Gene tagging and transgenic approaches for the study of legume nodule and root development

Experiments were carried out in the model legume L japonicus to determine the efficiency of promoter trapping and insertional mutagenesis using Agrobacterium tumefaciens (hypocotyl) and A. rhizogenes (hairy root) transformation systems. Three different binary vectors pAGUSBE^19, pGHMCS and pPCTVGUS, all with a promoterless gusA gene, were used for transformation. The frequency of promoter trapping was 1.9 % (6 out of 322 lines) in the hypocotyl system and 3.8 % (51 out of 1342) in the hairy root system. Some of the regenerated lines were sterile presumably due to somaclonal variation. Fourteen trapped lines showed root and nodule tissue-specific GUS expression. The trapped lines were divided into two classes based on the dependence of GUS expression upon rhizobium inoculation. GUS expression in lines CHEETAH, ARROWHEAD, MACHINE GUN, Lj335 and NEEL was not rhizobium inoculation dependent. Whereas, GUS expression in lines BAGEL, DONUT, FATA MORGANA, HYENA, TIMPA, Lj331-7, Lj331-2, and Lj343-3 was rhizobium inoculation dependent. CHEETAH expression was observed during both lateral root and nodule formation processes. FATA MORGANA, TIMPA, BAGEL, DONUT, HYENA, Lj33I-2, Lj331-7 and Lj343-3 expression was found only in nodules. MACHINE GUN and ARROWHEAD expression was detected only during root or lateral root formation process. Based on this result it was concluded that lateral root and nodule formation processes share at least some genetic control. A screening of the segregating generation (T2) of 209 transgenic lines resulted in the identification of 6 putative mutant phenotypes. These mutants were confirmed in the T3 generation for phenotypic reproducibility and segregation. However, none of the mutants were nodule or lateral root specific indicating that a larger number of independent insertion lines were needed to get observe such mutants. Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar gene encodes Phosphinothriein acetyl transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenie L. japonicus plants resistant to PPT were positive upon PGR using bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA locus into the plant genome. Typically, 15- 20% of transformed L. japonicus plants are lost to tissue culture-induced sterility when antibiotic selectable markers are used. All regenerated plants were fertile using PPT selection and void of visible abnormalities. The lack of tissue culture-induced variation, ease of PPT application and low cost of PPT make this protocol an attractive alternative for the regeneration of transgenic L. japonicus. Since several Lotus species are important forage crops, the production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production. Past studies of the effect of ethylene on nodulation have depended on the availability of ethylene insensitive mutants or application of precursors or inhibitors. Lotus japonicus plants expressing the dominant, negative etrl-1 allele from Arabidopsis thaliana were constructed and found to be ethylene insensitive by their lack of the triple response. Independent transgenic lines varied in their levels of insensitivity to ethylene. These lines were tested for their ability to be nodulated. The numbers of infection foci were enumerated using a Mesorhizobium loti strain constitutively expressing the lacZ gene. There was a direct correlation between the degree of nodulation and the level of ethylene insensitivity. Transgenic lines exhibiting a high level of insensitivity to ethylene were hypemodulated, compared to controls. In contrast, the level of nodulation in lines exhibiting mild insensitivity to ethylene did not differ significantly from controls. The position of infections was examined relative to root anatomy. Approximately 30% of the nodules or nodule primordia in transgenic plants were found between xylem poles, compared to only 5% in wild-type plants. The study indicated a role for ethylene in nodule initiation by influencing root cell infection by rhizobia and position of nodule initiation in relation to protoxylem pole. The hypemodulation of ethylene insensitive plants was nitrate sensitive. It is proposed that the hypemodulation due to ethylene insensitivity is due to defective \u27local autoregulation\u27 of nodulation. Since the hypernodulating plants grew similar to wild type, these findings may have agronomic importance

Studies on Gene Expression During Flower Development in Brassica napus

Floral development is central to the life cycle of the plant. It is the most complex example of tissue differentiation, and as such is appropriate to study in order to gain more knowledge of how a plant develops. In recent years the understanding of flower development has been greatly advanced by molecular and genetic studies of floral mutants of two species, Arabidopsis thaliana and Antirrhinum majus. In order to obtain a panel of genes which are expressed in the early stages of floral morphogenesis, it was decided to make and differentially screen a cDNA library. However the Arabidopsis plant is small and it is difficult to obtain sufficient tissue to allow the production of a floral apex cDNA library. Therefore Brassica napus, a closely related member of the Cruciferae, was used to construct a cDNA library from floral buds at an early stage of morphogenesis

Applications of molecular markers in genetic analysis

Restriction fragment length polymorphism (RFLP) and microsatellite molecular markers were used to map two soybean nodulation genes, enod2 and leghemoglobin (lbc3). In addition, a high annealing temperature DNA amplification fingerprinting (DAF) method was developed for DNA fingerprinting of soybean cyst nematode (SCN), Mychorrizae, aphid, centipedegrass, and bermudagrass samples. Recombinant inbred lines (RILs) as well as an F\u3c sub \u3e2 segregating population of soybean Glycine max (L. Merr) facilitated the mapping of two expressed sequence tags (EST) involved in early nodulation and subsequent nitrogen fixation in soybean. For the early nodulin gene enodl2, the parents of RILs, Minsoy and Noirl, showed a polymorphism (5.5 vs. 5.9 kb) after EcoRV digestion. RFLP patterns of 42 RILs were analyzed using the MAPMAKER program linking enod2 to the seed coat color gene, I, with a distance of 11.1 cM on linkage group U3 of RIL map. Enodl2 and I are located close to Rhg4 , a soybean cyst nematode (SCN) resistance gene, and a locus for seed coat hardness. The molecular marker pAllO and seed coat color were used to integrate enod2 on an F2 segregating population (72 plants) generated from a cross between cultivar Bragg and G. soja (Sieb and Zucc), PI468.397. Enod2 was mapped in the same order as on the RIL map but 18.5 cM from the I locus. A microsatellite from the 5\u27 region of enod2B was mapped in the same position, demonstrating that enod2B and not enod2A was mapped. An RFLP for lbc3 (leghemoglobin) segregated independently from enod2 and the nts-l supemodulating locus suggesting that in soybean, symbiotically significant loci (including rj1, rj2, and rj6) are not clustered. To overcome potential problems caused by mismatch priming and secondary DNA structure and taking advantage of high primer-template ratios used in DAF reactions, annealing temperature of 55°C were used with single short arbitrary oligonucleotide as well as mini-hairpin primers to provide high resolution DNA profiles of soybean. Initially, high annealing temperatures for three arbitrary octamer primers in polymerase chain reaction (PGR) were tested for DNA fingerprinting of two soybean cultivars, Minsoy and Noirl. Fifteen PGR programs differing in levels of annealing temperature (47, 55, and 60°C), denaturation, annealing, and extension time (30, 60, and 120 second), and presence/ absence of extension step (+/- 72°C) were tested. The number of bands (amplification products) ranged from 7 (Program 10) to 51 (Program 3). The average ramping temperature for heating and cooling were calculated 1.42 and 1.27 sec/°C, respectively. Intensity of the silver-stained bands in a 10% polyacrylamide gel was high for the most PCR programs. Program 15, DAF-15, (95°C/30 sec, 55°C/120, and 72°C/30 sec) generated a complex DNA fingerprinting profiles for tested primers in Minsoy and Noirl. These profiles contained an average of 42 sharp and highly intense bands using both octamer primers 8-4 and 8-8 for DNA amplification. Using high annealing temperature increased stringency of primer-template annealing, avoided potential mismatching and hybrid molecule formation, and consequently improved reproducibility of DNA fingerprinting. Newly-developed high annealing temperature DAF was used successfully and detected markers linked to the enod2 gene and analyzed DNA fingerprinting of soybean cyst nematode (SCN), Mycorrhizae, aphid, centipedegrass, and bermudagrass samples. RFLP patterns of 41 homozygous F2 individuals for enod2 gene were set into two bulks of 26 and 15 with RFLP patterns identical to their parental patterns Bragg and G. soja, respectively. Screening of the bulks B and S with 31 primers resulted in detection of four polymorphic bands using primers HpC29 and HpC30 and DAF-15 program. Due to low number of polymorphic bands in the B and S bulks, sub-pools were generated and screened. B1 and SI sub-pools were tested with total 196 primers of which 32 were used for screening of sub-pools B3 versus S2. Primers Hp30, HpC22 and HpC30 generated 1, 1 and 4 polymorphic markers, respectively, in the B3 vs. S2. The major screening was focused on the B1 versus SI sub-pools which resulted in screening of 196 mini-hairpin and unstructured primers of which a set of 9 primers detected 20 polymorphic bands. Primer HpD25 generated polymorphic bands with 920B1, 320B1, 220S1, and 185B1 base pairs which were reliable and reproducible. These bands are promising bands for further analysis such as cloning and generating SCAR markers in the region of genome containing the enod2 gene. Key Words: nitrogen fixation, RFLP, recombinant inbred lines, integration mapping, annealing temperature, PCR, DNA fingerprinting, arbitrary primers, soybean. Abbreviations: RFLP, restriction fragment length polymorphism; SCN, soybean cyst nematode; RJLs, recombinant inbred lines; CHS, chalcone synthase; QTL, quantitative trait locus; lbc3, leghemoglobin gene; DAF, DNA amplification fingerprinting; PCR, polymerase chain reaction

Molecular characterization of nodulation genes of Rhizobium fredii strain USDA193

A molecular study of nodulation (nod) genes involved in soybean-Rhizobium fredii USDA193 symbiosis was conducted. The nod genes, nod ABC and nod D, respectively, were located on two unlinked DNA fragments. These two fragments could confer nodulation ability of soybeans to symbiotic plasmid deleted strains of R. fredii or to Ti plasmid-cured strain of Agrobacterium tumefaciens. One of these DNA fragments (a 2.8 kb EcoRI fragment) itself was capable of extending the host range of heterologous rhizobia.;Transposon mutagenesis studies have identified the putative host specificity gene coded by the 2.8 kb EcoRI fragment. Conservation of this host specificity gene sequences in several rhizobia was examined.;Preliminary studies identifying other regions involved in the nodulation process have been carried out. Such studies indicated the possibility of more than one host specificity gene present on unlinked DNA fragments.;DNA homology studies between Rhizobium and Agrobacterium strains, using an insertion sequence IS66 originally isolated from the Ti plasmid of A. tumefaciens, identified the presence of IS66 homologues in R. fredii genome. Such sequences were found to be clustered near the nif gene sequences of R. fredii DNA. Possible roles of these insertion-like sequences in the evolution of R. fredii plasmids and in the generation of spontaneous deletions or genetic rearrangements have been discussed

Local auxin transport regulation in the nascent nodule - an overview in nodulating plants and an investigation into the cytokinin receptor, cre1 - mediated control of auxin transport in medicago truncatula

Legumes form a symbiotic relationship with a group of bacteria, collectively known as rhizobia. The bacterial symbiont fixes atmospheric nitrogen within root nodules, thus providing the host with an assimilative nitrogen source. Nodule formation involves a complex signalling pathway within the legume host. The plant hormone auxin is involved in nodule organogenesis, but how auxin regulates nodulation is still poorly described. Several studies have found increased auxin signalling in nodule primordia, but so far auxin metabolites have never been quantified during the early stages of nodulation. Therefore, the first aim of this thesis was to establish methods for auxin quantification in legume roots. The presumed build-up of auxin in nodule primordia has been predicted to be due to inhibition of auxin export from cells at the nodule initiation site, but the regulation of auxin transport has not been tested systematically in different legumes. Therefore, the second aim was to compare auxin concentrations and auxin transport changes during nodulation in different legumes. Third, the regulation of auxin transport and auxin accumulation was placed into the known signalling pathway of nodulation in the model legume, Medicago truncatula. Auxins are naturally present in low quantities in the root. We developed an LC-MS/MS method for the accurate and sensitive quantification of auxins in root tissues. The method was validated and produced sensitive limits of detection / quantification and correlation coefficients. To compare the role of auxin between indeterminate and determinate nodule types, we measured auxin transport and auxin content in M. truncatula (forming indeterminate nodules) and Lotus japonicus (forming determinate nodules). In addition to acropetal auxin transport, basipetal auxin transport was regulated in response to rhizobia inoculation in both legumes. Different auxins with distinct levels of abundance were detected in separate legumes, with some unique to the nodule tissues. Auxin concentrations increased at the early stages of nodule formation in M. truncatula, but not Lotus japonicus. The inhibition of acropetal polar auxin transport by rhizobia occurred only in indeterminate nodule-forming legumes and correlated with the ability of synthetic auxin transport inhibitors to induce pseudonodules in those legumes. Finally, we investigated the role of the cytokinin receptor CRE1 in modulating auxin transport during nodulation in M. truncatula. We found that cytokinin signalling through CRE1 is necessary for inhibition of acropetal auxin transport, increased auxin concentration and auxin signalling in response to rhizobia. The CRE1 receptor was also required for the correct induction of several flavonoids, which could act as endogenous auxin transport inhibitors. External application of those flavonoids rescued nodulation in the cre1 nodulation-deficient mutant. In conclusion, we demonstrated that the auxin transport machinery is a crucial component in the host legume that is regulated in response to rhizobia. Auxin transport changes could explain measured changes in auxin concentrations during nodule initiation of M. truncatula, but not L. japonicus. Auxin transport control is mediated by endogenous flavonoids, and both flavonoid induction and auxin transport control are regulated by cytokinin signalling in M. truncatula

Carbon Metabolism and Desiccation Tolerance in the Nitrogen-Fixing Rhizobia Bradyrhizobium japonicum and Sinorhizobium meliloti

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies

Application of Somaclonal Variation in Crop Improvement of Pigeonpea (Cajanus cajan (L.) Millspaugh

in the plant breeders perspective however, the boltom line, remains unimalely that the genetic variability recovered from regenerated plants should result In a phenotype that is agriculturally useful. The present reseorch program thus explored the possibility of exploiting agriculturally useful somaclonal variation for subsequent integration into plant breeding programmes and the following objectives were formulated: I. Develop tissue culture technology for Cajanus cajan (1.) Millsp. cv ICPL 87: a. Identity the best explant source. b. Optimize reproducible and high frequency regeneration, c. Irradiate in vitro cultures with gamma rays for additional variability. 2. Assess for somaclonal variation: a. Screen for plonk showing new variation coupled with statistical analysis d degree of variation in qualitative and quantitative charocten In the R2 and R3 generations. b. ldentw progenies with increased variaHon and selection of wurces d useful variation wlth stable inheritance. 3. Diagnostic: a. Investigation for somaclonal variation at the molecular level

Plant Development and Organogenesis: From Basic Principles to Applied Research

The way plants grow and develop organs significantly impacts the overall performance and yield of crop plants. The basic knowledge now available in plant development has the potential to help breeders in generating plants with defined architectural features to improve productivity. Plant translational research effort has steadily increased over the last decade due to the huge increase in the availability of crop genomic resources and Arabidopsis-based sequence annotation systems. However, a consistent gap between fundamental and applied science has yet to be filled. One critical point often brought up is the unreadiness of developmental biologists on one side to foresee agricultural applications for their discoveries, and of the breeders to exploit gene function studies to apply to candidate gene approaches when advantageous on the other. In this book, both developmental biologists and breeders make a special effort to reconcile research on the basic principles of plant development and organogenesis with its applications to crop production and genetic improvement. Fundamental and applied science contributions intertwine and chase each other, giving the reader different but complementary perspectives from only apparently distant corners of the same world

Genetic and molecular analyses of host symbiotic genes and an in vitro regeneration system for Cicer arietinum L

Ineffectively nodulating plant mutants PM405B, PM638A, and PM796B were used in molecular/genetic analyses of root nodule formation in chickpea. To establish the mode of inheritance of the mutant nodule phenotype in chickpea mutant PM638A, reciprocal crosses were made between PM638A and wild-type ICC640. The F\sb2 segregation data fit a 3 mutants: 1 wild-type monohybrid phenotypic ratio, indicating that ineffective nodulation is due to a monogenic dominant, nuclear mutation, tentatively designated as Rn\sb7 . Segregation analysis of F\sb3 progeny confirmed this model. Early (ENod2) and late (Lb) nodulin cDNAs were used as heterologous probes to identify and study the expression of corresponding chickpea genes. ENod2- and Lb-homologous sequences were detected in the chickpea genome by Southern analysis. Northern analysis of root or nodule RNA extracted at different developmental stages indicated that chickpea ENod2 and Lb genes behave as early and late nodulin genes, respectively, and are expressed in a developmentally regulated nodule-specific manner. Comparison of nodulin gene expression in wild-type and ineffective nodules lead to the following conclusions (1) the rn\sb4 (PM405B) and Rn\sb7 (PM638A) mutations do not prevent the expression of ENod2 gene; (2) the rn\sb4 mutation eliminated detectible levels of Lb mRNA. Rn\sb7 mutation reduced levels of detectible Lb mRNA, and rn\sb5 (PM796B) mutation did not reduce Lb gene expression. (3) The symbiotic process in PM638A (nod\sp+fix\sp-) is blocked at a later developmental stage as compared to that in PM405B (nod\sp+fix\sp-). To develop an in vitro regeneration system for chickpea, immature cotyledons were cultured on B5 basal medium with various growth regulators. Non-morphogenic callus formed in response to various auxins previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, different concentrations of zeatin induced formation of white cotyledon-like structures (CLS) at the proximal end of cotyledons. No morphogenesis, or occasional formation of fused, deformed CLS, was observed in response to kinetin or 6-benzyladenine (BA), respectively. Maximum frequency (64%) of explants forming CLS, was induced by 13.7 $\mu$M zeatin plus 0.2 $\mu$M indoleacetic acid. Shoots formed at the base of CLS, proliferated in medium with 4.4 $\mu$M BA or 46 $\mu$M kinetin, and required 4.9 $\mu$M indolebutyric acid or 5.4 $\mu$M naphthaleneacetic acid to produce roots