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Mark P. Bridgen

Traditional and biotechnological breeding techniques are being united to develop exciting new plants and to improve existing cultivated plants by introducing natural variability from germplasm resources. Intervarietal, interspecific and intergeneric crosses can be accomplished by using plant embryo culture techniques, sometimes also referred to as embryo rescue. Embryo culture involves the isolation and growth of immature or mature zygotic embryos under sterile conditions on an aseptic nutrient medium with the goal of obtaining a viable plant. The technique depends on isolating the embryo without injury, formulating a suitable nutrient medium, and inducing continued embryogenic growth and seedling formation. The culture of immature embryos is used to rescue embryos from hybrid crosses that were once thought to be incompatible because they would normally abort or not undergo the progressive sequence of ontogeny. The culture of mature embryos from ripened seeds is used to eliminate seed germination inhibitors, to overcome dormancy restrictions, or to shorten the breeding cycle. New and exciting cultivars of Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, have been bred by using zygotic embryo culture; these techniques and applications will be discussed.

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Charlotte R. Chan and Robert D. Marquard

Traditional seed propagation (warm/cold stratification) was compared to embryo culture of Chionanthus virginicus L. to determine if germination could be promoted and time necessary to produce a sizable plant could be reduced. Embryos of C. virginicus were extracted from immature fruit collected 9, 16, and 23 Aug. 1995 and grown in vitro on Anderson's rhododendron medium. They germinated in 4 weeks and were transferred ex vitro to flats. Mature fruit from the same source were grown simultaneously using warm/cold stratification. The two groups were evaluated periodically over a 2-year period for percent germination, plant size, and seedling success. The embryo-cultured plants had a lower survival rate (16% vs. 44%) and were more labor intensive. After 2 years, embryo-cultured plants were 13.4-fold the mass and 4.7-fold taller than traditionally grown plants. Ten-month-old cultured plants were comparable in size to 2-year-old plants grown traditionally.

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Richard T. Olsen, Thomas G. Ranney, and Zenaida Viloria

A series of studies were conducted to determine medium components necessary for ovule and embryo culture of ×Chitalpatashkentensis Elias & Wisura hybrids in order to improve recovery of interploid crosses. Ovules were collected at 2, 3, 4, 5, and 6 weeks after pollination (WAP) from selfed tetraploid × Chitalpa (S) and tetraploid × Chitalp × diploid Catalpabignonioides Walt. (3×) hybrids. Excised ovules were placed in petri dishes with Schenk and Hildebrandt (SH) medium and 0.7% agar, with or without coconut-water (2%) and three sucrose concentrations (20, 40, or 80 g·L-1). No ovules germinated for either cross in any treatment at 2, 3, and 4 WAP. Selfed ovules germinated at 5 WAP, in both 20 and 40 g·L-1 sucrose. At 6 WAP, 3× ovules germinated in 20 g·L-1 sucrose. Coconut water provided no apparent benefit. Embryos were apparent at 6 WAP, so a new study was initiated to compare ovule vs. embryo culture at this sample date. Excised embryos germinated in greater percentages than ovules, in all treatment combinations at 6 WAP. Germination in 80 g·L-1 sucrose was observed only for S embryos without coconut water. Greatest 3× germination (16.7%) was observed for embryos in 20 g·L-1 sucrose without coconut water. A final study was conducted to investigate the effect of gibberellic acid (GA3) on embryo germination. Embryos were harvested at 7 WAP for both crosses and grown in SH medium supplemented with 20 g·L-1 sucrose and 0, 1, 2, or 4 μm GA3. The addition of GA3, regardless of concentration, increased germination from 30.6% to 99.1% for S embryos and from 11.1% to 99.1% for 3× embryos.

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Denis Lauzer and Claire Laberge

To update and complete a collection of wild roses in the Montreal Botanical Garden, Canada, in vitro embryo culture was used to propagate several Rosa species that are only available as seeds and difficult to germinate conventionally. Using embryo culture, it was possible to overcome seed dormancy and to rapidly increase the number of species in the collection, and this from a very limited number of seeds obtained from botanical institutions located around the world.

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Jonathan W. Sinclair and David H. Byrne

Carbohydrate source of peach [Prunus persica (L.) Batsch] embryo culture media affects embryo growth and survival. The first objective of this study was to determine the effect of five carbohydrates (fructose, glucose, maltose, sorbitol, and sucrose) in Woody Plant Medium (WPM) on the germination and survival of peach embryos in vitro. Fructose, glucose, maltose, and sucrose in WPM resulted in better embryo germination and survival than sorbitol. Fructose (2% and 3%) produced greater survival than all other carbohydrates tested in smaller embryos (<10% ovule dry weight). However, sucrose was better than all other carbohydrates tested in the larger embryos (≥10% ovule dry weight). In addition, large embryos (>10% ovule dry weight) on fructose at 1% combined with glucose, maltose, sorbitol, or sucrose at 1% had equivalent or higher survival than did those on either 1% or 2% sucrose in conjunction with the same carbohydrates. Embryo survival on different carbohydrates varied with genotype. The second objective of this study was to determine the effect of three levels of MES buffer (0.0 mm, 4.5 mm, and 9.0 mm) on medium pH stability and embryo survival. MES buffer at 0.0 mm and 4.5 mm concentration produced significantly better embryo survival than 9.0 mm. The pH stability was better at MES 9.0 mm, however survival decreased significantly. Chemical name used: [2-(N-morpholino)-ethane sulphonic acid] (MES)

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Richard T. Olsen, Thomas G. Ranney, and Zenaida Viloria

×Chitalpa tashkentensis Elias & Wisura is a sterile intergeneric hybrid [Catalpa bignonioides Walt. × Chilopsis linearis (Cav.) Sweet]. To restore fertility in ×Chitalpa the following were evaluated: 1) oryzalin as a polyploidization agent, 2) fertility of induced polyploids, and 3) in vitro culture methods for embryo rescue of interploid crosses. Meristems of ×Chitalpa `Pink Dawn' were submerged in an aqueous solution of 150 μm oryzalin for 0, 6, 12, or 24 hours and ploidy analyzed via flow cytometry. As treatment duration increased, recovery of diploids decreased as mixoploids and shoot mortality increased. Two tetraploid shoots occurred in the 24-hour treatment. Four tetraploids and two cytochimeras were stabilized in total. Tetraploids flowered sparsely; however, cytochimeras flowered profusely and these were used to study fertility at the tetraploid level. Diploid ×Chitalpa `Pink Dawn' pollen was essentially nonviable, but cytochimera pollen stained and germinated equal to or greater than pollen of C. bignonioides and C. linearis `Bubba'. Cytochimera ×Chitalpa were selfed yielding tetraploid seedlings, crossed with C. bignonioides to yield triploids, but failed in reciprocal crosses with C. linearis `Bubba' and `Burgundy Lace'. To increase recovery of triploids, germination of triploid and tetraploid embryos was investigated, as either intact ovules or excised embryos, on Schenk and Hildebrandt (SH) basal salts supplemented with sucrose at 20, 40, and 80 g·L-1, presence or absence of 2% coconut-water, and gibberellic acid (GA3) at 0, 1, 2, or 4 μm, and harvested weekly beginning 2 weeks after pollination (WAP). Germination of triploids (cytochimera ×Chitalpa × diploid C. bignonioides) and tetraploids (selfed cytochimera ×Chitalpa) were greatest with excised embryos at 7 WAP on SH supplemented with sucrose at 20 g·L-1 and ≥1 μm GA3. Germination of triploids (diploid C. linearis × cytochimera ×Chitalpa) was <5% at 4, 5, or 6 WAP on the same medium as above. Oryzalin effectively induced polyploidy and restored fertility in ×Chitalpa `Pink Dawn'. Successful crosses between hybrid and parental taxa of different ploidy levels, coupled with embryo culture will facilitate a ×Chitalpa breeding program. Chemical names used: 4(dipropylamino)-3,5-dinitrobenzenesulfonamide (oryzalin).

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Juan Bernardo Pérez-Hernández and María José Grajal-Martín

been used for the maturation of mango somatic embryos, but reports on its influence on zygotic embryo maturation are absent. The aim of the present study was to establish a protocol for in vitro plant recovery through embryo culture and test its

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Yan Yao, Yao Kong, Ping Zhang, Hua Zhang, Hong-di Huang, and Guang-guang Li

accelerate flowering, the technique of young embryo culture in vitro has also been used to shorten the lifecycles by circumventing the requirements of seed maturation and postharvest dormancy ( Mobini and Warkentin, 2016 ; Zheng et al., 2013 ). The suitable

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Mark P. Bridgen

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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.