Agrobacterium-mediated transformation and regeneration of guayule

Abstract

In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations: BA -benzyladenine; EDTA -ethylene diamine tetraacetate; kanR -kanamycin resistance; MS salts -salts of Murashige and Skoog medium (1962); NAA -naphthalene acetic acid; NPT-II -neomycin phosphotransferase; SDS -sodium dodecyl sulfate

Preliminary examination of factors affecting Agrobacterium tumefaciens-mediated transformation of marula, Sclerocarya birrea subsp. caffra (Anacardiacease)

“…Addition of acetosyringone to the bacterial suspension also increased transformation efficiencies in tamarillo (Atkinson and Gardner, 1993), Norway spruce (Wenck et al, 1999) and guayule (Pan et al, 1996) and was essential for putative transgenic callus differentiation in black poplar (Confalonieri et al, 1995) and tea (Matsumoto and Fukui, 2002). Wounding has been reported to promote Agrobacterium-mediated transformation (Pan et al, 1996;Park et al, 1996;Santare´m et al, 1998;Tang, 2002;Weber et al, 2003). Wounding may enhance the accessibility of plant cells for Agrobacterium, stimulate the production of vir gene inducers and enhance the plant cell competence for transformation.…”

A simplified protocol for micropropagation of guayule (Parthenium argentatum gray)

“…This inverse relationship between shoot multiplication rates and shoot length in response to concentration of BA in the medium was also observed by Smith (1983). While several studies report the use of BA for shoot multiplication, optimal concentrations varied between 0.1 and 2.0 mg l Ϫ1 (0.44-8.8 mM; Pan et al, 1996;Trautmann and Visser, 1990;Smith, 1983;Staba and Nygaard, 1983). This indicates that BA concentration adjustments may be necessary to obtain the desired multiplication rate and shoot length for specific guayule lines or for differences in culture conditions.…”

“…Shoot cultures were initially established following the protocol of Pan et al (1996). Although adequate shoot proliferation occurred, shoots were rather short (5-10 mm) and sometimes hyperhydric (tissues had swollen, translucent appearance).…”

“…Shoot cultures of guayule lines N6-5 and hybrid line AZ101 (P. argentatum  P. tomentosum var. stramonium; Thompson and Ray, 1989) were initiated from aseptically germinated seedlings by the method of Pan et al (1996). After 2-3 wk, shoots with three or more nodes were excised from the rest of the seedling (above the primary leaves) and transferred to shoot multiplication medium consisting of MS basal medium (Murashige and Skoog, 1962) supplemented with 3% sucrose, 1.0 mg l Ϫ1 (4.4 mM) 6-benzylaminopurine (BAP), 0.025 mg l Ϫ1 (0.13 mM) a -naphthaleneacetic acid (NAA) and 0.8% TC agar (Carolina Biological Supply Co., Burlington, NC) in Magenta GA-7 boxes (Sigma Chemical Co., St Louis, MO).…”

“…Although these procedures did lead to regeneration of shoots and, in some cases, whole plants, induction of callus tissue often requires use of high growth regulator concentrations and can increase the occurrence of somaclonal variation (Phillips et al, 1994). Procedures for micropropagation of guayule, through in vitro multiplication of shoots followed by rooting and subsequent acclimation of plantlets, have been reported by only a few researchers (Pan et al, 1996;Trautmann and Visser, 1990;Smith, 1983). Although these methods have commonalities, they vary in terms of reported success rates as well as in terms of the time and labor that would be involved in applying these protocols to large scale micropropagation of plants.…”