Several methods have been published on shoot regeneration from watermelon cotyledon explants. The major differences in regeneration protocols include the light environment in which seeds are germinated and the cotyledon region used. The purpose of these experiments was to compare the two main protocols for plant regeneration and develop one general procedure. To fulfill this objective, seeds were germinated in vitro in darkness or 16-hr light photoperiod for 7 days. Cotyledon explants from four watermelon cultivars (`Crimson Sweet', `Minilee', `Sweet Gem', and `Yellow Doll') were prepared from both dark- and light-grown seedlings. Apical and basal halves were obtained by making a cut across the cotyledon width. Apical and basal quarters were made, for comparison, by cutting apical and basal halves longitudinally. All explants were incubated on shoot regeneration medium for 6 weeks followed by a 3-week cycle on shoot elonga-tion medium. The percentage of cotyledons with shoots was 1.7-fold greater for cotyledons derived from seedings incubated in darkness than those germinated in light. Shoot formation was about 10-fold greater for explants from cotyledon basal halves and quarters than apical halves and quarters. According to these results, the best watermelon regeneration protocol should consists of basal explants from in vitro-germinated seedlings incubated in the dark for 7 days.
Michael E. Compton
Organic competence of different explant sizes and locations on watermelon seedlings was determined by calculating the percentage of cotyledon explants that produced adventitious shoots. About 52% (214/412) of explants prepared from the proximal region of cotyledons formed shoots, whereas only ≈6% (24/411) of distal explants did so. Shoot formation was limited to the proximal end of basal explants but was not restricted to any specific region on distal ones. The percentage of explants that produced harvestable shoots was greater from basal halves than basal quarters in `Sweet Gem', `Crimson Sweet', and `Minilee', but explant size did not affect adventitious shoot regeneration of `Yellow Doll', resulting in significant interaction between cultivar and explant size. This study indicates that cultivars that respond poorly to in vitro procedures may have fewer cells competent for shoot regeneration, requiring special care during explant preparation.
Michael E. Compton
Fifty high schools were surveyed in northwestern Illinois, northeastern Iowa, southeastern Minnesota, and Wisconsin to determine the number of students interested in pursuing a horticulture degree at a 4-year university. Students were asked several questions pertaining to horticulture. About 45% of our surveys were returned. Of the 451 surveys received, about 47% of the high school freshman, sophomore, junior, and seniors indicated that they were interested in horticulture. About 41% of the students interested in horticulture wanted to work in landscaping, 20% greenhouse, 14% florist shop, and 7% in turfgrass management. About 70% of the students indicated that they wanted to own and operate their own horticultural business. Almost 53% of the students indicated that they would prefer an emphasis/minor in Agribusiness or Business Administration compared to plant and soil science (19%), biotechnology (14%), plant breeding and genetics (13%), or comprehensive horticulture (1%) in combination with their horticulture degree. The above information was used by our School of Agriculture and Depts. of Biology and Business and Accounting to develop a major in Ornamental Horticulture.
Michael E. Compton and Richard E. Veilleux
Genetic recombination rates of hybrid plants regenerated from three tissue culture. systems were compared by backcrossing regenerated plants with mutant parents and comparing the observed crossover frequencies with those expected based on control plants raised from seed. Increased recombination rates and map distances were observed among plants from micropropagated shoot tips (4.5%-5.9%), cotyledon calli (3.7%-8.5%), and thin cell layers (2.8%-6.5%) between the sunny (sy) and baby leaf syndrome (bls) markers which flank the centromere on chromosome 3. Conversely, a decrease in map distance was observed between bls and the solanifolia (sf) locus which is more distal to the centromere on the same arm of chromosome 3 as bls. Increased map distance among plants regenerated from micropropagated shoot tips, cotyledon calli, and thin cell layers was also observed between white virescence (wv) and anthocyanin reduced (are) loci on chromosome 2.
Laura R. Bahr and Michael E. Compton
Competence for in vitro bulblet regeneration was investigated for eight diverse Lilium genotypes (L. `Citronella', L. `Stargazer', L. `Stones', L. `Lovely Girl', L. pumilum, L. lancifolium, L. lancifolium `Orange Star', and L. speciosum var. rubrum). Explants established from bulb scales were cultured on lily bulblet regeneration medium [Murashige and Skoog (1962) modified by reducing the NH4NO3 to 0.825 g·L-1 and KH2PO4 to 0.170 g·L-1, and adding (per liter) 30 g sucrose, 0.1 g myoinositol, 0.4 mg thiamine·HCl, 80 mg adenine sulfate, 16 μm naphthaleneacetic acid, 2 mL·L-1 Plant Preservative Mixture (PPM), and 4.5 g·L-1 AgarGel at pH 5.7] for 8 weeks before transfer to sphagnum peat moss and 4 weeks refrigeration at 5 °C in darkness. All genotypes produced bulblets in vitro. However, the response rate varied among genotypes. Explants of Lilium `Lovely Girl', L. `Citronella', and L. speciosum var. rubrum were most responsive with ≈90% producing bulblets. The number of bulblets per responding explant ranged from 5.2 (`Stones') to 2.3 (L. lancifolium). When comparing in vitro and greenhouse bulblet production, about twice as many bulblets were produced by explants in vitro compared to halved scales incubated in the greenhouse. The percentage of responding explants ranged from 33% to 360% greater for six of the genotypes tested when propagated in vitro compared to bulb scales propagated in the greenhouse. Following refrigeration, bulblets were transferred to the greenhouse for sprouting. Over 80% of bulblets obtained from L. `Citronella', L. lancifolium `Orange Star', L. lancifolium, and L. `Stones' sprouted in the greenhouse. This study illustrates that a diverse range of lily species can be successfully propagated in vitro using a single medium formulation and is the first report of bulblet regeneration in vitro for L. `Citronella', L. `Stones', L. `Lovely Girl', and L. lancifolium `Orange Star'.
Jason M. Jaworski and Michael E. Compton
Cotyledon explants of five watermelon cultivars (`Desert King', `Mickylee', `Sangria', `Sweet Princess', and `Male Sterile') were prepared from 7-day-old in vitro-germinated seedlings. Explants were incubated on shoot regeneration medium for 6 weeks, followed by several 3-week cycles on shoot elongation medium. The five cultivars differed in their ability to form shoots within 9 weeks on the selected media. Shoot regeneration frequency was about 1.5to 2.9-fold greater for `Mickylee' (60%) than `Sangria' (47%), `Sweet Princess' (27%), `Male Sterile' (26%), and `Desert King' (24%). Rooting of elongated shoots (>2 cm) occurred within 2 weeks on medium containing 1 μM IBA and ranged from 25% (`Desert King') to 92% (`Sangria'). Plantlets were transferred to six-pack containers filled with soilless medium (1 Sunshine Mix: 1 coarse perlite) and covered with a transparent plastic lid. Plants were acclimatized to ambient conditions by gradually removing the lid over a period of 3 days after new growth was observed. The percentage of acclimatized plants ranged from 50% (`Sweet Princess' and `Mickylee') to 100% (`Male Sterile'). Acclimatized plants were transferred to the greenhouse and grown for at least 4 weeks before screening for ploidy variants. Ploidy of regenerated plants was estimated by counting the number of chloroplasts per guard cell pair. Plants with an average of 18 or more chloroplasts per guard cell pair were declared tetraploids. Plants with fewer chloroplasts per guard cell pair were declared diploids and discarded. Tetraploid plants were transferred to the field, grown to maturity, and self-pollinated for seed increase.
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.
Michael E. Compton, Brenda L. Fuchs, and Jack E. Staub
Cucumis hystrix Chakr. is a rare cucurbit species native to Asia. The species is valued by breeders because of its multiple branching habit and has been used in interspecific crosses with Cucumis sativus. However, individual C. hystrix plants have not been identified in the wild since 1990. Therefore, it was our objective to develop a micropropagation protocol that would allow us to clonally propagate plants in cultivation. Shoots tips (2 cm) were excised from a single C. hystrix plant grown in the greenhouse. All tendrils and leaves were removed before surface-sterilization in 1.25% NaOCl for 5 or 10 min and rinsed six times with sterile distilled water. Shoot tips were trimmed to 1 cm (meristem with two to three young leaf primordia) and placed into 25 × 125-mm test tubes containing 25 ml of initiation medium [MS plus (per liter) 100 mg inositol, 30 g sucrose and 5 g Agargel; pH 5.7-5.8]. PGR combinations tested were initiation medium with 1 μM BA, and initiation medium with 1.7 μM IBA, 0.5 μM kinetin and 0.3 μM GA3 (IKG). Explant survival was greater when shoot tips were surface-sterilized for 5 min (75%) compared to 10 min (33%). More axillary shoots formed when shoot tips were cultured in IKG medium (10.8) than in medium with BA (5.5). Shoots were considerably longer (10 mm) when cultured in medium with IKG compared to BA (1.5 mm). About 64% of shoots place in medium containing 8 μM NAA formed roots and were acclimatized to greenhouse conditions.
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).