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Ex vitro Survival and Early Growth of Alpinia purpurata Plantlets Inoculated with Azotobacter and Azospirillum
Abstract
The survival rate, shoot and root dry mass, shout number, plant growth, stem height and diameter, number of leaves and root length were measured in micropropagated plantlets of Alpinia purpurata (Red ginger) inoculated with Azospirillum sp. 11B and Azotobacter sp. Pachaz 008 at 10(7), 10(8) and 10(9) cells cm(-3) using a complete randomized experimental design. Inoculation ofA. purpurata plantlets with the Azospirillum sp. 11B or Azotobacter sp. PACHAZ 008 strains induced larger stem diameter, root dry mass, number of shoots and increased their survival rate from 77 to 100% compared to plantlets without inoculation, while other plant characteristics were not affected.
... Different effects were reported (Table 4). Increased plant biomass (shoot and root fresh weight and dry weight, plant height, leaf area, rhizom weight) (Kapoor et al., 2008;Rai, 2001), "better" rooting in vitro (number, length) (Larraburu and Llorente, 2015b), "better" acclimatization (survival rate, plant performance) (Duffy et al., 1999;Ovando-Medina et al., 2007), earlier flowering and increased flower number (Varma and Schuepp, 1995), induction of stress resistance (Nowak and Shulaev, 2003), biocontrol effects (Barka et al., 2000;Harish et al., 2008) or altered secondary metabolite profile are reported (Zabetakis, 1997). Table 4. Effects on plant performance after inoculation with microorganisms. ...
... Reference Increased plant biomass (shoot and root fresh weight and dry weight, plant height, leaf area, rhizom weight) Rai, 2001;Kapoor et al., 2008 "Better" rooting in vitro (number, length) Larraburu and Llorente, 2015b "Better" acclimatization (survival rate, plant performance) Duffy et al., 1999;Ovando-Medina et al., 2007 Earlier flowering, increased flower number Varma and Schuepp, 1995 Induction of stress resistance Nowak andShulaev, 2003 Biocontrol Barka et al., 2000;Harish et al., 2008 Altered secondary metabolite profile Zabetakis, 1997 Characterization of endophytes Endophytic microbes, esp. bacteria can be characterized by different biochemical properties in order to determine a possible plant growth promotion potential. ...
Micropropagated plants are facing different challenges under in vitro and ex vitro conditions: mixotrophic growth under low light conditions on artificial nutrient media, poor gas exchange in small vessels, abiotic stress, bad rooting, transplanting stress, low survival rate during acclimatization in greenhouse. Plants are superorganisms NDASH naturally colonized by myriads of bacteria, fungi and protists. Microbial endophytes are the dominant group of colonizers. Endophytes' behaviour can range from mutualism to antagonism and the use of endophytes in micropropagation can improve plant growth, yield, health and induce tolerance to abiotic and biotic stress. Micropropagated plants often harbor differences in their microbiome compared to plants conventionally grown and there is the risk of beneficial microbes being eliminated in micropropagation systems. A tool for the use of competent endophytes in micropropagation under in vitro and ex vitro conditions is “biotization” of plantlets with useful bacterial and fungal inocula. Fungal inocula, which are commercially used, are e.g., arbuscular mycorrhizal fungi in form of spores and extraradical mycelium on different carrier materials like expanded clay, vermiculite, sand or peat. Furthermore, representatives of the root fungal genus Trichoderma are applied as spores formulated in powder. Plant-growth promoting rhizobacteria of the important genera Bacillus, Pseudomonas, Azospirillum and Azotobacter in form of lyophilised endospores/bacterial cells in powder or liquid formulation are also available on the market. Inocula can be used as biofertilizers or biocontrol agents by dipping of microcuttings in bacterial suspensions or mixing of inocula with potting soils.
... The effect of symbionts on micropropagation has been mostly studied in others species such as medicinal and aromatic plants (Thomas et al. 2010;Prasad et al. 2013;Benson et al. 2014;Sharma et al. 2014;Verma et al. 2015), ornamentals (mainly orchids) (Ovando-Medina et al. 2007;Ye et al. 2014;Larraburu and Llorente 2015a, b), Prunus (Quambusch et al. 2016) or oil palm (Azlin et al. 2007). ...
... The E+ seeds were more successful than E− seeds in in vitro germination (83 ± 5 vs. 63 ± 6%), callus induction (78 ± 5 vs. 57 ± 6%), callus proliferation (average diameter of 21.5 ± 1.3 mm in two subcultures vs 17.3 ± 0.8 mm in three subcultures) and plant regeneration from callus (83 ± 7 vs. 30 ± 8%). These results indicate that E. occultans enhances significantly L. multiflorum (Larraburu and Llorente 2015b) and acclimatization (Ovando-Medina et al. 2007). The most studied fungal symbionts in micropropagation are mycorrhizal fungi (Azcón-Aguilar et al. 1996;Rai 2001;Padilla et al. 2006;Kapoor et al. 2008). ...
Italian ryegrass (Lolium multiflorum) is an annual grass considered as one of the most important temperate forage grasses in the world. However, it is recalcitrant to plant tissue culture techniques hindering its genetic manipulation. Epichloë occultans is an endophytic fungus associated with L. multiflorum. This symbiosis causes improvements in physiological and ecological traits of the host plants. The objective of this work was to study the effect of E. occultans on L. multiflorum micropropagation. We compared the response of endophyte-infected (E+) and endophyte-free (E−) seeds in different micropropagation stages. The E+ seeds were more successful than E− seeds in in vitro germination (83 ± 5 vs. 63 ± 6%), callus induction (78 ± 5 vs. 57 ± 6%), callus proliferation (average diameter of 21.5 ± 1.3 mm in two subcultures vs 17.3 ± 0.8 mm in three subcultures) and plant regeneration from callus (83 ± 7 vs. 30 ± 8%). These results indicate that E. occultans enhances significantly L. multiflorum micropropagation. The use of endophyte-infected (E+) seeds can be a solution to make this grass more amenable to different biotechnological tools, such as the genetic transformation.
... In vitro conditions compromise plantlets' leaf morphology and leaf anatomy expansionand these characteristics negatively impact the capacity for ex vitro acclimatization, but the degree to which plants are affected by the in vitro environment depends on the plant species 16,17&18 . Growth parameters of Alpiniapurpurata were increased mother plants compared to in vitro plants 19 . In vitro shoots and plantlets are tiny cell wall, sclerenchyma, collenchyma formationAbsent of cuticular wax, limited palisade layer, palisade development is affected by different light levels and reduced as a consequence of relatively low light intensity, xylem vessels were not completed, pericycle and endodermis were darkly 20,21&22 . ...
The in vitro experiment was carried to examine the effect of various MS strength culture medium (full or half strength) and growth regulators concentrations of BA at (0.0, 0.1 or 0.2 mg/l) on shootlet multiplication and zeatin, 2,4-D and NAA on callogenesis potentiality) on Hibiscus syriacus L. plants. The results showed that using MS (3/4 strength) medium supplemented with BA at 0.2 mg/l resulted in the highest shootlets number/explant (3.33shootlet/explant) and the highest number of leaves (7.67 leaf/shootlet).The in vitro plants showed increasing in number of xylem rows, number of vessels and length of vascular bundle as comparison with control (mother plants). For callus induction, zeatin and 2,4-D at 0.5% for each were favored (highest callus percentage/leaf explant (100%) was observed. The response of formed callus to grow as a result of using BA (0.2 mg/) in combination with 2,4-D or NAA (0.25, 0.5 or 1.0 mg/L) for three subcultures was recorded. The high concentration of 2,4-D (1.0mg/l) added to BA (0.2) was favored for callus growth in the third subculture.BA combined with 2,4-D at low concentration (0.25mg/L) had promotion effect on callus dry weight after three subcultures. All shootlet produced from above multiplication treatments were rooted on half strength MS free medium with 3 g/l activated charcoal. The highest survival percentage and longest roots of acclimatized plants were recorded for growth media peat + sand (1: 1) after transplanting (five weeks).
... The increase in root biomass is a beneficial factor for the plant in acclimatization and rooting processes, thus reducing losses in these processes. The use of rhizobacteria for this purpose has been reported by several authors, due to the increase in root biomass and greater area of nutrient uptake (Ovando-Medina et al., 2007). ...
Guarana [Paullinia cupana var. sorbilis (Mart.) Ducke] is a species of great economic and social important in Brazil, as it is the only commercial guarana producer in the world. The vegetative propagation method indicated for the culture is stem cuttings, which aims at productivity, tolerance, and uniformity of clonal cultivars, because
reproduction by seeds has slow germination and high genetic variability, which in traditional varieties is an undesirable factor. Genetic factors can interfere with the rooting capacity of the crop. Studies seek alternatives that can improve this condition and enhance the production system. Use of growth regulators, microorganisms that promote plant growth, variation of substrates and fertilization, have been strategies used. Preliminary tests on the rate of stem rooting and seed germination with the use of exogenous phytohormone did not demonstrate in relation to the non-application of these inducers. The use of rhizobacteria, which presents itself as a promising activity in many cultures, has not yet been demonstrated in the culture of guarana. On the other hand, the influence of different substrates on rooting has already shown consistent results as a function of rooting rate. Fertilizing the mother plants as recommended by the production system for the crop has proven to be an efficient procedure. There are still few studies aimed at improving the spread of guarana, demonstrating that new protocols need to
be explored, or that the protocols already used are reviewed from another perspective.
... A greater survival of micropropagated seedlings, also, was observed when inoculating with PGPRs other species cultivated in vitro, such as potato H. impetiginosus (Bignoniacea) , Photinia x fraseri (Rosaceae) (Larraburu et al., 2007), jojoba Simmondsia chinensis L. (Simmondsiaceae) (Larraburu et al., 2016a), tomato Lycopersicon esculentum P. Mill. (Solanaceae) (Nowak and Shulaev, 2003), grapevine Vitis vinífera L. (Vitaceae) (Barka et al., 2002), Alpinia purpurata K. Schum (Zingiberaceae) (Ayora-Talavera et al., 2007) and Prunus sp. (Rosaceae) (Russo et al., 2008). ...
Lisianthus (Eustoma grandiflorum (Raf) Shinn.) is an excellent ornamental cut variety because it has flowers with good vase life. Seed propagation is difficult due to the long production time (it takes up to 100 days for transplanting) and the small size of its seeds. In vitro germination may produce healthy and homogeneous seedlings and may be used as a model system to study the effect of plant growth promoting rhizobacteria. Azospirillum brasilense is widely studied for its ability to colonize and promote various plant species growth, especially by increasing root development and plant biomass. This work aimed to evaluate physiological, morphological and anatomical aspects involved in vitro germination of E. grandiflorum inoculated with Azospirillum brasilense Az39. Seeds of E. grandiflorum (F1 double big. Arena III Pink) were disinfected with 10% NaClO and culture in MS medium. Inoculation of A. brasilense Az39 was performed with 10⁶ and 10⁷ cfu on seed surface at sowing. Non-inoculated seeds were used as controls. Biofertilization generated earliness and higher germination percentages. Furthermore, bacterization with 10⁶ cfu significantly increased number of leaves, leaf area, fresh and dry weight of seedlings, length and number of roots, diameter of the vascular bundle, leaves thickness and root thickness, among other parameters. These results suggested that inoculation of lisianthus with A. brasilense Az39 could improve seedlings and lead to a shorter time production.
... As for the adequate distance to improve their development in this study, it was 50 cm and in low density conditions. In relation to the production of flowers, no flowers were produced during the study, which could be due to the fact that, during the flowering period, this plant under terrestrial conditions grows from 1.5 to 2 m tall in an approximate period of 8 months [32]. However, in this study very small plants (10 to 25 cm) were planted and they did not show the same growth behavior as in their natural state, given the new conditions to which they were exposed for development. ...
Wastewater treatment (WWT) is a priority around the world; conventional treatments are not widely used in rural areas owing to the high operating and maintenance costs. In Mexico, for instance, only 40% of wastewater is treated. One sustainable option for WWT is through the use of constructed wetlands (CWs) technology, which may remove pollutants using cells filled with porous material and vegetation that works as a natural filter. Knowing the optimal material and density of plants used per square meter in CWs would allow improving their WWT effect. In this study, the effect of material media (plastic/mineral) and plant density on the removal of organic/inorganic pollutants was evaluated. Low (three plants), medium (six plants) and high (nine plants) densities were compared in a surface area of 0.3 m 2 of ornamental plants (Alpinia purpurata, Canna hybrids and Hedychium coronarium) used in polycultures at the mesocosm level of household wetlands, planted on the two different substrates. Regarding the removal of contaminants, no significant differences were found between substrates (p ≥ 0.05), indicating the use of plastic residues (reusable) is an economical option compared to typical mineral materials. However, differences (p = 0.001) in removal of pollutants were found between different plant densities. For both substrates, the high density planted CWs were able to remove COD in a range of 86-90%, PO 4-P 22-33%, NH 4-N in 84-90%, NO 3-N 25-28% and NO 2-N 38-42%. At medium density, removals of 79-81%, 26-32, 80-82%, 24-26%, and 39-41%, were observed, whereas in CWs with low density, the detected removals were 65-68%, 20-26%, 79-80%, 24-26% and 31-40%, respectively. These results revealed that higher COD and ammonia were removed at high plant density than at medium or low densities. Other pollutants were removed similarly in all plant densities (22-42%), indicating the necessity of hybrid CWs to increase the elimination of PO 4-P, NO 3-N and NO 2-N. Moreover, high density favored 10 to 20% more the removal of pollutants than other plant densities. In addition, in cells with high density of plants and smaller planting distance, the development of new plant shoots was limited. Thus, it is suggested that the appropriate distance for this type of polyculture plants should be from 40 to 50 cm in expansion to real-scale systems in order to take advantage of the harvesting of species in these and allow species of greater foliage, favoring its growth and new shoots with the appropriate distance to compensate, in the short time, the removal of nutrients.
... Several studies reported that P. indica treated plantlets under in vitro conditions showed a better survival rate when transplanted from in vitro to ex vitro conditions (Das et al. 2014a;Sharma et al. 2014;Baishya et al. 2015;Vyas et al. 2008;Varma et al. 1999;Suthar and Purohit 2012). Alpinia purpurata plantlets inoculated with Azotobacter showed maximum survival percentage compared with control and enhanced the tolerance of transplanting shock (Ovando-Medina et al. 2007). In the present study, the plants inoculated singly with P. indica or A. chroococcum showed a less survival rate (70.2 or 66.6%) as compared with plants co-inoculated with both P. indica and A. chroococcum (83.3%). ...
Artemisinin and its derivatives have been used in artemisinin combination therapies (ACTs) for treatment of malaria. The drug artemisinin is produced in the leaves of Artemisia annua. The escalating demands of ACTs together with minor concentration of artemisinin in leaves (0.01–1.1%) of A. annua L. has necessitated advent of viable methods to meet this growing demand. In the present study, symbiotic microbes; Piriformospora indica (Pi) and Azotobacter chroococcum (Az) were used singly and in combination (Pi +Az) to investigate their effect on in vitro grown A. annua L. plants. For co-cultivation of A. annua L. plantlets with symbiotic microbes, four weeks old rooted plantlets were used. Results showed that interaction of in vitro raised plantlets with dual inoculants significantly enhanced the plant growth, photosynthetic pigments, total soluble sugar, soluble protein, flavonoids content compared to control plants. The plant growth and rest of the parameters were enhanced in the treatments with single inoculants and were higher in plants inoculated with Pi than Az. A significant enhancement of 2-fold in artemisinin content was also observed in treatments receiving both inoculants. The physiological and biochemical parameters in these tested treatments improved significantly in the order (Pi +Az) > (Pi) > (Az).
... (38), Pseudomonas fluorescens (39) и P. putida (40). Бактериальная инокуляция приводила к увеличению свежей и сухой массы побегов и корней, высоты растений, площади листовой поверхности и массы ризомы (19,20), улучшенному укоренению in vitro (число и длина корней) (30), лучшей адаптации (процент акклиматизации, внешний вид растения) (41), раннему цветению и росту числа цветков, повышению устойчивости к стрессу и иммунитета (42), кроме того, наблюдались различия в профилях метаболитов (43). ...
In vitro plant propagation is a developed biotechnology, however until now there are no effective protocols for many perennials, especially for trees. High contamination of mature explants during tissue culture initiation, low multiplication and rooting during following passages are the main challenges. Aseptic culture of explants is associated with stress due to tissue damage and exposition to aggressive disinfectants and antibiotics during initiation. These could be the reasons of virulence of endophytes in following propagation. Plant-associated microorganisms were until recently seen as a problem for micropropagation, leading to contamination of in vitro explants. However recent studies showed that colonization of endophytes often play crucial role for increasing viability of in vitro and ex vitro plants. Most endophytes affect positively plant growth, providing nutrients and exhibiting antagonism to pathogens, as well as decreasing stress effects on plants. Beneficial effects were obtained in using Beauveria bassiana (J. Akello et al., 2007), Piriformospora indica and other members of family Sebacinales (P. Sharma et al., 2014), Fusarium oxysporum (A.S.Y. Ting et al., 2008), Ophistoma- like fungi (M. Mucciarelli et al., 2003), Phialocephala fortinii (M. Vohnik et al., 2003), Trichoderma harzianum and other Trichoderma species (P. Franken et al., 2012). Of bacteria, Acetobacter diazotrophicus (C.O. Azlin et al., 2007), Achromobacter xylosoxidans (A. Benson et al., 2014), Azospirillum brasilense (E.E. Larraburu et al., 2015), Azotobacter chroococcum (E.E. Larraburu et al., 2007), Bacillus subtilis (M. Vestberg et al., 2004), B. megaterium (P. Trivedi et al., 2007), Burkholderia phytofirmans (E.A. Ait Barka et al., 2000), B. vietnamiensis (M. Govindarajan et al., 2006), Enterobacter sp. (M.S. Mirza et al., 2001), Klebsiella variicola (C.-Y. Wei et al., 2014), Microbacterium sp. (M. Quambusch et al., 2014), Pseudomonas fluorescens (J. Thomas et al., 2010) ? P. putida (R. Lifshitz et al., 1987) also can beneficially influence plants. But until now it is unclear which factor is a trigger switched endophytes from mutualism to virulence. The only way to control such a change is to develop optimal conditions (time of obtaining explants, culture media composition and pH, temperature, etc.) in view to save in vitro mutualism with benefit for both host plant and the endophyte. Studies of many perennials showed the in vitro biotization to be helpful in microclonal propagation and plant rooting. Particularly, arbuscular micorhyza, ectomicorhyzal fungi, ericoid micorhyzal fungi, and wide range of bacteria influence positively plant micropropagation. Bacterial and fungal endophytes could stimulate plant growth due to activation of plant protection mechanisms, induction of systemic resistance to pathogens, phytohormone synthesis and better transport of water and nutrients. In this, the difficulties of classification and obtaining pure cultures of microorganisms are the main problems faced with.
Date palm (Phoenix dactylifera L.) plants demand reclamation of new areas or renewal of the oldest trees. Different methods to produce these plants as seeds that gave only 50% of seedlings will be female. The dates from seedling plants are often smaller and of poorer quality, so the propagation of date palm plantlets will occur with different methods. In vitro culture produced true-to-type huge numbers of plantlets under control conditions that led to many differences in morphology, physiology, and anatomy characteristics. The acclimatization was done to modify these abnormalities, and the plantlets after acclimatization need to improve growth by different treatments. Offshoots are the other method for propagation. These offshoots that grow from the base of the tree need to improve new rooting at the trunk base by growth regulators substances. This work shed light on the different methods of date palm propagation, the crucial role of the acclimatization greenhouse stage, and improved plantlets planted in the open field.
O Hibiscus sabdariffa L., conhecido como vinagreira ou azedinha é uma hortaliça classificada como Planta Alimentícia Não Convencionai (PANC), exigente em alta fertilidade de nitrogênio. O uso de microrganismos para suprir a demanda do nutriente é uma alternativa viável. Com isso, o objetivo desta pesquisa experimental foi avaliar isolados de bactérias diazotróficas no crescimento vegetativo de hibisco. O delineamento estatístico utilizado foi em blocos casualizados, contendo 8 tratamentos e 5 repetições, totalizando-se 40 parcelas experimentais, sendo cada parcela um vaso contendo duas plantas. Os tratamentos foram constituídos por 6 diferentes mix bacterianos (B1, B2, B3, B4, B5 e B6) de estirpes mantidas em armazenamento controlado no Laboratório de Microbiologia Agrícola- UNIFENAS, acrescido de 2 controles, controle positivo (CP), sem inoculação, acrescido de fertilização completa, controle negativo (CN), sem inoculação e fertilização. A colheita da parte aérea e das raízes foi realizada após 90 dias do plantio, avaliando-se as principais medidas biométricas. O tratamento B4 utilizando as estirpes HNFB3B e HJR3, apresentou o melhor resultado nas variáveis biométricas de altura, número de folhas e biomassa radicular da planta.
Summary Sorghum plants were inoculated withAzospirillum brasilense or received an N-amended nutrient solution. Azospirillum inoculation increased plant dry weight and nitrogen assimilation by 25%. Most plant growth responses to Azospirillum were comparable to application of 2.0 mM N. Increased scavenging of nutrients, altered root permeability or nitrogen fixation are possible explanations for these effects.
An efficient method of micropropagation based on an increased percentage survival of explants and reduced phenol-induced browning
in wild strawberry has been developed. Serial transfer of nodal explants was carried out at 24-, 48- and 96-h intervals. Nodal
segments cultured on Murashige and Skoog medium supplemented with 6-benzyladenine (4.0 μM) and α-naphthalene acetic acid (0.1
μM) gave the best (94.4%) explant establishment and shoot number (22.3) per explant. Of the cytokinins tested, 6-benzyladenine
was found more effective than kinetin and N6-(γ,γ dimethylallyamino) purine. Excised shoots rooted on half-strength agar-gelled medium with 1.0 μM α-naphthalene acetic
acid. Rooted shoots with fully expanded leaves acclimatized successfully and about 70% of plantlets survived ex vitro.
Mixed inoculation of Vicia faba L. with four different Rhizobium/Azospirillum and Rhizobium/Azotobacter combinations led to changes in total content, concentration and/or distribution of the mineral macro- and micronutrients,
K, P, Ca, Mg, Fe, B, Mn, Zn and Cu, when compared with plants inoculated with Rhizobium only. The effects varied to a great extent among the Azotobacter and Azospirillum strains selected for combined inoculation.
Efficient micropropagation, callus culture and root culture protocols were developed for the medicinal plant Phyllanthus urinaria(Euphorbiaceae) using single node explants. Maximum multiplication (16–20 shoots per explant) was achieved on Murashige and Skoog media supplemented with 5.0 M kinetin. Murashige and Skoog and Anderson Rhododendron media promoted significant shoot culture growth in terms of numbers of shoots and nodes produced per explant. Rooting was achieved with 93–100% of the microshoots on Murashige and Skoog medium without growth regulators, although 1.25–5.0 M -naphthaleneacetic acid significantly increased the number of roots per explant. Regenerated plants were successfully acclimatized and 91% of plantlets survived under ex vitro conditions. Flowering was observed on micropropagated plants after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when single node explants were inoculated in the horizontal position on Murashige and Skoog medium supplemented with 5.0 M indole-3-butyric acid. Other auxins such as 2,4-dichlorophenoxyacetic acid and -naphthaleneacetic acid promoted moderate callus fresh weight increase, when used separately. Root cultures were successfully established on Murashige and Skoog medium containing 1.1 M -naphthaleneacetic acid. The optimized micropropagation, callus culture and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies.
The first international conference on bacterial contamination of plant tissue culture was held in 1987, since then there has been a series of conferences on the topic of the health quality of microplants. A consensus emerging from these conferences has been that: rigorous pathogen-indexing should be carried out on the stock plants. Where contamination is detected, pathogen-free escapes should be sought or thermo- or chemotherapy applied. In vivo progeny plants should be rigorously re-screened after therapy to ensure freedom from pathogens. Pathogenfree plants should be maintained under Stage 0 conditions. Cultures should be established preferentially from apical meristems. The excised meristem tips should be of minimal size, should be placed on bacterial expression media and should be rigorously indexed for bacterial contamination before clonal propagations begins. Where meristem culture combined with in vitro therapy is used to eliminate pathogens. Preferentially, progeny plants should be grown in vivo for pathogenindexing before clonal propagation begins. Mass clonal propagation from aseptic cultures should be monitored for laboratory contamination following HACCP guidelines. Contaminated cultures should be disposed of appropriately or attempts may be made to recover contaminated cultures by transfer to autotrophic culture. A weaning strategy should be devised to protect against the risks of damping-off pathogens. This can be based on promotion of microplant physiological quality and by elicitation of pathogenesis-related proteins and/or by biotization in vitro or immediately post vitrum. Ideally, microplants should be certified as to their freedom from known diseases of the crop and freedom from cultivable contaminant. These guidelines provide a framework for managing contamination in tissue culture. They make the clear distinction between the establishment of aseptic cultures and the management of laboratory contamination. The major problems are still associated with the first stage for there are few crops e.g. potato, where most if not all diseases are characterised and for which diagnostics and diagnostic tests are available. Even for temperate fruit crops e.g. rosaceous fruit crops, not all pathogens have been characterised, nor are commercial diagnostics available for all characterised pathogens. This problem is even greater for the less known crops which are the main subject for micropropagation. A further problem in e.g. woody plants, if the often reported re-emergence in the nursery of pathogens from plants exposed to thermotherapy and indexing negative in in vitro cultures. Here, contamination management in micropropagation will be discussed with emphasis on the development of broad spectrum (non-strain specific or 'quarantine') molecular diagnostics and on the validation of in vitro screening using serological and molecular diagnostics.
A postharvest treatment in hot water at 49 degrees C for 12 to 15 min eliminated >95% of ants, banana aphids and mealybugs infesting red ginger flowers, Alpinia purpurata (Vieill.) K. Schum. In a 'systems-approach' to quarantine security, preharvest chlorpyrifos applications combined with either a postharvest hot-water immersion at 49 degrees C for 12 min or 5-min insecticidal dip in a combination of fluvalinate 2.0 flowable (F) and insecticidal soap eliminated all pests, including aphids, mealybugs, thrips, soft scales, and ants. Phytotoxicity resulting from hot-water treatment was characterized by bract necrosis and shortened vase life. Flowers were more susceptible to heat injury if harvested during periods of high rainfall. Conditioning flowers in hot air at 39 degrees C for 2 h before hot-water treatment eliminated phytotoxicity.
The effect of an in vitro monoxenically produced arbuscular mycorrhizal fungus (AMF), i.e. Glomus intraradices on the growth and phosphorus content of micropropagated banana (Musa spp. c.v. Grand Naine) plantlets was investigated in a pasteurized and non-pasteurized Vertisol. The experiment was conducted as a 2×2 randomly factorial design with the Vertisol pasteurized or non-pasteurized and the plants inoculated or non-inoculated with the AMF. The plants colonized by AMF had larger shoot and root dry weight and P content than the non-mycorrhizal plants. The largest growth increment was observed in the plants inoculated during the weaning phase with the monoxenic AMF inoculum and subsequently grown in the pasteurized soil. The native AMF population also induced a marked increase in plant growth supporting further the efficiency of this natural population, whereas both inoculum sources in combination did not yield the largest growth increment. The latter suggests a competitive phenomenon between the introduced AMF and the other soil micro-organisms including the native AMF population. Our results demonstrate, for the first time, the potential benefits of a monoxenically produced AMF in micropropagated banana plants.
The greatest growth of wheat and rape plants in vitro was reached on media with 5 or 9 % sucrose, respectively. The highest
efficiency for transfer of these plants to ex vitro conditions was found at the same sucrose concentrations. The content of
endogenous non-structural saccharides (glucose, fructose, sucrose, starch and fructans) increased with increasing sucrose
concentration in the medium up to 10 %.
Alpinia purpurata K Schum inflorescence buds inoculated on Murashige and Skoog medium (MS) containing 10 M 6-benzyladenine with 5 M naphthaleneacetic acid gave rise to multiple shoot formation with a mean increase of 15 to 20 new shoots each 4 weeks. Plants could be stored for more than 6 months in flasks containing deionized water with 5 g 1-1 sucrose with minimal vegetative growth.
A successful system of direct organogenesis is described for the wild citrus tree, Citrus halimii Stone which used in vitro
seedling explants cultured on Murashige and Skoog medium supplemented with 0.4–11.1 µM 6-benzyladenine. Hypocotyl was the
best explant for multiple shoots regeneration. Maximum number of shoots was obtained on medium with 2.2–11.1 µM 6-benzyladenine.
Rooting of regenerated shoots was best on Murashige and Skoog medium supplemented with 2.7 µM α-naphthalenacetic acid. In
vitro and ex vitro rooted plantlets survived (on average 83.3%) after being transferred to the soil mixture consisting of
soil, sand and organic material (1: 1: 1) and kept in the glasshouse.