D. J. Gray and L.A. Hanger
Michael E. Compton and D.J. Gray
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).
Michael E. Compton and D.J. Gray
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.
D.J. Gray, D.W. McColley, and Michael E. Compton
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).
Michael E. Compton, J.W. Harris, and D.J. Gray
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.
Michael E. Compton, D.J. Gray, and G.W. Elmstrom
Tetraploid individuals were identified among plants regenerated from cotyledons of diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultured in vitro. Tetraploid and diploid plants were distinguished by counting the number of chloroplast per guard cell pair. The mean number of chloroplasts was 19 and 11 for tetraploid and diploid plants, respectively. Self-fertile tetraploids were obtained from the diploid cultivars Mickylee, Jubilee II and Royal Sweet. `Dixielee' and `Minilee' tetraploids failed to set fruit. Progeny obtained from self-fertile tetraploids were crossed with diploid pollinators to produce triploid hybrid seed. All triploid plants produced seedless fruit that was superior or equal to fruit produced by currently available triploid hybrids. This demonstrates that tissue culture can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.
M. Meyerson, C.M. Benton, and D.J. Gray
Micropropagation of Vitis bourquiniana Lenoir `Black Spanish', V. champini Planchon `Dog Ridge', Vitis hybrids (`Blanc du Bois', `Himrod', and `Niagara Seedless'), V. rotundifolia Michx. (`Carlos' and `Dixie'), and V. vinifera L. (`Autumn Seedless', `Cabernet Sauvignon', `Carignane', `French Colombard', `Ruby Cabernet', and `Tokay') was accomplished. Shoot tips taken from micropropagated plants in long-term culture were inoculated onto solidified C2D medium containing 5 μM benzyladenine. Culture times consisting of either one or two 4-week cycles were compared for effect on shoot number. A range of response among cultivars tested was noted. The best-responding variety was V. champini `Dog Ridge', with 5.8 shoots per apex. All other varieties were less prolific. When shoot micropropagation from nodal explants and apices was compared, so significant difference was noted. In vitro micropropagation offers rapid clonal production of grape and is a source of sterile leaf explant material for embryogenic cultures, which, in turn, are useful target for genetic transformation.
D.J. Gray, K.A. Labeau, and C.M. Benton
The development of grape somatic embryos (Vitis vinifera cv. Thompson Seedless) was studied using high-resolution light microscopy and scanning electron microscopy. Somatic embryos develop either from discrete embryogenic cell clusters (indirect somatic embryogenesis) or from previously formed somatic embryos (direct somatic embryogenesis). In both instances, embryo development begins when a small, isodiametric, densely cytoplasmic cell undergoes a series of organized divisions, which are identical to those observed during zygotic embryogenesis. Developing embryos pass through recognizable embryonic stages, remaining white and opaque through maturity. Upon germination, embryos begin to enlarge, become yellow, then green, and develop into morphologically correct plants. The cells of somatic embryos contain little starch, but abundant storage proteins. However, lipids comprise the primary storage compound. Some developmental abnormalities occur during embryogenesis, including overly enlarged hypocotyls and fewer or more than two cotyledons. In addition, relatively few somatic embryos grow into plants primarily due to inadequate shoot apical meristem development. These abnormalities are best attributed to inadequacies of the in vitro environment of medium in a culture vessel when compared to the in vivo environment of a seed.
D.J. Gray, R.N. Trigiano, and B.V. Conger
Orchardgrass is a member of the Poaceae, a family characterized by monocotyledonous-type embryos. This species is unique in that somatic embryogenesis can be induced from the cultured leaves of potted plants, which can be maintained conveniently in the greenhouse. Both embryogenic callus and isolated somatic embryos develop directly from cultured leaf sections. Embryogenic callus can be maintained indefinitely. Somatic embryos exhibit typical embryo morphology and germinate readily into plants. Exercises designed to lead students through aspects of culture initiation, maintenance, and plant regeneration are described.
R. Scorza, J.M. Cordts, D.J. Gray, D. Gonsalves, R.L. Emershad, and D.W. Ramming
Transgenic grape plants were regenerated from somatic embryos derived from leaves of in vitro-grown plants of `Thompson Seedless' grape (Vitis vinifera L.) plants. Somatic embryos were either exposed directly to engineered Agrobacterium tumefaciens or they were bombarded twice with 1-μm gold particles and then exposed to A. tumefaciens. Somatic embryos were transformed with either the lytic peptide Shiva-1 gene or the tomato ringspot virus (TomRSV) coat protein (CP) gene. After cocultivation, secondary embryos proliferated on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 40 μg·mL-1 kanamycin (kan). Transgenic embryos were identified after 3 to 5 months under selection and allowed to germinate and develop into rooted plants on woody plant medium containing 1 μm 6-benzylaminopurine, 1.5% sucrose, 0.3% activated charcoal, and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kanamycin (kan), positive β-glucuronidase (GUS) and polymerase chain-reaction (PCR) assays, and Southern analysis.