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Black shank, caused by Phytophthora parasitica var. nicotianae, is a widespread and severe disease of tobacco throughout the southeastern United States. Partial resistance derived from the cigar tobacco cultivar Florida 301 has been the primary means of reducing losses to the disease for many years. The recently released tobacco cultivar, Coker 371-Gold (C 371-G), was found to provide an additional source of resistance to P. parasitica var. nicotianae. Although the resistance in C 371-G is being used widely by breeders, the origin and inheritance of this resistance mechanism was unknown. Two populations of doubled haploid lines derived from C 371-G were used to determine that C 371-G possesses a single, dominant gene designated Ph, which confers a very high level of resistance to race 0 of P. parasitica var. nicotianae. A greenhouse inoculation procedure was developed that provided an efficient means of screening for the presence of this resistance gene prior to selection in the field, and confirmed that Ph provides complete resistance to race 0 but no resistance to race 1 of P. parasitica var. nicotianae. Because Florida 301 resistance is effective against both races of the pathogen that occur in the major tobacco growing areas of the United States, combination of these two sources of resistance should provide enhanced protection of new tobacco cultivars to P. parasitica var. nicotianae.

Flue-cured tobacco (Nicotiana tabacum) cultivar Coker 371-Gold (C 371-G) possesses a dominant gene, Ph, that confers high resistance to black shank disease, caused by race 0 of the soil-borne pathogen Phytophthora parasitica var. nicotianae. The origin of this gene is unknown. Breeding lines homozygous for the Ph gene were hybridized with NC 1071 and L8, flue-cured and burley genotypes known to possess qualitative resistance genes from Nicotiana plumbaginifolia and N. longiflora, respectively. The F1 hybrids were out-crossed to susceptible testers and the progenies evaluated in field black shank nurseries and in greenhouse disease tests with P. parasitica var. nicotianae race 0. Results showed that Ph was allelic to Php from N. plumbaginifolia in NC 1071. Testcross populations of hybrids between burley lines homozygous for Ph and L8, possessing Phl from N. longiflora, showed that Ph and Phl integrated into the same tobacco chromosome during interspecific transfer. Nevertheless, the two loci were estimated to be 3 cM apart. Random amplified polymorphic DNA (RAPD) analyses of the testcross progenies confirmed that recombination between the two loci was occurring. Forty-eight RAPD markers linked to Ph in doubled haploid lines were used in cluster analyses with multiple accessions of N. longiflora and N. plumbaginifolia, breeding lines L8, NC 1071, and DH92-2770-40, and cultivars K 326, Hicks, and C 371-G. A cladogram or region tree confirmed the data obtained from field and greenhouse trials, that Ph, transferred from C 371-G to DH92-2770-40, and Php in NC 1071 were allelic and originated from N. plumbaginifolia.

Host resistance is an important strategy for managing Globodera tabacum subsp. solanacearum and G. tabacum subsp. tabacum, important nematode pests of flue-cured tobacco (Nicotiana tabacum) in Virginia, and cigar wrapper tobacco (N. tabacum) in Connecticut and Massachusetts, respectively. Field research from 1992 to 2005 evaluated reproduction of G. tabacum subsp. solanacearum on genotypes with and without a chromosome segment from N. plumbaginifolia containing a gene (Ph

Johnson, C. S., Wernsman, E. A., and LaMondia, J. A. 2009. Effect of a chromosome segment marked by the Php gene for resistance to Phytophthora nicotianae on reproduction of tobacco cyst nematodes. Plant Dis. 93:309-315. Host resistance is an important strategy for managing Globodera tabacum subsp. solanacearum and G. tabacum subsp. tabacum, important nematode pests of flue-cured tobacco (Nicotiana tabacum) in Virginia, and cigar wrapper tobacco (N. tabacum) in Connecticut and Massachusetts, respectively. Field research from 1992 to 2005 evaluated reproduction of G. tabacum subsp. solanacearum on genotypes with and without a chromosome segment from N. plumbaginifolia containing a gene (Php) that conferred resistance to race 0 of Phytophthora nicotianae (causal agent of tobacco black shank). Ratios of G. tabacum subsp. solanacearum eggs/500 cm3 soil at the end versus the beginning of experiments (Pf/Pi) were significantly lower in cultivars and breeding lines possessing the Php-containing chromosome segment from N. plumbaginifolia compared with genotypes without the segment. Numbers of vermiform G. tabacum subsp. solanacearum juveniles in roots were similar among genotypes but numbers of swollen and pyriform nematodes were significantly lower for the known G. tabacum subsp. solanacearum-resistant cv. NC 567 and in genotypes possessing the Php gene compared with genotypes and cultivars without the gene. In a 2003 greenhouse test, the percentage of plants with visible G. tabacum subsp. tabacum cysts was also significantly lower for parental and progeny genotypes homozygous and heterozygous, respectively, for Php compared with similar lines without the gene. These results indicate a close linkage or association between a likely single, dominant gene (Php) for resistance to P. nicotianae and suppressed reproduction by G. tabacum subsp. solanacearum and G. tabacum subsp. tabacum. Further research to accurately elucidate the relationships among these genes could lead to significant improvements in tobacco disease control.

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.

Bulked segregant (BSA) and random amplified polymorphic DNA (RAPD) analyses were used to identify markers linked to the dominant black shank resistance gene, Ph, from flue-cured tobacco (Nicotiana tabacum) cv. Coker 371-Gold. Sixty RAPD markers, 54 in coupling and 6 in repulsion phase linkage to Ph, were identified in a K 326-derived BC1F1 (K 326-BC1F1) doubled haploid (DH) population. Thirty RAPD markers, 26 in coupling and 4 in repulsion phase linkage to Ph, were used to screen 149 K 326-BC2F1 haploid plants. Complete linkage between the 26 coupling phase markers and Ph was confirmed by screening 149 K 326-BC2F1 DH lines produced from the haploid plants in black shank nurseries. RAPD markers OPZ-5770 in coupling and OPZ-7370 in repulsion phase linkage were used to select plants homozygous for the Ph gene for further backcrossing to the widely grown flue-cured cultivar K 326. Black shank disease nursery evaluation of 11 K 326-BC4S1 lines and their testcross hybrids to a susceptible tester confirmed linkage between Ph and OPZ-5770. The results demonstrated the efficiency of marker-assisted selection for Ph using a RAPD marker linked in coupling and repulsion. Complete linkage between 26 RAPD markers and the Ph gene was confirmed in the K 326-BC5 generation, and RAPD phenotypes were stable across generations and ploidy levels. These RAPD markers are useful in marker-assisted selection for Ph, an important black shank resistance gene in tobacco.

Gene cloning and transformation can be used to circumvent linkage drag effects that can plague conventional interspecific gene transfers. These techniques can also be used to create desirable genetic linkages. Use of Nicotiana glutinosa L. N-gene mediated TMV (tobacco mosaic virus) resistance in flue-cured tobacco, N. tabacum L., has been limited due to linkage drag effects. Transformation was used to introduce the cloned N-gene into NC152, a chromosome addition line possessing a chromosome pair from N. africana. This chromosome has been proposed to be used as a designer chromosome into which numerous transgenes could be inserted to form a desirable linkage package. The system could be used to shuttle a large number of transgenes from genotype to genotype. One hundred thirty-six primary transformants possessing the N transgene were produced and hybridized with TMV-susceptible Petite Havana.' These may serve as valuable TMV-resistant breeding materials. For each independent transformant, BC 1 F 1 families which segregated for TMV resistance and the addition chromosome were generated. Data from cosegregation, transmission, and molecular analyses were used to conclude that one transformant possessed an insertion of the N-gene in the addition chromosome. By inserting N in the chromosome, we initiated construction of a disease resistance package by linking the TMV resistance gene with a potyvirus resistance gene(s) native to the chromosome. Occasional loss of the transgene, however, may be evidence of previously undetected interchromosomal recombination, and may have implications for use of this system in cultivar development.

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.

Random amplified polymorphic DNA (RAPD) analysis was conducted to map the Rk gene in tobacco which conditions resistance to races 1 and 3 of the root-knot nematode, Meloidogyne incognita. Resistant burley tobacco genotype NC 528, containing the Rk gene, and the susceptible cultivar Ky 14 were screened with 1,500 random decamers. A low rate of genetic polymor-phism (1 and F3) of maternally de-rived doubled haploid (MDH) lines of burley tobacco, developed from the cross NC 528 × Ky 14, were used to map the Rk gene. NC 528, Ky 14, three Rk-resistant (Rk-R) DNA bulks, andthree Rk-susceptible (Rk-S) bulks generated from F1-derived MDH individuals were screenedwith the primers that amplified bands polymorphic between Rk-R and Rk-S lines. A total of 67 F1MDH lines and 59 F3MDH lines were screened with the primers that amplified bands polymorphic between Rk-R bulks and Rk-S bulks to confirm linkage between candidate markers and the Rk gene. Sixteen RAPD markers were positioned at six loci in a map 24.1 centimorgans long. Six RAPD markers, including one identified in the F3MDH population, were mapped at the Rk locus.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.

Black shank, caused by Phytophthora parasitica Dast var. nicotianae [Breda de Haan] Tucker, is a common disease of tobacco (Nicotiana tabacum L.). Selection for genetic resistance to black shank within an F 1 -derived haploid population is more efficient than selection within an F 2 diploid population. Our objectives were to identify black shank resistance in three segregating haploid tobacco populations and to verify that disease resistance assessed on haploids corresponded to that of doubled haploids (DHs). Population 1 was derived from «McNair 944»×«ZA 692», Population 2 from «Coker 371-Gold» (C 371-G)׫K 326», and Population 3 from «VA 509»×breeding line MDH 410 [...]

The tobacco (Nicotiana tabacum L.) breeding line NC 152 is a doubled haploid that possesses an addition chromosome from N. africana [Merxm. and Buttler]. A gene on this chromosome confers potyvirus resistance (Poty(R)). Our objective was to use the addition chromosome as a base on which to construct a designer chromosome containing a foreign gene linkage package. A mutant dhfr gene conferring resistance to methotrexate (Mtx) was inserted into NC 152-haploid (n = 25) leaf tissue via Agrobacterium tumefaciens-mediated transformation. After chromosome doubling, 135 NC 152dhfr transformants (2n = 50) were pollinated with the potyvirus-susceptible (Poty(S)) cultivar 'McNair 944' (2n = 48). Linkage analysis was performed in the BC1 generation. Two transformants, NC 152dhfr-996 and NC 152dhfr-1517 exhibited complete linkage between Mtx resistance (Mtx(R)) and Poty(R). Segregants from these two transformants which were Mtx(R) and Poty(R) possessed 49 chromosomes, while Mtx sensitive (Mtx(S)) and Poty(S) progeny possessed 48 chromosomes. Eighty percent of the NC 152dhfr transformants transmitted the dhfr gene as one locus. Other foreign genes can be directed to the addition chromosome through transformation followed by selection for single loci with linkage to Poty(R) or Mtx(R). The integrity of both the foreign-gene linkage package and the rest of the genome will be maintained because recombination between the N. africiana and the N. tabacum chromosomes has not been observed.

Atypical quadrivalents, which appear to be the result or pairing between amplified homologous segments in nonhomologous chromosomes, have been found in tobacco (Nicotiana tabacum L.) doubled haploid (DH) × cultivar hybrids. The purpose or this study was to determine if the same amplified chromosomes are involved in these quadrivalents in different DH lines. Chromosome pairing was studied in hybrids between four DHs, of which two were derived from NC95' and the other two from C139' (.)

Amplification of DNA has been reported for another culture-derived doubled haploid (DH) lines of tobacco (Nicotiana tabacum L.). The purpose of this study was to evaluate the cytological changes associated with this DNA amplification. Crosses were made between three second-cycle DHs and their source cultivars, NC95 and C139. Examination of chromosome pairing in pollen mother cells (PMCs) revealed that two of the hybrids produced a single quadrivalent at metaphase I, whereas the other hybrid formed two quadrivalents. In all of the hybrids, quadrivalents either were in a ring, or consisted of two ring bivalents connected by a single strand of chromatin

Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha(-1)) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha(-1) (P < 0.05) and generated $1,365 ha(-1) more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.

This report describes a series of transposon tagging vectors for dicotyledonous plants based on the maize transposable element Ac. This binary system includes the transposase (Ts) and the tagging element (Ds) on separate T-DNA vectors. Ts elements include versions in which transcription is driven either by the endogenous Ac promoter or by the cauliflower mosaic virus (CaMV) 35S promoter. Ds tagging element includes a gene conferring methotrexate (Mtx) resistance for selection and a supF gene to facilitate cloning of tagged sequences. The Ds element is flanked by a CaMV 35S promoter and the beta-glucuronidase (GUS) coding sequence so that GUS expression occurs upon excision of the element. We have transformed these Ts and Ds elements into tobacco and demonstrated that the Ts is functional with either promoter, and that the artificial Ds elements are capable of transposition. The amount of excision was found to depend upon both the individual Ts and Ds primary transformants used. Somatic excision of Ds was seen in up to 100% of progeny seedlings containing Ts and Ds. Germinal excision was detected in up to 48% of the progeny of plants containing both elements. Hence, this system can generate a sufficient number of events to be useful in gene tagging.

Potato virus Y (PVY), susceptible tobacco (Nicotiana tabacum L.) cultivar, McNair 944, was subjected to in vitro anther culture to determine if genetic variability for virus resistance could be induced among resulting haploids. Five hundred and forty-five haploids were produced and inoculated with a highly necrotic strain (NN) of PVY. One haploid plant survived, even though it was infected with the virus. Selfed progenies of a chromosome-doubled plant of this variant, designated NC 602, proved to be highly resistant to the necrotic effects of the virus. An investigation into the genetic nature of this variant showed the resistance mechanism to be controlled by a single gene exhibiting incomplete dominance. Cytoplasmic and maternal effects were not involved in the disease resistance reaction. The variant was challenged with ten additional strains of PVY from an international collection, and it proved to be resistant to three (VAM-B, MM, and Spanish) strains. NC 602 was evaluated for five agronomic traits and concentrations of total alkaloids as nicotine and reducing sugars in cured leaf. The gametoclonal variant differed from McNair 944 only for cured leaf yield, where an 18.4% reduction was measured.

In tobacco (Nicotiana tabacum L.), anther-derived doubled haploid populations have been shown to exhibit large amounts of unexpected genetic variation and a severe depression in cured leaf yield when compared to conventionally inbred genotypes from comparable sources. A previous study had predicted that the yield depression observed in a doubled haploid population-derived from a near homozygous cultivar, NC95, might be overcome through a recurrent selection program. In the current study, progress from three cycles of full-sib family selection for improved yield in an anther-culture derived population of NC95 was measured, as well as the remaining genetic variation within the population. A design II experiment was conducted in the population following three cycles of selection. Results indicate that the NC95 yield level has been recovered in the third selection cycle population. Although most of the genetic variation in the population appears to be exhausted, the additive genetic variance among maternal half-sib families for yield is significant, and it appears that continued yield improvement can be made through recurrent selection. Significant additive-genetic variance for yield was found among maternal half-sib families but was essentially zero among the paternal half-sib families, suggesting that remaining genetic variation is not being transmitted through pollen. One possible explanation results from the phenomenon of DNA amplification that can occur during the anther culture process, and that may enable extraordinary recombinational events and reduce the viability of male gametes.

Tobacco lines expressing transgenes that encode tobacco etch virus (TEV) coat protein (CP) mRNA with or without nonsense codons give rise to TEV-resistant tissues that have reduced levels of TEV CP mRNA while maintaining high levels of transgene transcriptional activity. Two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). Here, we show that at early times in development, immune lines are susceptible to TEV infection and accumulate full-length CP mRNA. Therefore, immune lines also exhibit meiotic resetting, as is seen in the recovery lines, providing molecular evidence for a common mechanism of gene silencing and virus resistance in both cases. We also investigated the characteristics of two sets of low molecular weight RNAs that appear only in silenced tissue. One set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated with polyribosomes; the second set contains the 3[prime] end of the mRNA. Treating silenced leaf tissue with cycloheximide resulted in decreased levels of full-length mRNA and an increase in the levels of the low molecular weight RNAs, supporting a cytoplasmic decay mechanism that does not require ongoing translation. Surprisingly, mRNA from the transgene containing nonsense codons was associated with more ribosomes than expected, possibly resulting from translation from a start codon downstream of the introduced translational stop codons. We present a hypothesis for transgene/viral RNA degradation in which RNA degradation occurs in the cytoplasm while in association with polyribosomes.

Transgenic burley (cultivars KY14, NC3433-33, and TN90) and flue-cured (cultivar K326) tobacco with resistance to four necrotic isolates of potato virus Y (PVY) were constructed. These tobacco cultivars were transformed with a chimeric gene designed to express the coat protein (CP) from the necrotic Chilean isolate of PVY. CP expression among R 0 plants was undetectable by indirect enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Transgenic R 0 plants that harbored from one to five neomycin phosphotransferase II (NPT II) transgene loci were identified. Although the NPT II and the PVY CP chimeric genes were linked within a common T-DNA, no correlation was found between the number of NPT II and CP transgenes. Inoculation of 41 independent transgenic KY14 R 0 plants with PVY-Chilean identified eight resistant plants, while inoculation of 30 transgenic K326 R 0 plants identified six resistant plants. Inoculation of 17 transgenic NC3433-33 R 0 plants with PVY-Chilean identified six resistant plants, while inoculation of 50 transgenic TN90 R 0 plants with PVY isolate VAM B resulted in nine resistant plants. Progeny derived from PVY-inoculated but symptomless R 0 plants were also resistant to the Chilean, Europe H, M s N r , N-Canada, and VAM B isolates of PVY, demonstrating sexual transmission of the transgenes that condition resistance. We discuss the ability of the PVY-Chilean CP gene to protect transgenic tobacco K326, KY14, NC3433-33, and TN90 from infection by four necrotic isolates of PVY. The flue-cured tobacco cultivar K326, which carries a gene simultaneously conferring resistance against root-knot nematode and susceptibility to PVY strain M S N R , is protected against this strain when transformed with the PVY-Chilean CP gene.

Anther culture in tobacco (Nicotiana tabacum L.) has been associated with yield losses and DNA amplification without chromosome number changes among doubled haploid lines. The objectives of this study were to confirm the amplification phenomenon and to determine if increases in DNA are correlated with yield losses

The use of irradiated pollen to effect ‘egg-transformation’ in Nicotiana was tested using a nuclear chlorophyll-deficiency marker. Yellow-green (yg) plants of Nicotiana tabacum were pollinated with pollen from N. glutinosa which had received 50, 75, or 100 krads of gamma-irradiation. Progeny were scored for leaf color and chromosome number. Only one possible transformant was recovered; however, upon selfing, this plant segregated for leaf color. Thus, it could not be determined if this plant was the result of contaminant pollination, mutation, or a transformation event that occurred after diploidization. All other dark green plants had at least one intact chromosome from N. glutinosa. While no evidence in support of the egg-transformation theory was obtained from this study, cytological data concerning the use of irradiated pollen for haploid induction and for the rapid production of alien chromosome addition lines was presented.

Extranuclear temperature-sensitive lethality in Nicotiana tabacum L. was investigated at the ultrastructural level. Chloroplasts of temperature-sensitive and control plants were morphologically similar in plants grown at nonlethal temperatures (26 C day/20 C night); both possessed typical lamellar structure, starch grains, and plastoglobuli. At lowered temperatures (13 C day/8 C night), chloroplasts in temperature-sensitive plants gradually degenerated as evidenced by the disappearance of starch, increase in plastoglobuli, and the formation of numerous vesicles from the dilation of thylakoids. Chloroplasts in control plants continued to accumulate starch in several large grains, occupying most of the plastid volume by the end of the investigation. Mitochondria in both temperature-sensitive and control plants were morphologically unaltered by exposure to lowered temperatures. Ultrastructural evidence suggests that the site of inheritance of extranuclear temperature-sensitive lethality in the mutant is in t...

Summary Ultrastructural observations of extranuclear temperature-sensitive induced lethality in a mutant of Nicotiana tabacum L. have shown that chloroplasts in this mutant gradually degenerate in plants grown at reduced temperatures, while mitochondria remain intact (Nessler and Wernsman, 1979). To further elucidate the organellar site of temperature sensitivity in this same tobacco mutant, leaf chlorophyll content, rates of oxygen exchange and ribulose 1,5 bisphosphate carboxylase (RuBPCase) activities were examined in temperature sensitive and control genotypes at varying time intervals during 35 days exposure to lethal temperatures (13°C day/8°C night). Rates of photosynthetic oxygen evolution by leaf disks rapidly declined in mutant plants after one week at low temperatures and remained significantly lower than rates in controls. Total chlorophylls in both mutant and control plants gradually decreased with the increased time at reduced temperatures. This decrease in chlorophyll content of temperature sensitive plants was not sufficiently rapid to account for the sharp drop observed in photosynthetic rates and probably resulted from the disruption of chloroplast membranes. Mitochondrial function in mutant plants was apparently unaffected by prolonged exposure to lethal temperatures as evidenced by the almost constant rates of respiratory oxygen uptake seen throughout the experiment. RuBPCase activities on a leaf area basis were similar in mutant and control plants over the last four sampling dates (days 14–35); thus this enzyme does not appear to be the site of the temperature-sensitive lesion. The loss of chlorophylls, rapid decline in photosynthetic oxygen evolution and continued respiratory oxygen uptake reported here for mutant plants grown at lethal temperatures supports the concept that the chloroplast is the site of extranuclear temperature-sensitivity in this tobacco genotype.

Summary Three experiments were conducted to determine the influence of tobacco, Nicotiana tabacum L., genotypes on in vitro relative growth rates of callus (RGR) and the relationship of this parameter to mature plant characters, cured leaf yields, days from transplanting to flowering, and number of leaves per plant. Ten anther-derived dihaploid lines from the Coker 139 cultivar and three anther-derived dihaploid lines from NC 95 differed greatly in their in vitro growth rates in the first experiment. The correlation of in vitro RGR with mature plants characteristics was negative for all three parameters, but significantly different from zero only for leaf yields. Significant genotypic effects on RGR were detected among nine dihaploids of NC 95 and conventional cultivars NC 95 and C 139 in a second study, but correlations with mature plant characters were not significant. Dihaploid line NC 95 DH4 was common to both experiments and exhibited the highest RGR in both sutdies. In a third experiment 15 F 8 breeding lines, previously classified as producing high, moderate, or low cured leaf yields, exhibited significant variability for in vitro RGR. Genotypic variability for RGR was primarily a function of differences among lines within groups, as group means did not differ. All mature plant parameters were negatively correlated with RGR but nonsignificantly. The studies demonstrate that plant genotype has a pronounced effect on callus growth rate and that this characteristic is probably negatively related to mature plant leaf yields for fluecured tobacco. This relationship, as measured in these studies, is not sufficiently high to be of value in predicting leaf yields of varying genotypes.

Haploid and diploid plants of nine genotypes were grown in six replications of a split-plot design in two environments. The performance of the nine genotypes for green and cured leaf yields, total alkaloid and reducing sugar concentrations, plant height, and leaf number per plant was highly correlated between the two ploidy levels. Environmental effects on the haploid plants, and genotype X environment interactions did not mask variability among genotypes in the haploid condition

A negative genetic correlation between yield and total alkaloids (TA) in tobacco, L., populations causes decreases in yield when selecting for increased TA. A restricted selection index was employed in an ‘NC13’ ✕ ‘McNair 135’ population to increase TA and hold yield at the mean of the population. The genetic correlation in the base population was −0.67. After three cycles of full-sib family selection, TA had increased 2.5% of the mean per cycle and there had been no significant change in yield. The increase in TA was about 58% of that expected from direct selection for increased TA, where there would be an expected decrease in yield of 1.9% per cycle. Selection for increased TA also led to a correlated decrease in the ratio of particulate matter in smoke to TA in spite of a positive genetic correlation between particulate matter and TA.

The ADH and MDH lines were significantly different for all characters measured. The MDH lines were agronomically superior to ADH lines and more closely resembled the cultivars. The ADHs and MDHs yielded 79.9% and 91.5%, respectively, of the cultivars. A second cycle of MDH line production resulted in no further yield reduction. A model of heterozygosity and inbreeding depression in the parental cultivars could explain the results for yield of the MDH lines; it would not account for differences between ADH and MDH lines

Demand for flue−cured tobacco (Nicotiana tubacum L.) from basal leaf positions has decreased in recent years, and production systems which maximize higher quality, upstalk, leaf grades are needed. In this study we tested production regimes utilizing photoperiod−sensitive genotypes to prevent flowering and produce additional upstalk leaves as a substitute for discarded basal leaves. Normal and photoperiod−sensitive (mammoth) versions of four cultivars were grown in field experiments at two locations in 2 years. Apical meristems of mammoth genotypes were removed when plants had produced the same number of leaves as normal cultivars, four leaves more than normal in 1977, and two leaves more than noma1 in 1978. Mammoth genotypes allowed to produce the same leaf number as normal cultivars provided higher yields, with leaf quality equal or superior to the controls. Total alkaloids were lower than in normal cultivars in 1977, but equal in 1978. Reducing sugar contents were equal in both types in 1977, but significantly higher for mammoths in 1978. Permitting mammoth plants to produce four more leaves than normal, with the bottom four leaves removed from the plants and discarded, resulted in yield overcompensation with slight reductions in leaf alkaloids and increases in reducing sugars. Two additional top leaves on mammoth entries did not compensate adequately for yield reductions caused by discarding the bottom four leaves. Leaf alkaloids and sugar contents in the latter production regime did not differ from those of control cultivars. These regimes could reduce the production of downstalk tobaccos without significant yield reductions and would eliminate all premature flowering. The maintenance of apical dominance in mammoths would enhance axillary bud control.

Linear increases in mean yield cycle −1 were 1.43 g plant −1 for the population selected on stress nutrient agar and 0.99 g plant −1 when selection was on regular nutrient agar. The only other significant change detected was a decrease in total alkaloids of 0.65 mg g −1 cycle −1 for the regular nutrient agar selections. The lack of a decrease in total alkaloids accompanying the increase in yield of the stress agar selections suggests indirect selection of root modifications that increased the efficiency of total alkaloid synthesis

Two flue-cured varieties of N. tabacum were crossed to putative progenitor species and to distantly related species. Heterosis for yield, plant height, and number of leaves was largest for crosses to progenitor species, particularly to N. otophora and N. tomentosiformis. The magnitude of this heterosis appeared to be greater than estimates presented in the literature for crosses among varieties of N. tabacum. An additional study presented some evidence for the genomic basis of heterosis in crosses of N. tabacum with N. tomentosiformis and N. sylvestris.

Detection cytologique des regions heterochromatiques amplifiees chez les doubles haploides de N. tabacum.