Niranjan Baisakh

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.

Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.)

The response of a grass halophyte Spartina alterniflora at early stages of salt stress was investigated through generation and systematic analysis of expressed sequence tags (ESTs) from both leaf and root tissues. Random EST sequencing produced 1,227 quality ESTs, which were clustered into 127 contigs, and 368 were singletons. Of the 495 unigenes, 27% represented genes for stress response. Comparison of the 368 singletons against the Oryza sativa gene index showed that >85% of these genes had similarity with the rice unigenes. Moreover, the phylogenetic analysis of an EST similar to myo-inositol 1-phosphate synthase of Spartina and some selected grasses and halophytes showed closeness of Spartina with maize and rice. Transcript abundance analysis involving eight known genes of various metabolic pathways and nine transcription factor genes showed temporal and tissue-dependent variation in expression under salinity. Reverse northern analysis of a few selected unknown and ribosomal genes exhibited much higher abundance of transcripts in response to salt stress. The results provide evidence that, in addition to several unknown genes discovered in this study, genes involved in ion transport, osmolyte production, and house-keeping functions may play an important role in the primary responses to salt stress in this grass halophyte.

Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress

Plants capture solar energy and atmospheric carbon dioxide (CO2) through photosynthesis, which is the primary component of crop yield, and needs to be increased considerably to meet the growing global demand for food. Environmental stresses, which are increasing with climate change, adversely affect photosynthetic carbon metabolism (PCM) and limit yield of cereals such as rice (Oryza sativa) that feeds half the world. To study the regulation of photosynthesis, we developed a rice gene regulatory network and identified a transcription factor HYR (HIGHER YIELD RICE) associated with PCM, which on expression in rice enhances photosynthesis under multiple environmental conditions, determining a morpho-physiological programme leading to higher grain yield under normal, drought and high-temperature stress conditions. We show HYR is a master regulator, directly activating photosynthesis genes, cascades of transcription factors and other downstream genes involved in PCM and yield stability under drought and high-temperature environmental stress conditions.

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+‐ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K(+) /Na(+) ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na(+) and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa.

Overexpression of Arabidopsis and rice stress genes' inducible transcription factor confers drought and salinity tolerance to rice.

Rice yield is greatly affected by environmental stresses such as drought and salinity. In response to the challenge of producing rice plants tolerant to these stresses, we introduced cDNA encoding the transcription factors DREB1A and DREB1B under the control of the stress inducible rd29 promoter. Two different indica rice cultivars were used, BR29, an improved commercially cultivated variety from Bangladesh and IR68899B, an IRRI bred maintainer line for hybrid rice. Agrobacterium mediated transformation of BR29 was done independently with DREB1A isolated from rice and Arabidopsis and DREB1B isolated from rice, whereas biolistic transformation was done with rice- DREB1B in the case of IR68899B. Initial genetic integration was confirmed by PCR and Southern blot analysis. Salinity tolerance was assayed in very young seedlings. Drought stress tests were found to be more reliable when they were carried out at the pre-flowering booting stage. RNA gel blot analysis as well as quantitative PCR analysis was performed to estimate the transcription level under stressed and unstressed conditions. Agronomic performance studies were done with stressed and unstressed plants to compare the yield losses due to dehydration and salt loading stresses. Noticeably enhanced tolerance to dehydration was observed in the plants transformed with DREB1A isolated from Arabidopsis while DREB1B was found to be more effective for salt tolerance.

Ectopic expression of ADP ribosylation factor 1 (SaARF1) from smooth cordgrass (Spartina alterniflora Loisel) confers drought and salt tolerance in transgenic rice and Arabidopsis

Salinity and drought are two very important abiotic stressors that negatively impact the growth and yield of all sensitive crop plants. Genes from halophytes have been shown to be useful to engineer crop plants that can survive under adverse soil and water conditions. The present report establishes, for the first time, the physiological role of a class one ADP ribosylation factor gene (SaARF1) from the halophyte Spartina alterniflora (smooth cordgrass) in imparting salinity and drought stress tolerance when expressed in both monocot (rice) and dicot (Arabidopsis) systems. The Arabidopsis and rice plants overexpressing ARF1 are many-fold more tolerant to salt and drought than wild-type (WT) plants. The transgenics exhibited improved growth and productivity relative to WT through tissue tolerance by maintaining higher relative water content and membrane stability, and higher photosynthetic yield by retaining higher chlorophyll concentration and fluorescence under stress conditions compared to WT. These findings indicated that genes from halophyte resources can be useful to engineer and improve salt and drought stress tolerance in both monocot and dicot plants.

Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress.

Salinity is one of the most important environmental constraints limiting agricultural productivity. Considering the importance of the accumulation of osmolytes, myo-inositol in particular, in halophytic plants adaptive response to salinity, an effort was made to overexpress the SaINO1 gene from the grass halophyte Spartina alterniflora encoding myo-inositol 1-phosphate synthase (MIPS) in Arabidopsis thaliana. We demonstrated that SaINO1 is a stress-responsive gene and its constitutive over expression in Arabidopsis provides significantly improved tolerance to salt stress during germination and seedling growth and development. The transgenics retained more chlorophyll and carotenoid by protecting the photosystem II. The low level of stress-induced cellular damage in the transgenics was clearly evident by lower accumulation of proline in comparison to WT. Our results indicated that possible overaccumulation of MIPS enzyme in the cytosol protected the transgenic Arabidopsis plants overexpressing SaINO1 from the toxic effect of Na(+) under salt stress by reducing cellular damage and chlorophyll loss.

Golden rice: introgression, breeding, and field evaluation

Considerable progress has been made on the genetic engineering of rice for improved nutritional content involving micronutrients and carotenoid content. Golden Rice, developed by genetic engineering (Agrobacterium and biolistic transformation) was used in rice breeding for the transfer of high-nutritional value to the local rice cultivars. Simultaneously, commercial Asian indica rice cultivars were also developed with expression of high-carotenoid levels. The lines were developed based on POSITECH (PMI) selection system or made marker free by segregating out the marker gene from the gene of interest. Anther culture was used to develop the homozygous stable lines, which could be of much use in further introgress-breeding and in farmer’s field. Enhanced carotenoids levels (up to T3 generation) were observed in a number of lines compared to the T0-T1 seeds which could be due to transgeneration effect of growing under greenhouse versus field conditions. However, a few introgressed lines showed less carotenoid levels than the original lines used in the breeding process. Agronomic performance of introgressed lines, non-transgenic controls, and transgenic golden rice (IR64 and BR29) developed at IRRI showed acceptable and comparable data under identical limited field conditions (screenhouse data). Syngenta generated a new Golden Rice (US cultivar) containing high level of carotenoids grown in the field at Louisiana, USA is expected to be available to the public domain. Incorporation of genes for carotenogenesis in seeds by transgenesis or by introgression did not change any significant agronomic characteristics in rice plants. The ongoing and future study of bioavailability, quality, larger field testing and freedom to operate will ensure the benefit of Golden Rice to the people who need them most.

Molecular Diversity Among Members of the Saccharum Complex Assessed Using TRAP Markers Based on Lignin-Related Genes

In addition to the cultivation of sugarcane for sugar, the crop is considered seriously as an important bioenergy grass crop for its high biomass production ability. But, lignin is a serious bottleneck in the bioconversion of lignocellulosic biomass to ethanol. Hence, genetic relationships among 64 genotypes within the Saccharum complex were studied with respect to lignin-related genes using target region amplified polymorphic (TRAP) primers derived from caffeic acid O-methyltransferase (COMT), cinnamoyl alcohol dehydrogenase (CAD), cinnamoyl coA reductase (CCR), and ferrulate 5-hydroxylase (F5H) genes. While the average polymorphism detected by the TRAP markers was 43%, the markers derived from F5H gene (34%) were less polymorphic in comparison to those derived from COMT (46%), CCR (44%), and CAD (46%) genes. The lignin gene-based TRAP markers differentiated members of the Saccharum complex broadly according to previously established genetic relationships in the order of Miscanthus > Erianthus > Saccharum spontaneum > Saccharum robustum/Saccharum barberi/Saccharum sinense > Saccharum officinarum/cultivars. Principal coordinate analysis showed that 29% of the total variation was explained by the genotypes with respect to the lignin-related genes. The association of genetic variation revealed in this study with the biomass composition-related genes of the genotypes within a species will be helpful to design breeding strategies to develop superior energy cane cultivars with improved biomass quality of the sugarcane.

Spartina alterniflora Loisel., a halophyte grass model to dissect salt stress tolerance

Salinity is one of the most serious abiotic stresses affecting crop productivity worldwide. Improving tolerance to salinity in field crops is globally important because a majority of the world population relies on salt-sensitive crops such as rice, corn, and wheat for their daily calories. Although there is no salt stress sensor yet identified, different signaling components and tolerance mechanisms have been substantiated to a great extent in a glycophyte like Arabidopsis, and more recently in a few halophytes. With the rapid advances in genetics, genomics, and biochemical and transformation tools, it is now possible to explore the genetic and molecular basis of the unusually high level of salt tolerance in halophilic plants. We will focus on a halophyte grass, Spartina alterniflora, commonly known as smooth cordgrass, which possesses all known mechanisms of salt tolerance and subsequent exploitation of its genome information for crop improvement. A number of candidate genes encoding transcription factors, ion transport, osmoprotectants, antioxidants, detoxifying enzymes, etc. have been identified. Although recent efforts to develop salt tolerant cultivars that could retain the halophytic traits through transgenesis show some promise, further exploration is needed to test the contribution of single or multiple salt stress-related genes or regulatory factors from halophilic plants, including S. alterniflora, for possible utilization in crop improvement.