, in a cold greenhouse or under simpler facilities such as shade nets.  相似文献   

19.
Summary

Significant effects of seven basal media and three carbon sources (sucrose, glucose and fructose) on the induction of adventitious buds from embryos of Pinus sylvestris L. were observed. Moreover, hyperhydricity of expiants and shoot regenerants was observed on basal media containing fructose, especially with half-strength Murashige and Skoog (MS), MS, and woody plant medium (WPM). Expiants grown on a Gresshoff and Doy (GD) medium with sucrose produced the highest frequency of regeneration (81%) and with no hyperhydricity observed of developing adventitious shoots. Among three cytokinins tested including BA, BPA, and TDZ (at four concentrations each), 5 μM BA resulted in the highest regeneration frequency and mean number of adventitious shoots per embryo. Shoot régénérants were elongated after transfer to a GD medium containing 2 g-l–1 activated charcoal and no growth regulators. After one month, rooting was induced on 10% of expiants.  相似文献   

20.
Summary

Four species of the genus Kalanchoë (Crassulaceae), K. peltata, K. laxiflora, K. tubiflora and K. marmorata, were regenerated from leaf explants by direct organogenesis. Each species was tested on 19 media, all based on MS-medium. One medium was without growth regulators, the remaining 18 contained a combination of auxin and cytokinin. Auxin was indole-3-acetic acid (IAA): 1.1, 2.3 or 4.6 μM (0.2, 0.4 or 0.8 mg l–1). Cytokinin was either 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea(TDZ): 1.1, 2.3 or 4.5 μM (0.25, 0.5 or 1.0 mg l–1), or 6-benzylaminopurine (BAP): 1.1, 2.2 or 4.4 μM (0.25, 0.5 or 1.0 mg l–1). For each species an optimum level of growth regulators were obtained. One medium, called K22, containing 0.5 mg l–1 TDZ and 0.04 mg l–1 IAA, showed good shoot-generating capacity with all four species. Shoot elongation proved to be a problem only with K. marmorata. This could be bypassed by transferring shoots to a gibberellic acid (GA3)-containing medium, or by ventilating the containers. Shoots were rooted on MS-medium and rooted shoots were transferred to soil. K. laxiflora failed to root, but plantlets produced on the leaves were easily used for vegetable proliferation of the regenerated shoots. Eight additional Kalanchoë species and four species from other genera of Crassulaceae: Crassula, Echeveria and Sedum, were tested for regenerative capacity on K22-medium. From four Kalanchoë species and three other species, regenerated plants were established in soil. These results suggest that this medium has a high regenerative capacity within the Crassulaceae. No close dependency was found between systematic position and ability to regenerate on this medium.  相似文献   

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