About Bioline  All Journals  Testimonials  Membership  News  Donations

African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 3, Num. 2, 1995, pp. 171-180

African Crop Science Journal, Vol. 3. No.2, pp. 171-180, 1995





Purdue University, 1165 Horticulture Building, West Lafayette, IN 47907-1165, USA. 1 Pioneer Hi-Bred Intl., Inc., 7300 N.W. 62nd Avenue, P.O. Box 1004, Johnston, IA 50131-1004, USA

Code Number:CS95023
Size of Files:
    Text: 41.5K
    No associated graphics files


Transgenic fertile sorghum plants (Sorghum bicolor L. Moench) were obtained by microprojectile bombardment of immature embryo and immature inflorescence explants. Regeneration from the calli was through embryogenesis and organogenesis pathways, resulting in both the uniformly transformed and chimeric plants. Plant regeneration occurred on media supplemented with bialaphos. Microprojectile bombardment resulted in plants resistant to the herbicide and exhibited phosphinothricin acetyltransferase activity. The presence of bar, uMa and luc genes in the T[o] plants was confirmed by Southern blot analysis of genomic DNA. The herbicide resistance was inherited in T[1] plants.

Key Words: Bialaphos, explants, herbicide, regeneration, Sorghum bicolor, transgenic


Les plantes transgeniques fertiles de surgho (Sorghum bicolor L. Moench ) etaient obtenues par bombardement de microprojectiles d'embryons immatures et d'explants d'inflorescences immatures. La regeneration a partit de cales a lieu a travers l'embryogenese et l'organogenese qui resultent en la formation des plants uniformement transformes et des chimeres. La regeneration des plantes se fait sur des milieux ayant obtenu un supplement de bialaphos. Le bombardement des microprojectiles a abouti a la production des plants resistants aux herbicides et presentant l'activite phosphinothricine acetyltransferase.

La presence des genes bar, uida et luc sur les plants T[o] a ere confirmee a l'aide de l'analyse southern blot du DNA genomique. La resistance a l'herbicide etait herite par les plants en T[1].

Mots Cids: Bialaphos, explants, herbicide, regeneration, Sorghum bicolor; transgenique


The development of methods for genetic transformation of plants has made genes that are derived from recombinant DNA technologies accessible for practical use. This has allowed the genetic improvement of particular crop species by the use of genes obtained from totally unrelated species. The application of these methods has produced transgenic plants that are resistant to various bacterial (Martin et al., 1993), viral (Cuozzo et al., 1988; Nelson et al, 1988; Lawson et al., 1990) or fungal infections (Broglie et al., 1991; Liu et al, 1994), resistant to insect pests (Fischhoff el al., 1987; Vaeck et al., 1987; Perlak et al., 1993), tolerant to the application of various herbicides (Shah et al., 1986; De Greef et al., 1989), are male sterile (Mariani et al., 1990; Denis et al., 1993), have a higher nutritional content in the seed (Altenbachetal, 1989), have delayed fruit-ripening (Oeller et al., 1991), or have a greater degree of stress protection (Tarczynsk et al. 1993), due to the introgression of new genes.

The transformation of cereals has been delayed for many years by the lack of efficient transformation systems. Agrobacterium tumefaciens, the highly effective vector for dicotyledonous crops until recently (Hiei et al.,1974) either did not infect monocotyledonous plants, or did so very inefficiently (Gould et al., 1991; Shen et al., 1993),as described for asparagus (Bytebier et al., 1987) or rice (Reineri et al., 1990). Mediated by PEG (Lorz et al. 1985) or electropotation (Fromm et al., 1985), the introduction of DNA into protoplasts has been extensively examined for many cereals (Rhodes et al. 1988; Shimamoto et al., 1989; Datta et al., 1990). However. plant regeneration from protoplasts is very laborious, is strongly genotype dependent and apparently is only routinely applicable in the case of rice (Hayashimoto et al., 1990; Peng et al.. 1992).

Because of difficulties in obtaining transgenic monocotyledonous plants using Agrobacterium or protoplasts, other approaches have been attempted for introducing DNA into plant cells (Songstad et al., 1995). These include macroinjection (De la Pena et al. 1987), embryo imbibition (Topfer et al., 1990), agroinfection with gemini ,viruses (Grimsley et al., 1988), the pollen tube absorption pathway (Luo and Wu, 1988; Langridge et al., 1992), silicon-carbide fiber (Kaeppler et al., 1992), tissue electropotation (D'Halluin et al., 1992; Xu and Li, 1994), and microprojectile bombardment (Sanford, 1988; Klein et al., 1992; Christou, 1993; Morrish et al. 1993; Sanford et al., 1993). Of all these techniques particle bombardment has proved to be the most successful and reproducible, leading to the production of transgenic plants of a wide range of species, including most of the cereals and among them Sorghum bicolor (L. Moench) Casas et al., 1995; Maheshwarietal.,l995; Vasil 1995). This methodology allows the introduction of DNA into live cells or tissues, making it possible to evaluate transient expression of different gene constructs in intact organs, as well as the recovery of stably transformed tissues and plants.

DNA delivery utilizing microprojectile bombardment involves the acceleration of metal particles coated with DNA, such that they penetrate the cell wall and enter the cell proper. Bombarded cells transiently express the introduced gene and some of these cells incorporate the gene into the plant genome and become stably transformed. One can then select then select a population of transformed cells and eventually regenerate plants. One advantage of this methodology is that it can be used to target a wide range of tissues or organs, such as cell suspensions (Fromm et al., 1990; Gordon-Kamm et al. 1990; Cao et al., 1992; Somers et al., 1992) or calli (Bower and Birch, 1992; Vasil et al., 1992), immature embryos (Christou et al.. 1991; Casas et al., 1993; Weeks et al., 1993; Becker et al., 1994; Nehra et al., 1994; Wan and Lemaux, 1994). meristems (Bilang et al., 1993; Perez-Vicente et al.,1993; Ritala et al., 1994), pollen (Twell et al., 1989; Hamilton et al., 1992), microspore derived embryos (Creissen et al., 1990; Wan and Lemaux, 1994), tassel primordia (Dupuis and Pace, 1993), or leaf tissue (Reggiardo et al., 1991; Gallo-Meagher and Irvine. 1993). Using those various target tissues, stable transformation of all the major cereals including sorghum (Casas et al., 1995) has been accomplished.

Microprojectile mediated transformation systems consist of four major components. All of them are apparently essential for successful transformation. These include: l)a tissue culture component, 2) DNA delivery, 3).selection and 4)

molecular and genetic analyses of recovered plants.

This paper focuses on a transformation system that was developed in our laboratory and resulted in stable transformation of Sorghum bicolor utilizing microprojectile bombardment and selection under bialaphos selection pressure.


The choice of the explant or target tissue for

transformation and subsequent regeneration are the most critical factors influencing transformation via microprojectile bombardment. Over the last 20 years, several relatively efficient plant regeneration protocols have been developed for sorghum. These include regeneration from both immature and mature zygotic embryos (Gamborg et al.. 1977; Dunstan et al., 1978, 1989; Brar et al., 1979; Mackinnon et al.. 1986; Cai et al., 1987; Ma et al., 1987), from shoot segments of germinating seeds (Brar et al., 1979), from immature inflorescences (Bretell et al., 1980; Boyes and Vasil, 1984; Cai and Butler? 1990), and shoot tips (Bhaskaran and Smith, 1988; 1989; Bhaskaran et al., 1988). In our transformation research. immature embryos and immature inflorescences were utilized as explants for particle bombardment and plant regeneration. Protocols for plant regeneration via the embryogenesis pathway from both types of explants have been developed in Dr. L.G. Butler's (Dept. of Biochemistry, Purdue University, West Lafayette, Indiana, USA) laboratory, and after necessary modifications used in our laboratories to produce transgenic sorghum plants (Casas et al., 1993; 1995).

The plant regeneration protocol from immature embryos includes culture of immature zygotic embryos (12-15 days after pollination) onto a basal medium containing Murashige and Skoog (Murashige and Skoog, 1962) salts, modified B5 vitamins (Gamborg et al., 1977) and agar (8 g 1^-1) supplemented with asparagine (150mg l^-1), 10% coconut water, 2,4-D (2 mg l^-1), and sucrose (30 g 1^-1) ( 16 medium) for induction of embryo genesis and initiation of embryogenic callus. Selection and maintenance of embryogenic tissue and shoot and root development from organized structures are as described in details by Cai and Butler (1990). Media contains the basal constituents supplemented with 2 mg l^-1 2,4-D, 0.5 g l^-1 kinetin, 30 g 1^-1 sucrose for maintenance of embryogenic tissue ($2 medium) or 1 mg l^-1 IAA, 0.5 mg 1

kinetin, and 20 g l^-1 sucrose to facilitate shoot development. For root development, plantlets are transferred to RM medium containing 0.5 X Murashige and Skoog salts, 0.5 mg l^-1 NAA, 0.5 mg 1^-1 IBA, 20 g l^-1 sucrose and 8 g 1^-1 agar. Cultures of immature embryos and embryogenic tissue are grown in darkness, at 26 C and recultured every 2 weeks. Tissues on shoot regeneration medium are subcultured every 4 weeks and grown at 26^-1 C under a 16-hr photoperiod (1000-2000 lx from fluorescent, cool white light).

The plant regeneration protocol from immature inflorescences includes isolation of immature inflorescences (0.5 to 3 cm long) and after cutting into 0.5 cm large pieces, culture onto NS2 medium consisting of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al., 1977) 2.5 mg 1^-10 2,4-D, 0.5 mg l^-1 kinetin, 60 g 1^-1 sucrose. and 7 g l^-1 agar to induce embryogenic callus. Embryogenic callus can be maintained and plants from embryogenic tissue regenerated under culture conditions as described for immature embryo explants. After acclimation to low humidity, regenerated plants are transferred to a greenhouse and there grown to maturity.

It has to be emphasized. however, that genotype specific differences in responsiveness of primary explants to regeneration protocols have been found among sorghum genotypes utilized in our studies. The best responsive genotypes have been selected for further transformation research that utilized both types of explants, e.g. immature embryos (PS98012) and immature inflorescences (SRN39).


Besides the importance of target explant and plant regeneration, there are several features of the particle gun that influence the efficiency of the transformation process. These include the type, size and amount of microprojectiles used, the velocity of the microprojectiles and target distance, the amount of DNA per shot and the number of bombardments. Transient expression of reporter genes can be used to optimize these bombardment parameters. The pressure and expression of commonly used reporter genes can be determined by different methods. For the uida gene encoding for B-glucuronidase, histochemical or fluorometric assays described by Jefferson et al. ( 1986, 1987) are used. Furthermore, radiochemical or chemiluminescence methods are used to detect chloramphenicol acetyltransferase (Fromm et al. 1985) or luciferase reporter genes (Ow et al., 1986; Olssonet al., 1989), respectively. Another group of reporter genes includes transcriptional activators that regulate anthocyanin metabolism in maize, e.g. R (Ludwig et al., 1989), Cl Paz-Ares et al. 1987) and B-Peru (Chandler et al., 1988). The production and accumulation of anthocyanin pigment in a given tissue depends on the presence of a functional set of regulatory genes, which include Al, Bzl and C2. Expression of these genes can be easily monitored visually and, most important, nondestructively (Ludwig et al., 1990; Bowen, 1992).

TABLE 1. Particle bombardment parameters

Immature embryos:
Size of explants: 1-2 mm immature embryos
DNA amount: 0.15 ug of each plasmid DNA for cotransformation
    0.3 ug of DNA when single plasmid is used 
    Tungsten particles: 
    size: 1.7 um (M25) 
    amount: 0.15 mg 
Pressure: 1100 PSI
Distance: 4.5 cm for single shot
          7.5 cm for double bombardment

Immature inflorescences: 
Size of H: 1-3 cm
DNA amount: 0.2 ug of each plasmid DNA for co-

          0.4 4g of DNA when single plasmid is used 
          Tungsten particles: 
          size: 1.7 um (M25) 
          amount: 0.15 mg
Pressure: 1300 PSi
Distance: 4.5 cm for single shot
          7.5 cm for double bombardment

TABLE 2. Analysis of Transformation Event's T[o] Plants

Event              Number of insertions           Phenotypic
                   ---------------------          ----------- 
                         bar         luc           bar   luc
1119 (fertile)       l(EcoR l/bar)   NA            yes    NA 
1409 (sterile)       2 (EcoR ll/bar) NA            yes    NA 
1702-2H (fertile)    (EcoR ll/bar) 
                     (Sac ll/bar)    NA            yes    NA 
1752 (male sterile)  2 (EcoR l/bar)  NA            yes    NA 
2251 (fertile)       2 (Sac ll/bar)  1 (EcoRI/luc) yes    no   
2441 (fertile)       3 (Sac ll/bar)  3 (EcoRI/luc) yes    yes

NA - not applicable since bombardment plasmids did not contain
this gene

Both, uidA gene and R/Cl transcriptional regulators (driven by single or double CaMV 35S promoter with maize Adhl gene intron) have been successfully used in our sorghum transformation research as reporter genes to optimize particle bombardment parameters (Table 1) (for review see Sanford et al., 1993) and later for screening bombarded explants during early stages of plant regeneration.


A reliable selection procedure is a prerequisite for development of an efficient transformation system. An important feature of the selection system is its ability to facilitate the emergence of a developing transformant enclosed in a mass of untransformed tissue. As mentioned previously, both immature embryos and immature inflorescences are used as primary explants and plants are regenerated through in vitro induced somatic embryogenesis. For each type of explant, it is critical to develop an efficient selection strategy that utilizes selectable markers and an appropriate selective agent, antibiotic or herbicide, to identify and multiply transformed cells. Considering the probability that only a small percentage of the transiently expressing cells are likely to be stably transformed, it is important to use selection that confers a growth advantage to the transformed cells. To impose an efficient selection pressure, while maintaining the competence for regeneration, the dose, mode and time of selective agent application, as well as the duration of the selection need to be considered.

Several selectable marker genes conferring resistance to either antibiotics or herbicides have been isolated and are currently used for plant transformation (Table 2)(for review see Wilmink and Dons, 1993; Hinchee et al., 1994). In the case of monocotyledonous plants, the most common selection systems involves the use of the bacterial genes nptll, hph and bar that confer resistance to kanamycin, hygromycin and phosphinothricin, respectively. Even though kanamycin has been used to recover transformed calli and plants (Murry et al., 1993), cells of many monocotyledonous plants appeared to be quite resistant to this antibiotic (Klein et al., 1989; Hagio et al., 1991; Vasil et al., 1991). Better results have been obtained using geneticin (Bower and Birch, 1992; Nehra et al., 1994) and hygromycin (Christou et al., 1991; Walters et al., 1992). Nevertheless, the herbicides bialaphos or Basta were proven to be the best selective agents for cereal transformation and for this reason they were used in our early studies on sorghum and a selection protocol utilizing bar gene has been established in our laboratories for regeneration of transgenic plants from immature embryos and immature inflorescences.

As mentioned earlier the selection strategy is a compromise that involves the level of explant organization, the concentration of selective agent used, and the duration of selection pressure application. A selection strategy for each sorghum genotype has been developed experimentally based on detailed analyses of susceptibility of non-bombarded and bombarded explants to different doses of bialaphos at different stages of the regeneration process. For immature embryos we found that bialaphos at a concentration of 3 mg l^- 1 while applied at the beginning of the selection process would severely inhibit callus formation in bombarded material, probably due to accumulation of ammonia in the surrounding non-transformed cells. It was necessary to initiate selection on medium with 1 mg l^-1 of this herbicide. The formation of embryogenic callus from immature embryos on induction medium in the presence of a low concentration of bialaphos could be used as an indicator of possible transformants, since non-responsive embryos do not produce any callus later. A minimum of three more transfers on medium with increased concentration of bialaphos (3 mg l^-1), 2 weeks each, appeared to be necessary to identify putative transgenic callus. It became clear that maintenance of a continuous selection pressure is a critical factor to recover transgenic plants, because the sensitivity to the selective agent changes during cell growth and differentiation. It was frequently reported also for other plant species (Fromm et al., 1990; Christou et al., 1991; Bower and Birch, 1992) that plant regeneration in the absence of selection could result in the preferential recovery of escapes, untransformed material in large numbers. Furthermore, it has been proven that the process of root formation and elongation is one of the most sensitive stages of growth, and the application of selection pressure during this stage of culture allows the discrimination of true transgenic material (Cao et al., 1992; Somers et al., 1992; Li et al., 1993; Weeks et al., 1993; Wan and Lemaux, 1994). For this reason we included bialaphos also in regeneration (3 mg l^-1) and rooting (1 mg l^-1) media. Utilizing regeneration and bialaphos selection protocols described above, several transgenic sorghum plants have been regenerated in our laboratories from both immature embryo and immature inflorescence explants and characterized by molecular (Southern analysis) and biochemical (PAT. GUS and luciferase activity assays) methods. Furthermore, stability of the introduced genes in the following generations has been also proven using the same methodology as for primary transformants.

Although a selection system utilizing bar gene and bialaphos has proven to be reliable. it cannot be considered as a system for routine sorghum transformation, because of sorghum's ability to readily outcross with weed species. For this reason, selection strategy is one of the priorities of our current research. The nptll and hph genes conferring resistance to kanamycin and hygromycin, respectively, are now being considered as alternative selectable markers. Introduction of agronomically important genes (traits) and selectable marker genes into the plant genome by cotransformation of two separated plasmids, followed by segregating out the selectable marker gene in the progeny is considered as another strategy. Our preliminary studies on applying these strategies for sorghum are encouraging.


Sorghum research was supported by Pioneer HiBred International (Johnston, Iowa) and The Consortium for Plant Biotechnology Research, IIIc.


Altenbach, S.B., Pearson, K.W., Meeker, G., Staraci,L.C. and Sun, S.S.M. 1989. Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants. Plant Molecular Biology 13:513-522.

Becker,. D., Brettschneider, R. and Lorz, H. 1994. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant Journal 5:299-307.

Bhaskaran, S. and Smith, R.H. 1988. Enhanced somatic embryogenesis in Sorghum bicolor from shoot tip culture. In Vitro Cellular Development Biology 21:65-70.

Bhaskaran, S. and Smith. R.H. 1989. Control of morphogenesis in sorghum by 2,4-Dichlorophenoxyacetic acid and cytokinins. Annual Botany 64:217-2214.

Bhaskaran, S., Neumann, A.J. and Smith, R.H. 1988. Origin of somatic embryos from cultured shoot tips of Sorghum bicolor (L.) Moench. In Vitro Cellular Development Biology 24:947- 950.

Bilang, R., Zhang, S., Leduc, N., Iglesias, V.A., Gisel, A., Simmonds, J. Potrykus, I. and Sautter, C. 1993. Transient gene expression in vegetative. shoot apical meristems of wheat after ballistic microtargeting. Plant Journal 4:735-744.

Bowen, B. 1992. Anthocyanin genes as a visual markers in transformed maize tissues. In: GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression. Gallagher. S.R. (Ed.), pp. 163-177. Academic Press, Inc, San Diego.

Bower, R. and Birch, R.G. 1992. Transgenic sugarcane plants via microprojectile bombardment. Plant Journal 2:409-416.

Boyes, C.J. and Vasil, I.K. 1984. Plant regeneration by somatic embryogenesis from cultured young inflorescences of Sorghum arundinaceum (Desv.) Stapf. var. Sudanese (Sudan grass). Plant Science Letters 35:153-57.

Brar, D.S., Rambold, S., Gamborg, O. and Constabel, F. 1979. Tissue culture of corn and sorghum. Z. pflanzenphysiology 95:377-88.

Brettell, R.I.S., Wernicke, W. and Thomas, E. 1980. Embryogenesis from cultured immature inflorescences of Sorghum bicolor. Potoplasma 104:141 - 148.

Broglie, K., Chet. I., Holliday, M., Cressman, R., Biddie, P., Knowlton, S., Mauvais, C.J. and Broglie, R. 1991. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194-1197.

Bytebier, B., DeBoeck, F., De Greve, H., Van Montagu, M. and Hernalsteens, J.P. 1987. TDNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis. Proceedings of the National Academy of Sciences, USA 84:5345-5349.

Cai, T. and Butler, L. 1990. Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums. Plant Cell, Tissue and Organ

Culture 20:101-110.

Cai, T. Daly, B. and Butler. L. 1987. Callus induction and plant regeneration from shoot portions of mature embryos of high-tannin sorghums. Plant Cell, Tissue and Organ

Culture 9:245-252.

Cao, J., Duan, X., McElroy, D. and Wu, R. 1992.

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of

suspension culture cells. Plant Cell Reproduction 11:586-591.

Casas, A.M., Kononowicz, A.K-, Zehr, U.B., Tomes, D.T., Axtell, J.D., Butler, L.G., Bressan, R.A. and Hasegawa, P.M. 1993. Transgenic sorghum plants via microprojectile bombardment. Proceedings of National Academy of Sciences, USA 90:11212-11216.

Casas, A.M., Kononowicz, A.K., Bressan, R.A. and Hasegawa, P.M. 1995. Cereal transformation through particle bombardment. Plant Breeding Review 13:231-260.

Chandler, V.L. Radioella, J.p., Robbins, T.P, Chen, J. and Turks, D. 1988. Two regulatory genes of the maize anthocyanin pathway are homologous: Isolation of B utilizing R genomic sequences. Plant Cell 1:1175-1183.

Christou, p. 1993. Particle gun mediated transformation. Current Opinion in Biotechnology 4:135-141.

Christou, P., Ford, T.L. and Kofron, M. 1991. Production of transgenic rice (Orvza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9:957- 962.

Creissen, G., Smith, C., Francis, R., Reynolds, H. and Mullineaux, P. 1990. Agrobacterium and microprojectile- mediated vital DNA delivery into barley microspore-derived cultures. Plant Cell Reporter 8:680-683.

Cuozzo, M., O'Connel, K.M., Kaniewski, W., Fang, R., Chua, N. and Tumer, N.E. 1988. Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat protein or its antisense RNA. Bio/Technology 6:549-555.

Datta, S .K., Peterhans, A. Datta, K. and Potrykus, 1990. Genetically engineered fertile indica rice recovered from protoplasts. Bio/Technology 8:736-740.

De Greef, W., Delon, R., De Block, M., Leemans, J. and Botterm-nan, J. 1989. Evaluation of herbicide resistance in transgenic crops under field conditions. Bio/Technology 7:61-64.

De La Pena, A., Lorz, H. and Schell, J. 1987. Transgenic rye plants obtained by injecting DNA into young floral tillers. Nature 325:274-276.

Denis, M, Delourme, R., Gourmet, J.p., Mariani, C. and Renard, M. 1993. Expression of engineered nuclear male sterility in Brassica napus. Genetics, morphology, cytology, and sensitivity to temperature. Plant Physiology


D'Halluin, K., Bonne, E, Bossut, M., De Beuckeleer, M. and Leemans, J. 1992. Transgenic maize plants by tissue electroporation. Plant Cell 4: 1495-1505.

Dunstan, D.I., Short, K.C. and Thomas, E. 1978. The anatomy of secondary morphogenesis in cultured scutellum tissues or Sorghum bicolor. Protoplasma 97:251-260.

Dunstan, D.I., Short, K.C., Dhaliwal, H. and Thomas, E. 1979. Further studies on plantlet production from cultured tissues of Sorghum bicolor. Protoplasma 101:355-361.

Dupuis, I. and Pace, G.M. 1993. Gene transfer to maize male reproductive structure by particle bombardment of tassel primordia. Plant Cell Reproduction 12:607-611.

Fischhoff, D.A., Bowdish, K.S. Perlak, F.J., Marrone, P.G., McCormick. S.M., Niefermeyer, J.G, Dean, D.A., Kusano- Kreetzmer, K., Mayer, E.J., Rochester, D.E., Rogers, S.G. and Fraley, R.T. 1987. Insect tolerant transgenic tomato plants. Bio/Technology 5:807-813.

Fromm, M.E., Taylor, L.P. and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proceedings National Academy of Sciences, USA 82:5824-5828.

Fromm, M.E., Morrish, F., Armstrong, C., Williams, R., Thomas, J. and Klein, T.M. 2990. Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8:833-839.

Gallo-Meagher, M. and Irvine, J.E. 1993. Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment. Plant Cell Reproduction 12:666-670.

Gainborg, O.L., Shyluk, J.P. Brar, D.S. and Constabel, F. 1977. Morphogenesis and plant regeneration from callus of immature embryos of sorghum. Plant Science Letters 100:67-74.

Gordon-Kamm, W.J., Spencer, TM, Mangano, M.L., Adams, T.R., Daines, R.J., Start, W.G., O'Brien, J.V., Chambers, S.A.,Adams, Jr, W.R., Willetts, NG, Rice, T.B., Mackey, C.J., Krueger, R.W., Kausch, A.P. and Lemaux, P.G. 1990. Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603-618.

Gould, J., Devey, M., Hasegawa, 0., Ulian, E.C., Peterson, G. and Smith, R.H. 1991. Transformation of Zea mays L. using Agrobacterium tumefaciens and the shoot apex. Plant Physiology 95:426-434.

Grimsley, N.H., Ramos, C., Hein, T. and Hohn, B. 1988. Meristematic tissues of maize plants are most susceptible to agroinfection with maize streak virus. Bio/Technology 6:185-188.

Hamilton, D.A., Roy, M., Rueda, J., Sindhu, R.K., Sanford, J. and Mascarenhas, J.P. 1992. Dissection of a pollen-specific promoter from maize by transient transformation assays. Plant Molecular Biology 18:211-218.

Hayashimoto, A., Liu, Z. and Mural, N. 1990. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiology 93:857-863.

Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. 1994. Efficient transformation of rice (Oryza satira L.) mediated by Agrobacterium and sequence analysis of the boundaries of the FDNA. Plant Journal 6:271-282.

Hinchee, M.A.W., Corbin, D.R., Armstrong, Ch. L., Fry, J.E., Sato, S.S., DeBoer, D.L., Petersen, W.L., Armstrong, T.A., ConnorWard, D.V., Layton, J.G., and Horsch, R.B., 1994. Plant Transformation. In: Plant Cell and Tissue Culture. Vasil, I.K. and Thorpe , T.A. (Eds.), pp. 231-270. Kluwer Academic Publishers, Dordrecht.

Jefferson, R.A., Burgess, S.M. and Hirsch, D. 1986. B-Glucuronidase from Escherichia coil as a gene-fusion marker. Proceedings of the National Academy Sciences, USA 83: 8447-8451.

Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: B-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6:3901- 3907.

Kaeppler, H.F., Somers, D.A., Rines, H.W. and Cockbum, A.F. 1992. Silicon carbide fiber-mediated stable transformation of plant cells. Theoretical Applied Genetics 84:560- 566.

Klein, T.M., Arentzen, R., Lewis, P.A. and Fitzpatrick- McElligott, S. 1992. Transformation of microbes, plants and animals by particle bombardment. Bio/Technology 10:286-291.

Landgridge, P., Brettschneider, R., Lazzeri, P. andLorz, H. 1992. Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment. Plant Journal 2:631-638.

Lawson, C., Kaniewski, W. Haley, L., Rozman, R., Newell, C., Sanders, P. and Tumer, N. 1990. Engineering resistance to mixed virus iufection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in a transgenic Russet Burbank. Bio/ Technology 8:127-134.

Liu, D, Raghothama. K.G., Hasegawa, P.M. and Bressan, R.A. 1994 Osmotin overexpression in potato delays development of disease symptoms. Proceedings National Academy of Sciences, USA 91:1888-1892.

Lorz, H. Baker, B. and Schell. J. 1985. Gene transfer to cereal cells mediated by protoplast transformation. Molecular and General Genetics 199: 178-182.

Ludwig, S.R., Habera, L.F., Dellaporta, S .L. and Wessler, S.R. 1989. Lc, a member of the maize R gene family responsible for tissuespecific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proceedings of the National Academy Sciences, USA 86:7092-7096.

Ludwig, S.R., Bowen, B., Beach, L. and Wessler, SR. 1990. A regulatory gene as a novel visible marker for maize transformation. Science 247:449-450.

Luo, Z. and Wu, R. 1988. A simple method for the transformation of rice via the pollen-tube pathway. Plant Molecular Biology and Reproduction 6: 165-174.

Ma. H., Gu, M. and Liang, G.H. 1987. Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Mocnch, Theoretical Applied Genetics 73:389-394.

MacKinnon, C., Gunderson, G. and Nabors, M.W. 1986. Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum. Plant Cell Reproduction 5:349-351.

Maheshwari, N., Raiyalakshmi, K., Baweja, K., Dhtr, S.K., Chowdhry, C.N. and Maheshwari, S.C. 1995. In vitro culture of wheat and genetic transformation - retrospect and prospect. Critical Review of Plant Sciences 14:149-178.

Mariani, C., De Beuckeleer, M., Turettner, J., Leemans, J. and Goldberg, R.B. 1990. Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737-741.

Martin, G.B., BrommonschenkeI, S.H., Chungwongse, J., Frary, A., Ganal, M.W., Spivey, R., Wu, T., Earle, E.D. and Tanksley, S.D. 1993. Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262: 1432-1436.

Morrish, F., Songstad, D.D., Armstrong, C.L. and Fromm, M. 1993. Microprojectile bombardment: A method for the production of transgenic cereal crop plants and the functional analysis of genes. In: Transgenic Plants. Fundamentals and Applications. Andrew Hiatt (Ed.), pp. 133-171, Marcel Dekker Inc, New York.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology of Plants 15:473-497.

Nehra, N.S., Chibbar, R.N., Leung, N., Caswell, K., Mallard, C., Steinhauer, L., Baga, M. and Kartha, K. 1994. Self-fertile transgenic wheat plants regnerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant Journal 5:285-297.

Nelson, R.S., McCormick, S.M., Delannay, X., Dube,P., Layton, J., Anderson, E.J., Kaniewska, M., Proksch, R., Horsch, R., Rogers, S.G., Fraley, R.T. and Beachy, R.N. 198 8. Virus tolerance, plant growth, and field performance of transgenic tomato plants expressing coat protein from tobacco mosaic virus. Bio/Technology 6:403-409.

Oeller, P.W., Min-Wong, L., Taylor, L.P., Pike, D.A and Theologis, A. 1991. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254:437-439.

Olsson, 0., Escher, A., Sandberg, G., Schell, J., Koncz, C. and Szalay, A. 1989. Engineering of bacterial luciferase by fusion of luxB genes in Vibrio harveyi. Gene 81:335-347.

Ow, D., Wood, K.V., DeLuca, M., De Wet, J.R., Helinski, D.R. and Howell, S.H. 1986.Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856-859.

Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P.A. and Saedler, H. 1987. The regulatory CI locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO Journal 6:3553-3558.

Peng, J., Kononowicz, H. and Hodges, T.K. 1992. Transgenic indica rice plants. Theoretical Applied Genetics 83:855-863.

Perez-Vicente, R., Wen, X.D., Wang, Z.Y., Leduc, N., Sautter, C., Wehrli, E., Potrykus, I. and Spangenberg, G. 1993. Culture of vegetative and floral meristems in ryegrass: Potential targets for microballistic transformation. Journal of Plant Physiology 142:610-617.

Peflak, F.J., Stone, T.B., Muskoff, Y.M., Petersen, L.J., Parker, G.B., McPherson, S.A., Wyman, J., Love, S., Reed, G., Biever, D. and Fischhoff, D.A. 1993. Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Molecular Biology 22:313-321.

Raineri, D.M. Bottino, P., Gordon, M.P. and Nester, E.W. 1990. Agrobacterium-mediated transformation of rice (Oryza sativa L. ). Bio/Technology 8:33-38.

Reggiardo, M.I., Arana, J.L., Orsaria, L.M., Permingeat, H.R., SpitteIer, M.A. and VaIlejos, R.H. 1991. Transient transformation of maize tissue by microprojectile bombardment. Plant Science 75:237-243.

Rhodes, C.A., Pierce, D.A., Metler, I.J., Mascarenhas, D. and Detmer, J.J. 1988. Genetically transformed maize plants from protoplasts. Science 240:204-207.

Ritala, A., Aspegren, K., Kurten, U., SalmenkallioMarttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H. and Enari, T.M. 1994. Fertile transgenic barley by particle bombardment of immature embryos. Plant Molecular Biology 24:317-325.

Sanford, J.C. 1988. The biolistic process. Trends in Biotechnology 6:299-302.

Sanford, J.C., Smith, F.D. and Russell, J.A. 1993. Optimizing the biolistic process for different biological applications. Methods in Enzymology 217:483-509.

Shah, D., Horsch, R.B., Klee, H.J. Kishore, G.M., Winter, J.A.,Tumer, N.E., Hironaka, C.M., Sanders, P.R., Gasset, C.S., Aulent, S. Siegel, N.R. Rogers, S.G. and Fraley, R.T. 1986. Engineering herbicide tolerance in transgenic plants. Science 233:478-481.

Shen, W.H., Escurado, J., Schlappi, M., Ramos, C., Hohn, B. and Koukolikova-Nicola, Z. 1993. T-DNA transfer to maize cells: Histochemical investigation of B-Glucuronidase activity in maize cells. Proceedings of the National Academy Sciences, USA 90: 1488-1492.

Shimamoto, K, Terada, R., Izawa, T. and Fujimoto, H. 1989. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274-276.

Somers, D.A., Rines, H.W., Gu, W., Kaeppler, H.F. and Buschnell, W.R. 1992. Fertile, transgenic oat plants. Bio/Technology 10: 1589-1594.

Songstad, D.D., Somers, D.A. and Griesbach, R.J. 1995. Advances in alternative DNA delivery techniques. Plant Ceil, Tissue and Organ Culture 40:1 - 15.

Tarczynski, M.C., Jensen, R.G. and Bohnert, H.J. 1993. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 259:508-510.

Toplet, R., Gronenborn, E., Schafer, S., Schell, J. and Steinhiss, H.H 1990. Expression of engineered wheat dwarf virus in seed-derived embryos. Physiology of Plants 79:158-162.

Twell, D., Klein, T.M., Fromm, M.E. and McCormick, S. 1989. Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiology 91:1270-1274.

Vaeck, M., Reynaerts, A., Hofte, H., Jensen, S., De Beuckeller, M., Dean, C., Zabeau, M., Van Montagu, M. and Leemans, J. 1987. Transgenic plants protected from insect attack. Nature 328:33-37.

Vasil, I.K. 1995. Cellular and molecular genetic improvement of cereals. In: Current Issues in Plant Molecular and Cellular Biology. Terzi, M. et al. (Eds.), pp. 5-18. Kluwer Academic Publishers, Netherlands.

Vasil, V., Castillo, A.M., Fromm, M.E. and Vasil, I.K. 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10:667674.

Wan, Y. and Lemaux, P.G. 1994. Generation of large numbers of independently transfonned fertile barley plants. Plant Physiology 104:37-48.

Wilmink, A. and Dons, J.J.M. 1993. Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Molecular Biology Report. 11:165185.

Weeks, J.T., Anderson, O.D. and Blechl, A.E. 1993. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiology 102:1077-1084.

Xu, X. and Li, B. 1994. Fertile transgenic indica rice plants obtained by electroporation of the seed embryo cells. Plant Cell Reproduction 13:237-242.

Copyright 1995 African Crop Science Society

Home Faq Resources Email Bioline
© Bioline International, 1989 - 2022, Site last up-dated on 11-May-2022.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Internet Data Center of Rede Nacional de Ensino e Pesquisa, RNP, Brazil