In Vitro Regeneration from Internodal Explants and Somaclonal Variation in Chickpea (Cicer arietinum L)

Protocols have been developed for the in vitro regeneration of plants from calli derived from internode explants of chickpea (Cicer arietinum L) cv Pusa-372. Callusing was induced on both B5 and MS media supplemented with different combinations and concentrations of auxins and cytokinins, but shoot regeneration was achieved only in B5 medium supplemented with 4.0 mg l^?1 IAA and 0.5 mg l^?1 BAP after serial subculture of callus on media with increasing concentration of IAA and constant concentration of BAP. Rooting could not be achieved in in vitro regenerated shoots on any one of the media tried. Complete plantlets were, therefore, developed through grafting of the in vitro regenerated shoot on established root stock. The grafting methodology was found to be highly efficient and reproducible. The somaclones developed produced viable seeds which showed variability in terms of seed colour and seed weight. Thus, the protocols developed in this study remove one important bottleneck in the development of transgenic chickpea.     

鹰嘴豆种质资源对Ascochyta rabiei的抗性评价及遗传多态性分析

以25 个鹰嘴豆品系为试验材料,通过叶面喷雾的方式进行Ascochyta rabiei菌悬液室内外人工接种,评价不同鹰嘴豆种质资源的抗病性;同时利用RAPD方法进行基因型鉴定,采用NTSYSpc 2.10t软件对分子标记结果进行遗传相似性的统计分析并建立各品系间的亲缘关系聚类图,探讨不同鹰嘴豆品系对A.rabiei抗性与遗传多态性间的关系。通过室内和田间鹰嘴豆抗A.rabiei鉴定结果综合分析表明：在25个鹰嘴豆供试品系中,“系选 03”和“216”品系均表现出稳定抗性特性;北园春品系表现出稳定中抗特性。通过RAPD多态性引物对这25 个供试品系进行PCR扩增,共获得129 个扩增条带,其中多态性条带共有67 条,多态性比例达51.94%,遗传相似系数为0.3731-0.9254。结合抗病性和遗传多态性,经方差分析表明,本研究所采用的鹰嘴豆品系对A.rabiei的抗性强弱与其遗传相似性之间无显著相关性。     

Genetics of nodulation in Chickpea (Cicer arietinum L.)

Combining ability, components of genetic variance and graphic analysis revealed that nodulation in the cultivars of Chickpea (Cicer arietinum L.) under study, was predominantly under the control of non-additive gene action although substantial additive effect was also present. The crosses giving high specific combining ability effects also manifested highly significant positive heterosis. The parents F-61, Giza and Annegiri possessed mostly dominant alleles while Phule G-5, NEC-249 and N-31 possessed mostly recessive alleles having positive effect on nodule weight.     

Thidiazuron Induced Multiple Shoot Formation in Chickpea (Cicer arietinum L)

Thidiazuron (TDZ), a substituted urea with cytokinin-like activity stimulated shoot proliferation in chickpea (Cicer arietinum L). Embryonic axis with half portion of both cotyledons was more responsive explant, producing ～22 shootslexplant at 1.0 µM TDZ concentration but higher concentration of TDZ (5.0 µM) reduced both the shoot proliferation and growth. An incubation time of 12 h at 1.0 µM TDZ was sufficient for induction of multiple shoot formation.TD2 induced high frequency of shoot formation as compared to BAP and also minor salts of MS medium played an important role in increasing the number of shoots. Roots could be induced in these shoots in MS medium supplemented with 0.5 µM IBA.     

Low-Temperature Stress: Implications for Chickpea (Cicer arietinum L.) Improvement

Chickpea is the third major cool season grain legume crop in the world after dry bean and field pea. Chilling and freezing range temperatures in many of its production regions adversely affect chickpea production. This review provides a comprehensive account of the current information regarding the tolerance of chickpea to freezing and chilling range temperatures. The effect of freezing and chilling at the major phenological stages of chickpea growth are discussed, and its ability for acclimation and winter hardiness is reviewed. Response mechanisms to chilling and freezing are considered at the molecular, cellular, whole plant, and canopy levels. The genetics of tolerance to freezing in chickpea are outlined. Sources of resistance to both freezing and chilling from within the cultivated and wild Cicer genepools are compared and novel breeding technologies for the improvement of tolerance in chickpea are suggested. We also suggest future research be directed toward understanding the mechanisms involved in cold tolerance of chickpea at the physiological, biochemical, and molecular level. Further screening of both the cultivated and wild Cicer species is required in order to identify superior sources of tolerance, especially to chilling at the reproductive stages.     

鹰嘴豆种质资源农艺性状遗传多样性分析

以100份鹰嘴豆种质资源为材料,应用聚类分析和主成分分析方法,对15个主要农艺性状的遗传多样性进行分析。结果表明,参试材料存在广泛的遗传多样性。其中,多样性指数最高的是株高,其次是百粒重;性状变异系数最大的是单株荚数,其次是单株粒重;基于各种质间形态标记的遗传差异,将100份鹰嘴豆种质聚类并划分为4大类群。第Ⅰ类群可作为选育丰产中粒型和株高适中的品种,第Ⅱ类群可作为选育矮秆耐密及特异粒色(型)品种,第Ⅲ类群丰产性较差可作为选育子粒球型、光滑的品种,第Ⅳ类群可作为选育大粒型、适宜机械化收获的品种。9个数量性状的主成分分析结果表明,前4个主成分累计贡献率达73.91%,各主成分性状载荷值反映了主要数量性状的育种选择潜力。综合分析种质资源农艺性状,为鹰嘴豆的有效利用提供一定的科学依据。     

Molecular Analysis of Chickpea (Cicer arietinum L) Cultivars Using AFLP and STMS Markers

Fifteen AFLP and eighteen STMS primer pairs were employed to reveal genetic diversity and relationship in twenty-one cultivars of chickpea (Cicer arietinum L). Fifteen AFLP primer pairs generated 1804 amplicons, out of which 1732 amplicons (96%) were polymorphic and 600 amplicons (∼33%) were genotype specific. Eighteen polymorphic STMS primer pairs generated 64 amplicons with an average of 3.55 amplicons per primer pair. Polymorphic information content (PIC) varied from 0.52 to 1.0 for STMS markers. The genetic similarity between cultivars varied from 0.30 to 0.85 for AFLP and 0.22 to 0.83 for STMS markers. Dendrogram constructed after combining both AFLP and STMS markers data with Bootstrap analysis, grouped all the cultivars into four clusters. Association of varietal type and flower colour was observed as cultivars E 100Ymu and Nabin (Both Desi type and pink flower) clustered together in the dendrogram.     

Histology of Disease Development in Resistant and Susceptible Cultivars of Chickpea (Cicer arietinum L.) Inoculated with Spores of Ascochyta rabiei

Abstract Histological studies were performed on a compatible and an incompatible interaction between chickpea ( Cicer arietinum L.) plants and the fungus Ascochyta rabiei (Pass.) Labr. The time course of infection, development on leaflets and stems of susceptible (ILC 1929) and resistant (ILC 3279) plants was monitored by light or scanning electron microscopy with the aim to compare histological changes as the basis for further work on biochemical changes in this plant-pathogen interaction.
Spores of A. rabiei began to germinate from 12 hpi on and developed a polar germ tube; fungal colonization, secretion of a mucilaginous exudate and appressoria formation (1–3 dpi) were identical on both cultivars. Leaves of susceptible plants were invaded by the fungus directly through the cuticle, the fungus then spread subepidermally followed by a rapid collapse of the leaf tissue (4–6 dpi). Development of leaf spots and fungal pycnidia could be observed 6–8 dpi. The resistant cultivarrapidly responded (24–48 hpi) to fungal infection and cells of the palisade parenchyma exhibited autofluorescence. In later stages of the infection (4–5 dpi) fluorescent areas developed to small necrotic spots all over the leaflet. These necrotic areas, were the result of cell death and a subsequent change in the leaf structure and were characterized by the accumulation of phenolic compounds. Leaves of the resistant cultivar were invaded by the fungus to less than 5%.     

Characterization of Vicilin Seed Storage Protein of Chickpea (Cicer arietinum L.)

Vicilin, one of the major storage proteins of chickpea (Cicer arietinum L.) was purified and characterized during seed development. Vicilin was purified by zonal isoelectric precipitation followed by chromatography on DEAE-cellulose column. Vicilin on SDS-PAGE resolved into 5 major bands ranging in mol wt from 14 to 66 kD. More heterogenous pattern emerged on isoelectric focussing. This protein had high amount of amides and low amount of sulphur containing amino acids.     

Water Loss via the Glandular Trichomes of Chickpea (Cicer arietinum L.)

During late vegetative growth chickpea leaves and stems canbe covered with aqueous glandular droplets. If these dropletspersist at low humidities there may be substantial water lossvia the glandular trichomes Four solution culture experimentsin growth chambers tested for glandular water loss at differenthumidities. In the daytime, exudate persisted between relativehumidities of 55% and 95%, and the exudate water potential variedbetween - 2.0 M Pa and - 8.0 M Pa. Even by night, chickpea leaves,like wetted alfalfa leaves, were cooler than non-wetted alfalfaleaves or the ambient air. Daytime leaf temperatures were significantlyhigher in a mutant that produced fewer droplets than in itsnormal parent. It was concluded that water loss via the glandulartrichomes can be enough to lower leaf temperature by severaldegrees C within a wide range of atmospheric conditions. The exudate solutes, analysed to confirm the osmotic potentialmeasurements, were primarily malic, hydrochloric and oxalicacid. Without the strong acids a chickpea leaf, wet even ondry days, would be ripe for parasitic attack. Key words: Add exudate, leaf hairs, transpiration, leaf temperature     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Tolerance of Anoxia and Ethanol Toxicity in Chickpea seedlings (Cicer arietinum L.)

Controversy exists as to whether ethanol ever accumulates totoxic levels in anaerobic tissues of higher plants. In orderto manipulate the internal concentrations of ethanol and relatethese to anaerobic injury, seedlings of chickpea (Cicer arietinumL.) were incubated under strict anoxia in vessels in which theanaerobic atmosphere either remained static or else was circulatedwith that of a large anaerobic incubator. Incubation with acirculating, as compared with a static, anaerobic atmospheredoubled the time that the seedlings could be kept under anoxiaand emerge in subsequent survival testing in the glasshouse.Circulating the anaerobic atmosphere gave a 13-fold reductionin the accumulation of ethanol in the seedlings. Parallel experimentswhich varied the ratio of head space relative to seedling numberconfirmed that the dilution of the volatile products of anoxia.increasedsurvival. These products included carbon dioxide, ethanol andtraces of acetaldehyde. While carbon dioxide may play a rolein modifying glycolytic activity under anoxia, it is suggestedthat it is not directly toxic and that it is the reduction inethanol concentration in the seedlings as a result of head spacedilution that contributes to their increased longevity in circulatinganaerobic atmospheres. Key words: Cicer arietinum L., Ethanol, Anaerobic conditions     

Respiration and Phloem Translocation in the Roots of Chickpea (Cicer arietinum)

Root growth in chickpea (Cicer arietinum) has been studied fromthe early vegetative phase to the reproductive stage in orderto elucidate its growth and maintenance respiration and to quantifythe translocation of assimilates from shoot to root. A carbonbalance has been drawn for this purpose using the growth andrespiration data. The increase in the sieve tube cross-sectionalarea was also followed simultaneously. Plants growing in a nutrient culture medium were studied todetermine the relative growth rate (RGR) 5–60 d aftergermination. RGR declined from 113 to 41 mg d^–1 g^–1during the measurement period. Simultaneous with the RGR analysis,respiration rate was also measured using an oxygen electrode.The respiration rate declined as the plants aged and a drasticreduction was recorded following anthesis. The relationshipbetween RGR and respiration rate was used to extrapolate themaintenance respiration (m) and growth respiration (1/Y_EG).The respiration quotient (r.q.) of the roots was 1.2 and theQ₁₀ in the range 20–25 °C was 2·2. A carbon balance for the roots was constructed by subtractingthe carbon lost during respiration from that gained during growth.The roots were found to respire no less than 80% of the carbontranslocated. The increase in the cross-sectional area composed of sieve tubeswas measured near the root-shoot junction as the plants grew.Chickpea has storied sieve plates which simplifies these measurements.Their cross-sectional area increased during growth mainly becauseof an increase in sieve tube number. The diameter of individualsieve tubes remained constant. Specific mass transfer (SMT) values for seive tubes into theroots have been computed during various stages of growth. SMTvalues were relatively constant before anthesis (approx. 6·5g h^–1 cm^–2), but decreased following anthesis. Wedid not evaluate possible retranslocation from roots: any suchretranslocation would have the effect of increasing our SMTvalues. Chickpea, Cicer arietinum, legume, root, respiration, phloem, translocation, carbon balance, specific mass transfer, sieve-tube dimensions     

Mode of Infection of Ascochyta Blight of Chickpea Caused by Ascochyta rabiei

Process of infection and histological changes with Ascochyta blight of chickpea caused by A. rabiei (Pass.) Labr. were studied by light microscopy. Germ tubes from conidia of the fungus penetrate the stem tissue at the juncture of two epidermal cells and form subepidermal aggregates until the fourth day. On the sixth day, yellowing and necrotisation of host tissue coincides with formation of mature pycnidia. Fungus causes extensive damage to cellulosic cell walls of parenchymatous cortical and pith tissues in advance of invading hyphae indicating involvement of cell wall degrading enzymes. Lignified tissues, particularly xylem tracheary elements, remain intact.     

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.     

Vernalization in Chickpea (Cicer arietinum); Fact or Artefact?

Various cool treatments of imbibing seeds reduced the subsequenttimes taken to flower in two genotypes of chickpea (Cicer arietinumL.). These reductions were greater in the kabuli cv. Rabat thanin the desi accession ICC 5810. Nevertheless, in both genotypes,the hastening of flowering was entirely accounted for by thephotothermal time accumulated during each cool temperature pretreatment,provided it was recognized that the ceiling photoperiods wereapproximately 10 and 8 h d^–1, respectively; i.e. neithergenotype shows a true vernalization response. A thorough reevaluationof ‘ responsiveness to vernalization’ in the chickpeagermplasm might now be prudent. Cicer arietinum L, chickpea, vernalization, photothermal time, screening germplasm     

Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg²⁺ ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K_m of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO₃⁻ with S_0.5 value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, α-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca²⁺ and Mn²⁺. Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.     

Effect of Salinity on Growth, Nodulation and Nitrogen Yield of Chickpea (Cicer arietinum L.)

Chickpea cultivar ILC 482 was inoculated with salt-tolerantRhizobium strain Ch191 in solution culture with different saltconcentrations added either immediately with inoculation or5 d later. The inhibitory effect of salinity on nodulation ofchickpea occurred at 40 dS m^–1 (34.2 mol m^–3 NaCl)and nodulation was completely inhibited at 7 dS m^–1 (61.6mol m^–3 NaCl); the plants died at 8 dS m^–1 (71.8mol m^–3 NaCl). Chickpea cultivar ILC 482 inoculated with Rhizobium strain Ch191^spcstrwas grown in two pot experiments and irrigated with saline water.Salinity (NaCl equivalent to 1–4 dS m^–1) significantlydecreased shoot and root dry weight, total nodule number perplant, nodule weight and average nodule weight. The resultsindicate that Rhizobium strain Ch191 forms an infective andeffective symbiosis with chickpea under saline and non-salineconditions; this legume was more salt-sensitive compared tothe rhizobia, the roots were more sensitive than the shoots,and N₂ fixation was more sensitive to salinity than plant growth. Key words: Cicer arietinum, nodulation, N₂ fixation, Rhizobium, salinity     

Influence of L-Ascorbic Acid on Post-anoxic Growth and Survival of Chickpea Seedlings (Cicer arietinum L.)

Chickpea seedlings show diminished survival and reduction ingrowth when kept under anoxia for more than 48 h. Treatmentwith L-ascorbic acid before re-exposure to air after varyingperiods of anoxia shows that this anti-oxidant can improve seedlinggrowth and survival during the post-anoxic recovery period.Comparisons of growth during the recovery period with and withoutascorbic acid treatments, as well as observing the effects ofascorbic acid on K⁺ leakage from roots, suggest that post-anoxicinjury is a significant component of the damage that is causedto chickpea seedlings as a result of prolonged anaerobic incubation. Key words: L-ascorbic acid, anoxia, post-anoxia, growth