Marker-free transgenic (MFT) near-isogenic introgression lines (NIILs) of 'golden' indica rice (cv. IR64) with accumulation of provitamin A in the endosperm tissue

We have developed near-isogenic introgression lines (NIILs) of an elite indica rice cultivar (IR64) with the genes for β-carotene biosynthesis from dihaploid (DH) derivatives of golden japonica rice (cv. T309). A careful analysis of the DH lines indicated the integration of the genes of interest [phytoene synthase ( psy ) and phytoene desaturase ( crtI )] and the selectable marker gene (hygromycin phosphotransferase, hph ) in two unlinked loci. During subsequent crossing, progenies could be obtained carrying only the locus with psy and crtI , which was segregated independently from the locus containing the hph gene during meiotic segregation. The NIILs (BC₂F₂) showed maximum similarity with the recurrent parent cultivar IR64. Further, progenies of two NIILs were devoid of any fragments beyond the left or right border, including the chloramphenicol acetyltransferase ( cat ) antibiotic resistance gene of the transformation vector. Spectrophotometric readings showed the accumulation of up to 1.06 µg total carotenoids, including β-carotene, in 1 g of the endosperm. The accumulation of β-carotene was also evident from the clearly visible yellow colour of the polished seeds.     

Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet

The relationship between the expression level of the selectable marker gene and transformation frequency was investigated in transgenic sugar beets with five different promoters, a modified cauliflower mosaic virus 35S RNA promoter (E35S), an enhanced nopaline synthase promoter (ENOS), a modified mannopine synthase promoter (SMAS), a heat shock protein promoter (HSP80) and a chlorophyll a/b-binding protein promoter (CAB3), to drive the expression of the selectable marker gene. The selection system employed was based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable marker gene and mannose as selective agent. The selected transgenic shoots were analysed for PMI activity and the average activity for each promoter was found to be 5.9 (HSP80), 31 (SMAS), 38 (E35S), 49 (ENOS) and 61 (CAB3) mU/mg. The weakest promoter, HSP80, resulted in the lowest transformation frequency (0.30%), suggesting that this promoter was too weak to confer sufficient resistance to mannose. On the other hand, the strongest promoters, ENOS and CAB3, only gave intermediate transformation frequencies, 0.44% and 0.47% respectively, while the somewhat weaker SMAS promoter produced the highest transformation frequency, 0.89%. Thus, these data suggest that the activity of the selectable PMI gene should be above a certain threshold level; however, above this level, no simple correlation between the PMI activities, calculated as averages, and transformation frequencies could be deduced. However, extended data analysis by dividing the transgenic shoots into 4 groups according to their PMI activities (low(<10 mU/mg), medium (10–50 mU/mg), high (50–100 mU/mg) and very high (>100 mU/mg) expressers) revealed a significant positive correlation between the relative number of shoots having medium levels of expression and transformation frequency. This indicated that promoters that predominanthly give rise to intermediate expression levels of the selectable PMI gene result in high transformation frequencies.     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice

Transgenics for the expression of β-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and β-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T₁ progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T₁ progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T₁ progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 μg/g of polished seeds. High variation in the carotenoid accumulation was observed within the T₁ progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the β-carotene accumulation.     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Involvement of vitamin A in the photoperiodic induction of diapause in the spider mite Tetranychus urticae is demonstrated by rearing an albino mutant on a semi-synthetic diet with and without p-carotene or vitamin A

Abstract. Data are presented on the development from egg to adult of a wild-type and an albino strain of the spider mite Tetranychus urticae on a semi-synthetic liquid diet.The diet is a modification of the diet developed by Van der Geest et al. (1983).Apart from the standard diet without added carotenoids, diets supplemented with either β-carotene or vitamin A were tested.A high incidence of diapause was found in the wild-type strain under short-day conditions on all three diets.No diapause was found in the albino mutant under short days on the standard diet.Partial restoration of the photoperiodic response was obtained after addition of β-carotene to the diet, and full restoration was found after the addition of vitamin A.Diapause could be terminated in diet-reared diapausing females of both strains after a cold rest of 3 weeks at 4°C.The results lead to the conclusion that vitamin A or a derivative of vitamin A probably functions as the photoreceptor pigment for the photoperiodic induction of diapause in the spider mite.