No more recalcitrant: Chickpea regeneration and genetic transformation

Chickpea production is limited worldwide because of abiotic and biotic stresses. Efforts to overcome these production constraints through traditional breeding are difficult due to limited genetic variation. Novel regeneration is pre-requisite for genetic transformation offers the opportunity to overcome hybridization barriers and introduce novel genes for resistance. Although direct gene transfer via direct DNA transfer has been reported, Agrobacterium mediated transformation is the preferred method and standard protocols have been established for the production of transgenic plantlets derived from cocultivation of embryonic axes. This was soon adopted due to difficulties associated with regeneration of whole plants from callus. Only few reports have been reported using genetic transformation/transgene(s) against abiotic stress tolerance transgenic chickpea plants. Transgenic chickpea using bacterial codA gene tolerance against abiotic stresses have been developed. Chickpea improvement and application of genomics tools to the study of the chickpea genome will be enhanced through the use of genetic transformation

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA3) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement

Variation and long term regenerative capacity of two important tropical forage legumes: Cavalcade (Centrosema pascuorum cv. Cavalcade) and Stylo 184 (Stylosanthes guianensis CIAT184) in vitro

Shoots of Cavalcade (Centrosema pascuorum cv. Cavalcade) and Stylo 184 (Stylosanthes guianensis CIAT 184) from in vitro germinated seeds were cultured on Murashige and Skoog (MS) medium supplemented with 0 to 7 mg L-1 N 6-benzyladenine (BA) in combination with 0 to 0.5 mg L-1 napthalene acetic acid (NAA) for shoot induction and MS supplemented with 0 to 0.5 mg L-1 indolebutyric acid (IBA) for root induction. For Cavalcade, the medium containing 1 mg L-1 BA produced the best shoot multiplication with an excess of six shoots produced from a single shoot (over four weeks) with a mean height 2.0 ± 0.01 cm. Adventitious shoot regeneration was obtained directly from stem axes. For Stylo 184, the maximum shoot regeneration (29.5 ± 1.0 cm shoots/explant) and height (1.5 ± 0.1 cm) was achieved using 7 mg L-1 BA and 0.01 mg L-1 NAA. Direct and indirect shoot regeneration was obtained on the medium containing 1 mg L-1 BA and 0.01 mg L-1 NAA. The regeneration of shoots from callus of Stylo 184 varied between different genotypes and was high (2.6 to 5.8 shoots/explant) even after maintenance in culture of over three years. Both Cavalcade and Stylo 184 shoots were rooted on media supplemented with IBA (0 to 0.5 mg L-1) and readily transferred to soil (Stylo 184).Key words: Callus, forage legume, micropropagation, organogenesis, root induction

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species

Chickpea (Cicer arietinum L.)

Chickpea is one of the most important leguminous, cool-season, food crops, cultivated prevalently in the Asian Pacific region. In spite of its nutritional importance, its area of cultivation has been low, with virtually no increase. Conventional breeding has resulted in several important improvements in this crop, and recent advances in biotechnology such as plant tissue culture and genetic transformation can significantly contribute to better sustainability of this important food crop. Here, we describe an efficient Agrobacterium-mediated transformation protocol for chickpea using axillary meristem explants, which results in a high frequency of genetic transformation (70%) and recovery of valuable transgenic plants. The protocol is significant owing to its high reproducibility and recovery of the transgenics in a relatively short period (90–100 days)

Development and Analysis of Transgenic Chickpea for Resistant to Helicoverpa armigera (Hubner)

The production of chickpea has been reduced considerably for the past 2 to 3 decades mostly due to biotic and abiotic stress factors. On the global basis, annual yield loss due to these factors was estimated to be 11.2 million t, wherein 4.8 million t is being contributed by biotic factors alone (Ryan, 1997, www.icrisat.org). Amongst the biotic factors, the notorious pod borer Hel~coverpu armigera causes a severe pod damage of up to 90% accounting for about 10-33 % per cent yield failure, resulting in annual losses of over US $325 million (ICRISAT, 1992; Yadav et al., 2006). Often, the extent of losses caused by this pest has led to the total failure of the crop (Jayaraj, 1990). This serious threat has been ascribed to frequent and fast changes occurring in cropping pattern of agroecosystem and the polyphagous and cosmopolitan feeding nature of H. armiger

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

In vitro plant regeneration from cotyledonary nodes of recombinant inbred lines of lentil

An efficient and reproducible in vitro regeneration protocol for lentil was developed. For shoot regeneration, cotyledonary node explants of ten elite genotypes were cultured in an inverted orientation on different shoot regeneration media that consisted of Murashige and Skoog (MS) medium supplemented with 1mgL -1 6-benzylaminopurine (BAP) (M1), 1mgL -1 BAP+0.45mgL -1 indole-3-acetic acid (IAA) (M2), and 2mgL -1 BAP (M3). High percentages of shoot regeneration ranging from 80 to 100% on M1 and M3 media and from 50 to 100% on M2 medium were induced. M1 was the most efficient shoot regeneration medium for most genotypes tested. For rooting, in vitro and in vitro-in vivo methods were used. Low and variable rooting percentages ranging from 0 to 45% were recorded with in vitro-in vivo method. Efficiency of rooting on in vitro medium varied depending on the medium in which shoots had been previously regenerated and the genotype tested. When M1 medium was used, high rooting percentages (over 40%) for most genotypes except for microsperma genotypes were found. When the 10 genotypes were screened for good regeneration performance using M1 medium, 2 main clusters and 3 subgroups within one of the clusters were formed based on similarities respect of the number of regenerated shoots per explant and rooting percentages. Subgroup 1 composed by A1146 genotype produced the highest number of shoots per explant (6.17 shoots) and a high rooting percentage (60%) so was selected for further transformation and use as a potential commercial variety.Fil: Bermejo, Carolina Julieta. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Producción Vegetal. Cátedra de Mejoramiento Vegetal y Producción de Semillas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Espósito, María Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cravero, Vanina Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: López Anido, Fernando. Universidad Nacional de Rosario; ArgentinaFil: Cointry Peix, Enrique Luis. Universidad Nacional de Rosario; Argentin

Genetic engineering of chickpea (Cicer arietinum L.) with the P5CSF129A gene for osmoregulation with implications on drought tolerance.

Abiotic stresses including water deficit severely limits crop yields in the semi-arid tropics. In chickpea, annual losses of over 3.7 million tones have been estimated to be due to water deficit conditions alone. Therefore, major efforts are needed to improve its tolerance to water deficit, and genetic engineering approaches provide an increasing hope for this possibility. We have used transgenic technology for the introduction of an osmoregulatory gene P5CSF129A encoding the mutagenized Δ1-pyrroline-5-carboxylate synthetase (P5CS) for the overproduction of proline. A total of 49 transgenic events of chickpea were produced with the 35S:P5CSF129A gene through Agrobacterium tumefaciens-mediated gene transfer through the use of axillary meristem explants. Eleven transgenic events that accumulated high proline (2-6 folds) were further evaluated in greenhouse experiments based on their transpiration efficiency (TE), photosynthetic activity, stomatal conductance, and root length under water stress. Almost all the transgenic events showed a decline in transpiration at lower values of the fraction of transpirable soil water (dryer soil), and extracted more water than their untransformed parents. The accumulation of proline in the selected events was more pronounced that increased significantly in the leaves when exposed to water stress. However, the overexpression of P5CSF129A gene resulted only in a modest increase in TE, thereby indicating that the enhanced proline had little bearing on the components of yield architecture that are significant in overcoming the negative effects of drought stress in chickpea

Protein difference among the leaf explants determined for shoot regeneration and callus growth in Mulberry (Morus indica L.)

A comparison of protein profiles of leaves during different stages of shoot and callus induction showed similarities as well as differences in the expression of proteins.  A protein of 39 kDa was expressed in low levels in leaf explants and increased in intensity during induction of shoot organogenesis in both the cultivars. Analysis of protein patterns during organogenesis and callus proliferation from leaves by two dimensional gel analysis revealed the separation of 39 kDa protein into four spots during organogenesis with pI values ranging from 4.2-5.8.  However, the isoforms of 39 kDa protein with pI values of 4.2 and 5.8 were highly expressed in callus of M-5 cultivar in contrast to S-36 cultivar where only one isoform with pI value of 4.2 was detectable. The analysis of protein synthesis in different stages of development in the cultures may acts as markers to differentiate the group of specific isoforms