Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Micropropagation of a <Emphasis Type="Italic">Casuarina</Emphasis> hybrid (<Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> L. × <Emphasis Type="Italic">Casuarina glauca</Emphasis> Sieber ex Spreng) following facilitated seed germination

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.     

Jasmonic acid as modulator of lead toxicity in aquatic plant Wolffia arrhiza (Lemnaceae)

The present study was undertaken to test the influence of exogenously applied jasmonic acid (JA) upon the growth and metabolism of Wolffia arrhiza (Lemnaceae), the smallest vessel aquatic plant exposed to lead (Pb) stress. It was found, that JA acted in a concentration-dependent manner. Treatment with JA at the highest concentration 100 μM resulted in the enhancement of heavy metal toxicity leading to increase in metal biosorption and formation of lipid peroxides as well as decrease in fresh weight, chlorophyll a, carotenoid, monosaccharide and soluble protein content. In contrast, this phytohormone applied at 0.1 μM protected W. arrhiza fronds against Pb stress inhibiting heavy metal accumulation, restoring plant growth and primary metabolite level. Moreover, JA at 0.1 μM activated enzymatic (catalase, ascorbate peroxidase, NADH peroxidase) and non-enzymatic antioxidant (ascorbate, glutathione) system in W. arrhiza, and therefore, suppressed oxidative destruction of cellular components induced by heavy metal. The data suggest that JA plays an important role in the growth and metabolism of W. arrhiza exposed to abiotic stressor and its high adaptation ability to metal contamination of aquatic environment.     

Geographic distribution and genotypic composition of invasive hybrid watermilfoil (<Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> × <Emphasis Type="Italic">M. sibiricum</Emphasis>) populations in North America

The recent recognition of invasive hybrid watermilfoil (Myriophyllum spicatum × M. sibiricum) in North America has necessitated a more thorough evaluation of its overall distribution and occurrence in natural populations. A comprehensive survey of watermilfoil populations was conducted in five Minnesota lakes, three of which were suspected a priori to contain hybrid watermilfoil. DNA sequence data verified that hybrid plants between the nonindigenous M. spicatum L. and indigenous M. sibiricum Kom. occurred in three of the five lakes sampled. Myriophyllum spicatum was not detected in lakes where hybrids were prevalent. Further sampling of lakes in Idaho, Michigan, Minnesota, Wisconsin and Washington identified 30 additional hybrid watermilfoil populations. In only three of these populations the hybrid watermilfoil was found to co-occur with M. spicatum. To facilitate the field identification of the two parental species and their hybrid, morphological data from watermilfoil specimens collected across the United States were evaluated. We determined that leaf segment/leaf length measurements can effectively distinguish M. spicatum and M. sibiricum; however, hybrids are intermediate for these characters and such measurements frequently overlap with respect to their parental taxa. By incorporating a combined molecular and morphological approach to identifying watermilfoils, the hybrids can be identified readily and their distributions elucidated both within and between lakes. Because hybrids may respond differently to local ecological conditions than their parents, information on their presence and distribution should be of particular importance to management and conservation programs.     

感染内生真菌的羽茅对大针茅的化感作用

以羽茅(Achnatherum sibiricum)为材料,采用室内生物测定法研究感染不同内生真菌的羽茅浸提液对大针茅(Stipa grandis)种子萌发和幼苗生长的化感作用,并采用气相色谱-质谱(GC-MS)联用技术对羽茅浸提液中的化学成分进行分析。结果表明:(1)内生真菌感染可缓解宿主羽茅对群落优势种大针茅的化感作用;(2)内生真菌感染对宿主羽茅化感作用的影响与内生真菌种类有关,具体表现为相比于未染菌羽茅,感染Epichloё gansuensis可显著缓解宿主羽茅对大针茅的抑制作用,而感染Neotyphodium sibiricum、Neotyphodium gansuensis有缓解羽茅对大针茅化感作用的趋势,但未达到显著影响;(3)羽茅浸提液中含有2,4-二叔丁基苯酚、邻苯二甲酸、邻苯二甲酸二丁酯和硬脂酸甲酯等潜在化感物质,这4种物质相对含量的差异可能是不同内生真菌感染状态的羽茅产生不同化感效应的主要原因;(4)对羽茅浸提液中4种潜在化感物质的化感作用进行验证实验,结果表明,2,4-二叔丁基苯酚对大针茅的化感作用基本表现为低浓度无影响,高浓度抑制,邻苯二甲酸二丁酯的化感作用表现为"低促高抑"的效应趋势,而邻苯二甲酸和硬脂酸甲酯对大针茅均表现出"剂量效应"的化感作用趋势,即4种潜在化感物质在高浓度时均可显著抑制大针茅的种子萌发和幼苗生长。     

Assignment of <Emphasis Type="Italic">Lemna gibba</Emphasis> L. (duckweed) bioassay for <Emphasis Type="Italic">in situ</Emphasis> ecotoxicity assessment

To narrow the differences between the results obtained from radionuclides and heavy metal ecotoxicity investigations in the laboratory and in the abandoned uranium mines, a few standardised plant bioassay procedures were selected from the literature for testing with Lemna gibba L. The bioassay procedures were tested in situ and ex situ. The laboratory culturing was performed in batch and semicontinuous modes. The results revealed that most of the standardised plant bioassay procedures require modification for the L. gibba bioassay to predict the actual effects under field conditions. L. gibba performed relatively better in the field than laboratory batch cultures despite that the batch cultures had many-fold higher nutrient concentrations than in the field. For instance, the phosphorus concentration of the mine tailing water was 0.13 ± 0.09 μg l⁻¹ in the field, while the literature range for phosphorus in the laboratory culture media is 13.6–40 mg l⁻¹. L. gibba growth in the laboratory batch culture was influenced by speciation changes due to consumption of nutrients, CO₂ and O₂ phase exchanges, and excretion of organic substances by the test plants. Semicontinuous culture modes performed significantly better than batch cultivation even after 10× dilution of the nutrient solution. The growth behaviour revealed that L. gibba exhibited intrapopulation and probiotic interaction for best performance. Growth performance of L. gibba was influenced by the anions that balanced essential cations despite equal cation concentration in the culture media; e.g., the best growth was observed in culture media that had more SO₄²⁻ than Cl⁻. Water samples from the field had higher SO₄²⁻ concentrations than Cl⁻. The test vessel material, sterilisation and axenic culturing procedures also influenced the sensitivity of the bioassay. These, for instance, and a few others are neither described nor reported in most standard Lemna tests or the literature. Thus, this work presents results of a series of tests conducted on the selected methods. Common and possible errors and corrective measures in assigning L. gibba bioassay from laboratory population levels to field community levels are discussed.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 M. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.     

Morphogenic response of the alginate-encapsulated axillary buds from in vitro shoot cultures of six mulberries

Axillary buds obtained from in vitro shoot cultures of six mulberries (Morus alba L., M. australis Poir., M. bombycis Koidz., M. cathyana Hemsl., M. latifolia Poir., and M. nigra L.) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog (1962) nutrients (MS) and 4.4 μM benzyladenine (BA). Morphogenic response of encapsulated buds to various planting media such as MS medium + 4.4 μM BA, MS basal medium, soilrite mix + half-strength MS medium, garden soil + half-strength MS medium, soilrite mix + tap water and garden soil + tap water was evaluated. Encapsulated buds of M. alba, M. bombycis, M. latifolia and M. nigra exhibited shoot development in each of the six media tested whereas that of M. australis and M. cathyana responded only to the first four media. Analysis of variance revealed that the planting medium exhibited the greatest influence on shoot development. Of the six planting media evaluated, shoot development was highest in MS medium containing 4.4 μM BA and lowest in garden soil moistened with water. Of the six Morus species studied, one-step regeneration, i.e. both shoot and root formation, was recorded in M. alba, M. bombycis and M. latifolia. Rooted shoots were retrieved from encapsulated buds of these species on all planting media tested except the one that contained BA. Root development was significantly affected by the planting medium and the plant species with planting medium contributing the maximum amount (82%) of the total variation observed. Of the five planting media tested, the percentage of root development was highest in MS basal medium. Of the six Morus species studied, the best shoot and root development was observed in M. alba. Encapsulated buds of M. bombycis, M. latifolia and M. nigra stored for 90 days and those of M. alba, M. australis and M. cathyana for 60 days at 4 °C still regenerated shoots. Plants regenerated from the encapsulated buds were hardened off and transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from callus cultures ofNardostachys jatamansi

Callus cultures ofNardostachys jatamansi DC, an endangered medicinal and aromatic plant, were established using petiole explants on MS medium supplemented with 16.1 µM -naphthaleneacetic acid and 1.16 µM kinetin. Embryogenesis in these callus cultures took place only upon sequential subculture of the callus on media having gradually decreasing auxin (16.1 to 1.34 µM NAA) and simultaneously increasing cytokinin (1.16 to 9.30 µM kinetin) concentrations over a period of 7 months. Somatic embryo to plantlet conversion took place on a medium containing 9.30 µM kinetin and 1.34 µM NAA.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Factors Affecting Plant Regeneration from Tissue Cultures of Chinese Leymus (Leymus chinensis)

Chinese leymus (Leymus chinensis Trin.) is a perennial grass of the Gramineae, which is widely distributed in China, Mongolia and in Russian-Siberian. In order to explore the potential of biotechnology for genetic improvement of this forage grass, an efficient tissue culture system was established and the factors affecting plant regeneration were evaluated. Immature inflorescence segments 3–5 mm in length from eight accessions were cultured on N6 medium supplemented with 2.26–22.60 µM 2,4-D. The callus induction frequency ranged from 72.11 to 82.19%. Shoots were differentiated from the calli on N6 medium containing 4.65 µM kinetin and 4.44 µM BA. Viable regenerants were developed on hormone-free medium. Normal plants were obtained after natural vernalization in the field. The plant regeneration frequency in Chinese leymus was associated with different genotypes and different combinations of growth regulators in medium. The concentration of 2,4-D in the callus induction medium had a strong effect on successive plant regeneration. Relatively higher concentrations of 2,4-D (i.e., 9.04 and 22.60 µM) were more favorable to the plant regeneration than lower ones (i.e., 2.26 and 4.52 µM). This is the first report on plant regeneration in vitro in L. chinensis.     

In vitro plant regeneration of Arachis correntina (Leguminosae) through somatic embryogenesis and organogenesis

In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and organogenesis were developed using immature leaves as explants. Morphologically normal somatic embryos were obtained on culture media composed of 20.70 or 41.41 μM picloram (PIC) with the addition of 0.044 μM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively. The source of explants and the developmental stage of the leaves had a marked effect on somatic embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive producing up to 30% embryogenesis in MS+41.41 μM PIC. Embryos converted into plants after transfer to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) with or without 0.044 μM BA, achieving plant regeneration in the induction media. The highest percentage of bud formation was obtained on culture medium composed of␣MS+10.74 μM NAA+0.044 μM BA (12.5%). Roots were formed on all culture media tested. Regenerated plants were transferred to pots and grew well under greenhouse conditions.     

A nonchromatographic assay for sorbitol in chemically complex growth media containing sorbose

Summary Microbiologists who lack gas-chromatographic equipment cannot easily study the microbial conversion of sorbitol to sorbose, because there is no other proven method for quantitatively measuring sorbitol in complex growth media. The purpose of this study was to determine if the polyol assay suggested by Mäkinen in 1980 could be used to measure sorbitol depletion in chemically complex bacteriological-culture media containing sorbose. After we pretreated the culture medium to remove interfering phosphate complexes and slightly modified Mäkinen's assay, we were able to detect 0.1 mg/ml differences in sorbitol concentrations that varied between 0.9 and 1.5 mg of sorbitol/ml. The presence of sorbose in the complex medium did not affect the assay. Growing cultures ofGluconobacter oxydans were used to test this assay, because these bacteria reportedly oxidize sorbitol to sorbose and quantitatively release the sorbose into the growth medium. When samples of the culture medium removed during growth were centrifuged to remove cells and precipitated to remove interfering substances then tested with the mofidied Mäkinen assay, these cultures showed a sorbitol depletion rate of 4.9 (±0.1) mg/ml h^–1. The Fehling's assay on the same cultures showed a sorbose accumulation rate of 5.12 (±0.01) mg/ml h^–1. We concluded that the modified Mäkinen phosphate-interference assay can be satisfactorily used to quantitatively screen cultures for their ability to oxidize sorbitol.     

Allelopathic aquatic plants for aquatic weed management

This report presents, results of a feasibility study of use of allelopathic aquatic plants for aquatic weed management. In order to establish a list of potential allelopathic plants, we selected 16 aquatic plants native to the southeastern United States and subjected them to two bioassays — one involving lettuce seedlings and one involving the aquatic plantLemna minor as the target species. The lettuce seedling bioassay was selected because it is a widely used, experimentally simple assay to determine allelopathic activity. However, it uses lettuce, a terrestrial plant, as the target species, and thus may be less appropriate for use with aquatic plants. TheL. minor assay involves an aquatic plant as the target species and so is more appropriate for our goals, but it is experimentally much more complex and time-consuming. The plants selected for study wereBrasenia schreberi, Cabomba caroliniana, Ceratophyllum demersum, Eleocharis acicuiaris, Eleocharis obf usa, Hydrilla verticillata, Juncus repens, Limnobium spongia, Myriophyllum aquaticum, Myriophyllum spicatum, Najas guadalupensis, Nymphaea odorata, Nymphoides cordata, Potamogeton foliosus, Sparganium americanum, and Val/isneria americana.Nymphaea odorata (leaves and petioles) inhibited 78 % of lettuce seedling radicle growth and 98 % ofL. minor frond production. Brasenia schreberi inhibited 82 % of lettuce seedling radicle growth and 68 % of L. minor frond production. These results suggest thatN. odorata andB. schreberi are both highly inhibitory and are therefore candidates for use in aquatic weed management. Results also suggest that the simple lettuce seedling assay is a reasonable first “easy” one to use in an attempt to determine allelopathic potential of aquatic plants.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.     

Applications of the aquatic higher plant Lemna gibba for ecotoxicological assessment

Although higher plants represent a significant portion of the total biomass in some aquatic environments, their use in ecosystem evaluation has lagged behind that of other organisms. This is partly due to a lack of convenient aquatic higher plant systems that can be employed for ecotoxicological assessment. However, the aquatic C-3 monocot Lemna gibba has many attributes that makes it useful for ecosystem health assessment. In this report, using examples from the literature and our research, some of the applications Lemna has for environmental research are discussed. Toxicant impacts on Lemna can be readily assessed in terms of growth; the plants multiply quickly and changes in biomass (which doubles approximately every 2 days) can be accurately measured by counting leaves. The plants are small, allowing for simultaneous multiple replication. The small size also makes the lighting conditions easy to control; sunlight can be accurately simulated and specific spectral regions can be enhanced or deleted. Lemna is amenable to in vitro chlorophyll and photosynthesis assays, which make excellent companion endpoints for growth. The plants assimilate chemicals directly from the growth medium, facilitating controlled toxicant application. Furthermore, Lemna has a high bioconcentration capacity, indicating a potential for use in bioremediation technologies.     

In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.