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Genetic Mechanism and Chromosomal Location of Pollen-Specific Gene(s) in Gossypium.

Plant Genome research is directed primarily towards linking sporophytic traits with their major controlling genes. One of the major limitations in the genetic manipulation of plant reproductive systems is the paucity of information about genes regulating gametophyte development. Few reports are available about the genes responsible for pollen development. Cytogenetically deficient stocks are valuable tools for locating genes but have been used only to a limited extent for analysis of pollen development. Kindiger et al. (1990) used B-A translocation stocks to determine the cytological effects of chromosome deletions on pollen grain development in maize. Most major cytogenetic deficiencies in cotton (Gossypium hirsutum L.) induce sporophytic semisterility due to the production and segregation of genetically unbalanced microgametophytes. Such chromosome deficiencies are not pollen-transmissible because the chromatin-deficient pollen is normally not functional (Endrizzi et al., 1984; McCormick, 1993). Pollen hypoaneuploid for the respective chromosome are produced at a frequency of [+ or -] 50 % when a hypoaneuploid microsporocyte undergoes meiosis. Some effects of the genetic loci absent in such hypoaneuploid pollen can be detected by simply comparing the pollen deficient for a complete chromosome or chromosome fragment with the normal haploid pollen.

The mechanism by which non-viable pollen compete with viable pollen in effecting fertilization has not been investigated in detail because of the non-availability of suitable methods to characterize the pollen grain development. Electron microscopy (EM) has been used to characterize normal vs. abnormal pollen on the basis of morphology (Iwanami et al., 1988), but preparation and sectioning are costly, tedious, and labor intensive processes in EM. Confocal laser scanning microscopy (CLSM), optical sectioning, digital image acquisition systems, and 3-D reconstruction algorithms have eased the detailed 3-D morphological characterization of biological specimens (Fishkind and Wang, 1993). However, the application of the confocal microscope is limited in plant research primarily because of its dependence on the use of only fluorescent stains. The overall objectives of this research were (i) developing a suitable method to study pollen morphology by confocal microscopy; (ii) analyzing the genetic mechanism(s) underlying pollen morphological traits, and (iii) identifying the chromosomal location of gene(s) responsible for pollen development.


Pollen from G hirsutum cv. Texas Marker 1 (TM-1) and G. barbadense 3-79 were used as standards to compare with pollen from euploid [F.sub.1], monosomic [F.sub.1], monotelodisomic [F.sub.1], and backcross [BC.sub.n] [F.sub.1] chromosome substitution plants resulting from interspecific crosses between G. hirsutum and G. barbadense. These two inbred lines of the two cultivated tetraploid species were used because (i) they are morphologically distinct, so more variation in pollen morphology would be expected, (ii) aneuploid chromosome substitution stocks were available from these two lines, (iii) they are the only two cultivated tetraploid species, (iv) [TM.sub.1] is an inbred line that is considered as the genetic standard of G. hirsutum (Kohel et al., 1970), and (v) Line 3-79 is a double-haploid, i.e., also highly homozygous, and serves as a cytogenetic standard for G. barbadense types.

The monosomic stocks are interspecific [F.sub.1] substitution stocks where a particular G. hirsutum chromosome is missing although the homologous G. barbadense chromosome is present. The stocks are designated for the missing G. hirsutum chromosome; thus, H12 Sub [F.sub.1] indicates that the G. hirsutum chromosome 12 is lacking. On the other hand, the monotelodisomic stocks are designated by the particular chromosome arm that is present. Thus, Tel2 Lo Sub [F.sub.1] denotes that only the long arm of the G. hirsutum chromosome 12 is present. It is expected that if a gene is located on the missing chromosome of the monosomic stocks and if the same abnormal pollen morphology associated with the monosomic plant is also observed in a monotelodisomic plant then the location of the trait must be on the missing part of the chromosome in the hypoaneuploid stocks. In addition to the monosomic [F.sub.1] and monotelodisomic [F.sub.1] analysis, we also confirmed the results from the analysis of backcross [BC.sub.n][F.sub.1] chromosome substitution plants.

The pollen was collected from mature buds and flowers at anthesis from plants grown under controlled conditions in the greenhouse. Pollen grains from flowers at anthesis were dusted onto a slide and two to three drops of acridine orange fluorochrome staining solution (Verma and Babu, 1989) were added. Pollen grains from mature buds stored in Farmer's fixative solution were also studied. No differences in gross pollen grain morphology were detected between the fresh and fixed samples.

At least three to four buds-flowers were collected from each plant and five to six anthers were selected from each bud-flower and at least 1000 pollen grains were used for each analysis. Samples of pollen from monotelodisomic stocks and chromosome substitution lines were analyzed with or without prior knowledge of the chromosomal location in a blind study to avoid bias. In all cases, the morphological pattern of pollen from cytogenetically deficient plants was compared with those of pollen from normal [F.sub.1] plants and also with the two parents. Characteristic pollen morphologies were further confirmed with pollen collected from plants grown under two different greenhouse conditions (Texas A&M University and Alabama A&M University) to minimize the effect of environment.

The detailed pollen morphology was recorded with a Bio-Rad MRC 600 laser scanning confocal microscope (BIO-RAD Microscience Division, Cambridge, MA)(1). The microscope configuration included a scanning head attached to an upright Olympus BHS epifluorescence microscope allowing rapid switching from conventional to confocal imaging. A Krypton--Argon ion mixed-gas laser operating at 480, 560, and 647 nm, simultaneously, was used as the excitation source. The images were photographed directly from the computer (Gateway 2000 486/33C) display screen with an automated Focus Imagecorder Plus camera.

We used a monotelodisomic-based test to localize genetic effects of chromosome arms following the strategy of Saha and Stelly (1994). Data were analyzed statistically by LSD for mean comparison and Duncan's Multiple Range Test where appropriate (SAS Institute, 1985).


Distinguishing Interspecific Variation Based on Pollen Morphology

Results indicated that normal pollen grains in cotton were spherical in shape with uniform spines and numerous pores; these pores were arranged in a circular pattern around each pole (Fig. 1). Our results demonstrated that the pollen of TM-1 differed markedly from 3-79. Pollen grains of TM-1 are, on the average, smaller in size with more dense spines (DS) in comparison to 3-79 (Fig. 1, Table 1). Spines on pollen of 3-79 were larger but less numerous (SS) than on TM-1 (Fig. 1, Table 1). The diameter of the pollen grain, the average spine length and the number of spines in TM-1 were significantly different from 3-79 (Fig. 2, Table 1). Both TM-1 and 3-79 have a similar type of pollen aperture. We observed similar results in connection with pollen size and spine pattern in three other accessions of G. hirsutum and G. barbadense (unpublished data). The taxonomic significance of pollen morphology has already been documented in the studies of many plant species (Argue, 1993, 1980; Mukherjee, 1975).

Table 1. Interspecific variation in pollen morphology in cotton.

                              Equatorial              diameter
Type                      diameter([dagger])   CV    ([dagger])

G. hirsutum (TM-1)            100.9bc(#)       5        99.7c
G. barbadense (3-79)          117.9a           7.1     117.4a
Interspecific [F.sub.1]
   DS                          99.8c           5.4      99.1c
   SS                         107.1b           7.8     106b

Type                       CV    density([double dagger])    CV

G. hirsutum (TM-1)         4.1            8.3a              18
G. barbadense (3-79)       7.7            4.9c              24.2
Interspecific [F.sub.1]
   DS                      5.3            7.8b              20.4
   SS                      7.5            5.8bc             22.4

Type                      length([sections])    CV

G. hirsutum (TM-1)              12.1c          12.9
G. barbadense (3-79)            15.5a          10.5
Interspecific [F.sub.1]
   DS                           13.3b          15.8
   SS                           13.0b

([dagger]) Measurement in micrometers; n = 50.

([double dagger]) Average no. of spines/[10.sup.-3] [micro][m.sup.2]; n = 150.

([sections]) Measurement in micrometers; n = 100.

(#) Means in the column with a different letter are significantly different at P = 0.05 (Duncan's Multiple Range Test); Each mean/ coefficient of variation.

Saad (1960), in his survey of pollen grains within the Malvaceae, examined five Gossypium species including one G. hirsutum and 3 G. barbadense accessions. The diameter and spine length of pollen from these accessions was similar to our measurements, specific to the species, Heslop-Harrison (1968) reported that the patterning in the pollen grain wall in many species is so precise, distinctive, and consistent that it has formed the basis for pollen taxonomy and wall patterning is widely used in geological and paleobotanical analyses (Moore et al., 1991).

The role of spines may obviously seem to promote dispersal by entomophilous vectors (entomophily), but it has not been determined that dense spines with smaller size in Upland cotton have any species-specific advantage over Pima cotton in the outcrossing vectored by insects.

We also observed that a cytogenetically normal interspecific [F.sub.1] plant produced about twice the number of TM-1 type pollen (DS) than the 3-79 type (SS). This result suggested that TM-1 type pollen will have advantages in subsequent fertilization over the 3-79 type assuming that both type pollen have equal fitness in fertilization. Stephens (1949) reported similar rates of selective elimination of the donor parent genotype in interspecific backcrosses of cotton.

Developing a Suitable Method for Pollen Study

Cytogenetically deficient stocks are very useful tools for locating genes responsible for pollen development. Most cytogenetic deficiencies in cotton are not pollen transmissible because the chromatin-deficient pollen are normally not functional (Endrizzi et al., 1984). Several methods have been used to differentiate viable from non-viable pollen based on stain intensity in cotton (Aslam et al., 1964; Douglas, 1968; Gwyn and Stelly, 1989; Khatun and Flowers, 1995; Stelly et al., 1990). However, it is generally recognized that differentiation among pollen by such methods inadequately reflects inherent viability among the pollen and, at best, provides only a crude means of evaluating pollen viability and function (Barrow, 1983). Also, such methods do not typically reveal mechanisms by which pollen is rendered inviable, noncompetitive, or less competitive.

Gwyn and Stelly (1989), using translocation cotton heterozygotes and homozygotes, reported that viable pollen grains from heterozygotes were found to be larger, fully engorged with starch and fluoresced more uniformly brightly than non-viable pollen. Using image analysis of the confocal microscope composites, we were provided the opportunity to observe the morphology of the pollen in more detail and measure fluorescence intensity distributions by measuring the pixel intensity of the fluorescent pollen. However, we observed that some morphologically abnormal pollen from the [F.sub.1] and other plants also fluoresced very brightly with acridine orange staining. Barrow (1983) has questioned the validity of pollen stainability as an indicator of functional ability. The apparently high level of stained pollen from heterozygous translocation stocks in cotton (Aslam et al., 1964) also indicated that the genetically deficient pollen might be otherwise denoted as viable on the basis of the classification by staining intensity. Thus, our results with the specific stain acridine orange suggest that fluorescent evaluation based on staining intensity of pollen grains has limited utility in determining individual pollen viability and functional ability, although new fluorochromes may provide a solution, especially if linked to antibodies specific for pollen proteins. However, our observation is primarily based on pollen morphology with the assumption that morphologically normal looking pollen were viable, but not on any other type test of viability such as pollen germination or other functional ability.

Genetic Mechanism of Pollen Development

We observed that both TM-1 and 3-79 produced uniform pollen populations in which some of the pollen grains were without any spines (WS), Fig. 3. However, interspecific normal [F.sub.1] plants produced three different types of pollen: (i) a smaller "TM-1 type" with dense spines (DS), (ii) a larger "3-79 type" with dispersed spines (SS), and (iii) some pollen without spines (WS) (Fig. 3).


In cotton, even a highly inbred line such as TM-1, was shown to have almost 1% discernibly abortive pollen (Douglas, 1968). Similarly, we observed that pollen without spines accounted for 1.6% of TM-1 pollen and 1.1% of 3-79 pollen types. The cytogenetically normal [F.sub.1] plants produced 3.9% pollen without spines, possibly reflecting some interspecific incompatibility which induced more, pollen development without spines. We also observed that pollen without spines were produced in all cytogertetically deficient interspecific plants, both monosomic and monotelodisomic, ranging from 0.3 to 6.0%. For example, the monosomic [F.sub.1] plant deficient for G. hirsutum chromosome 12 and the interspecific substitution stock, H12 Sub [BC.sub.1][F.sub.1] had similar amounts of pollen without spines ranging from 5.8 to 6.0%, respectively. Even under ideal conditions, not every potential reproductive cell, especially the microspore, develops into a mature gametophyte. Thus, the differences in numbers of the universally present pollen without spines between interspecific [F.sub.1] plants, inbred parents, and cytogenetically deficient plants were probably an indicator of physical and environmental factors.

Chromosomal Location of Pollen Specific Gene(s)

Pollen of a monosomic [F.sub.1] plant deficient for chromosome 12 (H12 Sub [F.sub.1]) produced all three types of pollen observed in a normal interspecific [F.sub.1] as well as a very distinctive abnormal, semi-spherical type which had very few spines clustered unevenly on the surface (US, Fig. 2 and 3, Table 1). Other chromosomal substitution lines (Chromosome 1, 2, 3, 4, 6, 7, 9, 10, 16, and 20) did not exhibit significant levels of such abnormal pollen type (US). Backcross chromosome substitution lines deficient for chromosome 12 (H12 Sub [BC.sub.1][F.sub.1]) also produced a high percentage of abnormal pollen similar to the monosomic [F.sub.1] plant deficient for chromosome 12 (Fig. 3). It seemed that in the WS pollen produced by the normal [F.sub.1] plant and other lines, the spine development was stopped at a very early stage in development immediately after wall formation, whereas in the abnormal pollen produced by the chromosomal substitution lines for chromosome 12, the outer wall of the pollen with pores continued to differentiate and the abnormality occurred in a later stage, during the spine development.

Moreover, [F.sub.1] plants deficient for other chromosomes produced mostly the three different types of pollen, similar to those found in a normal [F.sub.1] interspecific plant. Observations of low percentage of abnormal pollen production, partial indehiscence, and partial sterility by aneuploids deficient for chromosome 12 have been reported previously (Gwyn and Stelly, 1989).

The pollen analyses from H12 Sub [BC.sub.1][F.sub.1] and H12 Sub [F.sub.1] plants both suggest that chromosome 12 has gene(s) that affect gross pollen morphology and spine development in cotton. In contrast to H12 monosomics, the Te12Lo monotelodisomic [F.sub.1] plant containing only the long arm of chromosome 12 (Te12Lo Sub [F.sub.1]) did not produce any abnormal pollen with uneven spines (US) like the monosomic plant deficient for chromosome 12 (Fig. 3). This Te12Lo stock is deficient for one copy of the short arm and is thus hemizygous for the 3-79 short arm. The results indicate that the responsible gene(s) were located in arm 12Lo.

Chromosome 12 and 26 Linkage Groups

It is interesting that we located the pollen-specific gene(s) onto chromosome 12 because chromosome 12 is one of the most genetically dissected linkage groups in cotton with many important genes located on it such as Le, Gl, N, and Pgm (Saha and Stelly, 1994). N determines the development of the fuzz fiber, Gl affects epidermal and sub-epidermal lysigenous gland formation, and Le is one of two loci that regulate hybrid lethality. The isozyme locus Pgm is the only biochemical marker mapped to the distal region of the long arm of chromosome 12 (Endrizzi et al., 1984; Saha and Stelly, 1994). Despite identifying over 500 molecular markers, the extensive RFLP map of Reinisch et al. (1994) did not associate any marker with chromosome 12.

Chromosomes 12 and 26 in cotton are considered homeologous chromosomes in tetraploid cotton (AD genome) because they contain duplicated loci and similar linkage groups (Endrizzi et al., 1984; Lee, 1981), Unfortunately, monosomic stocks for chromosome 26 were not available at the time of this experiment. However, pollen from Te26sh Sub [F.sub.1], a monotelodisomic plant lacking one copy of the long arm of chromosome 26, were studied (Table 1). The segregation pattern of the pollen from the plant was not significantly different than that from a normal [F.sub.1] plant.

If the long arm of chromosome 12 carries the functional pollen gene(s), then the corresponding genes on chromosome 26 must either be nonfunctional, absent, or without effect of interlocus complementation. Saha and Stelly (1994) reported a similar phenomenon of non-synteny with the PGM-7 locus in cotton. The results support the theory that since tetraploid cotton originated long ago (about 2 x [10.sup.6] yr), many of the duplicated loci have mutated during the course of time (Brubaker and Wendel, 1994; Reinisch et al., 1994). Similar evidence for evolutionary divergence in duplicated loci has been reported for hexaploid wheat and other plants (Hart, 1983; Wendel, 1989; Wendel et al., 1992).

Sporophyte vs. Gametophyte in Controlling Pollen Wall Formation

The formation of microspore-pollen walls is a complex process and has drawn the interest of scientists for many years over a fundamental question of whether the sporophyte or gametophyte genetically controls the pollen morpaology and its wall formation (Heslop-Harrison, 1968; Hormaza and Herrero, 1992; Moore et al., 1991). Hormaza and Herrero (1992) reported that three kinds of gene action could occur during pollen development: (i) expression only in the sporophyte (sporophytic gene expression), (ii) expression only in the gametophyte (microgametophytic expression), and (iii) expression controlled by both gametophyte and sporophyte. Sporophytic control functions could arise from control by the diploid spore mother cell (sporocytic) or control by other diploid tissues (sporophytic), such as the tapetum (Uffelen, 1991). However, whether or not the tapetum regulates microgametophyte development has still not been resolved, despite considerable investigation into gene expression in the tapetum (Schrauwen et al., 1996).

Heslop-Harrison (1968) reported from electron microscopic studies that the critical pattern-determining stage in pollen wall and spine formation occurred very early in development, implying that control is exerted by the sporophyte, i.e., the microsporocyte, and perhaps other micrmporangial tissues such as the tapetum. A more reasonable hypothesis was that both gametophytic and sporophytic control is present. Indeed, the patterns of wall sculpturing, which can be species-specific, are regulated b) the sporophyte (McCormick, 1993), while genes for spine density and spine length, which sort independently in Lycopersicon (Quiros, 1975), are thus apparently controlled by the gametophyte. Our results with three segregating pollen types from interspecific [F.sub.1] plants and the abnormal pollen morphology of the hypoploid pollen deficient for chromosome 12 clearly indicate that the pollen morphology, including the spine formation, in cotton is controlled, at least in part, by the haploid pollen nucleus. If microspore development was exclusively under sporophytic control, populations of pollen would be uniform and we would not have observed segregation of pollen of discretely variable exine types from the cytogenetically deficient [F.sub.1] stocks.

Gametophytic control, similar to our results, has been suggested by observations that 50% of the microspores from hypoploid stocks of maize showed specific abnormalities during microsporogenesis (Kindiger et al., 1990). The controversy of sporophyte vs. gametophytic control of wall pattern development can be resolved on the basis of genetic analyses similar to this study.

Abbreviations: CLSM, confocal microscopy; H12, monosomic for chromosome 12; H12 Sub [F.sub.1], monosomic [F.sub.1] chromosome 12 substitution from the cross of TM-1 (G. hirsutum) H12 x 3-79 (G. barbadense); Tel2Lo Sub [F.sub.1], monotelodisomic [F.sub.1] from the cross G. hirsutum Tel2 Lo x 3-79, where Tel2 Lo symbolizes disomy for 12 Lo and morosomy for 12 sh; Lo, long arm of the chromosome; sh, short arm of the chromosome.


This work was supported in part by Cotton Incorporated and a capacity, building grant from CSRS of USDA. We also appreciate the help of Drs. J. Jenkins and J. McCarty of USDA/ARS, Mississippi State, MS, and Dr. Rufina Ward, Department of Plant and Soil Science, Alabama A&M University for reviewing the manuscript. We also gratefully acknowledge the assistance of Drs. J. Reinecke and F. Callahan, USDA-ARS, Mississippi State, in figure preparation. Contributed by the Agricultural Experiment Station, Alabama A&M University, Journal No. 370.

(1) Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.


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A Kakani, S. Saha,(*) V. T. Sapra, A. Zipf, and D M. Stelly

A. Kakani, V.T. Sapra, and A. Zipf, Dep. of Plant and Soil Science, Alabama A&M Univ., Normal, AL 35762; S. Saha, USDA-ARS Crop Sci. Res. Lab., Box 5367, MS State, MS 39762 and Integrated Pest Management Unit, USDA-ARS, Mississippi State, MS 39762; D.M. Stelly, Dep. of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843. Received 11 May 1997. (*) Corresponding author (
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Author:Kakani, A.; Saha, S.; Sapra, V. T.; Zipf, A.; Stelly, D. M.
Publication:Crop Science
Date:May 1, 1999
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