Workshop on Soybean Biotechnology for Aluminum Tolerance on Acid Soils and Disease Resistance

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Aluminum Toxicity in Soybean Cell Cultures
(Central Research Institute for Food Crops, 2001-02) Rath, lngo; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Aluminum phytotoxicity is a major factor of the infertility of acid soils. The main toxic form of aluminum is the Al3* ion. Its occurrence strongly depends on the pH of the solution and the activity of other ions that can interact with Al3*. Simulating the mineral environment of aluminum-toxic soils in plant cell culture requires careful modifications to the standard culture medium. In principle, using such an aluminum-toxic medium it is possible to select for cell lines that show an increased resistance to aluminum Selection for aluminum resistance can be achieved by different strategies. In direct strategies cells are selected for their ability to actively grow in the presence of toxic concentrations of ionic aluminum. Rescue strategies involve a recovery of cells in normal medium that have survived a previous aluminum treatment. Different direct and rescue methods and required medium modifications for soybean cell cultures are presented and discussed with regard to arising problems and possible solutions. Aluminum ions cause a multitude of harmful changes to various cellular process. Despite intensive research a primary target of aluminum phytotoxicity has not been identified so far. Al1* can enhance the iron-mediated peroxidation of membrane lipids. In soybean cell cultures this stimulation of lip id peroxidation coincides with aluminuminduced cell death. Experiments with lipophilic antioxidants showed that soybean cells survived in the presence of cytotoxic levels of aluminum in the culture medium when lipid peroxidation was suppressed. This leads to the conclusion that aluminum toxicity In soybean cell cultures is at least partly mediated by an enhancement of lipid peroxidation The underlying mechanism of Al1* action could involve the increased formation of reactive oxygen species. In soybean cell cultures, however, aluminum treatment did not lead to a measurable formation of reactive oxygen species. Therefore, it seems more likely that direct interactions between A!1* and membrane lipids lead to changes in membrane structure that facilitate lipid peroxidation processes. The importance of different toxic effects of aluminum on soybean cells are discussed.
Comparative Studies on Inducible Antimicrobial Defense Reactions in Soybean Cultivars
(Central Research Institute for Food Crops, 2001-02) Groten, Karin; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Plants react to microbial attack with a number of defence mechanisms which include a rapid formation of reactive oxygen species (ROS), cross-linking of cell wall proteins and strengthening of the plant cell wall, induction of the phenylpropanoid pathway, synthesis of phenolic compounds, accumulation of antimicrobial compounds named phytoatexins, synthesis of fungal wall degrading enzymes {glucanases, chitinases) and a rapid localized cell death. These defence responses can also be Induced by application of various biotic and abiotic substances (elicitors) to cell suspension cultures. Most analyses of defence reactions were performed on a few cuttivars and elicitors. There is little knowledge about the reactivity of different cultivars against various stimuli. Therefore, suspension cultured soybean (Glycine max [L] Merr.) cells of four cultivars (Wills, Lumut, Kalmit, Dokb RC) were treated either with crude cell wall extracts of the fungal pathogens Phytophlhora sojae, Rhizoctonia solani, Athelia mlfsii (Pmg-, Riso-, Aro-elicitor, respectively) or with two isolates of the bacterial pathogen Pseudomonas syrmgae pv. glycinea (Psg01'02). Cells of all four cultivars showed the same range of defence reactions but time courses and intensities differed significantly between the cultivars. Furthermore, the reactivity of the cultivars depended on the tested elicitor. A common response to el citation is the rapid release of ROS called oxidative burst. Comparing the amount of H^O; induced by the fungal elicitors three cuttivars produced significantly less H^Oj after application of Pmg-elicitor than with Riso-elictor, only Doko RC showed equal values for both elicitors. These differences in the expression of an oxidative burst corresponded with a rapid and transient alkalinization of the ceil culture medium after elicitation. Those elicitors that caused a strong oxidative burst were also capable of Inducing a greater alkalinization. Nevertheless there is no causal relationship beetween the two reactions as could be shown by specific inhibitors of the oxidative burst. Activation of the phenylpropanoid metabolism is an important step for the synthesis of various secondary compounds (i.e. [iso]-flavonoids, Itgnin-like substances, soluble phenolics). Phenylaianine ammonia-lyase (PAL), the key enzyme of phenylpropanoid metabolism, was activated after elicitor-treatment. The enzyme activity was stronger after addition of Riso-elicitor than after application of bacterial suspensions. The intensity of PAL-activity correlated with an increase of soluble phenolic compounds in the culture medium but not with an accumulation of the isoflavonoid phytoalexins. These results suggest that soybean cultivars recognise the same stimuli but differ In the intensity of elicltor-induced defence reactions. This is probably due to a difrent recognition of the elicitors at the plasma membrane.
Current Progress of Research on Soybean Diseases in Indonesia
(Central Research Institute for Food Crops, 2001-02) Machmud ...[at a], Muhammad; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Recent field surveys indicated that the major soybean disease in Indonesia are soybean stunt, peanut stripe, cowpea mild mottle, bacterial blight, bacterial pustule, rust, and anthracnose. In 1990-1999, research on soybean diseases have been done on the following aspects: (a) field surveys, (b) etiology of the diseases, (c) ecobiology of the pathogens such as strain identification, (d) development of techniques for detection of the pathogens from seeds and other plant parts, and (e) control of the diseases through development and screening of soybean genotypes resistant to the pathogens and biological control such as using antagonistic microbes. This paper is an overview of research progress on soybean diseases in Indonesia that have been done in the past ten years.
Genetic Diversity Evaluated with Molecular Markers in International and Indonesian Soybean Germplasm
(Central Research Institute for Food Crops, 2001-02) Schenkel ...[at al], Werner; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
To assess future breeding strategies for the development of soybean cultivars tolerant to acid soil a analysis of genetic diversity in Indonesian and international soybean germplasm was performed. Fourty-five of Indonesian, African, European, US, Brazilian and genebank origin were analysed. Genetic diversity calculations were based on data from molecular marker analysis. In total 25 RFLP, 22 RAPD, and 269 AFLP loci were evaluated. Based on genetic distances calculated from binary data, a multidimensional scaling and hierarchical cluster analysis was performed. Multidimensional scaling demonstrated that the Indonesian genepool is separated from all other accessions. This was confirmed by hierarchical cluster analysis which revealed a structure of six clusters within the investigated germplasm. The clusters could be correlated to the origin of genotypes. All but two Indonesian cultivars build a single cluster that contains no foreign genotypes. The other clusters are comprised by accessions of different ohgin. Nevertheless clusters clearly correspond to origin, if cultivars from southern USA are pooled together with Brazilian cultivars. These results were confirmed by available pedigree information. Seven ancestors contribute 63% of the genes of Indonesian cultivars released since 1974. Eight genotypes cover 74% of the genome of cultivars of southern USA and contribute strongly to the Brazilian genepool. The same genotypes cover only 16% of the genes of northern USA cultivars. Preliminary data of field trials suggest, that genetic similarity to the Indonesian genepool is positively correlated to field performance under Indonesian climatic conditions. Significance of molecular genetic information could be demonstrated and should be used to plan strategies of future breeding programs.
Improvement of Soybean Yields under Acid Soil Conditions in Indonesia
(Central Research Institute for Food Crops, 2001-02) Sunarlim ...[at al], Novianti; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Acid soils usually are deficient in major nutrients such as phosphorous, potassium, calcium, and magnesium, toxic to exchangeable aluminum and low cation exchange capacity. Soybean cultivate grown in this soil usually produced low yields. The soybean yield can be improved by growing soybean varieties tolerant to the acid soil, soil management, and nutrient management. Results of several experiments indicated that some varieties were more tolerant to acid soil than others. Some improved varieties were developed to obtained varieties tolerant to acid soil. Application of manure in acid soils increased organic matter content and increased soybean yields. Artificial soil conditioners did not affect the soybean yield, because they only affected soil physics but not the soil chemical properties. Plant inoculation with Rhizobium in an areas where soybean had not previously been grown increased nodule weight, percentage of nitrogen content, and seed yield. In some experiments, plant inoculation with Rhizobium In an areas where soybean had been grown increased soybean yield, but in other experiments did not affect the yield. Results of nitrogen fixation experiments using 1SN showed that total nitrogen fixed by soybean plants was 63.2 kg/ha or 45.4% from the total plant requirement of nitrogen. Liming at the rate of 1.0 to 1.5 x exchangeable aluminum was enough to obtain a good soybean yield. Anorganic fertilizers (P and K) were needed in the acid soils. The rate of P fertilizer was between 46 to 92 kg P2O5/ha, while and K fertilizer was 60 kg K2O/ha.
In Vitro Organogenesis of Soybean and Agronomic Characterization of Obtained Plants
(Central Research Institute for Food Crops, 2001-02) Sianipar ...[at al], Nesti F.; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Organogenesis of soybean through shoot bud induction was done using cotyledonry nodes of soybean cultivars Sicinang and Sriyono as sources of explants. A Murashige-Skoog (MS) medium supplemented with thidiazuran increasing from 0.005 to 0.05 mg/l plus NAA or 2,4-D 0.01 mg/l was used in the study. The shoots obtained were subcultured and the original cotyledonary nodes were cut off after subculturing the shoots. Growths and productions of the Fi and F2 in vitro derived plants were recorded. The results indicated that increasing concentration of thidiazuron from 0.005 to 0.05 mgfl and the presence of either NAA or 2,4-D 0.01 mg/l in the medium were beneficial to shoot regeneration. During the induction period, the average number of shoots obtained were 5.6 from cultivar Sicinang and 5.2 from cultivar Sriyono. The shoots were then cut off and subcultured into a fresh medium containing 0.01 mg/l thidiazuron. in the first subcultures, the numbers of shoots ranged from 2.8-6.2 shoots per explants, while the cotyledonary node produced 21-25 shoots. After the third subcultures, the cotyledons dried out and no further buds were formed. Plants derived from in vitro cultures of both subcultures I and II were shorter than the original seed derived plants. The seed derived plants were also mature earlier and more tolerant to shading of up to 70% shading. The plants obtained from in vitro subculture seem to be more suitable to be grown intercropped with other plants
Isolation of Genes Involved in Soybean Response to Al Toxicity under Low pH Condition
(Central Research Institute for Food Crops, 2001-02) Ermoiayev, Vladimir; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Al toxicity in plants is one of the major limitation of crop production on acid soils. Because of complex interaction between Al and a plant, it is very likely that there are 3 number of different mechanisms used by plants to confer Al resistance. Genetic studies of Al resistance have shown it to be a dominant, multigenic traits controlled by one or a few major genes and several minor genes. The aim of our investigations was to study such gene activities involved in molecular mechanisms of plant stress resistance. Therefore our research started with application of Differential Display RT PCR (DD) approach to find some differences in mRNA population between roots and suspensions cultured cells of sensitive (Malabar and Lumut) and tolerant (Tambora and Wills) soybean (G/ycrne max L. Merril) lines under stress and non-stress conditions. After set of DD experiments 12 clones specific to mRNA of stress treated tolerant lines were obtained and sequenced. Marathon cDNA amplification was used to prolong the DD fragments. Eight prolonged PCR fragments were obtained and enhanced expression of corresponding mRNA under stress conditions was verified by Reverse Northern Blotting. In order to isolate complete protein coding cDNA sequence, selected fragments were used to screen ^-ZAP cDNA library produced from root tip and suspension cultured cell cDNA from tolerant soybean lines. After putative search, clones were obtained. Their differential expression under conditions of aluminum stress was confirmed by Reverse Northern or Northern Blotting experiment. The clone II-58 1b2 is coding for protein of 168 amino acids. It has 80% homology to translation ally controlled tumor protein from plants. The clone II-63 2a3 is coding for protein of 319 amino acids having 77% identity with inosine-5'-mo no phosphate dehydrogenase from Arabidopsis thaliana. The clone 1-31 bb is coding for protein of 539 amino acids having 35-42% identity with ATP-dependent transporter family. In addition, several genes of probable interest in Al resistance mechanisms were tested for their A I-responsiveness by Reverse Northern Blotting. Eight genes, for example G-protein, a-subunrt, glutathione-S-transferase, calreticulin, ma I ate dehydrogenase, metallothionein MT1, either gave equal signal or did not give any signal at all. For soybean malate synthase, Walii and Wah4 (wheat aluminum induced) genes as well as for PEP carboxylase turned out to be possible to prove that their expression was enhanced under conditions of Al stress. In order to study influence of obtained genes on plant resistance to Al stress It is planned to over express selected genes in soybean protoplas and Nicotians plumbaginifolia as a model organism.
Marker Techniques as Tools for Efficient Plant Breeding
(Central Research Institute for Food Crops, 2001-02) Wenzel G. ...[at al]; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Today, in plant biology molecular genetics opens up an innovative spectrum even under the applied aspect of plant breeding. The most spectacular approach is surely the area of .gene transfer which at the same time is under severe and controversial discussion. One reason for this conflict is the fact that presently no convincing economic or ecological successes due to a gene transfer have been applied e.g. in food or feed production. There is still a need of more physiological knowledge as one prerequisite for useful gene transformations, Here, the lack of cloned genes may soon be overcome by improved genome analysis. Such analysis demands molecular marker techniques. These are predominantly RFLP {restriction fragment lenght polymorphism). RAPD (random amplified polymorphic DNA), AFLP (amplified fragment lenght polymorphism), and SSR (single sequence repeats or micro satellites). Such tools are not only useful for the identification of genes phenotypically difficult to characterise but also during marker assisted selection (MAS). By MAS it is possible to select in populations classically produced by combination breeding, those individuals which express desired characters even if they are very rare. Particularly for the complex quantitatively inherited traits – like e.g. aluminum tolerance of soybean - the use of such a technique will speed up the ' process to produce adapted genotypes. On the short and medium term basis marker techniques will contribute more to plant breeding than gene transfer. Like any other molecular technique it requires, however, a great deal of classical breeding work, thus molecular markers are not replacing classical approaches but are improving the breeding efficiency.
Micropropagation of F, Hybrids Soybean [Glycine max (L.) Merrill] Tolerance to Aluminum
(Central Research Institute for Food Crops, 2001-02) Handayanl ...[at al], Tltin; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
A. reproducible method Tor multiplication of shoot from cotyledone node of soybean (Glycine mex) has been developed. Progeny from seed recovered from regenerated plants appear normal. The technique for micropropagation of F, hybrid soybean plants is described. By culturing cotyledone node obtained from 5 day old seedlings on modified B5 medium, average success multiply shoot were obtained. Best regeneration was from cotyledone node explants obtained on modified B5 medium with NAA and picloram. The optimal concentration of NAA for the induction of regeneration to be 0.1 mg/l. A few buds were induced in the 0.01 mg/l picloram treatment but at the higher levels the entire explant call used. When the explant was placed directly on 85 medium standard, the production of buds and small shoots was not inhibited. However, when the explants were placed directly onto B5 medium with BAP, resulting buds and shoots had a low rate of survival. No multiplication shoot occurred when coconut milk was added on B5 medium. The cotyledone nodes explant were continued to grow but the growth showed faster than on B5 medium standard. Plantlets were grown to maturity under greenhouse condition.
An Overview Soybean Production in Indonesia
(Central Research Institute for Food Crops, 2001-02) Sumarno; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
Performances of Soybean Breeding Lines in Dryland Acid Soils
(Central Research Institute for Food Crops, 2001-02) Arsyad ...[at al], Darman M.; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
In 1995, a field trial was conducted in North Lampung to evaluate soybean germ pi asm in an effort to develop soybean cultivars suitable for acid soils in Sumatera. Thirty soybean genotypes were selected from 350 germ pi asm accessions based on their agronomic performances. These genotypes were then reevaluated, and four genotypes were selected. Twelve single-cross combinations were made, and pedigree as well as bulk methods were used for inbreeding from F2 to Fs generations. A number of 80 Fg breeding lines were evaluated and selected for tolerance to acid soils in North Lampung. Selected 10 F7 lines along with check cultivars Wilis, Slamet, and Sindoro were reevaluated in Lampung and North Sumatera provinces. A breeding line K3911-66 was identified as the best line with good adaptability to both trial sites. Lines D3578-4 and D3623-22 well adapted to North Sumatera and Lampung conditions. Those breeding lines were considered as promising lines that need to be evaluated further In other locations and seasons.
Somatic Embryogenesis in Different Soybean Varieties
(Central Research Institute for Food Crops, 2001-02) Mariska ...[at al], Ika; Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian
National demand for soybean (Gfytine max L. Merrill) is higher that its production, so that Indonesia needs to import the commodity. The government has planned to extensity the crop to acid sort areas, which has not been managed at the maximum capacity. However, many problems faced in acid soil, particularly high content of aluminum and low pH, which inhibit the growth of plant Soybean variety tolerant to the condition is still limited. To improve the tolerance to aluminum, a study was conducted through in vitro selection. One of the problems needs to be accomplished is the method of cell regeneration, since no standard (repeatable) method has been established. Therefore, it is necessary to study the effect of genotypes (varieties), source of exptants, t^ne of subculturing/physiologycal condition of mother plant and other factors on the success of somatic embryogenesis The study was conducted in two steps. In the first step, 7 sources of explants respectively collected from 10 soybean varieties were tested for their response to callus induction on 18 formulation media and to embryo maturation on 25 formulation media, Callus induction media were Murashige and Skoog (MS) or Phillips and Collins (PC-L2) in the combination with 2.4-0 (0-40 mg/I), picloram (0-0.01 mg/f), 8A (0-0.5 mg/l), kinetin (0-0.1 mg/l), NAA (0-10 mg/l), and several amino acids. Meanwhile for somatic embryo maturation basal media MS or PC-L2 had been used in the combination with 2,4-0 (0-0 05 mg/l). BA (0-0 5 mg/l), NAA (0-0.3 mg/l), zeatm (0-1 mg/l). GA3 (0-0.1 mg/l), manitoi, and charcoal. The best explants from first expenment were then used in second experiment. Results showed that 5 varieties (Bromo, Tambora, Wills, Black Manchu, and Argomulyo) were responsive to the treatments. From 18 media formulations. 5 formulae gave good results for embryonic callus, which developed into embryosomatic structures. Culture in MS media with high concentration of auxin, NAA (10 mg/i) or 2,4-0 (40 mg/l) and amino acid, followed by subculture in the media with low concentration of 2,4-D produced embryonic callus which was able to develop to bipolar embryo. For maturation and germination, somatic embryo structures were tubcuKured on a media without euxtn. The best methods of somatic embryogenesis produced in this study were repeatable and relatively produced high regeneration ability.

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