embryos has been used to overcome early embryo abortion in interspecific hybrids ( Ledbetter et al., 1998 ; Liu et al., 2007 ; Stanys, 1998 ). The success of raising plants from immature or aborted embryos depends on their stage of maturity at culture
Arancha Arbeloa, Ma Elena Daorden, Elena García, Pilar Andreu, and Juan A. Marín
Juan Bernardo Pérez-Hernández and María José Grajal-Martín
less than 1% ( Usman et al., 2001 ); and 3) severe natural fruit drop that causes premature loss of many of the scarce fruits derived from putative successful hand crosses ( Bally et al., 2009 ). Therefore, in vitro culture of immature embryos has been
Mark P. Bridgen
143 WORKSHOP 20 (Abstr. 691-693) Underpublicized, Underutilized, and Innovative Plant Tissue Culture Techniques Wednesday, 26 July, 10:00 a.m.-12:00 noon
Ching-yeh Hu, Lee Wang, and Bernard Wu
Embryo culture can by-pass yew (Taxus) seed dormancy and produce large population of seedlings to be screened for the anticancer drug, taxol, production. Immature linear embryos from seeds of T. baccata, T. brevifolia. T. cuspidata, and T. media were dissected and cultured. B5 medium supported the best embryonic growth during the initial two week's culture for T. cuspidata and T. baccata. T. brevifolia grew faster on MS medium. Weak embryo dormancy was encountered in T. brevifolia and T. cuspidata from the mature seeds but not from the immature ones. No embryonic growth had been observed in T. media dissected from mature seeds due to strong dormancy. Developing embryos were subsequently transferred to 1/2X B5 medium for germination. Rooting percentage in the mature seed derived T. brevifolia embryos increased from 12.5 to 63.6 when 30 μM GA3 was added to the initial medium. Several hundreds of seedlings of T. baccata. T. brevifolia and T. cuspidata had been acclimatized to the greenhouse conditions. The taxol content of resultant T. cuspidata seedlings was 0.027% (dry weight), while that of T. brevifolia obtained from the wild twig was 0.030%.
Pedro A. Sansberro, Hebe Y. Rey, and Luis A. Mroginski
Plants of Ilex argentina L., I. brasiliensis (S.) L., I. brevicuspis R., I. dumosa R., I. integerrima (V.C.) L., I. microdonta R., I. pseudoboxus R., and I. theezans C.M. were obtained by immature embryo culture. Heart-stage zygotic embryos were removed from immature fruits and cultured aseptically on quarter-strength Murashige and Skoog medium with 3% sucrose, 0.65% agar, and 0.1 mg·L-1 zeatin. Cultures were incubated at 27±2°C for 4 weeks, in darkness and subsequently transferred to a culture room with a 14-hour photoperiod (116 μmol·m-2·s-1) for another 4 weeks. Seedlings with two leaves, derived from germinated embryos, were successfully transplanted to pots containing 1 peat: 1 perlite: 1 sand (v/v) and were maintained in greenhouse conditions. From 95% to 100% of transplanted seedlings survived. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).
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.
Paula P. Chee
An embryo culture method overcomes the lengthy dormancy requirement of Taxus L. spp. (yew) seeds. When zygotic embryos excised from mature T. brevifolia L. seeds were cultured in darkness for 4 weeks on one of three basal salt media (B5, Litvay, and Murashige and Skoog), radicle emergence and seedling development was highest on B5 basal salt medium. After 1 to 2 weeks on B5 basal salt medium, seedling development of T. brevifolia, T. cuspidata L., T. baccata L., and T. baccata stricta L. ranged from 2% to 36%. BA at 2.25 μm had no effect on radicle emergence; 22.5 μm prevented it. Embryos excised from mature or nearly mature seeds had the highest frequency of radicle emergence and seedling development. Cultured embryos developed seedlings in only 8 to 10 weeks. Chemical name used: N 6-benzyladenine (BA).
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.
D.W. Ramming, R.L. Emershad, P. Spiegel-Roy, N. Sahar, and I. Baron
Immature grape embryos from early ripening genotypes of Vitis vinifera were successfully cultured in vitro on Difco orchid agar or a modified White's agar medium. Germination was increased in vitro for five genotypes from 0%, 7%, 11%, 12%, and 16% in vivo to 15%, 24%, 23%, 34%, and 24%, respectively. Subculturing embryos onto liquid culture from seeds that failed to germinate on agar also was possible. Differences in germination rates, as affected by pollen, were significant. This method will allow accelerated development of early ripening cultivars by allowing breeders to use such genotypes as females, as well as males.
Ana Maria Sabja, David W.S. Mok, and Machteld C. Mok
Interspecific hybrid embryos resulting from crosses between Phaseolus species generally fail to reach maturity, and embryo rescue techniques are required to recover plants. To determine if ovary (pod) culture could permit interspecific hybrid embryo development, pods of P. vulgaris L. × P. acutifolius A. Gray and P. vulgaris L. (control) were cultured upright, supported by glasswool, in modified Murashige-Skoog liquid medium. The weight of seeds and length of embryos in P. vulgaris pods increased significantly during culture. However, usually only one or occasionally two seeds located at the middle positions of the pods developed to maturity. The same pod culture procedure promoted precocious germination of early cotyledonary-stage P. vulgaris × P. acutifolius embryos without further maturation of the embryos. These findings confirm that developmental arrest of the interspecific hybrid embryos in vivo is due to intrinsic deficiencies.