African Crop Science Journal, Vol. 3. No.2, pp. 171-180, 1995
NEW VISTAS ARE OPENED FOR SORGHUM IMPROVEMENT BY GENETIC
A.K. KONONOWICZ, A.M. CASAS, D.T. TOMES^1, R.A. BRESSAN and
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 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.
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.
TISSUE CULTURE COMPONENT
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).
DNA DELIVERY BY PARTICLE BOMBARDMENT
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- transformation 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 expression --------------------- ----------- 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.
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