Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).
Knowledge of the chromosome number in Rubus would be valuable when planning crosses and identifying plants, etc., however, preparation of tissue for microscopic evaluation and chromosome counting is difficult and time-consuming. Flow cytometry offers a more-efficient approach to this task. DNA flow cytometry was used to determine the nuclear DNA content in 22 Rubus genotypes. The genotypes represented a range of reported chromosome numbers from 2x to 12x. Six of the genotypes were representatives of Rubus ursinus, which is reported to have both 8x and 12x forms. Samples of nuclei were prepared from leaf discs of newly emerged and mature leaves following published protocols with some modifications. The DNA content was estimated by comparison of the fluorescence of Rubus nuclei with an internal DNA standard. There was an increase in nuclear DNA content concurrent with the increase in chromosome number. In these studies DNA flow cytometry could differentiate genotypes that differed by 2x, such as 6x and 8x, but could not reliably distinguish genotypes that differed by 1x, such as 7x vs. 8x or 6x. Aneuploids cannot be differentiated at this time.
`Marion' is the most important blackberry cultivar in the world, primarily due to its outstanding processing characteristics. Ideally, `Marion' could be enhanced via transformation while maintaining its fruit quality. As a successful in vitro regeneration is the prerequisite for genetic transformation, a series of experiments were conducted to optimize the conditions for in vitro regeneration for `Marion' blackberry. Parameters studied included types (three cytokinins: BA, kinetin, and zeatin; four auxins: IBA, IAA, NAA, and 2,4-D) and concentrations of plant growth regulators, explants (leaf and petiole), medium formulations (MS, WPM, and BMM), and the duration of TDZ pretreatment (3 to 6 weeks) of in vitro-grown stock plants. The highest shoot regeneration rate (65.7%) and highest number of shoots (5.1 shoots/explant) were obtained under the following conditions: stock plants were pretreated with TDZ (1 mm) for 3 weeks, followed by leaf explants dark pretreatment for 1 week on the regeneration medium (WPM with 5 mM BA and 0. 5 mm IBA). After dark treatment, regeneration plates were placed under 16-h photoperiod at light intensity of ≈50 μmol·m-2·s-1 at 23 + 2 °C for 4 weeks.
Experiments focusing on plant growth regulators' concentrations and combinations, mineral salt formulations, and TDZ pretreatment formations were conducted to optimize in vitro shoot regeneration from leaf and petiole explants of `Marion' blackberry. Optimum shoot formation was obtained when stock plants were incubated in TDZ pretreatment medium for 3 weeks before culturing leaf explants on regeneration medium (Woody Plant Medium with 5 μm BA and 0.5 μm IBA) in darkness for 1 week before transfer to light photoperiod (16-hour photoperiod at photosynthetic photon flux of ≈50 μmol·m-2·s-1) at 23 °C ± 2 °C for 4 weeks. Under these conditions, ≈70% of leaf explants formed ≈40 shoots per petri dish that could be harvested and rooted to form plantlets. Chemical names used: N6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); gibberellic acid (GA3); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); N-phenyl-N'-1,2,3-thidiazol-5-ylurea [thidiazuron (TDZ)].