Development of a temporary immersion system (RITA®) for mass production of sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. interspecific hybrids)

Commercial micropropagation of sugarcane is largely determined by the clonal fidelity and the cost of plants produced. Rapid production of plants in vitro reduces the frequency of offtypes in many species. By exploiting the concept of transverse thin cell layer culture, we have developed a rapid, high frequency direct plant regeneration system, called SmartSett®, for commercial sugarcane cultivars grown in Australia. Similar to conventional micropropagation, labour remains the major cost of this plant production system. Hence, to reduce the labour component, we have integrated the SmartSett® system with the RITA® temporary immersion bioreactor. Thin transverse leaf sections or fragmented leaves cultured on agar-based SmartSett® shoot induction medium were used as the starting material for RITA®. Shoot initiation on semi-solid medium prior to transferring to RITA®, culture immersion frequency, explant size and genotype determined the productivity (number of plants produced per unit culture) of the system. Results obtained with cultivar Q165 indicate that explants cultured for 45 d on SmartSett® shoot induction medium were the most prolific, producing on average 275 shoots per vessel after 45 d of culture in RITA with 1 min immersion every 12 or 24 h. Using the fragmented tissue, 14-d-old explants and 3-mm leaf tissue fragments were the most productive. Experiments with three cultivars (Q117, Q165 and Q205) showed that RITA® culture conditions need to be optimised for each cultivar for maximum plant production.     

Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF. Section Editor: C. P. Vance     

Sugarcane shoot formation in an improved temporary immersion system

A new protocol was established for sugarcane cv. C-1051-73 shoot formation in a temporary immersion system. The two-step protocol involves shoot formation in 50 ml of culture medium per explant and 1.0 mg l-1 paclobutrazol for 30 days followed by shoot elongation by exposure to 1.0 mg l-1 gibberellic acid for 15 days. The multiplication rate was doubled in comparison with the conventional micropropagation protocol (Jiménez et al., 1995) and the cost has been reduced by 46%. Three additional sugarcane varieties have been micropropagated according to this new protocol and results are comparable. Temporary immersion-derived plants have also been compared with conventionally propagated plants in sugarcane fields for more than 9 months and their agricultural indicators of performance are similar. This revised version was published online in June 2006 with corrections to the Cover Date.     

Field performance of artificial seed-derived sugarcane plants

This study shows the behaviour of sugarcane plants cv. CP-5243 derived from artificial seed compared with traditional and isolated bud methods. Artificial seed-acclimatised plants were planted in field conditions simultaneously with two-control treatments previously germinated: macropropagated plants derived from stems of three buds and axillary buds isolated from field-grown plants. Plants from artificial seed were taller and had a smaller diameter at 8 months, but these differences disappeared at 12 months. With respect to sugar analysis and yield, no differences in all parameters evaluated were found between artificial seed-derived plants and plants derived from the two other methods.     

Artemisia judaica L.: micropropagation and antioxidant activity

Artemisia judaica L., an Egyptian medicinal plant used in the treatment of gastrointestinal disorders, was mass-propagated and grown using solid, paper-bridge-support liquid, liquid-flask and bioreactor cultures. The liquid-flask culture using 50 ml MS liquid medium in 250 ml flask yielded significantly greater shoot proliferation than either solid cultures or paper-bridge-support liquid cultures. Increasing flask capacity from 100 to 500 ml improved shoot proliferation and growth. Mass-propagation efficiencies of various bioreactor systems, viz. temporary immersion reactors and bubble column reactors, were also compared. The temporary immersion bioreactor was found to have significant advantages for A. judaica shoot proliferation. The shoot cultures from the temporary immersion reactor formed complete plantlets when subcultured onto a medium containing 1 micromoll(-1) indole-3-butyric acid (IBA), and mature plants were established, acclimatized and thrived in standard greenhouse conditions. Assays of antioxidant activity and total flavonoid content of in vitro and in vivo grown tissues were evaluated as gross parameters of medicinal efficacy. Significantly higher antioxidant activity and flavonoid contents were observed in the tissues of mature greenhouse-grown plants. The efficient in vitro production systems developed in this study provided sterile, consistent tissues for investigation of bioactivity and germplasm conservation of A. judaica.     

Alleviation of hyperhydricity of sugarcane plantlets produced in RITA® vessels and genotypic and phenotypic characterization of acclimated plants

The effect of employing a RITA® system in one or both of somatic embryo induction and germination stages was investigated, and it was deemed far superior to a semi-solid (agar) substrate in terms of in vitro plant yields for sugarcane genotype N41. Approximately 18,000 plants/leaf roll were obtained in vitro in 12 weeks, when both culture stages were undertaken using temporary immersion, compared with approximately 2000 plants/leaf roll produced on semi-solid medium. However, due to hyperhydricity, only ~ 34% of the plants produced in RITA® survived acclimatization. To overcome this, and realize the potential yields of the RITA® system, various culture conditions were investigated, viz. nutrient and sucrose supplies, a rockwool substrate and the immersion regime. Of these, increasing the resting time between immersions from 1 min/12 h to 1 min/72 h, and lowering MS nutrient to 1/2 strength, proved the most beneficial, resulting in 60% acclimation success. Genetic fidelity of these plants was investigated by AFLP analyses where only 0–0.9%, of polymorphic bands were scored compared with the conventionally- propagated N41 control. Phenotypic characterization of plants grown in the field for 6 months showed that, although all in vitro derived plants had a reduced stalk diameter relative to the control, there were no significant differences regarding stalk mass, height and population.     

Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif- mutants strains

The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field by comparing plants inoculated with wild-type and Nif mutant MAd3A in two independent experiments. The wild-type and Nif mutant strains colonized sugarcane plants equally and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had a higher total N content 60 days after planting than did plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane might be a significant mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement was observed in plants inoculated with either wild-type or Nif- mutants, suggesting the additional effect of a plant growth promoting factor provided by A. diazotrophicus. A 15N2 incorporation experiment demonstrated that A. diazotrophicus wild-type strains actively fixed N2 inside sugarcane plants, whereas the Nif- mutants did not.     

Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems

A procedure for the mass propagation of pineapple plants (Ananas comosus L. Merr) using a temporary immersion technique is described. This procedure involved three distinct phases in the automated temporary immersion system: shooting, bud differentiation and elongation. To establish this protocol, we used in vitro shoots obtained from established liquid culture as starting materials. Three culture methods (solid, liquid and temporary immersion) were compared. Temporary immersion increased the multiplication rate and fresh and dry weight after 42 days. Conventional micropropagation (liquid medium) and temporary immersion were compared in combination with paclobutrazol. Paclobutrazol promoted the formation of compact bud clusters with limited leaf development. The highest multiplication rate (106) was found when ex-plants were cultured in shooting medium (MS+2.1 mg/l BA+0.3 mg/l NAA) supplemented with 1 mg/l PB for 7 weeks. A 10-l temporary immersion bioreactor was used to test two approaches during elongation stage: reduction of the shoot-formation period or decrease of the initial number of explants. The highest number of competent and uniform plants (191.8 plant/l) was achieved when shoots were cultured for 4 weeks in shooting medium supplemented with PB. Received: 4 February 1998 / Revision received: 22 June 1998 / Accepted: 14 August 1998     

Sugarcane micropropagation and phenolic excretion

Sugarcane shoot formation was followed using a temporary immersion system. Plant fresh weight, plant dry weight, shoot number and phenolic excretion to the culture medium were recorded during shoot formation. Shoot number increased for 30 days of culture but formation of new shoots was greatly reduced from 31 to 40 days. Phenolic excretion also increased during the first 20 days of culture (gallic acid represented 82% total phenolics) and decreased during the last 10 days (31–40 days of culture). The most intensive period of phenolic excretion (11–20 days) preceded the most intensive period of shoot formation (21–30 days). The same relationship does not seem to exist between the accumulation of fresh and dry weights. Subculture onto fresh medium at the beginning of proliferation (10 days after culture initiation) was detrimental to shoot formation in the subsequent period (11–20 days). However, such a detrimental effect could be avoided if gallic acid was added to the medium. Addition of cysteine to the culture medium reduced both excretion of phenolics and shoot formation but not fresh weight. The use of temporary immersion systems, the increase of culture medium volume per initial explant and the addition of paclobutrazol promoted both phenolic excretion and sugarcane shoot formation. Results presented here indicate a relationship between phenolic excretion and shoot formation but not with accumulation of plant weight. This revised version was published online in June 2006 with corrections to the Cover Date.     

Foliar Iron Fertilization of Peach (Prunus persica (L.) Batsch): Effects of Iron Compounds, Surfactants and Other Adjuvants

During a survey of nitrogen-fixing Burkholderia associated with sugarcane in Tamil Nadu, some endophytes were isolated on PCAT medium. Isolation was based on the use of the selective PCAT medium. Four isolates were studied, all belonging to the genus Burkholderia. One of them, MG43 was consistently more active in reducing acetylene and was identified as Burkholderia vietnamiensis. This isolate was used to inoculate micro-propagated sugarcane plantlets in a comparison with two other diazoptrophs, viz. Gluconacetobacter diazotrophicus^T and Herbaspirillum seropedicae^T. Inoculated plants and uninoculated controls were used in a pot experiment followed by two field experiments under different rates of nitrogen fertilisers. MG43 and G. diazotrophicus performed best in sugarcane, their natural host. Biomass increase due to MG43 inoculation reached 20% in the field. Inoculated plants were heavily colonised by the inoculated bacterium (up to 115,000 CFU g⁻¹ root fresh weight). Inoculation by a combinaison of the three strains performed less well than inoculation by a single MG43 suspension. Ecological implications are discussed, as well as the potential of these bacteria to provide a feasible alternative to higher N fertilisers rates in a low input and long term sustainable rural economy.     

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.     

Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor

Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%-1.5% of total soluble protein (TSP), but still produced 2.4-7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP). This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts.     

The dynamics of nutrient utilization and growth of apple root stock ‘M9 EMLA’ in temporary versus continuous immersion bioreactors

The present study investigated the dynamics of nutrient utilization and various growth and physiological parameters during in vitro proliferation of apple root stock ‘M9 EMLA’ in two different bioreactor systems, i.e. temporary and continuous immersions. Individual shoots obtained from temporary immersion system had higher dry mass and were of better quality than those obtained from continuous immersion. In continuous immersion bioreactor, apple shoots appeared to utilize more nutrients from liquid culture medium than that from temporary immersion. The shoot growth was limited by the availability of phosphate and nitrogen in continuous immersion system. The shoots produced in temporary immersion bioreactor showed higher photosynthetic rate, maximum quantum yield of photosystem-II and slow but steady rate of nutrient absorption, indicating the occurrence of higher photomixotrophic metabolism. The study also showed that high level of antioxidant scavenging enzymes in shoots grown in continuous immersion system induced physiological changes to foster adaptation to stresses.     

剥粒菠萝组织培养及快速繁殖的研究

剥粒菠萝是目前菠萝品种中最适于鲜食的良种之一。本研究以剥粒菠萝为材料,通过10种培养基的诱导筛选、繁殖生根,从中选出最适宜剥粒菠萝组培快繁的一整套稳定、高效的生产程序,即：芽诱导培养基为MS+6_BA 3 mg/L（单位下同）＋NAA 0.1,增殖培养基为MS+6-BA4+NAA 0.1,生根培养基为1/2MS+6-BA 2+NAA 0.2+活性炭0.1%。该组培技术的成功,为规模化生产剥粒菠萝提供了快速繁殖种苗的技术保障,有利于促进南方地区名优水果的发展,有利于南方农业结构的产业化调整。     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

Use of bioreactors for large-scale multiplication of sugarcane (Saccharum spp.), energy cane (Saccharum spp.), and related species

The objective of this study was to set up a plant micropropagation facility to mass propagate sugarcane, energy cane, and related clonally propagated species. An efficient methodology for micropropagation of energy cane and perennial grasses using temporary immersion bioreactors was developed. Several different methods of tissue culture initiation, multiplication, and rooting were evaluated for several varieties of sugarcane (Saccharum officinarum L.) and sugarcane-related species such as Erianthus spp., Miscanthus spp., and Sorghum spp. × sugarcane hybrids, all from a germplasm collection. Apical meristem cultures were initiated for all genotypes that were micropropagated, when liquid or semisolid Murashige and Skoog (MS) medium was used, which was supplemented with 0.1–0.2 mg L⁻¹ BAP, 0.1 mg L⁻¹ kinetin, 0–0.1 mg L⁻¹ NAA, and 0–0.2 μg L⁻¹ giberellic acid. These cultures produced shoots between 4 and 8 wk after initiation. Shoot regeneration from leaf rolls or immature inflorescences was observed as early as 4 wk after initiation. Shoot multiplication was successful for all genotypes cultured in MS medium with 0.2 mg L⁻¹ BAP and 0.1 mg L⁻¹ kinetin. Energy cane had a significantly higher combined multiplication rate when grown under four or five LED lamps than when grown under three LED lamps, or under fluorescent lights in a growth chamber. The addition of 2 mg L⁻¹ NAA produced faster and better rooting in all of the genotypes tested. Shoots produced well-developed roots after one cycle of 15–21 d in the bioreactors. The maximum number of plantlets produced per bioreactor was 1080. Plantlets developed a vigorous root system and were ready to be transplanted into the field after 2 mo. A protocol was standardized for different energy cane clones that were recommended for their biomass production and cell wall composition. Different tissues were used to speed up or facilitate tissue culture initiation. Visual assessment of micropropagated plants in the field did not show any off-types, based on gross morphological changes of plant morphology or disease reaction, compared to plants of the same genotype derived from a traditional propagation method (stem cuttings). This is the first report of energy cane and Miscanthus spp. micropropagation using the SETIS bioreactor.

     

Optimization of plantain (<Emphasis Type="Italic">Musa</Emphasis> AAB) micropropagation by temporary immersion system

The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 M was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.     

Biomass production of Cymbopogon citratus (D.C.) stapf., a medicinal plant, in temporary immersion systems

Summary In vitro plants of lemon grass were established, starting from shoot apices derived from plants cultivated under field conditions. The effect of the immersion frequency (two, four, and six immersions per day) on the production of biomass in temporary immersion systems (TIS) of 1 liter capacity was studied. The highest multiplication coefficient (12.3) was obtained when six immersions per day were used. The maximum values of fresh weight (FW; 62.2 and 66.2 g) were obtained with a frequency of four and six immersions per day, respectively. However, the values for dry weight (DW; 6.4g) and height (8.97cm) were greater in the treatment with four immersions per day. The TIS used in this work for the production of lemon grass biomass may offer the possibility of manipulating the culture parameters, which can influence the production of biomass and the accumulation of secondary metabolites. We describe for the first time the in vitro production of Cymbopogon citratus biomass in TIS.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.