Evaluation of the Effect of Pollen Irradiation on Karyotype Variability in M1 Cotton Plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearrangements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M₁ plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M₁ after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

Improvement of cotton fiber quality by transforming the<Emphasis Type="Italic"> acsA</Emphasis> and<Emphasis Type="Italic"> acsB</Emphasis> genes into<Emphasis Type="Italic"> Gossypium hirsutum</Emphasis> L. by means of vacuum infiltration

A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene -glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.Communicated by D. Bartels     

Evaluation of the effect of pollen irradiation on karyotype variability in cotton plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearragements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M1 plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M1 after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

Evaluation of the Effect of Pollen Irradiation on Karyotype Variability in M2 Cotton Plants

The karyotypes of biomorphologically abnormal cotton (Gossypium hirsutum L.) plants obtained in M₂ after pollination with pollen irradiated at dose rates 10, 15, 20, and 25 Gy were studied. Various genomic and chromosomal mutations were detected in 57 M₂ families. The primary monosomics isolated in M₂ were found to be cytologically more stable and more viable, since they had higher meiotic index, pollen fertility, and seed formation. In M₂, a decrease in the number of plants with multiple karyotype aberrations and interchromosomal exchanges with high frequency of multivalent formation was observed. The multivalent configurations had different types and chromosome disjunctions. Their pollen fertility was higher than in translocants found in M₁. Desynapsis often occurred in M₂, including plants with chromosome deficiency or rearrangements. The variation in the number of univalents in various cells was found to result from different expression of synaptic genes. The results indicate stabilization of karyotypes, increase in cytologic stability and viability, and the absence of sterility in aberrant plants.     

The origin, identification, and plant morphology of five rice monosomics.

Five different monosomics of rice (Oryza sativa L.) were obtained by treatment of pollen with gamma irradiation, as a by-product of attempts to determine the cytological loci of certain marker genes, i.e., mature pollen carrying normal alleles at all loci was given gamma rays and used for pollinating strains that were homozygous for recessive marker genes. The monosomics showed distinguishable morphological features and had complete seed sterility. Cytological studies revealed that one monosomic was tertiary, the others primary. The tertiary monosomic was related to chromosome 10. Two primary monosomics for chromosomes 10 and 11 were identified. At metaphase I, the tertiary monosomic showed the chromosome configurations 1 III + 10 II, 11 II + 1 I, and 10 II + 3 I, and all primary ones showed the configuration 11 II + 1 I. All five monosomics showed very poor crossing ability and were not transmitted to the few progenies observed. A few trisomic plants were found in the progenies of a cross between monosomic and normal pollen in one monosomic. This is the first time that many monosomics in rice have been characterized. This information will be useful in studies of rice aneuploidy and cytogenetics. Key words : rice, monosomics, morphology, cytology, transmission, trisomics.     

Cytogenetic studies inClarkia,section primigenia. III. Cytogenetics of monosomics inClarkia amoena

Naturally occurring monosomic plants, with 13 instead of the usual 14 somatic chromosomes, have been found in several populations ofClarkia amoena subsp.huntiana (Onagraceae). These plants show no obvious phenotypic differences from their 14 chromosome sibs. Three types of meiotic pairing were found amongst 7 monosomic strains: 4 bivalents+chain of 4+univalent, 3 bivalents+chain of 4+chain of 3, and 2 bivalents+one heteromorphic rod bivalent+ring of 4+chain of 3. All are basically translocation heterozygotes of a peculiar kind composed of two genomes, one with 6, the other with 7 chromosomes. Both genomes can be transmitted through pollen and eggs, but because of the nature of the meiotic divisions, gametes with 6 chromosomes are in functional excess. Self-pollination of 5 of the monosomic strains does not give 12 chromosome nullisomic progeny. The nullisomics produced by the other 2 strains are weaker and later flowering than their 13 or 14 chromosome sibs, and are partially or completely sterile. The 6 chromosome genomes are hence usually inviable when homozygous. Crosses of the monosomics to a standard cytological strain, and intercrosses between monosomic strains, have allowed analysis of the end arrangements of chromosomes. Six different 7-chromosome genomes and 3 different 6-chromosome genomes have been identified. The translocation scheme proposed to account for the origin of the 6-chromosome genomes involves partition of most of the genetic material of one chromosome amongst two others plus the loss of a small centromere-bearing chromosome. This loss accounts for the lethality of the 6-genomes when homozygous. The fact that vigorous, healthy nullisomics are formed whenever two monosomics of different geographical origin are crossed indicates that the 3 monosomic genomes have had an independent origin, since they obviously complement one another's genetic deficiencies. The hybrid nullisomics are fairly fertile, and if formed in nature might serve as the starting point for a new race or species with a reduced basic chromosome number.     

Pollen irradiation and gene transfer in Capsicum

Summary The aim of the experiment was to study the possibility of facilitating the gene transfer and reducing the number of required backcrosses through pollen irradiation and subsequent selection of F₁M₁ plants containing a very high proportion of sterile pollen as male parent for backcrossing. Anthers of a donor line, C-3-1, were irradiated with 1,500 rad -rays and the pollen used for pollination of a recipient genotype W-8 which posseses a number of recessive marker genes. Five F₁M₂ plants containing more than 80% sterile pollen grains and one semi-sterile plant were selected and used for backcross to W-8. The segregation pattern of four characters expressed in the first backcross generation [W–8×(W–8×C–3–1)] was assessed and compared with the non-irradiated control. A changed segregation pattern was observed (in some cases even non-transfer of a paternal allele) as well as a shift towards more plants possessing the investigated maternal alleles. A scheme for backcross procedure in combination with pollen irradiation is discussed.     

Cytogenetic Analysis of Cotton Monosomics

Eleven monosomics in cotton that were obtained in the progenies of three disomic desynaptic plants were cytologically characterized. The transmission of the monosomes in progeny was shown in the 26 monosomic plants. In 23 plants the frequency of monosomics was ranged between 14.29 and 41.67 %. Three monosomics usually occurred in much lower frequencies (from 3.03 to 5.00 %). Various transmission rates indirectly pointed out different monosomes as a specific chromosomes of cotton genome. Three telochromosomes and one isochromosome were isolated from the progenies of the four monosomics. Using translocation test it was recovered that seven monosomes of different monosomics are homologous to one of the chromosomes of six translocation lines of our collection.     

Effects of low-efficiency pollinators on plant fitness and floral trait evolution in <Emphasis Type="Italic">Campanula americana</Emphasis> (Campanulaceae)

Floral visitors vary in their pollination efficiency and their preferences for floral traits. If low-efficiency pollinators decrease the amount of pollen available to higher efficiency visitors, then low-efficiency visitors may actually have negative fitness consequences for the plants that they visit. We used experimental arrays in two populations to determine the floral preferences and the fitness effects of low-efficiency (or ugly) pollinators on Campanula americana. These ugly pollinators (halictid bees) preferentially visited flowers with pollen over flowers that had had their pollen removed. C. americana pollen color varies quantitatively from light tan to dark purple, and we found that natural variation in pollen color influenced the magnitude of halictid preferences for flowers with pollen. In general, preferences for flowers with pollen were stronger when the ugly pollinators foraged in arrays of flowers with tan-colored pollen than in arrays with purple-colored pollen. When plants received few visits by efficient Bombus pollinators, visits by ugly pollinators significantly decreased siring success relative to plants where visits by ugly pollinators were prevented. In contrast, ugly pollinators did not influence siring success when higher efficiency pollinators were more abundant. Thus, the relationship between low-efficiency pollinators and the plants that they visit varies from commensalistic to antagonistic depending on the presence of other pollinators in the community. Our findings suggest that the negative fitness effects and floral preferences of low-efficiency or ugly pollinators may contribute to the maintenance of a pollen color polymorphism in C. americana.     

Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

Cytogenetic effects of the treatment of cotton seeds with thermal neutrons

After irradiation of cotton seeds with thermal neutrons, primary and tertiary monosomics and also plants with translocations were detected among offsprings M1 and M2. The interchanges had high frequency of multivalents at metaphase-I of meiosis. Genetic analysis of chlorophyll deficient cotton mutants has shown that recessive monogenic factors caused new mutations of the types xantha and viridis. New viridis mutant was also characterized by the heterozygosity of interchromosome translocations.     

Endosperm responses to irradiated pollen in apples

Summary The cytological effects of pollen -irradiation at 50 and 100 krad on both embryo and endosperm development were studied in Malus × domestica. Fruit and seed set were reduced by increasing doses of pollen irradiation, while embryo sacs resulting from the treatments differed in number and morphology of endosperm nuclei and in the presence or absence of an embryo. Nuclear abnormalities, distinguished from normal nuclear behaviour in embryo sacs derived from unirradiated pollen, included enhanced numbers of polyploid restitution nuclei, bridges between nuclei, excluded metaphase chromosome fragments and disrupted mitotic synchrony. Generally, a high dose of pollen irradiation (100 krad) generated an all-or-nothing response in the embryo sac, either creating highly abnormal embryos and/or endosperms which aborted, or showing relatively normal development. Callus produced from excised cellular endosperm differed in average genome size, that derived from 100 krad pollen being smaller than that from unirradiated pollen.     

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a Double Gene/Single Selection (DGSS) plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than Single Gene/Single Selection (SGSS) vector (aphA-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops ($ 120 billion US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.     

Insect-induced wind pollination of the palm Chamaedorea pinnatifrons and pollination in the related Wendlandiella sp.

In Peruvian Amazonia pollination ecology of two palms, Chamaedorea pinnatifrons (Jacq.) Oerst. and Wendlandiella sp. was studied from October 1988 to January 1990. Both palms are dioecious understory species that form locally dense populations.In C. pinnatifrons both sexes flower synchronously during the dry season. Prior to anthesis, the pendulous male inflorescence is inhabited by numerous thrips (Thysanoptera) and Ptiliidae (Coleoptera). Staminate flowers open by a small basal slit between the petals. At anthesis pollen is shed and the movements of the insects inside the flowers trigger pollen release in small clouds. Thus, the powdery pollen becomes airborne and finally air currents act as a vector, carrying pollen to the inconspicuous female plants, which usually are not visited by insects. The term insect induced wind pollination is suggested for this pollination mode. Wendlandiella flowers during four months in the dry season. Male and female plants were not visited by insects. The dry condition of the pollen indicates that anemophily is the pollination mode in Wendlandiella. Fructification is rare but the plants show intense vegetative propagation.The significance of anemophily in the tropical lowland rain forest is discussed.     

Suppression of heterotrimeric G-protein β-subunit affects anther shape,pollen development and inflorescence architecture in tobacco

The role of the heterotrimeric G-protein -subunit in plant development was studied in transgenic tobacco (Nicotiana tabacum L.) plants with reduced -subunit levels due to the antisense expression of the -subunit mRNA. The antisense plants had aberrant anther shape and produced non-germinating pollen. The anthers were sporadically transformed to petals, whereas other floral organs were not affected. The pollen grains were smaller than the wild-type pollen and had abnormal cell walls. The architecture of mature antisense plants was altered. The plants had long branched panicles and short stems. These data suggest that the -subunit of the plant heterotrimeric G-proteins is involved in the regulation of the reproductive phase of the tobacco life cycle, particularly in stamen development and pollen maturation.     

Are nectar-robbers mutualists or antagonists?

As exploiters of plant-pollinator mutualisms, nectar-robbers remove rewards (nectar) without providing pollination services. Though one might expect nectar-robbing to be costly to plants, it may instead benefit plants by indirectly increasing pollen dispersal. I investigated the direct effects of nectar-robbing bees (Xylocopa californica) on floral rewards and behaviors of pollinators visiting desert willow (Chilopsis linearis) and indirect effects of robbing on the reproductive success of the plant. Nectar-robbers reduced nectar; while unrobbed and robbed flowers were equally likely to contain nectar, nectar volumes were smaller in robbed flowers with nectar. Apis mellifera (honeybees), ineffective pollinators in terms of pollen deposition, avoided robbed flowers. In contrast, Bombus sonorus (bumblebees), effective pollinators, did not avoid robbed flowers. While bumblebees tended to spend less time in robbed flowers, the time that they spent in flowers was not correlated with pollen deposition. Using powder mimicking pollen, I found that on some days, powder was dispersed farther or to more flowers from robbed flowers, indicating that robbing may sometimes benefit plants by increasing male reproductive success. Powder movement suggested that the effect of robbing on male reproductive success ranged from costly to beneficial. The outcome for flowers that were marked early each morning was a function of prevalence of robbing and abundances of effective pollinators, but not a function of spatial variability among trees in prevalence of robbing or the abundance of ineffective honeybees. Unlike powder dispersal, female reproductive success, measured by fruit set and the number of pollen tubes growing in styles, was not affected by robbing. Thus, robbers did not reduce plants female reproductive success either directly by damaging flowers or indirectly by reducing pollen deposition by pollinators. Overall, this study indicates that nectar-robbers were not often costly to plants, and sometimes even benefited plants.     

Pollen in packrat middens

Fossil packrat middens are used to reconstruct late Quaternary environments in the arid Western U.S.A. Airborne pollen is contributed to middens both directly from the air, and through adherence to plant macrofossils. I examined the filtration of atmospheric pollen by Larrea tridentata leaves (resinous, with sparse recumbent hairs), Cercidium microphyllum twigs (glabrous with recumbent hairs) and Sphaeralcea ambigua leaves (covered with stellate hairs) and evaluated the potential biases of scavenged pollen on the interpretation of pollen records from middens. Pollen grains collected from the surfaces of the plants were most numerous during the spring when (airborne) pollen concentrations were high. Of the species investigated, the Sphaeralcea leaves filtered the greatest number of spring pollen grains from the air (3419 grains cm^?2); captured pollen was very low during other seasons. Sticky Larrea leaves continuously captured airborne pollen ( = 246 grains cm^?2). Pollen captured by smooth Cercidium was consistently low ( = 31 grains cm^?2). Only the spring pollen was filtered in sufficient numbers to obtain minimum pollen counts of 200 grains by all 3 taxa. The plants frequently captured greater proportions of their own pollen. Pollen content of packrat middens will reflect the season of plant macrofossil collection and the species composition of the plants incorporated.     

Molecular cloning of alpha-amylases from cotton boll weevil,Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of -amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two -amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5 and 3 RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several -amylase inhibitors from plants were assayed against A. grandis -amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and -AI1 inhibitors on A. grandis -amylase activity. This work suggests that genetic engineering of cotton to express -amylase inhibitors may offer a novel route to A. grandis resistance.     

Further attempts to induce “egg transformation” using irradiated pollen

Summary Numerous pollination treatments involving heavily irradiated (40–100 krad) pollen of diverse plant species failed to produce any clear cut egg transformants of the type reported by Pandey in Nicotiana. Genetic stocks of pea, rapeseed, and apple, bearing multiple Mendelian markers, were employed to detect any possible transformation events. For each plant species, an optimal level of irradiation was determined which would allow normal pollen tube growth leading to fertilization, but which would prevent the formation of normal hybrids due to the pulverized condition of the chromosomes contributed by the irradiated pollen. Pollination treatments included selfing, pollination with donor pollen mixed with self pollen, pollination with irradiated donor pollen mixed with self pollen, pollination with irradiated pollen followed by a delayed self pollination, and pollination with irradiated pollen by itself. None of these treatments produced clearly transformed seedlings. The total number of potential transformation events screened was in excess of 6,046 including 2,268 for pea, 3,309 for rapeseed, and 469 for apple. It is concluded that if egg transformation occurs outside of Nicotiana it is a rare event, and its frequent occurrence in Nicotiana must be, at best, an isolated phenomenon.Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper No. 3557     

Transformation of pollen embryo-derived explants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient -glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, -glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.