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 first documented by Charles Bonnet in the late 18th century, and it has been commercially practiced for more than half a century (Bridgen, 1994). Further research by Hannig (1904) refined the technique and laid the groundwork that would allow future scientists to bypass physical and/or late-onset chemical seed dormancy, shorten breeding cycles, examine seed/embryo viability, and develop hybrids from previously incompatible crosses (Bridgen, 1994). In plant breeding programs, ER is particularly critical because it can circumvent seed abortion of wide crosses, resulting in the retention of novel genotypes (Conger, 1981; Dunwell, 1986). Embryo rescue has also been widely used to efficiently propagate threatened and/or endangered species because it has been noted to improve germination rates (Lakshmi et al., 2010; Stephenson and Fahey, 2004).
Butterfly weed (Asclepias tuberosa) is one of 106 species that are indigenous to North America (Stevens, 1945; Woodson, 1954). Although milkweed (Asclepias) species have many ornamental and ecologically important traits, there is little commercial production outside of niche native plant growers. Limited commercial production is thought to result from limited seed set because milkweed species frequently display late-term seed and pod abortion, possibly due to complex reproductive structures (Broyles, 2002; Kephart, 1981; Shannon and Wyatt, 1986). The predominant theory regarding the failure of intraspecific and interspecific seed development in milkweed species is that late-term embryo abortion is attributed to postfertilization rejection, whereby fertilization of the endosperm is successful but the egg (precursor to the zygote) remains unfertilized. Gametophytic and sporophytic self-incompatibility systems typically experience abortion between a few hours to a few days after pollination; however, in milkweed species, late-term abortion can occur several weeks to 2 months after fertilization (Lipow and Wyatt, 2000; Seavey and Bawa, 1986; Sparrow and Pearson, 1948; Whiting, 1943). Embryo abortion is difficult to overcome; however, by prematurely harvesting seeds, removing the embryo, and aseptically culturing the embryo on nutrient medium, pod abortion and developmental failure could be circumvented.
There are no published ER protocols available for milkweed species. Several studies document embryogenesis protocols for milkweed species from cell cultures, leaf tissue, or nodal explants (Groet and Kidd, 1981; Kim et al., 2004; Pramanik and Datta, 1986; Sahai et al., 2010). Embryogenesis, compared with ER, takes longer because embryonic tissue must first be dedifferentiated, multiplied via callus formation, and then redifferentiated with the aid of plant growth regulators (PGRs) (Kim et al., 2004). Although large nutritional differences in ER protocols compared to those of embryogenesis protocols exist, a study of embryogenesis of indian ipecac (Tylophora indicia) found that this species, also in the milkweed family (Asclepiadaceae), preferred a relatively lower nutrient content compared with other taxa (Sahai et al., 2010). A half-strength MS medium increased explant regeneration of indian ipecac by more than 20% when applied at embryo maturation compared with other commonly used media (Sahai et al., 2010).
ER may be a commercially viable mechanism to overcome seed pod abortion if the embryo is excised from the seed at a point when it has reached the autotrophic (self-sufficient) stage but before seed pod abortion. However, an ER protocol for butterfly weed has not been documented; therefore, medium type, days of stratification, seed/explant cleaning methods, embryo culture environmental conditions, and the effect of embryo maturity on success are unknown. The primary objectives of this project were to 1) determine if it is possible to rescue embryos in the autotrophic stage through the development of an ER methodology; 2) assess the optimum embryo maturity to harvest seed and excise embryos for maximum growth of germinated embryos; and 3) determine if ER methods offer an improvement over seed germination rates using traditional seed germination in soilless substrates determine. Based on previous research of another milkweed species (indian ipecac), we hypothesized that media with less nutrition would be superior for successful germination of autotrophic embryos. We also predicted that germination rates of rescued embryos would exceed those observed with traditional commercial settings, and that embryos harvested in the heterotrophic phase of development would fail to produce viable plantlets.
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