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- Author or Editor: D.J. Gray x
Orthodox seeds enter an arrested growth phase during their final stages of development that follows closely after seed coat hardening, reduction of water content, and maturation of embryonic and storage tissues. This resting phase may last for a number of years, depending on species and environmental conditions (2), and is the major factor accounting for the efficient storage and handling qualities of seed. A similar resting phase induced in synthetic seed will be essential for propagation of many annual crop plants, especially those grown at high density over large areas (e.g., com, soybean, wheat, etc.) where planting efficiencies must be optimum. Additionally, a resting phase will be needed if synthetic seed is to be used for germplasm preservation.
The prospect of using plant somatic embryos produced in tissue culture as synthetic seeds has been a subject of increased interest, as evidenced by recent news articles (e.g., refs 7, 8, and 12). This interest is partly stimulated by the now more than 150 agriculturally important species for which somatic embryogenesis has been obtained (14). For example, somatic embryogenesis is now routine in soybean (1, 11) as well as many grasses and cereals (3, 15). In addition, recently reported methods for embryogenesis and plant regeneration from tissue cultures of European larch (10), Norway spruce (5, 6), and sugar pine (4) may represent long-awaited break-throughs for conifers.
Potential applications of synthetic seed technology to agriculture are numerous, but certain milestones are ahead. The driving force behind commercialization must be the added benefit conferred by use of synthetic seed-derived plants, and this benefit will vary greatly in magnitude depending on specific applications. Increased crop value must be great enough to justify new research and development costs that are not encountered in traditional seed production. Anticipated profit for a few high-value applications will fuel basic advances in technology that will be adaptable to many other crops and applications.
‘Orlando Seedless’, the first seedless grape with resistance to Pierce’s disease (PD), has been released by the Univ. of Florida (Fig. 1). It was developed from the grape breeding program at the Agricultural Research and Education Center in Leesburg (3). ‘Orlando Seedless’ is productive and long-lived in Florida, and was unaffected by the recent cold winters in Florida (− 10°C) that killed citrus and damaged muscadine grapes (Vitis rotundifolia).
Cotyledon explants of four watermelon [Citrullus lanatus (Thunb.) Mataum. & Nakai] breeding lines (F92U8, SP90-1, SP90-2, and SP90-4) were prepared from mature seed or from 2-, 4-, 6-, 8-, or 10-day-old seedlings. Explants were incubated on shoot regeneration medium for 8 weeks followed by 4 weeks on shoot elongation medium. The four genotypes differed in their ability to produce shoots at each explant age. The highest frequency with which F92U8 (66%) and SP90-2 (60%) explants produced shoots was for 2-day-old seedlings. Fewer explants formed shoots when established from mature seed or seedlings older than 2 days. In contrast, the percentage of SP90-4 explants that produced shoots was highest when cotyledons were obtained from 4-day-old seedlings (40%), but the response was less than the optimum for F92U8 and SP90-2. SP90-1 cotyledon explants exhibited the poorest response of the four breeding lines (<11% produced shoots), with little difference in response among the explant ages tested. The number of shoots per responding explant also depended on the age of the explant source. Explants from 2- to 4-day-old seedlings produced the most shoots. Fewer shoots formed on cotyledons from mature seed or seedlings older than 4 days.
Adventitious shoots were obtained from watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai] cotyledons incubated on a modified Murashige and Skoog medium containing BA. Initial experiments comparing the effects of BA (0, 5, 10, or 20 μm) and IA4 (0, 0.5, or 5 μm) demonstrated that BA was required for adventitious shoot formation but its concentration in the medium was not critical. The addition of IAA to medium with BA increased callus production and inhibited shoot formation. However, the percentage of responding explants in the best treatment was <30%. Therefore, the manner in which cotyledon explants were prepared and seedling age at the time of explantation was examined to improve the organogenic response. The percentage of explants with shoots was improved by using explants that consisted of cotyledon bases (43%) or cotyledons cut in half longitudinally (39%). A lower percentage (16%) of cotyledons cut longitudinally into four pieces produced shoots. Explants taken from the apical half of cotyledons failed to regenerate shoots. Shoot formation was improved further by using explants from young seedlings. The percentage of explants with shoots was >90% for `Minilee', 64% for S86NE, and 50% for `Jubilee II' when explants were prepared from 5-day-old seedlings. Explants from nongerminated embryos or seedlings germinated for 10, 15, or 20 days produced fewer shoots. The effect of several cytokinins on shoot organogenesis was then examined using the optimized protocol. The percentage of explants with shoots and the number of shoots per explant were about two to four times higher when 5 to 10 μm BA was used compared to the most effective kinetin (20 μm) or thidiazuron (0.1 μm) concentration. The percentage of explants with shoots and the number of shoots per explant were greater for diploid (57% and 2.2, respectively) than for triploid (22% and 0.6, respectively) or tetraploid (20% and 0.8, respectively) lines. Chemical names used: N -(phenylmethyl)-1 H -purin-6-amine (BA); 6-furfurylaminopurine (kinetin); N -phenyl-N' -1,2,3-thiadiazol-5-ylurea (thidiazuron); 1 H -indole3-acetic acid (IAA).
A protocol for high-frequency somatic embryogenesis in Cucumis melo L. was developed using `Male Sterile A147 as a model cultivar. Basal halves of quiescent seed cotyledons were cultured on embryo induction (EI) medium containing concentration ranges of the auxin 2,4-D and the cytokinins BA, Bin, TDZ, or 2iP before transfer to embryo development (ED) medium. Medium with 2,4-D at 5 mg·liter-1 and TDZ at 0.1 mg·liter-1 was superior, with 49% of explants responding and an average of 3.3 somatic embryos per explant (6.8 somatic embryos per responding explant). More explants produced embryos when incubated on EI medium for 1 or 2 weeks (30% and 33%) than for 3 or 4 weeks or with no induction. However, 2 weeks was 2.9 times better than 1 week in terms of number of embryos per explant. One week of initial culture in darkness, followed by a 16 hour light/8 hour dark photoperiod, produced more responding explants (26%) than two or more weeks in darkness or no dark period at all; but 1 and 2 weeks of darkness resulted in a similar number of embryos per explant (2.1 and 2.8). Sucrose concentration in EI and ED media had a highly significant effect on embryo induction and development. EI medium with 3% sucrose resulted in more embryogenic explants than EI medium with 1.5% or 6% sucrose. However, treatments with 3% sucrose in EI medium and 3% or 6% sucrose in ED medium produced significantly more embryos per explant (8.5 and 11.9) than other treatments. Treatments did not affect embryo induction directly and regeneration per se but, instead, frequency and efficiency of somatic embryo development. The optimal treatments were tested with 51 other commercial varieties. All varieties underwent somatic embryogenesis, exhibiting a response of 5% to 100% explant response and 0.1-20.2 embryos per explant. Chemical names used: N-(phenylmethyl)-lH-purin-6-amine (benzyladenine or BA); N-(2-furanylmethyl)-lH-purin-6-amine (kinetin or BIN); N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ); N-(3-methyl-2-butenyl)-lH-purin-6-amine (2iP); (2,4-dichlorophenoxy) acetic acid (2,4-D).
Ploidy of in vitro watermelon plantlets was estimated by painting the lower epidermis of leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from shoot-tip cultures of known diploid and tetraploid cultivars were used to establish the mean number of chloroplasts per guard cell pair for in vitro plantlets. Leaves from diploid and tetraploid plantlets had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method was used to estimate ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 10.6% of regenerated shoots were classified as tetraploid while still in vitro. Putative tetraploids were transplanted to the field and self-pollinated. A majority of polyploids identified in vitro were true breeding, nonchimeric tetraploids. This study demonstrate that FDA can be used to estimate ploidy of in vitro shoots of watermelon prior to acclimatization and transfer of plants to the greenhouse or field.
In ovulo embryo rescue provides an attractive alternative to conventional methods of breeding for seedless grape (Vitis) by allowing recovery of progeny from abortive ovules of seedless × seedless crosses. This technique theoretically increases the proportion of seedless progeny that can be obtained in one breeding cycle (1). Proper inheritance studies can be accomplished by heretofore impossible direct combinations of seedless cultivars.