Breeding objectives and requirements for producing transgenics for major field crops of India

To identify crop improvement objectives in twelve important field crops (rice, wheat, maize, sorghum, pearlmillet, pigeonpea, chickpea, mungbean, cotton, potato, mustard and soybean) that are grown extensively in India, we conducted a survey amongst plant breeders, pathologists, entomologists and agronomists specializing in each of these identified crops. A questionnaire was sent to around fifteen scientists actively involved with each crop with the following queries: (1) Identification of problems with the crop at the regional level in terms of priority, (2) Identification of problems with the crop at the national level in terms of priority, (3) Which are the most extensively grown cultivars of the crop at the regional and at the national levels?, (4) What steps could be taken to raise the yield of the crop (heterosis breeding, pure-line breeding)?, Do you know of combiners that would give high heterosis in the crop?, (5) Do you know of germplasm sources that could be used for meeting some of the breeding objectives?, (6) What is your assessment of need for transgenics (a) for nutritional enhancement, (b) for resistance to biotic stresses, (c) for resistance to abiotic stresses, (d) for herbicide resistance and (e) for value addition

Some theoretical and practical possibilities of plant genetic manipulation using protoplasts

Protoplasts capable of division and plant regeneration are now available for a large number of vegetable, oil and forage crops. However, routine hybrid production is not possible due to methodological limitations in selection and culture of hybrid cells. Recent improvement in techniques are the use of a double mutant as a universal hybridizer and the use of fluorescence activated cell sorter to recover hybrid cells. Interest is also centered on limited gene transfer by protoplast fusion. We propose a model of generating triploid plants by somatic cell fusion to transfer limited genomic information from an alien plant to a crop plant. Somatic hybridization has some novel features but, in practice and conception, it is an extension of the methods of sexual hybridization. By contrast, genetic transformation is a radically different approach to plant genetic manipulation. The success of this approach will depend upon how readily genotype can be related to phenotype in a tangible way so as to ascertain what biochemical and developmental activity is controlled or modulated by a DNA sequence

A four-element based transposon system for allele specific tagging in plants- theoretical considerations

The two-element transposon constructs, utilizing either Ac/Ds or Spm/dSpm, allow random tagging of genes in heterologous model species, but are inadequate for directed tagging of specific alleles of agronomic importance. We propose the use of Ac/Ds in conjunction with Spm/dSpm to develop a four-element system for directed tagging of crop-specific alleles. The four-element based construct would include both Ds and dSpm along with relevant marker genes and would function in two steps. In the first step dSpm(Ds) stocks (a minimum of two) would be crossed to a line containing transposases of Spm and unlinked integrations would be selected from segregating population by the use of a negative selection marker to develop stocks representing integration of dSpm(Ds) at a large number of locations in the genome. Selections would be made for a line in which dSpm(Ds) shows partial or complete linkage to the allele of interest. In the second step selected line would be crossed to a line containing Ac transposase to induce transpositions of Ds element to linked sites thereby exploiting the natural tendency of Ds element to jump to linked sites. Unlinked jumps of dSpm(Ds) and linked jumps of Ds could be monitored by appropriate marker genes. The proposed model would allow tagging of allele of interest in chromosome addition lines and also help in the efficient use of genic male sterility systems for hybrid seed production by tightly marking the fertility restorer gene with a negative selection marker

Somatic hybridisation using a double mutant of Nicotiana tabacum

Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers

Design and commissioning of a crude oil fouling facility

This thesis describes the design, construction and commissioning of a major new facility for the study of the fouling of heated surfaces across which crude oil streams pass. The objective is to simulate the behaviour of surfaces in heat exchangers, typically those in a crude pre-heat train. The thesis presents a review of previous work in this area and discusses the design assumptions for the new facility. Safety has been a prime consideration and the safety philosophy and the main features of the safety systems are described. The thesis continues with a description of the construction of the facility and the installation of the instrumentation systems. The instrumentation is designed to monitor the performance and safety of the facility and to retrieve the data for use in assessing modelling methods. The rig was commissioned using a heat transfer fluid (Paratherm™) as a substitute for the oil and the commissioning tests are described. The rig has both tubular and annulus test sections. Though the tubular section models more closely the normal heat exchanger configurations, the use of the annulus section offers a number of advantages which include more accurate temperature measurement, the ability to remove the test section to examine the fouling layer and the ability to make continuous in situ measurements of fouling layer thickness. Using the annulus test section, however, raises the difficulty of retrieving data for the shear stress exerted by the fluid on the fouling layer on the inner tube of the annulus (this shear stress is recognised as an important parameter in many models) and of estimating the effective roughness of the fouling layer so that its effect on pressure drop in tubular geometries can be calculated. To address these predictions problems, an analytical method has been implemented and CFD studies have been carried out to explore the features of this analytical model. Chemistry aspects of the fouling process are also very important and these are also considered in the thesis. The research facility and additional associated tools and techniques form a unique and potentially extremely valuable asset which has great potential in improving the understanding and interpretation of crude oil fouling phenomena

Development of 2,4-D-resistant transgenics in Indian oilseed mustard (Brassica juncea)

Transgenic lines resistant to the herbicide 2,4- dichlorophenoxyacetic acid (2,4-D) were developed in mustard (Brassica juncea), a major oilseed crop grown in more than six million hectares of land in North India. The developed construct contained the tfdA gene, encoding the enzyme 2,4-D monooxygenase, cloned downstream to the 35S promoter along with a leader sequence from RNA4 of alfalfa mosaic virus (AMV leader sequence), for improved expression of the transgene in plant cells. Southern analysis of T0 transgenics confirmed six out of 24 transgenics to be single copy events, from both the flanks of T-DNA. Selfed progeny derived from single copy tfdA lines germinated normally and rooted in medium containing 2,4-D at concentrations as high as 2.5 mg l–1 compared to the wild-type seedlings which did not root even at a concentration of 0.5 mg l–1. The tfdA transgenic lines were also sprayed with commercially available 2,4-D herbicide at concentrations ranging from 10 to 1000 mg l–1 under field conditions. Wild type plants were affected by levels as low as 10 mg l–1 and were completely killed at a concentration of 50 mg l–1. The four transgenic lines tested in the study were resistant to herbicide concentration of 500 mg l–1. The available transgenic lines can be used for testing the potential of 2,4-D in weed control including the control of parasitic weeds (Orobanche spp) of mustard and for low-till cultivation of mustard

Development of transgenics in Indian oilseed mustard (Brassica juncea) resistant to herbicide phosphinothricin

Transgenic lines resistant to herbicide phosphinothricin (PPT) were developed in mustard (Brassica juncea), a major oilseed crop grown in more than 6 million hectares of land in North India. Seedling-derived hypocotyl explants were transformed with a disarmed Agrobacterium tumefaciens strain GV3101. The developed constructs contained the bar gene encoding the enzyme phosphinothricin-acetyl-transferase (PAT) which inactivates phosphinothricin (PPT) by acetylating it. The expression of the bar gene was controlled either by the double enhancer version of CaMV35S promoter (35Sdebar) or a CaMV35S promoter with a leader sequence from RNA4 of alfalfa mosaic virus introduced at the 5' end of the bar gene (35SAMVLbar) or without (35Sbar) it. Plant viral leader sequences have been shown to be translational enhancers. In vitro selections for transformed plants were carried out on a medium containing PPT. Transgenic shoots were recovered at a frequency of 23% with 35Sdebar gene construct and at a frequency of 16% with 35SAMVLbar containing construct. Transformation frequencies were low with 35Sbar construct. Individual transgenics with 35Sdebar and 35SAMVLbar constructs were tested for copy number on both the right and left border flanks of T-DNA by Southern hybridization. Single copy transgenic lines were further analysed for transcript levels of the bar gene by Northern blotting and for protein levels by PAT assays. Wide variation in expression levels were observed, particularly amongst the transgenics containing the 35Sdebar construct. Single copy transgenics were selfed to develop homozygous lines which could be used for the study of resistance to herbicide PPT at the field level and to correlate this protection with expression levels observed through molecular analysis. Herbicide- tolerant lines could be used for testing the possibility of low-till or no-till cultivation of mustard in the rain-fed areas where it is extensively grown

Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile-restorer combinations for heterosis breeding

We report in this study, an improved method for identifying male sterile-restorer combinations using the barnase-barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line × tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66-90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and ~30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) × tester (barstar) crosses, wherein only two viable male sterile–restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T0 generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T1 progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation

Development of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea)

Transgenic lines containing the barstar gene (encoding for Barstar an intracellular inhibitor of the ribonuclease, Barnase both from Bacillus amyloliquefaciens) have been developed in Indian oilseed mustard, Brassica juncea, to develop a complete male sterility/ restoration system for heterosis breeding in this crop. Transgenics were also raised using a modified sequence of the barstar gene based on parameters known to influence transgene expression in heterologous systems. The wild type and modified barstar lines were analysed for their restoration capabilities by crossing them with agronomically suitable male sterile barnase lines developed earlier in our laboratory. Of 30 different combinations of crosses tested between three male sterile barnase lines and several single-copy barstar lines, only one combination was found to restore male fertility among F1 progeny. Subsequent analysis of F2 progeny derived from such F1 restored events (containing both barnase and barstar genes) revealed stable inheritance of both genes in the segregating population thereby indicating proper functionality of the same. Further, pollen viability in restored events was found to be comparable to that observed in transgenic lines containing the barstar gene alone, indicating efficient restoration by the barstar protein in the presence of the ribonuclease. The male sterile line and its corresponding restorer identified in the present study constitute a complete, functional male sterility/restorer system in B. juncea and the traits can be diversified into appropriate combiners for heterosis breeding