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 superior 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

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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. 1990). Young leaves (one or two) from some T, plants were harvested to extract total protein for the acetylation assay following the procedure described by Weeks et al. (1993). A total of 5 ng total protein was loaded onto each lane of a What- man (Whatman Inc., USA) LHP-KDF linear high-performance TLC plate.

Southern Blot Analysis Genomic DNA from nontransformed (control) and some putatively transformed T, and T2 plants was extracted according to the CTAB method (Doyle and Doyle 1990). Undigested DNA, plasmid DNA, and genomic DNA of some T, plants restricted with BamVH or ///ndlll were electrophoresed on \% agarose (Boehringer Mannheim GmbH) gels. Because of weak hybridization signal in Southern analysis of the dwtl line, genomic DNA from some T, and T2 plants was amplified by PCR using two primers (barl: 5'-ACCATCGTCAACCACTACATCGAG-3'; bar?. S'-AGGCTGAAGTCCAGCTGCCAGA-S1) specific to the bar gene. The PCR reaction mixture (50 (j.1 volume) consisted of 10X PCR buffer (with 2.0 mM MgCl2, pH 9.0), primers each at 20 p.M, dNTPs at 1000 JJLM, and Taq at 2.0 units; 35 thermal cycles (each cycle consists of 94°C for 1 min., 60°C for 2 min., and 72°C for 1 min.) of amplification were used. Primers specific to the gus gene were used and its PCR amplification conditions were followed as described by Walters et al. (1992). The PCR-amplified DNA (20 fjJ) was electrophoresed on 1% agarose gels. The DNA from the agarose gels was transferred to Hybond-N nitrocellulose membranes (Amersham) using a PosiBlot pressure 'blotter (Stratagene). The mem- Figure 2. Transformation of durum wheat (a) Expression of gus 2 days after bombardment, (b) Stable expression branes were prehybridized for 3 h and hy- of gus In To flower organs at different developmental stages, (c) GUS activity in brush end portions of T, mature bridized overnight at 42°C following the seeds of line dwt2. (d) Results of spot application of herbicide glufosinate at 120 mg/1 L-PPT on T8 plants (left two samples are from non-translormed plants), (e) Results of spraying of glufoslnate ammonium at 120 mg/1 L-PPT on standard procedures described by Ausu- T, plants of line dwt4. bel et al. (1992). The gus or bar probes

Bommrneni et al • Transgentc Durum Wheat 477 were labeled with KP using a random abcdefgh primed DNA labeling kit (Promega) (Bom- mineni et al. 1997). The hybridized membranes were washed four times at 45°C with 0.1% SSC and 0.1% SDS and autora- diographed.

Results Analysis of Plants Recovered from Selection Medium Two days after bombardment, a high tran- sient expression of gus was observed in the cells of scutella (Figure 2a). On average, 116 GUS foci per scutellum were observed with pBARGUS. Of the 245 bom- Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 barded scutella, five (2%) produced plantlets that showed resistance to the herbicide glufosinate ammonium and later matured into fertile plants. No plants were recovered from 55 nonbombarded scutella after culturing In selection medium. Flow- ers of putative transgenlc plants (Figure 2b) and endosperm of brush end portions of some mature seeds (Figure 2c) showed positive for GUS activity. On spot application of glufosinate at 120 mg/1 L-PPT concentration, the To transformed plant (line Figure 3. Phosphlnothrlcln acetyitransferase activity In protein extracts of leaves from nontransformed and dwt2) showed no damage and remained transformed durum plants (T,). Nontransformed durum cultlvars Medora (lane a) and Monroe (lane b) and transgenlc plants from line dwt2 (lanes c-h). green 10 days after application, whereas the leaves of nontransformed plants were damaged (Figure 2d). Seeds were collected from all five lines (dwtl to dwt5), ger- i j minated, and seedlings at the two-leaf stage were sprayed with the herbicide. One hundred four of the 1048 (i.e., 10%) T, plants showed resistance to the herbicide (Table 1) and remained green. On the other hand, leaves of non-transformed plants wilted and dried after herbicide application (Figure 2e). The herbicide-resistant plants were then allowed to mature to collect selfed T2 seed from them. The functional activity of the bar gene in T, plants of line dwt2 was assessed by the PAT assay (Figure 3). Samples from six plants showed different levels of PAT activity. Whereas samples from five plants

2.0- Figure 4. Southern blot analysis of transgenlc (T, plants of lines dwtl and dwt2) and control plants. Sam- ples (15 |ig DNA) In lanes a-f were hybridized with a "P-labeled gut fragment and samples In lanes g-j were hybridized with a bar fragment, (a) Undigested genomlc DNA from nontransformed plant, (b) ///nduT-restrlcted DNA from transgenlc line dwt2. (c) SamHl-restrlcted DNA from line dwtl. (d) and (e) pBARGUS plas- 1.0- mld restricted with BamW at 15 pg (lane d) and 20 pg (lane e). (f) Undigested DNA from transgenlc line dwtl. (g) and (h) DNA restricted with BamHi (two different plants of line dwtl). 0) DNA restricted with HimW (line dwt2). Q) pBARGUS plasmid DNA (5 pg) restricted with //indlD. Arrows Indicate fragments hybridized to gut and bar.

478 The Journal of Heredity 1997:88(6) bcdefghij klmno eny. The hybridization of an approximately 1.8 kb fragment with a gus fragment after PCR amplification in these plants also indicated coinheritance of bar and gus in the T2 progeny (Fig. 6). However, one of the T2 plants showed no hybridization of either gus or bar fragments (Figure 6, lane h), whereas two plants exhibited only hybridization of either gus (Figure 6, lane i) or bar fragments (Figure 6, lane g).

Discussion 4.3 " The results presented here demonstrate a procedure for durum wheat transformation by microprojectile bombardment of Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 scutella. All of the five To plants that were 2.0" selected for herbicide resistance exhibited histochemical expression of gus. Selected plants (TJ from lines dwtl, dwt2, dwt3, and dwt4 showed integrations of both gus and bar genes in the durum genome (Fig- ures 4 and 5). However, approximately 10% of T, plants survived after herbicide 1.0- application in the following generation. This may be either because To plants were chimeric, resulting from bombardment of scutella 2 days after preculture on callus induction medium, or because the herbi- Figure 5. Southern blot analysis of T, transgenlc plants [three plants each from dwt3 (lanes b, c, d, and h, I, J), cide application (two applications) was and dwt4 (lanes e, f, g, and k, I, m)]. DNA samples (10 (ig) were hybridized with "FMabeled gus fragment (lanes too high. a-g) or with bar fragment (anes h-o). (a) Undigested genomlc DNA from nontransformed plant. CMS) BamtH- restricted DNA from lines dwt3 (lanes b-d) and dwt4 (lanes e-g). (hMD BamHl-restrlcted DNA from line dwt3. An effective selection procedure is es- (kMm) //indlD-restricted DNA from line dwt4. (o) and (o) pBARGUS plasmld restricted with tfmdlll at 15 pg (lane sential for screening the tissues or plant- n) and 30 pg (lane o). lets having the Introduced genes. An op- timal screening method depends on the (Figure 3, lanes c, d, e, g, and h) showed lane c). Hybridized DNA fragments with selective agent used, the introduced positive for PAT activity, one sample less than 3.5 kb represented rearranged or gene(s), and the time of selection after its showed a low level of PAT activity (Figure incomplete integrations. introduction into plant cells. The bar gene 3, lane f) which was almost equivalent to Hybridization fragments of approxi- was successfully introduced to produce the background level of control plants mately 1.4 kb after restricting DNA with herbicide-resistant plants of hexaploid (Figure 3, lanes a and b). BamVtt or approximately 2.1 kb after spring wheat (Becker et al. 1994; Nehra et HindW restriction (Figure 1) would indi- al. 1994; Vasil et al. 1992, 1993; Weeks et Integration of Transgenes cate the integration of the bar gene in the al. 1993), maize (Gordon-Kamm et al. Because of the low leaf mass of To plants, durum genome. Hybridized DNA frag- 1990), rice (Christou et al. 1991), barley it was not possible to extract sufficient ge- ments in Figure 4 (lanes g and h) and Fig- (Ritala et al. 1994; Wan and Lemaux 1994), nomlc DNA for Southern analyses. There- ure 5 (lanes h-j) with BamHI digestion, and oat (Somers et al. 1992). Essentially fore, DNA from T, plants of lines dwtl, and in Figure 4 (lane I) and Figure 5 (lanes two factors influenced the selection for dwt2, dwt3, and dwt4 were used in the k-m) with MndHl demonstrated the inte- bar gene: (1) the type of herbicide used Southern blot analysis to confirm the in- gration of the bar gene in the durum ge- (bialaphos or glufosinate ammonium), tegration of gus and bar genes into the du- nome. and (2) the time and developmental stage rum wheat genome (Figures 4 and 5). An of explants used for selection. Although undigested DNA sample from the trans- Figure 6 illustrates a preliminary bialaphos was used in most reports, we formed plant (line dwtl) showed the in- Southern analysis of T2 plants of line dwtl have successfully used glufosinate for de- tegration of the gus gene into the high mo- after amplification of genomic DNA with tecting the bar gene in durum wheat. lecular weight genomic DNA (Figure 4, gus- and bar-specific primers by PCR. Fig- We found that the time of selection is lane f). Hybridized DNA fragments of more ure 6 is a composite of two individual critical. Initially we applied the selection (5 than 6.0 kb from HindUl or BamHI restric- Southern blots that were hybridized with mg/1 WPT) at 7 or 10 days after micropro- tion demonstrate Integrations of intact gus labeled gus or bar gene probes. An ap- Jection, but were unable to continue the so- into the durum genome (Figure 4, lanes b proximately 0.6 kb fragment hybridized matic embryogenesis to recover putative and c; Figure 5, lanes b-g). At least two with a bar fragment, confirming the stable transformants. The herbicide-damage to copies of gus DNA were observed from integration of the bar gene in the durum the callus while in the selection medium Southern analysis of line dwtl (Figure 4, genome and its transmission to the prog- probably inhibited further tissue develop-

Bommirieni et al • Transgentc Durum Wheat 479 1990; Somers et al. 1992; Vasll et al. 1992; T, 5 Weeks et al. 1993). Sterility could be in- duced during long-term tissue cultures or abcdefghij the regeneration process (see Bommineni and Jauhar 1997b for a detailed review). With shorter culture procedures for so- Mr(kb) matic embryogenesis and regeneration, that is, 3-4 weeks, the scutellar callus was 23.1- successfully used to recover self-fertile transgenic wheat (Becker et al. 1994; Neh- ra et al. 1994). These studies suggest that rapid regeneration procedures are important for genetic transformation of cereals. 6.5- Our results with durum wheat are in ac- cord with these reports, as we successful- Downloaded from https://academic.oup.com/jhered/article/88/6/475/840829 by guest on 25 September 2021 ly produced self-fertile plants from calli of bombarded scutella. Most T, plants produced one or two tillers and therefore 4.3- only a few samples of the lines dwtl, dwt2, dwt3, and dwt4 were used in the Southern analysis and PAT assay. Although 10% herbicide-resistant transgenic plants were recovered in T, generation, based on the presence of hybridized fragments, our preliminary progeny anal- 2.2- ysis with few samples suggested colnher- itance of gus and bar genes in the T2 generation (Figure 6). Six of the nine plants analyzed showed the presence of both gus and bar fragments. In addition, as illus- trated in Figure 4 (lane c), Southern analysis of amplified sample of the same T, plant (line dwtl) confirmed the presence of two gus fragments (Figure 6, lane a). However, their T2 progeny showed the presence of only one gus fragment. The re- maining seed samples of all transgenic lines will be planted to study the segregation of bar and gus transgenes in the T2 generation. A combination of histochemical GUS assays, herbicide sprays, and PCR analysis will be adopted to screen T2 and T3 plants and study the segregation of bar and gus genes. We have demonstrated, for the first time, successful transformation of durum Figure 6. Southern analysis of T, and T, plants (line dwtl) after PCR amplification of genomic DNA with ollgo- wheat, an important tetraploid cereal. nucleotide primers specific to gus and bar fragments; the amplified DNA with gus or bar primers was electropho- This work was done on an agronomically resed separately and hybridized to a BP-labeled gus or bar fragment, respectively, and two blots were overlapped to expose on a single autoradlograph. (a) T, plant; (b)-Q) Plant* from T, progeny, (k) Nontransformed control. important durum cultivar, Medora, for which the entire transformation protocol was established. Using isolated scutella as ment. Selection for herbicide resistance ported In rice (Christou et al. 1991) or bar- target tissue, we obtained self-fertile trans- was more effective when applied after dif- ley (Wan and Lemaux 1994), it is compar- genic durum wheat. The development of ferentiation of small green somatic embry- able to that reported in hexaploid wheat transformation procedures for agronomi- os. Similar observations were reported in (Becker et al. 1994; Nehra et al. 1994; Vasil cally superior cultlvars will open up new hexaploid wheat (Becker et al. 1994; Nehra et al. 1992; Weeks et al. 1993) and maize avenues for the enhancement of existing et al. 1994). With this selection procedure (Gordon-Kamm et al. 1990). durum germplasm using tools of modern we were able to produce, on average, two Some sterility was reported in trans- biotechnology. We are using this proce- transgenic plants from 100 scutella bom- formed cereals resulting from bombard- dure to introduce genes of agronomic im- barded. Although this transformation fre- ment of callus or suspension cultures portance into several other important du- quency is considerably lower than that re- (Fromm et al. 1990; Gordon-Kamm et al. rum cultivars.

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