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
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.
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.
Fabio De Pasquale, Salvatore Giuffrida, and Francesco Carimi
Minigrafting was used for rescue of tissue culture regenerants of the following four species of Citrus: sour orange (C. aurantium L. `AA CNR 31'), sweet orange [C. sinensis (L.) Osb. `Valencia Late'], lemon [C. limon (L.) Burm. `Femminello Comune'] and mandarin (C. deliciosa Tenore `Tardivo di Ciaculli'). The grafting was carried out with different scion types including shoots, roots, inverted roots and somatic embryos. This material was obtained in vitro from embryogenic style-derived callus. Seedlings of open-pollinated sour orange (C. aurantium L.), Cleopatra mandarin (C. reshni Hort. ex Tan.) and `Troyer' citrange [C. sinensis Osb. × Poncirus trifoliata (L.) Raf.] were used as rootstocks. Minigrafting of shoots, roots, inverted roots and embryos regenerated in vitro allowed successful rescue of these four species. Percentages of successful minigrafts ranged from 100% (shoots) to 2.5% (inverted roots). The probability of successful graft unions increased with the age of the rootstock. The final mean canopy leaf area (120 days after grafting) ranged from 5.2 cm2 (`Tardivo di Ciaculli' mandarin grafted on 6-month-old Cleopatra mandarin) to 157.9 cm2 (`Valencia Late' sweet orange grafted on 18-month-old Cleopatra mandarin). In this work we examined some of the variables which influenced minigrafting and we determined the efficacy of this method for rescue of in vitro regenerants of Citrus. This method is also suggested as a technique to produce a high percentage of viable plants from in vitro regenerants difficult to root.
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.
Elisabet Claveria, Jordi Garcia-Mas, and Ramon Dolcet-Sanjuan
Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.
Akira Sugiura, Takeshi Ohkuma, Young A Choi, Ryutaro Tao, and Mihoko Tamura
To produce nonaploid Japanese persimmon (Diospyros kaki L.f.) by artificial hybridization, we surveyed the natural occurrence of unreduced (2n) pollen among hexaploid cultivars and sorted them from normal reduced (n) pollen. The sorted 2n pollen was crossed with a hexaploid female cultivar and the resultant embryos were rescued by in vitro culture techniques to obtain plantlets. Three out of six male-flower-bearing cultivars (2n = 6x = 90) produced 2n pollen at rates of 4.8% to 15.5% varying with the cultivar, which was estimated by both pollen size and flow cytometry. After sorting giant (2n) from normal pollen grains by using nylon mesh, they were crossed with a hexaploid female cultivar. The seeds obtained from pollination with normal pollen were perfect, but those obtained from pollination with giant pollen were mostly imperfect, with embryo growth being suspended at the globular stage. Although the rate of survival was very low, some embryos at the globular stage were rescued successfully and grown in vitro. Both flow cytometric analysis and chromosome counting proved that the plantlets obtained were nonaploid.