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D.J. Gray, J.A. Mortensen, CM. Benton, R.E. Durham and G.A. Moore

Ovules of seedless bunch grapes (Vitis spp.) fertilized by controlled pollination increased in size during berry development. More ovules cultured 10 days or 60 to 70 days after pollination became brown compared to those cultured at 20 to 40 days. Cultured ovules developed with and without endosperm. Globular to torpedo stage embryos were recovered. More embryos and plants were recovered from ovules cultured at 40 or 60 days than at 10 or 20 days after pollination. Pollen parent significantly affected both embryo and plant recovery at certain sampling times. BA incorporated into medium significantly increased embryo germination percentage. Electrophoretic analysis of glucosephosphate isomerase in progeny showed that 67% to 88% were hybrids of controlled crosses. Of four vines that fruited thus far, two were seedless. Seedless progeny had smaller seed traces than either parent. Chemical name used: N-(phenylmethyl) -1H-purin-6-amine (BA).

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David W. Ramming, Richard L. Emershad and Carol Foster

Various in vitro conditions for culture of ovules prior to extraction and culture of immature embryos of peach [Prunus persica (L.) Batsch] and nectarine [Prunus persica (L.) Batsch var. nucipersica Schneid.] were investigated. Culture vessels consisting of test tubes, petri dishes, and polycarbonate jars were tested along with various types of support and nutrient media. Agar support was superior to liquid media with filter paper supports. Agar produced the largest embryos with 90% to 93% being converted into plants compared to liquid with only 1% to 12% embryo conversion. The best ovule orientation and support was with the micropyle down and pushed halfway into an agar-gelled medium. In experiments two and three, test tubes with vertical ovule orientation (micropyle end of ovule pushed into agar) produced larger embryos, the largest plants and the greatest percentage of embryos that converted into plants (60% and 91%). Petri dish treatments were less successful in embryo conversion than test tubes and polycarbonate jars. The addition of activated charcoal (AC) to an agar-gelled medium produced significantly larger embryos with a similar conversion rate. The addition of an agar-gelled medium to culture vessels reduces preparation time compared to filter paper supports, and placing each ovule within a test tube eliminates cross contamination, making immature embryo culture more successful.

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Ming Cai, Ke Wang, Le Luo, Hui-tang Pan, Qi-xiang Zhang and Yu-yong Yang

. macrophylla and H . arborescens . Putative hybrid plants were obtained from ovule cultures initiated from seedlings on cotyledonary stage, but the hybridity of these plants was not verified by a genetic analysis ( Kudo and Niimi, 1999 ). In an effort to

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Justin A. Schulze, Jason D. Lattier and Ryan N. Contreras

A tissue culture protocol was developed to germinate immature Prunus lusitanica seeds in vitro. The study was conducted by first identifying the best media for germination, followed by investigating effects of seed conditioning. In Expt. I, seeds were collected 12 weeks after pollination (WAP) ± 1 week and placed on media after removing the pericarp. Eight different MS media (Murashige and Skoog, 1962) were tested (M1–M8) containing two concentrations each of 6-benzylaminopurine (BA), gibberellic acid (GA3), and sucrose. The longest shoots resulted from M4 (1.45 µm GA3, 6 µm BA, and 30 g·L−1 sucrose), followed by M1 (0 µm GA3, 3 µm BA, and 30 g·L−1 sucrose). Radicle and shoot emergence was greater than or equal to 90% for M1, M3, and M4 after a stratification treatment. In Expt. II, M1 was used to test root and shoot emergence at 6, 9, and 12 WAP, with and without cold stratification. Little success was seen 6 and 9 WAP, with only callus development in 6 WAP, nonstratified seed. Cold stratification increased shoot emergence in the 12 WAP group from 4% to 28%, appearing to be critical for shoot emergence. If the cotyledons are retained on the seed, future efforts to expedite breeding of P. lusitanica using in vitro germination should not be collected before 12 WAP and will benefit from cold stratification before germinating on M1 or M4. Chemical names: 6-benzylaminopurine (BA), gibberellic acid (GA3).

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Josephina G. Niederwieser, H.A. van de Venter and P.J. Robbertse

Techniques are described to determine whether embryos are formed in ovules of incompatible crosses between Ornithogalum (L.) plants, and to rescue embryos in cases where the development of embryos is halted following fertilization. By using Herr's clearing liquid, it can be ascertained within 5 hours whether hybrid embryos have been formed. Such embryos can be rescued by culturing them in ovulo on basal medium containing 70 g sucrose/liter and no added growth regulators. The embryos' requirement for sucrose changes as they develop; therefore, cultured ovules are transferred after 14 days to a medium containing 10 g sucrose/liter, where germination occurs.

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Mohammed Elsayed El-Mahrouk, Mossad K. Maamoun, Antar Nasr EL-Banna, Soliman A. Omran, Yaser Hassan Dewir and Salah El-Hendawy

formation in black cumin ( Peterson, 1973 , 1974 ). However, to our knowledge, there are no reports on haploid induction in black cumin. Therefore, this study is the first report on haploid production of black cumin from in vitro ovule culture. The aim of

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Jodie L. Ramsay, Donald S. Galitz and Chiwon W. Lee

Influences of culture media and sucrose concentrations on plant regeneration from Easter lily (Lilium longiflorum L. cv. Ace) ovary tissues were investigated. Pistils excised from unopened flower buds (3-5 cm long) were sectioned and cultured on either B-5 medium or Murashige and Skoog (MS) medium containing 2%, 5%, or 10% sucrose, with 1 mg·L-1 2,4-D and 2 mg·L-1 BA. Callus formation was most prolific on MS medium containing 5% sucrose. Shoot differentiation was higher on MS medium than on B-5 medium. Rooted plants were transferred into soil medium and grown in a greenhouse. Root tip smears showed that 35% of the regenerated plants showed a variation in chromosome numbers from 10 to 25 per cell, while the rest of the regenerants showed the normal 2n = 2x = 24 chromosomes per cell. The mixoploid condition also existed in different root cells of the same regenerated plant. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); 6-benzylaminopurine (BA).

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Roy N. Keys, Dennis T. Ray and David A. Dierig

Guayule (Parthenium argentatum Gray), a latex-producing perennial desert shrub and potential industrial crop for semiarid regions, exhibits reproductive modes ranging from sexual, self-sterile diploids to predominantly apomictic, self-compatible polyploids. The objectives of this study were to develop and evaluate a rapid, simple technique for characterizing apomictic potential (percentage of ovules that produce apomictic embryos) in guayule breeding lines. Initial in vivo experiments were based on an auxin test that permitted quantification of apomictic frequency in grasses. In our trials, floral application of NAA or IBA resulted in embryo production similar to that of open-pollinated controls, but 2,4-D inhibited embryo production. Breeding lines could be separated based on embryo production using an in vivo auxin test; however, accuracy of the results was questionable because 1) pollen release and insect activity within isolation bags prevented distinguishing between sexual and apomictic embryos, and 2) high temperatures and large humidity fluctuations could have affected results. Thus, in vitro flower culture was investigated using liquid medium, because it would provide better control of these factors. Flowers developed normally in vitro, except that pollen was not released from the anthers; therefore, any embryos produced in vitro were considered to be apomictic. Embryo production was similar on both Nitsch and Nitsch and Woody Plant Media. Addition of growth regulators inhibited embryo production. Embryo production was tested on Nitsch and Nitsch medium without growth regulators for seven breeding lines. Based on statistical analyses, four classes of apomictic potential were identified, ranging from none (sexual) to high. Chemical names used: 2,4-dichlorophenoxy acetic acid (2,4-D); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA).

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Jana Murovec, Natasa Stajner, Jernej Jakse and Branka Javornik

A codominant marker for homozygosity testing and species discrimination needed in breeding programs was developed and applied to different Mimulus L. species and cultivars. Degenerative primers used to amplify intron 10 of topoisomerase 6 subunit B (top6B) in distant species also amplified the locus in all analyzed Mimulus species. The sequences obtained revealed the presence of a microsatellite motif and were used to design a specific microsatellite primer pair, Mim-top6B, for Mimulus species. The microsatellite marker showed a high degree of polymorphism in Mimulus species, and the heterozygous nature of most M. aurantiacus Curtis cultivars. The marker was further used to analyze putative doubled haploids of M. aurantiacus and showed that all but one was heterozygous, indicating their hybrid origin.