Interspecific hybridization in the genus Gossypium through embryo culture

Summary With embryo rescue and culture techniques interspecific hybrids have been obtained amongst diploid cultivated (Gossypium arboreum, G. herbaceum) and wild species (G. stocksii, G. anomalum) of cotton. The early abscission of the young bolls was prevented by repeated application of growth regulators followed by the culture of immature hybrid embryos (15 days after pollination). The best growth and development were obtained on modified Murashige and Skoog's medium with supplements. The plants were stouter when the cultures were first incubated in the dark for 15–25 days, followed by exposure to light. The hybrid plants transferred to soil developed further and matured, and were more or less intermediate between their respective parents.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Relationship between molecular marker heterozygosity and hybrid performance in intra- and interspecific hybrids of cotton

The present study was conducted to investigate the relationship between parental molecular marker diversity and hybrid performance in both intra‐ and interspecific hybrids of cotton to evaluate the feasibility of predicting hybrid performance using molecular markers. Three cytoplasmic male sterile (CMS) lines were crossed with 10 restorer lines to produce 22 F₁ hybrids during 2003. Of 22 F₁s, 14 hybrids were intraspecific (Gossypium hirsutum × G. hirsutum) and eight interspecific (G. hirsutum × G. barbadense). These 22 F₁ hybrids and their parents were evaluated for yield and fibre quality traits at Zhejiang University, Hangzhou, China during 2004 and 2005. Genetic distances (GD) among the parents were calculated from 56 random‐amplified polymorphic DNAs (RAPD) and 66 simple sequence repeat (SSR) marker data, and their correlation with hybrid performance and heterosis were analysed. The parents could be discriminated into G. hirsutum and G. barbadense clusters by cluster analysis based on both RAPD and SSR markers data. The correlation (r = 0.503, P ≤ 0.05) was calculated between GD_rapd (GD based on RAPD markers) and GD_ssr (GD based on SSR markers). Correlation of GD with hybrid performance and heterosis differed considerably between intra‐ and interspecific hybrids. The correlation between GD and hybrid performance was non‐significant for most of traits within the hybrids of G. hirsutum species. However, it was significantly and positively correlated for fibre length, fibre strength and elongation in interspecific hybrids. The relationship between GD and heterosis was observed to be positively significant for boll weight within hybrids of G. hirsutum with significant and negative correlations for fibre length and elongation. In conclusion, the power of predicting hybrid performance using molecular markers in cotton is low. But, the relationship between SSR marker heterozygosity and hybrid performance can be used to predict fibre length during interspecific hybrid cotton breeding.     

Heterosis for symbiotic nitrogen fixation in Phaseolus interspecific hybrids

Summary Interspecific hybrids of Phaseolus vulgaris and Phaseolus acutifolius or Phaseolus filiformis have been evaluated for evidence of heterosis for symbiotic nitrogen fixation activity. Hybrids showing composite leghemoglobin profiles were selected for comparison. Rooted cuttings from hybrids and their respective parents were used to estimate nitrogen fixation (acetylene reduction) rates and to determine nitrogen accumulation during the growth period. Some hybrids of Phasolus vulgaris × Phaseolus acutifolius and Phaseolus vulgaris × Phaseolus filiformis had significantly (P<0.01) higher nitrogen fixation rates and in the latter case, accumulated more total nitrogen, than either parent. The interspecific hybrids also showed more complex leghemoglobin profiles than either parent.     

Heterosis in CMS hybrids of cotton for photosynthetic and chlorophyll fluorescence parameters

Summary The characterization of photosynthetic, chlorophyll fluorescence parameters and yield and yield components was compared between two newly developed CMS cotton hybrids (H1, H2) and their parents, as well as currently planted hybrid and conventional cultivars (C1, artificially produced hybrid CCRI 29 and C2, conventional cultivar CCRI 12) under the field condition. The results showed that P_n, F_v/F_m, Φ_PSII and qP of hybrids was significantly higher than that of C1 and C2. Furthermore, heterosis was found on photosynthetic parameters in hybrids over their parents at the first four growth stages, while only heterosis over mean of parents (HOMP) was detected in chlorophyll fluorescence parameters. It was clearly shown that one male-sterile line M2 was higher than the hybrids in F_v/F_m, Φ_PSII and qP over all growth stages. Significantly positive correlations (P < 0.01) were found between LAI or Yield and P_n, C_s, T_r, F_v/F_m, Φ_PSII, or qP, while significantly negative correlations between C_i and LAI or Yield, and no significance between qN and Chl a, C_s and qP were found. It was concluded that CMS hybrid cottons showed better potential to maintain relatively higher photosynthetic ability during the growth, which contributes to the increased lint yield.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Heterosis of interspecific soybean hybrids for traits related to N2 fixation

Summary Three greenhouse experiments were conducted to compare the performance of soybean (Glycine max (L.) Merr.), wild soybean (G. soja Sieb. et Zucc.), and soybean x wild soybean hybrids for traits relating to N₂ fixation including nodulation, acetylene reduction, nodule leghemoglobin concentration, and nitrogen (N) accumulation and dry matter (DM) accumulation. In all three experiments G. max generally exceeded G. soja in nodulation, acetylene reduction, and N and DM accumulation while the soybean possessed higher nodule leghemoglobin concentration. In Experiment I, the mean of the hybrids did not differ significantly from the G. max parent in nodule mass, acetylene reduction activity, nodule leghemoglobin concentration, or DM accumulation. The hybrids did exceed the soybean parent in N accumulation, thus demonstrating high parent heterosis. In Experiments IIA and IIB with a more carefully chosen set of G. soja parents, high parent heterosis of individual crosses was common. Across the three experiments average high parent heterosis was 34, 28, and 28%, respectively, for nodule mass, N accumulation, and DM accumulation. If one accepts the assumption that hybrid vigor results from the accumulation of dominant alleles, then these alleles could theoretically be accumulated via selection in a homozygous genotype. If this is true than the results of the experiments reported here suggest that interspecific soybean x wild soybean crosses could serve as sources of homozygous lines which would exceed currently available soybean cultivars in nodule mass, and N and DM accumulation.This work was supported in part by the USDA Competitive Grants Program, Grant 82-CRCR-1-1039 and Cooperative Agreement 58-32U4-2-370 between the U.S. Department of Agriculture and the Agronomy Department, University of Maryland, Scientific Article No. A-4648, Contribution No. 7644 of the Maryland Agricultural Experiment Station.     

Heterosis in yield,fiber quality,and photosynthesis of okra leaf oriented hybrid cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Leaf shape is an important factor affecting canopy structure, photosynthetic characteristics, and yield. Introducing okra leaf shape is considered an important strategy in cotton heterosis breeding. The objective of the study was to evaluate the heterosis in okra leaf cotton hybrid F₁s, which were developed by crossing two sterile near isolines and three restorer near isolines. A normal leaf check hybrid F₁, Zhongza 29, and four newly developed okra leaf oriented hybrid F₁s, including the super-okra (severely cleft and narrowly lobed leaf), okra (deeply cleft and narrowly lobed leaf), semi-okra (intermediate leaf lobe), and sub-okra (large leaf lobe) hybrids were selected in this experiment. Okra leaf oriented cotton hybrids significantly reduced leaf area index (LAI) (P < 0.0001), thus more light penetrated into the lower canopy. The LAIs for the super-okra and okra hybrids, however, were too small to supply adequate photosynthate, thus resulting in early maturity, short boll filling period, and low yield. As the LAI increases, the semi-okra and sub-okra hybrids showed certain heterosis. The semi-okra hybrid showed 7–12% heterosis over the check hybrid in lint percent, 6–11% in boll weight, 2–147% in canopy light intensity, 1–10% in net photosynthetic rate (Pn), and −3 to 3% in lint yield. As well, all tested okra leaf hybrids improved fiber quality such as micronaire compared with the normal leaf check hybrid. The results suggested that adopting okra leaf parental lines in cotton breeding provided potentials in increasing fiber quality with comparable lint yield.     

Introgression of a gene for delayed pigment gland morphogenesis from Gossypium bickii into upland cotton

The presence of gossypol and its derivatives above the WHO/FAO standards (0.02–0.04%) in cotton seed oil and meal limits its usage as food and feed. To the contrary, the presence of pigment glands filled with gossypol and its derivatives helps to protect cotton plants from phytophageous pests. Thus a desirable cultivar would have glandless seeds on a glanded plant. This paper describes results on the successful introgression of this trait from Gossypium bickii into cultivated upland cotton. Five different tri‐specific hybrids (ABH1, ABH2, ABH3, ABH4 and ABH5) were obtained by crossing the amphidiploid F₁ (G. arboreum × G. bickii) with different gland genotypes of G. hirsutum as male parent. The hybrids were highly sterile, and their chromosome configuration at meiosis metaphase 1 (M1) in pollen mother cell (PMC) was 2n = 52 = 41.04 I + 4.54 II + 0.57 III + 0.04 IV. All five hybrids were similar in morphological characters, except for the gland expression and gossypol contents. The hybrid (ABH3) derived from genotype Gl₂Gl₂gl₃gl₃ of upland cotton (a single gene dominant line) had completely introgressed the target trait of G. bickii. While ABH1 and ABH2, which derived from recessive (gl₂gl₂gl₃gl₃) or dominant (GlGl) glandless upland cotton genotypes, had glandless seeds too, but the density and size of the glands on the plant were reduced significantly.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic effects on biomass yield in interspecific hybrids between Brassica napus and B. rapa

This study was conducted to estimate the genetic effects on biomass yield in the interspecific hybrids between Brassica napus and B. rapa, and to evaluate the relationship between parental genetic diversity and its effect on biomass yield of interspecific hybrids. Six cultivars and lines of oilseed B. napus and 20 cultivars of oilseed B. rapa from different regions of the world were chosen to produce interspecific hybrids using NC design II. Obvious genetic differences between B. rapa and B. napus were detected by RFLP. In addition, Chinese B. rapa and European B. rapa were shown genetically differences. Plant biomass yield from these interspecific hybrids were measured at the end of flowering period. Significant differences were detected among general combining ability (GCA) effects over two years and specific combining ability (SCA) effects differences were detected in 2000. The ratios of mean squares, (σ² _GCA(f) + σ² _GCA(m)) / (σ² _GCA(f) + σ² _GCA(m) + σ² _SCA), were 89% and 88% in 1999 and 2000, respectively. This indicates that both additive effects and non-additive effects contributed to the biomass yield of interspecific hybrids and the former played more important role. Some European B. rapa had significant negative GCA effects while many of Chinese B. rapa had significant positive GCA effects, indicating that Chinese B. rapa may be a valuable source for transferring favorable genes of biomass yield to B. napus. Significant positive correlation between parental genetic distance and biomass yield of interspecific hybrids implies that larger genetic distance results in higher biomass yield for the interspecific hybrids. A way to utilize interspecific heterosis for seed yield was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of aneuploids of the cotton hybrid G. barbadense × <Emphasis Type="Italic">G. hirsutum</Emphasis> L. via intergeneric pollination with <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis>

The possibility to induce embryo development after pollination of F₁ interspecific cotton hybrids (Gossypium barbadense × Gossypium. hirsutum) with pollen from Abelmoschus esculentus was investigated to determine if wide-cross hybrid, haploid or other types of progeny might result. Small numbers of progeny (Pa) were indeed recovered after numerous alien pollinations and in-planta development or in-vitro culture. The Pa plants were characterized phenotypically and studied cytogenetically and microscopically to help establish their origin documenting their reproductive basis. Root-tip chromosome counts and meiotic Metaphase-I analyses revealed that chromosome numbers among cells of the Pa plants ranged from 33 to 44 and that the differences in chromosome number among cells of the same plant ranged from 1 to 3, indicating somatic instability. Flow cytometric analysis also indicated the aneuploid nature of Pa plants. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that alien pollinations may have resulted in parthenogenetic (Pa) egg cell development, or some other unusual reproductive events. The production of wide-crosses and high degrees of aneuploidy could be of use for several types of genomic studies, e.g., functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.     

Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue

Black rot is a bacterial disease of Brassica oleracea caused by Xanthomonas campestris pv. campestris. Resistance to the major black rot races 1 or 4 has been identified in related Brassica species including B. carinata and B. napus. In this study, two B. juncea accessions (A 19182 and A 19183) that are resistant to races 1 and 4 of Xcc were used as maternal and paternal parents to generate interspecific hybrids with B. oleracea cultivars. Interspecific hybrids were recovered using the embryo rescue technique and confirmed through inheritance of paternal molecular markers. Twenty-six interspecific hybrid plants were obtained between A 19182 and B. oleracea cultivars, but no interspecific hybrids were obtained using A 19183. Although interspecific hybrid plants were male sterile, they were used successfully as maternal parents to generate backcross plants using embryo rescue. All hybrid and BC₁ plants were resistant to black rot races 1 and 4.     

Interspecific hybrid of Helianthus annuus × H. simulans: Characterization and utilization in improvement of cultivated sunflower (H. annuus L.)

The first success at interspecific hybridization between cultivated sunflower(H. annuus) and a diploid perennial species, H. simulans is reported. The F₁s from both direct and reciprocal directions exhibited dominance of the wild species phenotype and were pollen sterile. Meiosis was irregular in the F₁ hybrids and both univalents and multivalents were observed. Multiplication of the interspecific hybrids was achieved through in vitro culture of nodal sections on Murashige & Skoog medium supplemented with 0.5 mg l^-1 benzyladenine. Fertility of the interspecific hybrids was improved by subjecting the in vitro proliferating shoots to 0.001% colchicine incorporated in the shoot multiplication medium. The amphiploids serve as fertile bridges and facilitate interspecific gene transfer through conventional breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

Risk assessment of outcrossing of transgenic rapessed to related species:

Summary The risk for a gene dispersal is reported for reciprocal crosses between a transgenic rapeseed variety resistant to the herbicide phosphinotricin and five related species. The first stages after pollination were cytologically observed and fertilized ovaries were established in in vitro culture for the production of interspecific hybrids. A similar classification was observed for the index of pollination compatibility and embryo yield. From the 243 embryos produced, 109 plantlets were obtained in a greenhouse. All the interspecific combinations tested were able to produce hybrid plants. A higher number of hybrids was obtained when rapeseed was used as the female parent. The hybrids had the expected triploid structure except for two amphidiploid, B. napus × B. oleracea, and one amphidiploid, B. napus × S. arvensis, plants with 56 chromosomes. The triploid hybrids were sterile or partially fertile but two of the amphidiploid plants, B. napus × B. oleracea, were fully fertile. The cytoplasm source did not seem to affect the fertility of the hybrids.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

Resistance of lettuce (Lactuca) to the leaf aphid Nasonovia ribis nigri. 1. Transfer of resistance from L. virosa to L. sativa by interspecific crosses and selection of resistant breeding lines

Summary Interspecific crosses were carried out between the three Lactuca species L. virosa, L. serriola and L. sativa to transfer resistance to the leaf aphid Nasonovia ribis nigri from L. virosa to the cultivated lettuce. L. sativa, L. serriola was used as an intermediate parent between the other two species. Many irregularities were observed in the interspecific hybrids, ranging from premature dying of F₁ plants resulting from a kind of bastard necrosis till complete male and female sterility and deviating microsporogenesis. Using in vitro culture and after several backcrosses male and female fertile plants were obtained with a L. sativa habit and with resistance to the leaf aphid.     

Genotypic diversity enhances recovery of hybrids and fertile backcrosses of Phaseolus vulgaris L. × P. acutifolius A. Gray

Summary Homozygous and heterozygous Phaseolus vulgaris and P. acutifolius parental genotypes were hybridized to determine if genotypic diversity would aid gene transfer between these species. Certain P. vulgaris female parents resulting from diverse intraspecific crosses increased the frequency of species hybrids obtained. From a total of 19 self-sterile hybrids, 20 backcross-1 (BC₁) lines (P. vulgaris recurrent parent) were produced from 4 partially female-fertile hybrids, each of which had a heterozygous P. vulgaris female parent. Heterozygous P. acutifolius male parents did not influence the frequency at which interspecific hybrids could be produced but apparently improved female-fertility. Fertility of the F₁BC₁ generation was highly variable. The frequency of fertile individuals increased in each subsequent backcross generation (F₁BC₂ and F₁BC₃).Purdue University Agricultural Experiment Station Publication 9946.