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African Crop Science Journal, Vol. 8. No. 2, pp. 129-136 INTERSPECIFIC HYBRIDISATION OF LATHYRUS SATIVUS (GUAYA) WITH WILD LATHYRUS SPECIES AND EMBRYO RESCUE G. ADDIS and R.K.J. NARAYAN1 (Received 27 October, 1998; accepted 2 February, 2000) Code Number: CS00013 INTRODUCTION The genus Lathyrus is classified under the family Leguminosae of the tribe Vicieae. It includes about 150 species which are distributed in the temperate regions of the Northern hemispheres, and extends into tropical East Africa and South America (Kupicha, 1983). A survey of species included in the genus Lathyrus has identified a number of non protein amino acids which are either detrimental or harmless when consumed by man and animals. Bell (1964) has classified 53 Lathyrus species on the basis of the distribution of toxic and non toxic non protein amino acids. In this survey, 38 species showed either the non toxic amino acid lathyrine or the major toxic amino acid, b-N-oxalyl-L-a,b-diaminopropionic acid (ODAP), which causes human neurolathyrism. The survey also showed that the group of species which included L. sativus accumulated ODAP in its seeds, but did not accumulate lathyrine and vice-versa. Lathyrus sativus is the most widely cultivated and economically important species in the genus Lathyrus. At present, the species is widely cultivated in tropical and subtropical regions such as India (Geda et al., 1995), Bangladesh (Hussain and Chowdhury, 1995) and Ethiopia. In Ethiopia, it is known by different vernacular names such as guaya (Amharic), gaayyo (Oromigna), and Sebere (Tigrigna). The agricultural importance of L. sativus can not be overemphasised. The crop can withstand extreme conditions of drought and water logging and grows well in clay soils (Vertisols) that are deficient in nitrogen and phosphorus (Araia, 1994), and requires few inputs for production. It contains 30% crude protein, 27.8% starch, 22% fibre, 1% lipids and 3.3% ash w/w of dry seed (Combes et al., 1989). During drought and famine, it becomes a staple diet for large sections of the rural population in India, Bangladesh and Ethiopia. Despite the economic importance of the crop, excessive consumption of L. sativus seeds for prolonged period of time causes human neurolathyrism, a neurological disease which is characterised by corticospinal dysfunction. The affected individuals are left with permanent disability ranging from inability to walk, to complete paralysis of the lower limbs and in extreme cases death (Ross and Spencer, 1989). The symptoms are well known by local people in Ethiopia. The vernacular name "Sebere" means "it breaks" and indicates the consequence of consuming seeds of L. sativus. As indicated earlier, the amount of the neurotoxic substance ODAP varies among Lathyrus species. The intraspecific variation in ODAP content ranges from 0.0259% w/w in L. sativus var. 8246 (Campbell and Briggs, 1987) to 1.145% w/w of dry seed in L. sativus var. Jamalpur (Kaul et al., 1986). Attempts have been made to breed a variety of L. sativus which is safe for human consumption. However, conventional breeding efforts have been unsuccessful. Interspecific hybridisation between L. sativus and wild Lathyrus species, which contain lathyrine, might produce novel segregants devoid of ODAP. These interspecific hybrids could then be employed in a conventional breeding to produce L. sativus varieties devoid of ODAP. Moreover, these hybrids would be useful in the study of the genetic and biochemical mechanisms controlling the biosynthesis of ODAP. MATERIAL AND METHODS Seed sample collection. Seeds of L. angulatus, L. annuus, L. aphaca, L. articulatus, L. cicera, L. inconspicuus, L. nissolia and L. tingitanus were obtained from collections at the University of Wales, Aberystwyth; L. gorgoni, L. marmoratus, L. pseudo-cicera and L. stenophyllus were requested from the University of South Hampton, and seeds of L. sativus var. 8507 (high toxin variety) were obtained from the University of Ghent. All species which were used in this investigation were inbreeding annuals with the same chromosome number, 2N = 14 (Narayan, 1982; Kupicha, 1983). Natural outcrossing rate in L. sativus is 2.16% (Chowdhury and Slinkard, 1997). However, such information is lacking for the wild species. Germination of seeds and growth of plants in a glass house. Seeds of L. sativus var. 8507 and 12 wild Lathyrus species (Table 1) were sown in multitrays and kept in a heated glasshouse maintained at 26 °C. The seedlings were later transplanted into 10 cm diameter pots. L. sativus was grown in five different batches with an interval of one week between batches. This practice was adopted in order to match the flowering of L. sativus with wild Lathyrus species, the flowering dates of which could not be predicted.
Emasculating and crossing of female parents. Emasculation and pollination were conducted from March to June in the University of Wales, Aberystwyth. Interspecific crossing was carried out between L. sativus and the 12 wild Lathyrus species with or without ODAP. In all combinations, reciprocal crosses were attempted. The pattern of flowering and anthesis in each species was studied prior to crossing. Flowers were collected at six developmental stages; early-bud (no petal colour evident), mid-bud (slight petal colour in kell, no colour in standard or wings), late-bud (colour in all petals), early-flower (standard partially separated along the suture), mid-flower (standard fully separated), and full- flower (all petals fully expanded). Pollen viability was determined by germinating the pollen on an artificial medium as described by Fell et al. (1983). This study showed that the early-bud stage is optimal for emasculation in hand-cross pollination. Although some pollen dehiscence was observed in some of the species at this stage, not more than 2% of the released pollen germinated on an artificial medium. These flowers therefore involved little risk of self pollination when used as female parent. Mid- or late-stage flowers produced the highest levels of pollen germination and were satisfactory sources of pollen. Therefore, the 10 staminal tubes and anthers were removed during early-bud stage in the morning using sterile microforceps (very few flower damages were observed from the higher number of emasculations made in the study). The emasculated flowers were loosely covered with cotton wool to protect the petals from desiccation and accidental cross pollination. Male and female parents were kept in different glass houses. Pollinations were carried out the morning following emasculation (Coulombe et al., 1990). Fresh matured anthers were collected from male parents (mid- or late- stage flowers) using sterilised forceps and rubbed onto the stigma of emasculated flowers. Hybrid seeds were sown again to observe whether they were fertile or sterile. Interspecific hybrid embryo rescue. The interspecific hybrid ovaries (Table 2) were surface sterilised by soaking them in 10% sodium hypochlorite solution for 10 minutes. The ovaries were then thoroughly washed with sterilised distilled water. Fertilised ovules were subsequently removed with sterilised forceps and placed on the media. Sterilisation, dissection and inoculation of plant material were performed in a laminar flow cabinet to prevent microbial contamination.
Media used in the study. The first media used in this investigation was prepared according to Yamada and Fukuka (1986). It contained 3.875 gm-1Gamborge B5 salts, 70 gm-1 sucrose, 2 gm-1 casein hydrolysate and 8 gm-1 agar. The pH of the solution was adjusted to 5.8, and autoclaved at 15 psi (120 °C) for 15 minutes. After dispensing the media into sterilised screw capped test tubes and placing the fertilised ovules on the media, the tubes were placed in a growth chamber which was maintained at 26 - 27 °C under continuous illumination. After three weeks, the embryos were subcultured to a fresh medium of the same composition, except that the concentration of sucrose was reduced to 10 gm-1. After 21 days in this medium, proliferating tissues were subdivided and cultured in a new growth medium containing 4.71 gm-1 MS, 8 gm-1 Difcobacter agar, 30 gm-1 sucrose, 1 mg1-1-naphthalene acetic acid (NAA) and 0.5 mg-1 N6-benzyladenine (BA). After 30 days in this medium, actively growing explants were transferred to shoot inducing medium which contained MS medium, 0.5 mg-1 indole acetic acid (IAA) and 1 mg-1 BA. To initiate root growth, explants were transferred to half strength MS medium containing 1 mg-1 indole butyric acid (IBA) after 40 days in the shoot initiating medium. Nuclear DNA of parents and hybrids. Seeds from two parent Lathyrus species and reciprocal interspecific hybrids were germinated under aseptic conditions. Primary root tips were excised and fixed in 4% formaldehyde for two hours. They were fixed again in freshly prepared 1:3 acetic acid-ethanol for 24 hours. After washing in two changes of distilled water (10 minutes each change), the root tips were hydrolysed for one hour using 5N HCl at room temperature, rinsed for one minute with distilled water and stained for one hour in Feulgen solution (pH = 2.2) consecutively. Stained root tips were washed in three changes of sodium metabisulphite solution (10 minutes each change) and transferred to distilled water. From each of the species and hybrids, three root tips were used. Stained root tips were dissected and squashed on to microscope slides in a drop of 50% glycerol. Photometric measurement of DNA was made using Vickers M85 microdensitometer. For each root tip, absorbance readings were made for fifteen 2C nuclei. The mean absorbance value for each slide was then calculated and the mean values for each species and hybrid were calculated as an average of the three replications (Table 3). All arbitrary absorption units were converted into absolute amounts using L. sativus (2C = 16.78 picogram) as standard (Narayan, 1985).
Chromatographic analysis of ODAP. Five µl of ethanol extracts from seeds of parent species and hybrids as well as pure ODAP were spotted on Silica gel-60 Thin Layer Chromatographic (TLC) plates without a fluorescent indicator. The TLC plate was placed in a chromatank which was saturated with a solvent system containing n-butanol, acetic acid and water in a ratio of 4:1:1 (v/v), respectively. The chromatogram was run for six hours at room temperature. It was then dried at room temperature and sprayed with ninhydrin reagent, which was prepared by mixing 95 ml of 0.2% (dissolved in n-butanol) with 5 ml 10% aqueous acetic acid. The plates were then heated at 110 °C for 10 min to identify the positions of amino acid spots. RESULTS AND DISCUSSION Interspecific hybridisation. About 1263 emasculations and pollinations were made between L. sativus and 12 wild Lathyrus species and the results are summarised in Table 1. Species differed in their response to emasculation and pollination when crossed with L. sativus. Aborted embryos, empty pods, shrunken and fertile seeds were observed in different interspecific hybrid combinations. The outcome of crossing depended largely on choice of the species and female parent. Based on results of this experiment, the species fell in four groups. The first group consists of L. angulatus, L. annus, L. aphaca and L. articulatus. No pods were developed when these species were crossed with L. sativus. This was also true in the reciprocal crosses. Except for L. articulatus, all the species contain the non-protein amino acid, lathyrine (Bell, 1964). The reference value for lathyrine was 0.14. The second group consists of L. inconspicuus, L. nissola, L. stenophyllus and L. tingitanus. Pods were developed when L. sativus was used as female parent. Similarly, L. tingitanus and L. inconspicuus contain lathyrine (Bell, 1964), however, the pods dried at different stages of development without developing viable seeds. The third group included L. cicera, L. gorgoni and L. marmoratus. Reciprocal crosses of these species with L. sativus developed pods. However, percent pod development and average life of the combinations were different as indicated in Table 1. Mature seeds were not observed in these crosses. The fourth group was represented by L. pseudo-cicera. In this cross, hybrid seeds were obtained with relative ease when either of the species was used as a female parent, and the rate of success in the interspecific hybridisation was comparable to the interaspecific hybridisation of varieties of L. sativus. The F1 seeds were germinated and developed into mature plants. These hybrid plants developed viable seeds which suggested that there was normal synapsis in the hybrid plants, an indication of regular meotic behaviour in the hybrid plants and close similarity between the two Lathyrus species. In the present investigation, only one of the 12 attempted reciprocal crosses was found to be successful. These results are in agreement with the findings of various researchers (e.g. Senn, 1938; Yamamoto et al., 1986), and further confirms that Lathyrus is a genus in which it is difficult to make interspecific hybrids. Combinations of strong reproductive barriers, such as structural differences of chromosome compliments which prevent homoeologous pairing, genetic incompatibility factors which are known among higher plants, difference in nuclear DNA amounts which range from 6.86 picogram (pg) in L. minitus to 34.20 pg in L. palustries (Narayan, 1985; Narayan, 1991), as well as the presence of the non-protein amino acid lathyrine which is known to affect pollen germination and pollen tube growth during crossing between Lathyrus species (Simola, 1967), must have played significant roles in the cross incompatibilities. Recent studies have revealed that few successful crosses between taxonomically related Lathyrus species have produced viable offsprings (Khawaja, 1988). Gerdazi (1990) also reported that interspecific cross pollination of L. sativus (male parent) and L. amphicarpus (female parent) produced viable seeds. L. pseudo-cicera and L. amphicarpus which successfully developed viable offsprings when crossed with L. sativus are closely related to each other taxonomically. These three species are classified under section Lathyrus in the infrageneric structure of Lathyrus (Kupicha, 1983). Nuclear DNA of parent species and hybrids. Nuclear DNA amounts for L. sativus, L. pseudo-cicera and their interspecific hybrids are given in Table 3. The results indicate that there is very little 2C DNA variation between the parent species. As would be expected, the interspecific hybrid progenies also show 2C DNA values closely similar to that of the parent species. Analysis of variance showed no statistically significant difference (P < 0.05) among nuclei within root tips, among root tips (plants) within replications, as well as among the parent species and their hybrids. Taxonomic similarity (which is merely based on morphology and habit of the species), between L. sativus and L. pseudo-cicera (Kupicha, 1983) as well as their similarity in genome size (nuclear DNA amount) show that the species are closely related to each other. This may explain the success of the reciprocal crosses to develop viable offsprings. This also suggests that classification of the genus Lathyrus should be revised based on multidisciplinary studies which include cytogenetics, biochemistry and anatomy of plants. Comparison of ODAP between parent Lathyrus species and hybrids. Based on the area and colour intensity of the amino acid spots that correspond with pure ODAP on the TLC chromatogram, the relative amounts of ODAP in the parent species and hybrids were scored visually. It was found that the amount of ODAP in L. pseudo-cicera and hybrids (L. sativus x L. pseudo-cicera and L. pseudo-cicera x L. sativus) were comparable to that of L. sativus var. 8507 which is known for its high ODAP content (Fig. 1). The reference value for ODAP was 0.04. This indicates that interspecific hybrid offsprings of L. sativus and L. pseudo-cicera are not suitable for selection of segregants with no or very low neurotoxin content. Interspecific hybrid embryo rescue. Failure to produce viable interspecific hybrid seeds from fertilised embryos prompted attempts at rescuing aborted embryos through in vitro culture. A total of 161 fertile embryos from different crosses were used and results are summerised in Table 2. Different hybrid embryos reacted differently to embryo rescue. With the exception of the hybrid embryos of L. sativus (female parent) with L. tingitanus, L. inconspicuus and L. stenophyllus which showed cell proliferation, the hybrid embryos developed into calli. Embryo culture was successful in crosses involving L. cicera (female parent) and L. sativus (male parent). Unfortunately L. cicera contains the neurotoxin non protein amino acid (Bell, 1964). No segregants were found that could have been used to develop a variety of L. sativus with low or no neurotoxin ODAP. The agronomic importance and nutritional benefit of L. sativus would suggest continued production of this unique legume. Therefore, efforts to develop a variety of L. sativus which is safe for human consumption is a plausible solution in the foreseeable future. It is recommended that special attention should be given to interspecific hybridisation of L. sativus and wild Lathyrus species which contain lathyrine. Our research indicates that modifications in the methods of the embryo rescue might produce mature plants which could be useful in breeding programmes and research aimed at elucidating the mechanisms which control the biosynthesis of ODAP and lathyrine. ACKNOWLEDGMENTS The principal author would like to thank The World Health Organisation for the Fellowship assistance and The Department of Traditional Medicine for selection to pursue the study. We thank Dr. F. Lambein for sending us seeds of high toxin variety of L. sativus and Dr. D.N.Roy for pure ODAP used in this investigation. REFERENCES
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