Transgenic Durum Wheat by Microprojectile Bombardment of Isolated Scutella V. R. Bommineni, P. P. Jauhar, and T. S. Peterson A blolisUc transformation method was developed, for the first time, for durum wheat (Triticum turgldum L., 2n = 4x = 28; AABB) cultivar Medora using isolated scutella Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 as target cells, gus as a reporter gene, and bar (herbicide resistance gene) as a selectable marker. An average of 116 GUS foci per scutellum were observed 2 days after bombardment. After selection for herbicide resistance by adding 5 mg/1 L-phosphinothrlcin (L-PPT) to the medium during regeneration and spot application of 120 mg/1 L-PPT on the leaves of regenerated plants, we Identified five resistant plants from a total of 245 scutella bombarded. All these plants were fertile. Of the 1048 T, seeds germinated from five lines (dwt1, dwt2, dwt3, dwt4, and dwt5), a total of 104 T, plants were recovered that showed resistance to the herbicide glufoslnate when sprayed at a concentration of 120 mg/1 L-PPT. Some of the herbicide-resistant T, plants exhibited phosphlnothricin acetyltransferase (PAT) enzyme activity, indi- cating the presence of the bar gene In the transgenlcs. The Integration of gus and bar genes Into the genomes of durum wheat was further confirmed by Southern analysis. Development of this transformation procedure with an agronomlcally su- perior durum cultivar will open up new avenues for the enhancement of the existing germplasm through biotechnology. Plant breeders and cytogenetlcists have However, the usefulness of genetic engi- achieved a certain degree of success in neering for germplasm enhancement of transferring superior agronomic traits durum wheat (Triticum turgidum L, 2n = from related wild grasses into both tetra- Ax = 28; AABB genomes) has not been ex- ploid and hexaploid wheats by intergener- plored. A major limitation to durum wheat ic hybridization (Bommineni and Jauhar transformation has been the lack of an ef- 1997a; Jauhar 1993; Jiang et al. 1994). Al- ficient method of in vitro regeneration by though wide hybridization is an effective somatic embryogenesis. Therefore we ini- means of introducing desirable alien genes tiated work on development of in vitro cul- into wheat, it has several limitations, for ture techniques and established a rapid example, transmission of unwanted alien regeneration protocol using isolated scu- chromosomes and adverse genetic Inter- tella of four agronomically Important du- actions leading to sterility. Thus to intro- rum cultivars (Bommineni and Jauhar duce a single desirable alien gene into 1996). Using this regeneration protocol, wheat by sexual means is extremely tedi- we standardized a durum transformation ous and time consuming. However, bio- procedure using isolated scutella as target technological approaches facilitate the in- material and a herbicide resistance gene, troduction of desirable alien genes asexu- bar (Thompson et al. 1987). Details of the From the USDA-ARS, Northern Crop Science Labora- ally into plants. transformation procedure are described tory, Fargo, ND 58105-5677 (Bommineni and Jauhar) and the Department of Plant Sciences, North Dakota Genetic transformation by microprojec- and its implications in crop improvement State University, Fargo, North Dakota (Peterson). We tion has been demonstrated in most ce- discussed in this article. thank Drs. 0. D. Anderson, L Dahleen, and N. D. Wil- liams for their suggestions during the preparation of reals (Bommineni and Jauhar 1997b; Mor- the manuscript, and 0. D. Anderson and A. E. Blechl rish and Fromm 1992), Including spring Materials and Methods for their technical help In the PAT assay. We also thank wheat (Triticum aestiuum L, 2/i = for = 42; Paul Mayland of AgrEvo for providing the herbicide. Mention ol trademark or proprietary product does not AABBDD) (Becker et al. 1994; Nehra et al. Plant Material and In Vitro Culture constitute a guarantee or warranty of the product by 1994; Vasil et al. 1992, 1993; Weeks et al. Seed of an agronomlcally important du- the USDA or Imply approval to the exclusion of other products that also may be suitable. Address corre- 1993). These workers successfully used a rum wheat cultivar, Medora, was obtained spondence to P. P. Jauhar at the address above or variety of embryogenic target tissues such from Dr. E. Ellas of the Department of Plant e-mail: [email protected]. as callus and scutellum tissue to establish Sciences, North Dakota State University, Journal of Heredity 1997^8:475-481; 0022-1503/97/J5.00 transformation protocols for spring wheat. Fargo. Seedlings were raised in pots (12 475 Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 Figure 1. Schematic representation of pBARGUS (Fromm et al. 1990) used In durum wheat transformation. cm diameter) in the greenhouse. Proce- cles (BioRad) using the procedure de- but was supplemented with 5 mg/1 L-phos- dures for the isolation of scutella from im- scribed previously (Bommineni et al. phlnothricin (L-PPT) [glufosinate ammo- mature embryos, in vitro induction and re- 1993). A blolistic helium device (BioRad) nium (600 mg/1), kindly provided by Paul generation media, and other growth con- was used to mlcroproject the DNA-coated Mayland of AgrEvo, Fargo, North Dakota]. ditions were reported earlier (Bommineni gold particles into the isolated scutellum The green plantlets developing on selec- and Jauhar 1996). After surface steriliza- cells. The DNA was microprojected twice tion medium were then transferred to half- tion of immature caryopses, the scutella at 9.0 cm flight distance, 1100 psi helium strength hormone-free MS medium (same were isolated by removing the embryonic pressure, and 26 in. (66 cm) Hg vacuum. as callus induction medium, but lacking axes. The isolated scutella were then The bombarded scutella were then in- hormones and having 3% sucrose and placed on the callus induction medium, cubated in the dark at 25 ± 2°C. Two days 0.8% agar) for another 2 weeks before which consisted of Murashige and Skoog after bombardment, the actively growing planting in pots (12 cm diameter) in the (1962) medium (MS medium) supplement- scutella explants were transferred to fresh greenhouse. Mature seeds were obtained ed with 2 mg/1 2,4-D, 3% sucrose, 100 mg/1 callus induction medium (5-7 scutella per from putatlvely transformed plants casein hydrolysate, and 100 mg/1 myo-ino- 15 X 100 mm petri dish). Some scutella through selfing and were planted to pro- sitol. The medium was solidified with 0.8% were analyzed randomly by the histo- duce subsequent generations. agar (Sigma Chemical Co.). The scutella chemical GUS assay (Bommineni et al. were incubated in the dark at 25 ± 2°C for 1993, 1997). Bombardment experiments 2 days before biolistlc bombardment. were repeated five times with a total of 245 Herbicide (Glufosinate) Application scutella, and nonbombarded scutella The leaves of plants (To) regenerated from L-PPT containing selection medium were Gene Expression Vector served as controls. spot painted with 120 mg/1 L-PPT solution DNA from the pBARGUS plasmid vector (with 0.1% Tween-20) and observed for (Fromm et al. 1990) was used in the trans- Regeneration of Plantlets herbicide damage after 1 week. In the sub- formation of durum wheat. It consists of a Seven days after bombardment, the scu- sequent generation (T[), all plants were bar gene under the control of cauliflower tella with embryogenic call! were trans- sprayed twice at 1 week intervals at the mosaic virus 35S promoter (CaMV 35S) ferred to a incubator with white fluores- two- to three-leaf stage with L-PPT (120 with the maize alcohol dehydrogenase 1 cent lights for a week (Bommineni and mg/1) solution. Selfed seed was collected (Adhl) intron 1 in the 5' region and a no- Jauhar 1996). The embryogenic calll were from resistant plants. Some of the seed palene synthase (nos) terminator (Figure then transferred to regeneration medium (line dwtl) was planted to produce the T2 1). This plasmid vector also contains a gus to differentiate into somatic embryos. The generation. reporter gene controlled independently by regeneration medium consisted of callus a maize Adhl promoter with its intron 1 induction medium without 2,4-D but sup- and terminated by a nos sequence. plemented with 1 mg/1 each of BA and IAA, Enzymatic Assays and 2% sucrose, solidified with 0.8% aga- Leaf, flower, and brush end portions of ma- Microprojectlon of pBARGUS rose (Sigma Chemical Co.). The green so- ture seeds of To plants were used to test Two days after preculture incubation on matic embryos differentiated in 2-3 gus expression with the histochemlcal GUS induction medium, 20-25 scutella were weeks. They were isolated and transferred assay (Bommineni et al. 1994). The sam- transferred to fresh callus induction me- to selection medium for six weeks or until ples were Incubated overnight in X-gluc dium and placed in the center of a petri they developed Into green plantlets. The solution (0.05% w/v) (McCabe et al. 1988) dish (15 x 100 mm). pBARGUS plasmid selection medium consisted of regenera- to identify GUS-positive material. The GUS- DNA was coated onto 1.0 jim gold parti- tion medium without casein hydrolysate positlve samples were soaked for a day in 476 "[Tie Journal ct Heredity 199788(6) Table 1. Herbicide resistance In T, and T, planti Number of Number of plants (Ti) Transgenic seeds recovered resistant Une from To plants to herbicide dwtl 6 1* dwt2 84 6* dwt3 358 26 dwt4 273 53 dwt5 327 18 * PAT positive plants. absolute ethanol prior to photomicrogra- phy. Phosphinothricin acetyhransferase (PAT) Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 assay was carried out using the procedure of silica gel thin layer chromatography (Spencer et al.