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Yinping Shi, Qiangsheng Wang, Guangfang Zhou, and Congyi Sui

Plant mutation induced with colchicine, disturbance of chimeras has long been unsolved. Authors used embryo culture in vitro induced with colchicine for inducing genome of embryonic cells of diploid apple to be doubled, cell doubled differentiated into adventitious shoots, and then were culture into plantlets. By morphological preselection, plants induced hundreds of genotypes had been obtained. To identify ploidy variation of three histogenic layers of shoot apices, sections of shoot apices of 284 plants were identified. Two-hundred-forty-nine tetraploid plants were selected. Entire mutants accounted for 98%, chimeras 2%. This proved that induction in vitro could indeed eliminate disturbance of chimeras and was a new induction technique simply and effectively. Accurate rate of morphological preselection was confirmed by 87.7% by sections of shoot apices. The identification of ploidy of mutated plants of apple in vitro induced with colchicine, the method of combining morphological preselection with sections of shoot apices had advantages over that of chromosome count. First, the method is simple, saving time and labor, with a high success rate and reliable results. Second, whether the mutated plants were chimeras and chimera structures could be known. Main criteria of identifying ploidy by sections of shoot apices are the size of cells, nuclei, and nucleoli and numbers of nucleoli of three histogenic layers of shoot apices. Morphological characters of tetraploid were dumpy, thick, and strong stem with short internodes; small petiole angle; broad-round thick leaves with dark green color; round leaf base; thick and sharp-pointed sawteeth; protruding and clear main vein.

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Hajime Araki, Shoji Hayashi, Takashi Harada, and Toshiro Yakuwa

Dioscorea is one of the important tuber crops in Asia and dioecious. D.opposita cv.Yamatoimo (female, round tuber shape, high eating quality, low yield) and cv.Nagaimo (male, high yield, low eating quality) are typical cultivars in Japan. Much labor is necessary for harvesting tuber of cv. Nagaimo because this tuber is slender and brittle, and elongates to 60-70 cm underground. Some hybrid plants between the mentioned 2 cultivars were successfully grown and obtained through artificial pollination under high temperature conditions and embryo culture. The variations of leaf and tuber shape in hybrid yam were estimated by image processing and principal component analysis. A large range of variation in leaf shape was recognized, and a more round-shape(cordate) leaves and longer and narrower leaves in comparison with the parent cultivars were observed. In tuber shape, the variations with regard to fullness and elongation were recognized. The fullness ranged from the level of cv.Nagaimo (thin) to that of cv.Yamatoimo (thick), and slender, ellipsoid and clump shape tubers were observed. A correlation between leaf shape and tuber shape was not recognized. A relationship existed between the shape of tuber harvested in the 1st year (1990) and 2nd year(1991) after transferring in vitro plantlets to soil.

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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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Mark Bridgen

Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, has been bred at the Univ. of Connecticut since 1985. In vitro procedures have been integrated with traditional breeding techniques to create new and exciting cultivars. Embryo culture has been used to generate interspecific, intraspecific, and intergeneric hybrids that would not have been possible with traditional breeding. Somaclonal variation has been used to create new plants from spontaneous and induced mutations, but, in most cases, the plants have not been acceptable commercially. Chromosome doubling with colchicine has been used for fertility restoration of sterile diploids. Somatic embryogenesis has also been studied quite extensively; somatic embryos are easily obtained from zygotic embryos of Alstroemeria. In vitro fertilization procedures are currently being studied in order to hasten embryo development after hybridization has occurred. Because Alstroemeria plants are slow to propagate by traditional rhizome division, micropropagation is used to multiply new cultivars rapidly. Because the production of pathogen-free plants is one of the goals of our breeding and new plant introduction programs, meristem culture and thermotherapy are also being studied. All of these techniques will be described during the workshop.

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Hiroshi Iwanami, Nobuyuki Hirakawa, Hiroyasu Yamane, and Akihiko Sato

Crosses between seedless cultivars had been conducted to produce seedless table grape efficiently by combining with ovule and embryo culture in Vitis vinifera L. But very few plants grew normally in this method. Four plant growth regulators (Cycocel, B-Nine, Uniconazole-P, Ethrel) were applied to shoots 4 weeks before anthesis to develop the seeds of two seedless cultivars `Flame seedless' and `A1706'. Correlation was significant in each cultivar between the shoot length at anthesis and the number of seed traces per berry in all combined treatments. Analysis of covariance revealed that the number of seed traces per berry was significantly higher when the shoots were applied with Uniconazole-P (240 ppm) than B-nine (2000 ppm), Cycocel (500 ppm) and Ethrel (400 ppm) in `Flame seedless' and Uniconazole-P and B-nine than Ethrel in `A1706'. Ovules of these two seedless cultivars crossed with seedless cultivar `Perlette' after the application of four plant growth regulators were cultured on half-strength MS medium with 10 μm IAA and the percentage of developed embryos in ovules was higher when the shoots were applied with Uniconazole-P and B-nine than Cycocel, Ethrel in `Flame seedless' and B-nine than others in `A1706'. These results indicate that the use of certain plant growth regulators promotes the embryo development.

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Zenaida Viloria and Jude W. Grosser

Interploid hybridization was conducted using `Key' lime [Citrus aurantifolia (Cristm.) Swing.], `Lakeland' limequat hybrid [C. aurantifolia × Fortunella japonica (Thumb.) Swing.], Palestine sweet lime (C. limettioides Tan.), `Etrog' citron (C. medica L.), and seven lemon [C. limon (L.) Burm. F.] varieties as female progenitors and five allotetraploid somatic hybrids {`Hamlin' sweet orange [C. sinensis (L.) Osbeck] × `Femminello' lemon (C. limon)]; `Key' lime × `Valencia' sweet orange (C. sinensis); `Valencia' sweet orange × rough lemon (C. jambhiri Lush); Milam lemon (purported C. jambhiri hybrid) × `Femminello' lemon (C. limon); and `Valencia' sweet orange × `Femminello' lemon} and two autotetraploids [`Giant Key' lime (C. aurantifolia) and `Femminello' lemon] as pollen progenitors. A few tetraploid × diploid crosses were also performed. Thirty-five parental cross combinations were accomplished in 2000, 2001, and 2002. The breeding targets were seedlessness, cold-tolerance, and disease resistance. Triploid hybrids were recovered through embryo culture. Generation of triploid citrus hybrids was affected by several factors including sexual compatibility, cross direction, embryo developmental stage, pollen viability, as well as horticultural practices and climatic conditions. Efficiency of triploid hybrid production was higher in diploid × tetraploid crosses than the reciprocal. Many more triploid hybrids were generated from lemon seed progenitors compared to the other acid citrus fruit progenitors. `Todo el Año', `Lisbon', and `Limonero Fino 49' showed the highest sexual compatibility. Embryo germination rate and normal plant recovery were also higher in lemons as compared to the other seed progenitors. Low winter temperatures might have affected the hybrid production efficiency from tropical acid fruit progenitors. A total of 650 hybrids (mostly triploid) were transferred to soil. The novel genetic combinations of these progenies should be valuable for the genetic improvement of acid citrus fruit (lemons and limes).

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Rodomiro Ortiz, Dirk Vuylsteke, Hutoshki Crouch, and Jonathan Crouch

. International mailing address: IITA, c/o L.W. Lambourn and Co., Carolyn House, 26 Dingwall Road, CR9 3EE, England. Paper No. IITA/96/JA/64. The authors thank Mr. Leo Oragwa for assistance in embryo culture and the crew led by Ms. Josephine Okoro for data

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David W. Ramming

demonstrate that plants produced from embryo culture were from fertilized eggs (zygotic) and not from maternal tissue (somatic). ‘Thompson Seedless’ fruit is white in color and neutral in flavor, whereas the paternal parent, ‘Concord’, has blue-colored fruit

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Craig A. Ledbetter

-skinned with a large berry size, firm flesh, and a small seed trace. The cross was performed in 2004, with immature berries being harvested ≈6 weeks after pollination and established in in ovulo embryo culture. Seedlings from this planned cross were field

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Bekir Şan, Adnan Nurhan Yildirim, and Fatma Yildirim

heating Nat. Areas J. 17 313 323 Baskin, J.M. Baskin, C.C. 2004 A classification system for seed dormancy Seed Sci. Res. 14 1 16 Bewley, J.D. 1997 Seed germination and dormancy Plant Cell 9 1055 1066 Bridgen, M.P. 1994 A review of plant embryo culture