of the seedcoat ( Striem et al., 1992 ). In California and now widespread in private industry, breeders continue to select for nonperceived seed trace (reduced size and seedcoat hardness), and use embryo rescue techniques. To advance table grape
Laise S. Moreira and Matthew D. Clark
Mary Lewis, Matthew Chappell, Donglin Zhang, and Rebekah Maynard
Embryo rescue (ER), or the excision and culturing of immature zygotic embryos from developing seeds, is conducted under aseptic conditions to obtain viable and pathogen-free plantlets ( Bhojwani and Razdan, 1986 ; Morel, 1960 ). The technique was
Neil O. Anderson, Peter D. Ascher, Richard E. Widmer, and James J. Luby
The generation time (0.75 to 1.5 years) in perennial, hexaploid chrysanthemums [Dendranthema grandiflora Tzvelv. (Chrysanthemum morifolium Ramat.)] impedes the rate of progress for sexual breeding programs in creating new clonal cultivars, inbred lines for hybrid seed production, and genetic studies. Modifications to the crossing environment and embryo rescue were evaluated to minimize the chrysanthemum generation cycle. One greenhouse chrysanthemum clone was outcross-pollinated using a bulk pollen source. Following emasculation, inflorescences were either left in situ or the peduncle bases were placed in styrofoam boards floating on a solution of 1% sucrose and 200 ppm 8-HQC under laboratory conditions. Embryogenesis occurred at a faster rate under laboratory conditions as tested with histological techniques; the heart stage appeared as early as the second day after pollination, compared with 11 days using in situ methods. Total embryogenic development time ranged from 25 (laboratory seed development) to 52+ days (in situ ripening). In a second test, embryo rescue (ER) significantly improved percent seed set, percent germination, and percent of progeny reaching anthesis relative to normal development. ER progeny from both garden parents were significantly earlier in total generation time than corresponding non-ER siblings. Laboratory seed development and ER were then used sequentially to obtain an average progeny generation time of =100 days, thus allowing for three generations per year. The potential impact of these two techniques on breeding chrysanthemums and other perennial crops with long generation times is discussed.
Ron G. Goldy and Dana F. Moxley
A laboratory exercise is outlined and discussed for embryo culture of bean, corn, and pea embryos. Fresh, inexpensive material is generally available for these crop species throughout the year. The exercise gives students experience in embryo excision and exposure to some benefits of embryo rescue. Embryos from the three species are identified easily and can be removed without magnification, and data can be obtained within 3 weeks after culture. Further investigations using embryos are suggested.
Fred G. Gmitter Jr. and Jude W. Grosser
Although no longer as glamorous as it was a few decades past, the routine application of embryo rescue techniques, leading to plant recovery, is a valuable tool for citrus cultivar improvement. Embryo rescue approaches can be used to generate useful variation or to capture various kinds of spontaneous genetic variation. Embryo rescue, by in vitro culture of undeveloped, and presumably unfertilized, ovules in colchicine-supplemented media is a practical method of producing tetraploid clones, which are used then in crosses with diploids to produce seedless triploid hybrids. This same approach, i.e., in vitro culture of undeveloped ovules, is also used to recover plants from chimeric sectored fruit exhibiting economically important mutations for fruit characteristics, and for producing potentially variant somaclones. Seedlessness is an important objective for fresh citrus fruit cultivar improvement, and triploidy following 2x × 4x hybridizations is one approach being exploited for this objective. When monoembryonic diploid seed parents are crossed with tetraploid pollen parents, however, normal seed development is not usually possible. Embryos must be excised from abortive seeds fairly early in development and cultured appropriately to ensure the recovery of sufficient numbers of 3x offspring from these crosses, to increase the likelihood of identifying superior seedless hybrids. These applications will be described in some detail, and progress toward breeding objectives are highlighted.
Hector G. Nunez-Palenius, Daniel J. Cantliffe, Harry J. Klee, and Don J. Huber
Embryo abortion and empty seeds after self-pollination occur in some transgenic (ACO antisense) `Galia' male parental lines. An embryo-rescue system in this melon was developed to save potential viable embryos. To obtain the best and reliable embryo-rescue technique, several parameters were used including an improved (five new supplements) nutrient medium (named E-21) from the E-20A basic medium (Sauton and Dumax de Vaulx, 1987), an inoculation system (removing the embryo from the seed or intact seed), and the use of different fruit harvesting dates of the wild type and a transgenic `Galia' male parental line. Fruits of wild type (WT) and transgenic (ACO gene in antisense orientation) `Galia' male parental line were harvested at 4, 10, 17, 24, and 30 days after pollination (DAP). Fruits were surface sterilized by dipping in a 20% commercial bleach solution for 30 minutes. Subsequently, seeds were removed from fruit under sterile conditions. These seeds were either used to dissect the embryos or placed directly with the hilum facing E-20A or E-21 medium. Seedlings from all treatments were transferred to E-21 elongation medium, incubated 4 weeks, and transferred to soil to evaluate growth. The efficiency of this technique was greater when the time after pollination (4, 10, 17, 24, and 30 DAP) to rescue the embryos was increased. Thus, 30 DAP was the best time to rescue the embryos. The number of rescued embryos using E-21 medium was greater than with E-20A. We did not find any significant differences in survival efficiency rate between WT and transgenic embryos. We have obtained a competent embryo-rescue technique for WT and transgenic `Galia' male parental line, which can be applied to rescue valuable GMO hybrid-melon embryos.
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.
Natalia R. Dolce, Luis A. Mroginski, and Hebe Y. Rey
produce low or no germination, in vitro techniques may greatly enhance germination and growth rates by optimizing culture conditions and media ( Fay, 1992 ). The embryo rescue technique has been used for over 50 years to accelerate the maturation of
Lauren E. Kurtz, Mark H. Brand, and Jessica D. Lubell-Brand
first 2 weeks after pollination, at which time seed development appeared to arrest and seeds started to become soft. For this reason, on 24 Jan. 2020, embryo rescue was attempted for the remaining seeds that were firm. The majority of developing seeds
David W. Ramming, Richard L. Emershad, and Ronald Tarailo
Hybridizations between seedless Vitis vinifera L. genotypes and V. rotundifolia (Michx.) were made specifically to introgress the seedless trait into the disease-resistant background of V. rotundifolia. The seedless, gynoecious P79-101 was hybridized with three V. rotundifolia parents, producing a total of 102 ovules. From these ovules, 44 embryos developed, producing 20 plants. Isozyme analyses and morphological traits confirmed that 19 of the plants were hybrids. Sixteen were planted in an experimental vineyard at California State Univ. Fresno. One seedling, C41-5, produced seedless fruit that appeared to be stenospermocarpic based on fruit and aborted seed size. Fruit weight was slightly less than that of `Thompson Seedless' (stenospermocarpic) and at least twice that of parthenocarpic fruit of `Black Corinth' and C41-7, a seeded hybrid with many parthenocarpic fruit. Aborted seeds of C41-5 were larger than, but not significantly different from, those of `Thompson Seedless', while parthenocarpic fruit from `Black Corinth' and C41-7 had aborted seeds that were smaller than those of C41-5. Seed weight of C41-7 averaged almost 10 times that of aborted C41-5 seeds. This is the first report of a stenospermocarpic, seedless hybrid of V. vinifera × V. rotundifolia.