S. R. Bhat

Molecular and genetic analyses of transgenic plants: Considerations and approaches

Plant transformation has become an important experimental tool for investigations into various aspects of plant biology such as physiology, genetics, developmental biology, molecular biology etc. The precision and simplicity of the approach coupled with the power of resolution of details at the molecular level have led to its adoption to answer hitherto intractable problems. In addition, the feasibility of mobilizing and expressing foreign genes into plants has opened up whole new era of genetically engineered crop plants capable of defending against biotic and abiotic stresses and producing better quality products or novel compounds of pharmaceutical and industrial value. There are, however, aspects of plant transformation that are not well understood and cause considerable variation among independent transgenics produced under identical conditions. In this article we discuss various factors that contribute to variation among transgenics and suggest experimental approaches for genetic and molecular analyses of transgenics to derive meaningful conclusions.

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

Abstract A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F1 plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis.

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard.

Cytoplasmic male sterility in alloplasmic Brassica juncea carrying Diplotaxis catholica cytoplasm: molecular characterization and genetics of fertility restoration

Abstract The present study was aimed at characterizing cytoplasmic male sterility (CMS) and identifying the fertility restorer gene for CMS (Diplotaxis catholica) Brassica juncea derived through sexual hybridization. The fertility restorer gene was identified by crossing the CMS line with progeny plants derived from somatic hybrids of B. juncea and D. cathoilca. The CMS line is comparable to the nuclear donor B. juncea in all respects except for flower and silique characteristics. In CMS plants, the flowers have smaller nectaries, and anthers are converted into petals or tubular structures. Gynoecium exhibits a crooked style and trilocular ovary. Seed fertility was reduced in the CMS line. Genetic segregation data indicated that a single, dominant, nuclear gene governs fertility restoration. Restored plants showed a high female fertility and lacked gynoecium abnormalities. In fertility-restored plants, petal development was found to be variable; some flowers had the normal number of four petals, while others had zero to three petals. Interestingly, the trilocular character of the ovary was found to co-segregate with CMS and became bilocular upon male-fertility restoration. Thus, this trait appears to be affected by the interaction of nuclear and mitochondrial (mt) genomes. Restriction fragment length polymorphism analysis indicated that mt-genome of D. catholica is highly divergent from that of B. juncea. However, in Northern analysis, out of eight mt genes studied, an altered transcript pattern was recorded for only atpA. In fertility-restored plants, the atpA transcript became shorter, thereby showing its association with CMS.

Chloroplast substitution overcomes leaf chlorosis in a Moricandia arvensis-based cytoplasmic male sterile Brassica juncea

Abstract A male sterile Brassica juncea line based on Moricandia arvensis cytoplasm was developed previously by backcrossing the somatic hybrid M. arvensis+B. juncea, and the gene for restoring fertility was introgressed. The CMS line is very severely chlorotic because of the presence of alien chloroplasts and flowering is delayed by 30–40 days, making it unsuitable for the exploitation of heterosis. We have resorted to another cycle of protoplast fusion between green fertile B. juncea and chlorotic male sterile B. juncea, and developed green male-sterile plants. Molecular analysis revealed that in green male-sterile plants chloroplasts of M. arvensis origin were substituted by those from B. juncea, giving rise to intergeneric cytoplasmic hybrids with mitochondria of M. arvensis origin. With the development of dark-green male-sterile plants, the CMS fertility restoration system is suitable for the production of hybrid mustard.

An improved cytoplasmic male sterile (Diplotaxis berthautii) Brassica juncea: identification of restorer and molecular characterization

We report the development of an improved cytoplasmic male sterile (CMS) system of Brassica juncea carrying cytoplasm of the wild species Diplotaxis berthautii. Flowers of the CMS line are smaller than the euplasmic line but have improved nectaries. Anthers are slender and fail to extend to the level of stigma. Female fertility of the CMS line is comparable to the euplasmic line. Fertility restorers of Moricandia arvensis and D. catholica-based alloplasmic CMS systems of B. juncea were found capable of restoring male fertility to this new CMS line. The fertility restoration is monogenic and gametophytic. Southern analysis showed that the cytoplasm of the CMS line is different from euplasmic B. juncea and other CMS systems restored by the same restorer lines. Northern analysis of the CMS, fertility restored and euplasmic lines using eight mitochondrial gene probes revealed altered atpA expression associated with male sterility. Cleaved amplified polymorphic sequence (CAPS) markers were identified for the plastid gene psbB, which could be useful for a quick identification of this CMS line.

Promoter Trapping in Arabidopsis Using T-DNA Insertional Mutagenesis

A new promoter trap vector was constructed based on the juxtaposition of T-DNA right border to coding sequence of GUS. The new vector pRN-1 carried an intron in the GUS coding region. Promoter trap vectors pGKB5 and pRN-1 vectors were used to transform Arabidopsis ecotype Columbia using the floral dip transformation system. The transformants were selected on appropriate selection media and the primary transformants were confirmed by PCR using gene specific primers. Approximately 50 % of the T2 lines segregated for a 3:1 ratio indicating presence of T-DNA at single locus. Approximately 15% of the transformed lines showed expression of GUS. Morphological mutants for male sterility and dwarfism were also identified in the T2 population. A T-DNA tagged line was identified in T2 with GUS expression specifically in the floral parts. The number of T-DNA loci in this line was confirmed by Southern blot hybridization. T-DNA flanking region isolated from this line suggested insertions into chromosome 2 at two closely linked loci. The results demonstrate that the population generated can be used effectively to identify and characterize gene regulatory elements.

Characterization of Upstream Sequences of the LOJ Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in Arabidopsis thaliana

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Genetic diversity analysis of elite pearl millet inbred lines using RAPD and SSR markers

Pearl millet [Pennisetum glaucum (L) R Br] is one of the widely grown cereal crops in the arid and semi-arid regions of Africa and India. We undertook a study to ascertain the genetic diversity in 21 elite inbreds (parental lines of 13 pearl millet hybrids in India) using 20 Random Amplified Polymorphic DNA (RAPD) and 21 Simple Sequence Repeat (SSR) markers. Based on Polymorphism Information Content (PIC) and unique banding profiles, 6 RAPD primers OPD12, OPA16, OPB6, OPA19, OPB5 and OPB1, and 3 SSR markers Xpsmp2208, Xpsmp2223 and Xpsmp2220, were found to be highly discriminative. The PIC values ranged from 0.28 to 0.48 for the RAPD and from 0.24 to 0.60 for the SSR markers. Cluster analysis and principal component analysis of the combined dataset of RAPD and SSR markers indicated moderate genetic divergence among the elite pearl millet germplasm, besides unraveling the genetic relationships among the male sterile lines and the restorers.