Genetical control of alkaloid production in Lupinus mutabilis and the effect of a mutant allele Mutal isolated following chemical mutagenesis

Summary A new mutant allele, described here as mutal, which reduces the alkaloid content in dry matter of lupinus mutabilis has been identified following seed treatment with ethyl methane sulphanate. The allele, when homozygous, reduces the alkaloid content from levels of > 2.0 per cent found in seed dry matter of existing populations to 0.2–0.3 per cent and produces plants with vegetative and seed tissues that are organoleptically sweet. Component alkaloids in plants homozygous for mutal differ in respect of the percentage composition of sparteine and of lupanine, as well as possibly of oxa-sparteine and 4-hydroxylupanine, but none is eliminated in genotypes homozygous for the mutant allele. Alkaloid concentration, in so far as not under the control of mutal, has low heritability but lines were isolated following two generations of successive selection which possessed reduced alkaloid levels. These were interpreted to have arisen as a result of recombination of alleles affecting reduction in alkaloid level at several loci. the mutant, mutal, has been established in a pure breeding line which represents a crucial additional step in the evolution of Lupinus mutabilis towards full status as a crop plant.     

A molecular approach to combat spatio-temporal variation in insecticidal gene (<Emphasis Type="Italic">Cry1Ac</Emphasis>) expression in cotton

Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression in roots, cotton seed and other cotton products and by products.     

The inheritance of chemical phenotype in Cannabis sativa L. (II): Cannabigerol predominant plants

This paper aims to clarify the genetic mechanism that is responsible for the accumulation of cannabigerol (CBG) in certain phenotypes of Cannabis sativa L. CBG is the direct precursor of the cannabinoids CBD, THC and CBC. Plants strongly predominant in CBG have been found in different fibre hemp accessions. Inbred offspring derived from one such individual were crossed with true breeding THC predominant- and CBD predominant plants, respectively. The segregations in the cross progenies indicate that CBG accumulation is due to the homozygous presence of a minimally functional allele, tentatively called B₀, at the single locus B that normally controls the conversion of CBG into THC (allele B_T) and/or CBD (allele B_D). The fact that CBG accumulating plants have so far been found in European fibre hemp populations that are generally composed of B_D/B_D plants, and the observation that the here investigated B₀ allele possesses a residual ability to convert small amounts of CBG into CBD, make it plausible that this B₀ is a mutation of normally functional B_D. Therefore, B₀ is considered as a member of the B_D allelic series encoding a CBD synthase isoform with greatly weakened substrate affinity and/or catalytic capacity.     

Linkage relationships of genes for leaf morphology and double flowering in Matthiola incana

Summary The inheritance and linkage relationships of a leaf morphology gene of Matthiola incana were investigated. The allele for sinuate leaf shape, c, was found to be recessive to the allele for normal entire leaf, C. The c allele was tightly linked to the recessive allele for double flowering, s. The recombination frequency between the two loci was close to zero. The mode of inheritance of the C gene was in accordance with the hypothesis that a pollen lethal gene is responsible for the constant 1:1 segregation ratio of double-flowered (= male sterile) to single-flowered (= fertile) plants in most M. incana breeding lines. The sinuate leaf allele seemed to reduce the frequency and delay the flowering of double-flowered plants. The importance of the C gene as a double flowering marker in the cultivation and the breeding of M. incana is discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 1128-E, 1993 series.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rich allelic variations of Viviparous-1A and their associations with seed dormancy/pre-harvest sprouting of common wheat

The allelic variations of Vp-1B have been confirmed to have close association with seed dormancy (SD) and pre-harvest sprouting (PHS) of Chinese wheat in previous research, but little was known regarding whether the alleles of two other orthologs of Vp1 on 3AL (Vp-1A) and 3DL (Vp-1D) are also present and related to these traits. In view of this, 11 primer pairs flanking the whole sequences of these two orthologs were designed to investigate their allelic variations. The results identified six alleles of Vp-1A using the primer pair A17-19 among 81 wheat cultivars and advanced lines, which were designated as Vp-1Aa, Vp-1Ab, Vp-1Ac, Vp-1Ad, Vp-1Ae, and Vp-1Af. Except for Vp-1Ac, the other five alleles were proven novel, but no allelic variation was found in Vp-1D. On sequence analysis of alleles of Vp-1A, five deletions were observed, all occurring in the same region holding many TTC repeats. Of the six alleles detected in this study, four (Vp-1Aa, Vp-1Ac, Vp-1Ae, and Vp-1Af) were generally distributed in varieties exhibiting higher average germination index (GI, range 0.46–0.56) and spike sprouting (SS, range 39.6–49.4%); however, the alleles Vp-1Ab and Vp-1Ad were distributed in genotypes carrying higher SD (GI 0.19–0.26) and stronger PHS resistance (SS 12.3–17.2%). On Spearman correlation analysis, the allele Vp-1Ab had significantly negative correlation with GI (−0.479) and SS (−0.542) at the 0.01 level, and the three alleles Vp-1Aa, Vp-1Ac, and Vp-1Ae had significantly positive correlation with GI [0.311 (0.05 level), 0.401 (0.01 level), and 0.294 (0.05 level)] and SS [0.283 (0.05 level), 0.309 (0.05 level), and 0.266 (0.05 level)]. The other alleles, including Vp-1Ad and Vp-1Af, also exhibited correlation, albeit not significant, with these two traits. This negative correlation showed that Vp-1Ab helped to improve SD and PHS tolerance, but Vp-1Aa, Vp-1Ac, and Vp-1Ae appeared to exert the opposite effect. To further confirm the association between alleles of Vp-1A and the two traits, a recombinant inbred line (RIL) population with 157 lines was genotyped using the primer pair A17-19, developed from the cross between Wanxianbaimaizi (Vp-1Ab) and Jing411 (Vp-1Ac). General linear model analysis indicated that variation in Vp-1A had a significant (P < 0.001) association with the two traits, explaining 23.4% of the variation in GI and 16.7% of the variation in SS in the population across three crop seasons.     

Race specific resistance against Phytophthora infestans in potato is controlled by more genetic factors than only R-genes

Summary For RFLP mapping of R-genes, determining resistance to specific races of Phytophthora infestans in tetraploid potato, it is necessary to develop well segregating populations at the 2x level. During mapping studies, evidence was obtained that more genetic factor(s) are involved in the expression of R-genes than conventionally believed. Two experiments are described in which such an additional genetic factor was suppressing or enhancing the expression of unknown R _nand R _ifactors. R _nand R _iappeared to be present in the investigated plant material, containing R4 and R10, or in one of the susceptible crossing parents. In a third experiment, the expression and the segregation of the well known R1 gene was influenced by an additional genetic factor. In that case there were indications for a dominant suppressor. This was established by the selection of susceptible plants carrying a RFLP allele of probe GP21 closely linked to R1. In three of the four F₁ populations, resulting from crosses between such susceptible plants and susceptible tester plants, resistnat progenies were found. The resistance appeared to be R1-specific. This clearly indicates that in three of the four investigated susceptible plants, the R1 gene was still present but not expressed.     

Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR

The Pi-ta gene from indica introgressed into japonica rice has been used to control the blast disease caused by the fungal pathogen Magnaporthe grisea (Herbert) Barr. (anamorph Pyricularia oryzae Cav.) worldwide. A single nucleotide length polymorphism (SNLP) was identified at the intron region of the Pi-ta gene to develop a codominant Pi-ta gene marker suitable for genotyping with an automated machine. The DNA primer specific to the resistant Pi-taallele was labeled with blue dye (FAM, 6-carboxyfluorescein) as a forward primer, the DNA primer specific to the susceptible pi-ta allele was labeled with green dye (HEX, 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) as another forward primer and the DNA primer identical to both Pi-ta/pi-ta alleles was unlabeled as the reverse primer for polymerase chain reaction (PCR). Using these three primers, a 181-bp blue peak in homozygous resistant and a green peak of 182–183 bp in homozygous susceptible, and both peaks in heterozygous plants were produced by PCR. The utility of marker was verified using a segregating F₂ population, inbred cultivated lines, dominant markers and pathogenicity testing. A codominant Pi-ta marker was thus developed for effective Pi-ta assisted selection for crop improvement. Using highly homologous competitive primers for allele detection by PCR can benefit the study of genome organization of the complex locus. This revised version was published online in August 2006 with corrections to the Cover Date.     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.     

A partially male-sterile mutant line of soybeans,Glycine max (L.) Merr.: inheritance

Summary A partial male sterility system in the soybean (Glycine max (L.) Merr.) germplasm population AP6(SI)CI was found to be controlled monogenically by a recessive allele, msp. Observations of msp msp plants in different environments suggested that environmental conditions significantly affect expressivity of the msp allele with respect to male sterility. We obtained no experimental evidence of cytoplasmic effects on msp expression. Background genotypes, however, seem to affect msp expressivity through their determination of flowering dates and resultant interactions with varying environmental conditions.Homogeneous populations of partially male-sterile plants can be generated by increasing families of msp msp plants in fertility-inducing environments, if measures are implemented to prevent the introduction and/or build-up of fertile genotypes that arise from natural cross-pollinations.Joint contribution: North Central Region, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, and Journal Paper No. J-9596 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Identification of segregation-distortion-neutral alleles to improve pollen fertility of indica-japonica hybrids in rice (Oryza sativa L.)

The genetics of resistance to Ascochyta blight (Ascochyta fabae f. sp. fabae) was studied in two populations of faba bean (Vicia faba). Plants of a resistant population, ILB 752, and a susceptible one, NEB 463, were screened for their reaction to the pathogen and the results were quantified on a scale of 0–5. Crosses were made between plants both within and between accessions and the F₁ and F₂ generations assessed in a field trial 21 and 45 days after inoculation. Disease scores were greater at 45 days than at 21 days and they were not significantly affected by the presence of susceptible spreader rows in part of the trial. ILB 752 carried a major dominant gene conferring resistance while NEB 463 carried the recessive allele for susceptibility. Furthermore, a minority of plants of NEB 463 appeared to carry at least one pair of complementary recessive genes, also conferring resistance. Most of the plants of ILB 752 were homozygous for the dominant resistance gene and a few were heterozygous. Reciprocal crosses behaved identically, indicating the absence of maternal effects in the expression of Ascochyta blight resistance in faba beans. The results show that it is important to confirm the level of heterozygosity for the resistance genes in this partially outbreeding species before crossing is commenced. The major dominant gene for resistance, identified in ILB 752, has clear potential for use in breeding for Ascochyta blight resistance. The minor genes identified in NEB 463 also show the potential for accumulating resistance through mass selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of alleles at two loci controlling resistance to Phomopsis stem blight in narrow-leafed lupin (Lupinus angustifolius L.)

The genetics of resistance to Phomopsis stem blight caused by Diaporthe toxica Will., Highet, Gams & Sivasith. in narrow-leafed lupin (Lupinus angustifolius L.) was studied in crosses between resistant cv. Merrit, very resistant breeding line 75A:258 and susceptible cv. Unicrop. A non-destructive glasshouse infection test was developed to assess resistance in the F₁, F₂, selected F₂-derived F₃ (F_2:3) families, and in selfed parent plants. The F₁ of Unicrop × 75A:258 (and reciprocal cross) was very resistant, and the F₂ segregated in a ratio of 3:1 (resistant: susceptible), which suggested the presence of a single dominant allele for resistance in 75A:258. In Merrit × Unicrop (and reciprocal), the F₁ was moderately resistant, and the F₂ segregated in a ratio of 3:1 (resistant: susceptible). Thus Merrit appeared to carry an incompletely dominant resistance allele for resistance. The F₁ of Merrit × 75A:258 (and reciprocal) was very resistant and the F₂ segregated in a ratio of 15:1 (resistant: susceptible), which supported the existence of independently segregating resistance alleles for resistance in 75A:258 and Merrit. Alleles at loci for early flowering (Ku) and speckled seeds (for which we propose the symbol Spk) segregated normally and independently of the resistance alleles. Resistant F₂ plants gave rise to uniformly resistant or segregating F_2:3 families, whereas susceptible F₂ plants gave rise only to susceptible F_2:3 families. However, the variation in resistance in the F₂ and some F_2:3 families of crosses involving 75A:258, from moderately to extremely resistant, was greater than that expected by chance or environmental variation. We propose the symbols Phr1 to describe the dominant resistance allele in 75A:258, and Phr2 for the incompletely dominant resistance allele in Merrit. Phr1 appears to be epistatic to Phr2, and expression of Phr1 may be altered by independently segregating modifier allele(s). This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of the recessive crossability allele kr1 from Chinese Spring into the winter wheat variety Martonvásári 9

Summary The recessive of crossability allele kr1 was transferred from the spring wheat variety Chinese Spring (CS) into the winter wheat variety Martonvásári 9 (Mv9) by backcrossing the Mv9 × CS hybrids with Mv9. The Mv9 variety possesses dominant Kr1 alleles and is heterogeneous at the kr2 locus, so that some individual plants carry recessive kr2 alleles. The selection of plants possessing the recessive kr alleles from the (Mv9 × CS)Mv9 BC₁ generation was carried out according to the seed set achieved when pollinated with rye (Secale cereale L. cv. Mercator). The partial dominance of the Kr alleles made it possible to differentiate between plants heterozygous at the Kr1 locus and Kr1Kr1 homozygous dominant plants. Two selfed consecutive progenies were tested by pollination with rye to select the homozygous recessive kr1kr1kr2kr2 plants and to check the result of the selection after each backcross.As a result of three backcrosses with Mv9 and two selfings after each backcross the selected progenies had 61.6% seed set with rye tested on sixty individual plants. These data confirm that after the third backcross the selected Mv9 kr1 line carries necessive crossability alleles Kr1 and Kr2, but the genotype is 93.75% Mv9.     

Genetics of two mechanisms of resistance to Meloidogyne naasi (FRANKLIN) in an Aegilops variabilis Eig. accession

Summary Accession No. 1 of Aegilops variabilis has complete resistance to the root knot nematode, Meloidogyne naasi. F2 segregation in a cross between this and two susceptible accessions showed that one dominant gene named Rkn-mn1 prevented development of galls on the roots and consequently of female nematodes. The study of the numbers of females in galls on the F2 plants allowed detection of a recessive gene, Rkn-mnAv, suppressing development of J2 larvae into females. The presence of Rkn-mnAv also resulted in a decrease of the level of galling. Rkn-mn1 has already been introduced into wheat. The interest in transferring also Rkn-mnAv is discussed in relation to extending durability of the nematode resistance.     

Embryonal recombination and germline inheritance of recombined FRT loci mediated by constitutively expressed FLP in tobacco

FLP site-specific recombination has been shown in transgenic plants to excise DNA sequences between target FRT sites, and thereby activate transgenes in plants. In previous reports, crossing of tobacco plants expressing FLP recombinase from a CaMV-35s promoter with plants containing the target FRT sites, hybrid plants with deletion sectors were generated, which were infrequently transmitted to progeny. In this report we evaluate the occurrence of recombination in F₁ hybrid seed derived from crosses of different FLP and FRT-reporter target lines and the germinal transmission of recombined loci from these hybrids to F₂ progeny. Twenty hybrids were generated from crosses of independent five FLP-active lines and four FRT-reporter target lines. In one hybrid, FLP deletions occurred at an early stage, prior to seed maturation, and the deletions from this hybrid were more efficiently transmitted to F₂ progeny. The demonstration of FLP-mediated recombination activity and germinal inheritance of the recombined FRT loci are supported by both molecular and enzymatic evidence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytogenetic and morphological characteristics of new types of diploids (2n=22, 24, 40) derived from consecutive selfing of aneuploids in brassica crops

Summary New types of diploids in Brassica crops were synthesized after three consecutive selfing of aneuploids derived from backcrossing of sesquidiploids (2n=29, AAC) with B. campestris (2n=20, AA). The cytogenetic and morphological characteristics of plants with 2n=22, 24 and 40 in the S₃ generation were analyzed in order to establish the extent in which these addition and polyploid lines were stabilized. A high frequency of 11_II (79.7%), 12_II (84.6%) and 20_II (100%), were observed at metaphase _I of pollen mother cells in 2n=22, 24 and 40 plants, respectively. The chromosome configuration at methaphase _II also indicates that a certain level of stability has been attained cytogenetically. Although pollen stainability was relatively high, the seed set percentage was still low. Variation in morphological characteristics indicate the incorporation of one or more chromosome pairs from the C genome of B. oleracea. Other diagnostic characters such as the formation of determinate inflorescence, branching from the base of the stem, and the shift from self-incompatibility to self-compatibility must have resulted from the interaction between A and C genomes. Thus plants with 2n=22, 24 and 40 have been stabilized to some extent and can be developed into new breeding lines of Brassica. It is suggested that limited pollination could be effective in increasing the seed fertility of these plants.     

An allelic series at the Co-1 locus conditioning resistance to anthracnose in common bean of Andean origin

In this study, we characterized the genetic resistance of the Andean bean cultivars Kaboon and Perry Marrow and their relation to other sources of anthracnose resistance in common bean. Based on the segregation ratio (3R:1S) observed in two F₂ populations we demonstrated that Kaboon carries one major dominant gene conferring resistance to races 7 and 73 of Colletotrichum lindemuthianum. This gene in Kaboon is independent from the Co-2 gene and is an allele of the Co-1 gene present in Michigan Dark Red Kidney (MDRK) cultivar. Therefore, we propose the symbol CO-1 ² for the major dominant gene in Kaboon. The Co-1 is the only gene of Andean origin among the Co anthracnose resistance genes characterized in common bean. When inoculated with the less virulent Andean race 5, the segregation ratio in the F₂ progeny of Cardinal and Kaboon was 57R:7S (p = 0.38). These data indicate that Kaboon must possess other weaker dominant resistance genes with a complementary mode of action, since Cardinal is not known to possess genes for anthracnose resistance. Perry Marrow, a second Andean cultivar with resistance to a different group of races, was shown to possess another resistant allele at the Co-1 locus and the gene symbol Co-1 ³ was assigned. In R × R crosses between Perry Marrow and MDRK or Kaboon, no susceptible F₂ plants were found when inoculated with race 73. These findings support the presence of a multiple allelic series at the Andean Co-1 locus, and have major implications in breeding for durable anthracnose resistance in common bean. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of a mutation in the male fertility gene of the preferentially transmitted Aegilops sharonensis chromosome 4S1 and its application for hybrid wheat production

Summary Wheat plants nullisomic for chromosome 4B are male sterile due to the absence of the male fertility gene Ms1. However, plants in which chromosome 4B has been substituted by the preferentially transmitted chromosome 4S¹ of Ae. sharonensis are male fertile due to the compensating effect of Ms4 on the alien chromosome. This substitution line has been mutated and three recessive mutation of Ms4 have been selected. Plants homozygous for these mutations are male sterile. The implication of these mutations for hybrid wheat production is discussed.     

The controlled introgression of Festuca arundinacea genes into Lolium multiflorum

Summary Using phosphoglucoisomerase (PGI/2) as a genetic marker, it has been shown to be possible to transfer genes from Festuca arundinacea into diploid Lolium multiflorum using the pentaploid hybrid L. multiflorum (4x) x F. arundinacea (6x). The pentaploid hybrid was sufficiently fertile to be used in reciprocal crosses with diploid. L. multiflorum. When used as the male parent, only two backcross generations were then required to reconstitute the diploid genotype. Intergeneric recombinants including a F. arundinacea PGI/2 allele were found among the diploid BC₂. Cytological data indicates that although the majority of chromosome associations involve only homologous Lolium chromosomes, associations involving Lolium and Festuca chromosomes also occur.Interpollinating the pentaploid hybrids prior to commencing a backcrossing programme increases the number of cycles of recombination and improves the chance of recovering intergeneric recombinants. The crossing programme described is proposed to be an effective method of introducing F. arundinacea genes into L. multiflorum.