Abbreviations: NAA, a-naphthaleneacetic acid; 2, 4-D,2,4-dichlorophenoxy-acetic acid; PGR, plant growth regulators; ZYMV, Zucchini yellow mosaic virus. 1 Postdoctorate Research Assistant. To whom reprint requests should be addressed. 2 Associate
Michael E. Compton and D.J. Gray
Mahmoud A. Hassan and Harry Jan Swartz
An efficient protocol for plantlet regeneration using petioles and internodes of two blackberry cultivars has been developed for use in genetic transformation. Maximum shoot regeneration was induced on MS medium supplemented with 5 μM Thidiazuron (TDZ) and 0.5 μM IBA. Preconditioning the source shoots on 0.5 μM TDZ containing MS medium did increase the number of regenerated shoots/explant, but did not affect the regeneration percentage. The antibiotic, kanamycin, significantly reduced the growth and regeneration of explants at 10 mg/L or higher. In contrast, cefatoxime at 100-500 mg/L increased explant growth and percentage regeneration.
Mohamed F. Mohamed, P. E. Read, and D. P. Coyne
Regeneration in vitro from the embryonic axis in Phaseolus sp. has not been reported. Two embryo sizes, 0.3-0.4 mm and 0.6-0.7 mm long at 10-12 and 21 days after pollination, respectively, were excised from 4 P. vulgaris (P.v.) and 2 P. acutifolius (P.a.) genotypes. The embryonic leaves and radicale were removed, and 0.1-0.2 mm of the embryonic axis was cultured on Gamborg's B5 medium with 0, 5, 10 and 20μ MBA. The cultures were incubated in the dark at 25°C for 2 weeks followed by 1 week in continuous cool white light (25μ MS-1m2) before transferring to the second medium (0, 2μ MBA and 2μ MBA + 4μ MGA3). The tissues from the larger embryos initiated a single shoot without PGR in 30% of 1 P.v. explants and 30-60% in 2 P.a. The other 3 P.v. formed roots only. Multiple shoots were initiated in all P.v. (15-60%) and in 2 P.a. (60 and 70%) with 5 or 10μ MBA. The tissues from the smaller embryos had single shoots for all genotypes (30-60%) without PGR. Multiple shoots were initiated in 50-80% and 75-90% of the explants from P.v. and P.a., respectively, with 5 or 10μ MBA. Excess callus formed with 20μ MBA and regeneration decreased. After 3 weeks on the second medium, 6-8 shoot s/P. v. and up to 15-20 shoots/Pa. explants were observed.
A detached-leaf bioassay was used to evaluate peach [Prunus persica (L.) Batsch] regenerants derived from zygotic embryo callus cultures of cultivars Sunhigh (susceptible to leaf spot) and Redhaven (moderately resistant to leaf spot) for resistance to Xanthomonas campestris pv. pruni [(E.F. Sm.) Dews], the causal agent of bacterial leaf spot. Regenerants obtained from calli produced on two `Sunhigh' embryos, #61 and #156, and on three `Redhaven' embryos were evaluated. Sixty-four percent of the regenerants derived from `Sunhigh' embryo #156 and 13% of the regenerants derived from `Sunhigh' embryo #61 demonstrated significantly greater spot resistance than `Sunhigh'. Regenerants with resistance greater than `Redhaven' were also obtained from both `Sunhigh' embryos. The frequency of variation in the `Sunhigh' seedling population, with respect to the response to bacterial leaf spot, was not so great as that exhibited by the regenerants derived from `Sunhigh' embryo #156. None of the `Redhaven' seedlings or any of the regenerants derived from `Redhaven' embryos were more resistant than `Redhaven'. These studies suggest that the frequency of somaclonal variation is genetically determined and that screening for somaclonal variation may be a feasible approach to obtaining leaf spot-resistant peach plants.
Roger L. Vallejo and Jane E. Polston
Cultured cotyledon and leaf pieces of five cultivars of Lycopersicon esculentum Mill. were tested in six culture media for their ability to produce shoots for transformation studies. The no. of tissue pieces with callus/total tissue pieces, quality of callus (size and vigor), no. of tissue pieces with shoots/total tissue pieces, and shoot quality (size and vigor) were measured. Cultivars tested were `Campbell 28', `Flora-Dade', `UC82b', and two breeding lines, Fla.7171 and Fla.7324. The six media used were Murashige and Skoog medium supplemented with six combinations of indole acetic acid (IAA) and cytokinins: A) 1 mg/l IAA + 1 mg/l kinetin, B) 0.5 mg/l IAA + 2 mg/l kinetin, C) 0.02 mg/l IAA + 1 mg/l zeatin, D) 0.2 mg/l IAA + 2 mg/l zestin, E) 1 mg/l IAA + 2.5 mg/l BAP (6-benzyl amino purine), and F) 0.2 mg/l IAA + 1 mg/l BAP. Standard procedures were followed for culturing 4 - 5 mm pieces of cotyledon and leaves. Callus and shoot regeneration were greater, less variable and faster, in cotyledon than in leaf pieces. Media C and F gave the highest rates of callus and shoot production, respectively, in cotyledon tissue. Medium E gave the highest rate for both callus and shoot production in leaf discs. The best rates of shoot production were achieved with cotyledon tissue from cultivar UC82b cultured on media C (85.3%) and F (77.2%).
Dennis P. Stimart and John C. Mather
Cotyledons from developing embryos 6 to 8 weeks old of Liatris spicata (blazing star) were cultured on Murashige-Skoog (MS) medium containing 0, 0.4, 4.4, and 44.4 μ M benzyladenine (BA) or 0, 0.2, 2.2, and 22.2 μ M thidiazuron (TDZ) to induce adventitious shoot formation. The highest percent of cotyledons forming shoots with highest shoot counts was on medium containing 2.2 μ M TDZ. Vitreous shoots formed on medium with 22.2 μ M TDZ. Callus derived from cotyledons and cultured on medium containing 4.44 μ M BA or 2.2 μ M TDZ formed adventitious shoots with highest shoot counts on 4.44 μ M BA. Adventitious shoots derived from cotyledons and callus were rooted on MS medium with 5.0 μ Mindole-3-butyric acid, acclimatized and grown ex vitro. All micropropagated plants appeared similar to each other.
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.
Grzegorz Bartoszewski, Cesar V. Mujer, Katarzyna Niemirowicz-Szczytt, and Ann C. Smigocki
A Lycopersicon esculentum Mill. (tomato) cDNA clone with high similarity to a Nicotiana plumbaginifolia Viv. (tobacco) cytochrome P450 gene was isolated using 5' and 3' rapid amplification of cDNA ends (RACE). The isolated cDNA (GenBank Accession No. AF249329) has an open reading frame of 1494 base pairs (bp) and encodes a protein of 498 amino acids with 75% identity to the N. plumbaginifolia cytochrome P450 (CYP72A2) and 45% to a Catharanthus roseus G. Don (Madagaskar periwinkle) CYP72A1 protein sequence. By Southern-blot analysis, one or two highly homologous genes were detected in the L. esculentum genome. Expression of the cloned P450 gene was regulated by circadian rhythm and enhanced by wounding. Leaf transcripts were detected in the light but not dark. Highest transcript levels were observed 3 hours after mechanical wounding. No increase in expression was seen in response to applications of zeatin as with the N. plumbaginifolia gene. Of the tissues analyzed, shoot tips and young leaves and fruit had the highest detectable transcript levels. Attempts to transform more than 1400 cotyledon explants of L. esculentum with sense or antisense CYP72A2 gene constructs produced no transgenic plants.
Xiaojuan Zong, Brandon J. Denler, Gharbia H. Danial, Yongjian Chang, and Guo-qing Song
., 2010 ). For most cultivars of Prunus species, leaf explants are preferable to seed-derived explants for maintaining an identical genetic background of the cultivars because of their heterozygosity and self-incompatibility. To date, plant regeneration
L. Xu, G.F. Liu, and M.Z. Bao
inherited traits. In L. styraciflua , plants have been regenerated via adventitious shoots from mature leaf and petiole segments ( Brand and Lineberger, 1988 ) and hypocotyl segments ( Kim et al., 1997 ), and via somatic embryogenesis from hypocotyl