Printer Friendly

Applications of biotechnology in turfgrass genetic improvement.

Turfgrass refers to grass species suitable to be used as turf, i.e., a covering of vegetation, plus the matted, upper stratum of earth filled with roots and rhizomes (Beard, 1973). About 40 grass species are used as turf because of their ability to persist under regular mowing (Duble, 1996). The characteristics of low growth habit, prostate and creeping tendency, high shoot density, and medium fine leaf textures have been used in selecting grasses for turf. While contributing to soil development, stabilization, and improvement, and erosion control, turfgrasses are also used to beautify the earth, enrich our lives, and provide recreation and enjoyment for people. Turfgrass is maintained on lawns, estates, parks, golf courses, playing fields and public grounds, which cover about 14 million hectares of the land in the United States (Duble, 1996). The turfgrass industry also plays an important role in the U.S. economy, with more than $580 million in annual seed sales, second only to hybrid seed corn (Zea mays L.) (Kidd, 1993; Lee, 1996). Since the establishment of turfgrass culture in North America and the rest of the world, cultivar improvement has always been a central issue. As the result of the persistent efforts of breeders, significant achievements have been made in turfgrass breeding. More than 245 warmseason and cool-season cultivars have been registered since 1946 in the USA (Lee, 1996). These achievements have contributed to the establishment and growth of the turfgrass industry and the development of turfgrass science (Beard, 1973).

As in most other crops, turfgrass improvement traditionally has relied on conventional breeding methods, in which the accessible genetic material is restricted by sexual reproduction. This situation raises the necessity of exploring other approaches for more efficient achievements. Modern plant biotechnology is based on our current understanding of biological systems at both the cellular and molecular levels and has become increasingly associated with genetic engineering and other in vitro cellular manipulations of plants (Krans, 1989). Biotechnology is being used to supplement and complement traditional methods of plant improvement by bridging the individual gene pools in nature (Vasil, 1995) and thereby creating opportunities for breeders to utilize the genetic material from heterologous sources. Introgression of genetic traits from unrelated plant species or even other kingdoms has become a realistic breeding objective (Asay and Sleper, 1989; DeBlock, 1993).

Plant biotechnology has been applied to turfgrass improvements during the past two decades. Turfgrass biotechnology in the context of the biotechnological progress of grass species has not been reviewed. The achievements made in turfgrasses can be categorized as applications of molecular markers to assist breeding practices, in vitro culture, genetic engineering, and the use of fungal endophytes to improve turfgrass performance. This paper provides an overview of the recent advances in these areas.


Genetic study by using markers remains the most powerful approach to the understanding of hereditary transmission (Beckmann and Soller, 1986), which constitutes the basis for most modern breeding practices. Breeding practices improve the traits of cultivars by selecting individuals according to desirable criteria. To achieve this goal, Mendelian genetic markers that are associated with agronomic traits to be selected for are essential. The turfgrass industries need to identify cultivars and breeding lines in order to control the quality of germplasm and protect breeders' rights (Wu and Lin, 1994). However, the paucity of available markers has constrained efforts for detailed analysis of genomes of many important agricultural species (Beckmann and Soller, 1986), and therefore has hampered progress in cultivar improvement. For more than 20 yr, macromolecules have been exploited as genetic markers to meet the need for more accurate identification for genetic study of turfgrasses. Compared with morphological traits, molecular markers are discrete traits and are less subject to the infiuence of physiological and environmental variations. Two types of molecular markers have been used in turfgrasses: protein-based and DNA-based markers.

Protein-Based Methods

The use of electrophoretic patterns of total protein extracts for cultivar identification of turfgrasses was first investigated by Wilkinson and Beard (1972). Cultivars of bentgrass (Agrostis spp.) and Kentucky bluegrass (Poa pratensis L.) were distinguished on the basis of electrophoretic separation of leaf proteins on polyacrylamide gels. A limitation of this method is that consistency and reproducibility of the leaf protein extracts are affected by variations in growing conditions. Seed proteins are less susceptible to effects from fluctuations and, therefore, generate more informative electrophoretic patterns for turfgrasses as demonstrated by Clark et al. (1989) in studies of bentgrasses.

The first isozyme marker used in turfgrasses was peroxidase (Moberg, 1972), which was used for cultivar identification of creeping bentgrass (Agrostis palustris Hudson) and Kentucky bluegrass. Wehner et al. (1976) examined peroxidase isozymes by using improved equipment and procedures. The informative value of peroxidase banding patterns as affected by gel variability, intraplant variability, as well as age and off-type, was evaluated. The uniqueness of individual peroxidase patterns depended on the number of bands, mobility, and intensity ratio differences. Isozyme mobility is the most stable and reliable indicator of cultivar differences. Other isozyme markers including esterases and phosphoglucomutases have been successfully tested for red fescues (Festuca rubra L. subsp, rubra) (Villamil et al., 1982) and Kentucky bluegrass (Wu et al., 1984; Weeden and Emmo, 1985; Wu and Jampates, 1986).

The most systematic application of isozyme polymorphisms in turfgrasses involved multiple loci, such as phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), glutamate oxaloacetate transaminase (GOT), and triosphosphate isomerase (TPI), which generated unique banding patterns for several creeping bentgrass cultivars (Warnke et al., 1997). This study also exploited isozyme patterns to create a genetic distance matrix between the cultivars and therefore provided the basic data for Unweighted Pair-Group Method of Analysis (UPGMA) that suggested the possible phylogenetic linkages among cultivars. In addition to cultivar identification and lineage inference, isozyme markers have been successfully used to provide strong evidence to elucidate inheritance patterns in tetraploid creeping bentgrass (Warnke et al., 1998).

Isozyme markers provide a convenient and inexpensive tool for turfgrass genotyping. Nevertheless, the lack of polymorphisms makes isozyme markers unsuitable for resolving closely related cultivars or breeding lines. In addition, the quality and quantity of isozymes and proteins may be subject to variations due to plant growth and development and environmental effects (Wu and Lin, 1994).

DNA-Based Markers

Genetic characterization of organisms has been dramatically improved by the use of DNA-based markers. Genetic polymorphisms revealed by DNA analyses are much more informative than protein-based markers because they are variations in the genetic material. DNAbased markers are also greater in number and almost immune to environmental and physiological variability. Turfgrass researchers have begun to explore DNAbased markers for marker-aided breeding, cultivar identification, and germplasm protection.

Several approaches have been developed to detect DNA polymorphisms that characterize individual organisms. Restriction Fragment Length Polymorphisms (RFLPs) were first described for mapping the human genome (Botstein et al., 1980; Wyman and White, 1980) and later applied in plants. The RFLP method was developed to detect DNA restriction fragments of different lengths. Restriction Fragment Length Polymorphism fragments, produced by digestion of DNA samples with restriction endonucleases, are separated on the electrophoresis gels and detected by DNA hybridization with specific probes. Restriction Fragment Length Polymorphisms are enormous in number because any change to the DNA sequence involving restriction enzyme recognition sites or the length between sites generates polymorphisms. The fact that RFLPs behave in a codominant manner makes these markers ideal for genetic study of turfgrass, which consists of species using different reproductive strategies, because it allows genotyping of a locus in plants derived from any mating scheme (Tanksley et al., 1989). The RFLP markers generated from nuclear and chloroplast DNAs (cpDNAs) of turfgrass species have provided consistent evidence for accurate identification and clarification of phylogenetic relationships (Ohmura et al., 1993; Yaneshita et al., 1993a,b). A wide range of variation among cpDNAs of turfgrass was revealed by RFLP analysis (Yaneshita et al., 1993a). Since the genome structure of chloroplasts is conserved among plants (Palmer et al., 1988), and transmitted maternally in most plants, RFLP analyses of cpDNAs provide a simple and reliable tool for tracing evolutionary linkages along maternal lines. The combination of RFLPs of cpDNA and phenetic analysis was used to construct a dendrogram showing the clustering pattern among turfgrass and cereal species (Yaneshita et al., 1993a). The phylogenetic relationships suggested by this clustering were consistent with those suggested by traditional taxonomy and with the current turfgrass grouping scheme, e.g., cool-season (C3 type) and warm-season (C4 type) turfgrasses, based on ecological habitats, agronomical aspects, and physiological traits. The RFLPs of nuclear genomes and cell organelle genomes can also be used in turfgrass to analyze the effects of in vitro manipulations on genetic stability of plants. This technique has been widely used in other grass species, such as meadow fescue (Festuca pratensis Hudson) (Valles et al., 1993).

In addition to RFLPs of low-copy number sequences, some repetitive DNA sequences were found stable and species-specific among some turf species by PerezVicente et al. (1992). This group used species-specific DNA clones as probes to characterize the genomes of species in genera Lolium, Festuca, and their hybrid through Southern blot and in situ chromosome hybridization. Repetitive DNA markers could be used to study genome evolution and species divergence at the molecular level. Despite the important utilities of RFLPs in turfgrass studies, generation of these markers requires prior DNA sequence knowledge for making proper probes.

Since Random Amplified Polymorphic DNA (RAPD) was developed by Williams et al. (1990) and Welsh and McClelland (1990), the Polymerase Chain Reaction-based (PCR-based) method has drawn much interest in turfgrass research. This is a simple and convenient process for detecting polymorphisms in the absence of specific nucleotide sequence information (Williams et al., 1990). DNA markers generated by the RAPD procedure have remarkable potential for investigating genetic variations in turfgrass species (Callahan et al., 1993; Huffet al., 1993; Wu and Lin, 1994; CaetanoAnolles et al., 1995). Cultivar identification of high resolution (97%) and high reproducibility (80%) was achieved for buffalograss [Buchloe dactyloides (Nutt.) Engelm.] with RAPD-generated DNA profiles of DNA amplification fingerprinting (DAF) (Caetano-Anolles et al., 1991 a,b). A simple and fast DNA extraction protocol developed by Edwards et al. (1991) has made the RAPD method more convenient and accessible to turfgrass researchers. This method has been proven reliable in turfgrasses (Sweeney and Danneberger, 1994). High levels of polymorphic DNA profiles were detected by DAF in bermudagrass species (Cynodon spp.) by digesting DNA templates with restriction enzymes prior to amplification or by using arbitrary mini-hairpin primers (Caetano-Anolles et al., 1995). Capillary electrophoresis was superior to traditional slab gel methods in resolving DAF profiles (Caetano-Anolles et al., 1995). RAPD markers have simplified the task in identifying closely related cultivars and are able to provide informative data for phylogenetic analysis. This is especially true for inbred or clonely propagated species because RAPD markers revealed extensive variation among, but little variation within, populations (Huff et al., 1993). With the use of a statistical analysis method, Analysis of Molecular Variance (AMOVA), RAPD markers are also useful for characterizing closely related populations of the outcrossing turfgrass species, such as buffalograss (Huff et al., 1993) and perennial ryegrass (Loliurn perenne L.) (Huff, 1997), in which the significant withinpopulation variation makes it difficult to distinguish individual populations from each other on the basis of morphological traits and agronomical performance. The utility of RAPD markers was explored in Kentucky bluegrass in determining the genetic origins of aberrant plants derived from facultative aposporous apomixis using the improved procedure with silver-stained polyacrylamide gels (Huff and Bara, 1993). The finding that RAPD markers from bulk samples of perennial ryegrass were not simply the sum of amplification products of the individual plants (Sweeney and Danneberger, 1994) should effect the proper use of RAPD for homogeneous vs. heterogeneous populations of turfgrass species. The flexibility of RAPD markers in plant profiling has also been recognized. Different resolutions can be achieved by empirically choosing specific primers or a set of primers to deal with species or cultivars of diverse genetic structures, e.g., homogeneous inbred or heterogeneous outbred (Huff et al., 1993). Random Amplified Polymorphic DNA analysis would be of great value for almost any research or breeding program in which monitoring, identification, and genetic mapping of cultivars are involved.

Another class of PCR-based DNA markers is Simple Sequence Repeat (SSR) polymorphisms or microsatellites. Simple Sequence Repeat polymorphisms are generated by the variation in the number of the tandem repetitive di-, tri-, or tetranucleotide units present. This variation leads to the length polymorphism of the DNA fragments consisting of SSRs, which can be detected with PCR using pairs of primers flanking each SSR. Applications of SSRs in plants have been focused on linkage mapping, which was reported for soybean [Glycine max (L.) Merr.] (Akkaya et al., 1992; Morgante and Olivieri, 1993; Morgante et al., 1994; Rongwen et al., 1995), rice (Oryza sativa L.) (Zhao and Kocher, 1992, 1993; Wu and Tanksley, 1993), maize (Tautz, 1989; Senior and Heun, 1993), barley (Hordeum vulgare L.) (Saghai-Maroof et al., 1994; Becker and Heun, 1995; Liu et al., 1996), and several other crop species. Simple Sequence Repeat markers reveal high allelic variation throughout the entire genomes (Becker and Heun, 1995; Brown et al., 1996). In addition to their codominant behavior and Mendalian inheritance (Saghai-Maroof et al., 1994), they provide a better choice than RFLPs to detect genetic variation in self-fertilized species such as barley, which showed little variation in RFLPs (Becker and Heun, 1995). This feature also makes SSRs particularly valuable for self-fertilized turf species such as seashore paspalum (Paspalum vaginatum Swartz), for which SSR markers were successfully used to generate DNA typing for ecotypes and cultivars (Liu et al., 1995; Brown et al., 1997)

Nevertheless, the development of SSR markers is considered time consuming and expensive. The primary method involves constructing genomic libraries, screening by hybridization with SSR probes, followed by sequencing positively hybridizing clones (Condit and Hubbell, 1991). Since primer designing is based on the sequences flanking SSRs, for those well-characterized species such as Arobidopsis thaliana, rice, and maize, the sequence information obtained by screening the public sequence databases is sufficient for developing SSR primers (Brown et al., 1996). Without prior information of the genomic sequence, it is possible to amplify SSRs in one species by using primers developed in other related species, as demonstrated by Zhao and Kochert (1993) who used a rice PCR primer pair to amplify a SSRcontaining product in maize and bamboo (Bambusa vulgaris Schreber), though the results from using primers across species are difficult to predict (Brown et al., 1996).

The other novel PCR-based assay, Amplified Fragment Length Polymorphism (AFLP), was developed to selectively amplify and detect the restriction fragments from endonuclease digestion of the genomic DNA (Zabeau and Vos, 1993). This class of DNA markers has been used to facilitate genetic mapping for soybean (Prabhu and Gresshoff, 1994; Maughan et al., 1996), rice (Mackill et al., 1996; Yong et al., 1996), barley (Becker et al., 1995), citrus (Citrus spp.) (Orford et al., 1995), and potato (Solanum tuberosum L.) (Meksem et al., 1995). In AFLP assays, site-specific adapters are used to ligate to the restriction fragments. The primers are designed to be complementary to the adapters at their 5' ends with selective sequences of nucleotides at their 3' ends. Only those restriction fragments with nucleotides ranking the restriction sites that match both the adapters and the selective nucleotides of the primers can be amplified to generate discrete bands through fractionation in electrophoresis gels. The AFLP markers reflect restriction size variation as RFLP markers. However, more genetic loci can be analyzed simultaneously with higher polymorphisms by each experiment of AFLPs than that of RFLPs (Powell et al., 1996). In addition, AFLP assays take less time and need neither prior sequence knowledge nor a radioactively labeled probe. The flexibility of AFLP assays is similar to RAPD in that the selective nucleotides on the primers can be adjusted to maximize the polymorphisms. Because of these features, AFLPs are promising for rapid identification and mapping in plant species like turfgrasses, for which little sequence knowledge is afforded by their lower priorities in the economy.

DNA-based markers are also effective for etiological studies of turfgrass diseases. They have been used to detect pathogens in the infected plant tissues. Since many ectotrophic fungi are difficult to isolate and culture on artificial media, high diagnostic specificity is a top priority to identify numerous fungal isolates from infected plants. DNA probes for Leptosphaeria korrae (J.C. Walker & A.M. Sm.), a cause of spring dead spot of bermudagrass, were used to detect this pathogenic fungus in host plants (Tisserat et al., 1991). Detection sensitivity for another spring dead spot pathogenic fungus, [Ophiosphaerella herpotricha (L.) Pers.], has been substantially improved by selecting cloned DNA probes that hybridize to highly repetitive DNA sequences in the fungal genome to obtain stronger signals than with low-copy probes (Sauer et al., 1993). In addition to DNA probes, PCR is a rapid procedure to diagnose turfgrass patch diseases caused by the root-rotting fungi Ophiospaerella korrae (J.D. Walker & A.M. Sm.) R. Shoemaker & C. Babcock and O. herpotricha (Fr.:Fr.) Sacc. & Roum. (Tisserat et al., 1994). By using OKITS (specific for O. korrae) and OHITS (specific for O. herpotricha) primers, which were identified by sequence analyses of these fungal genomes, the rDNA internal transcribed spacer regions that are species-specific can be selectively amplified by PCR cycling and detected.

Construction of DNA-based genetic maps for crops will greatly facilitate genetic improvements in crops (Vasil, 1995). However, only two reports have been published about generating physical maps of cpDNA for turfgrass (Katayama and Yaasunari, 1993; Yaneshita et al., 1993a). The ongoing Rice and Maize Genome Mapping project may become valuable for generating DNA-based linkage maps of turfgrasses.


Plant regeneration systems are critical in turfgrass biotechnology. An efficient in vitro regeneration is a necessary step to recover genetically altered material, somaclonal variations, haploids, and existing germplasm with unique features, as well as for micropropagation and aseptic storage of valuable germplasm.

Cell and Protoplast Cultures

A common difficulty with in vitro culture of turfgrasses and other grass species is the lack of natural ability for secondary growth by cambium or cambium-like tissues in mature and differentiated explants. Slow progress in early programs was largely due to the improper use of mature and differentiated tissues as the explant material (Vasil and Vasil, 1994).

The key strategies to establish regenerable cell cultures for grass species were reviewed and generalized by Vasil (1995) as follows: (i) choose explants made of meristematic tissues and undifferentiated cells such as immature embryos or seeds, leaf base meristems, and meristematic segments of young inflorescences; (ii) use culture medium supplemented with high concentrations of strong auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 3,6-dichloro-o-anisic acid (dicamba) for inducing embryogenic calli; (iii) use embryogenic calli-derived cell suspensions for protoplast isolation. These strategies have led to the development of successful regeneration systems for all major turfgrass species.

A pioneer study in plant regeneration of grass species was the establishment of an embryogenic culture of Italian ryegrass (Lolium multifiorum Lam.) from immature embryos (Dale, 1980). This study and that of rice and sorghum [Sorghum bicolor (L.) Moench] provided the first observations of somatic embryogenesis in grass species (Vasil and Vasil, 1994). Efforts have been made to establish regenerable cultures from diverse turfgrass explant material (Table 1). Although direct somatic embryogenesis was achieved in orchardgrass (Dactylis glomerata L.) from mesophyll cells (Hanning and Conger, 1982; Conger et al., 1983; Trigiano et al., 1989), plant regeneration from embryogenic callus is the single most important path for turfgrass, as many major turfgrass species have been regenerated in this way. The culture medium requirements for turfgrasses are similar to that for other grass species. Generally high salt nutrient solutions such as the Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), the most frequently used medium for turfgrass, and modified CC-medium (Potrykus et al., 1979; Nielsen and Knudsen, 1993) with 3 to 12% sucrose and high concentrations of strong auxins such as 2,4-D and dicamba have been used. Cytokinins, such as 6-benzylaminopurine (6-BA) and N-(2-furanylmethyl)-1H-purin-6-amine (kinetin), at low concentrations, in combination with auxins were often used in turfgrass species to promote callus initiation. Addition of casein hydrolysate to the culture medium was found to be beneficial to embryogenic callus initiation (Artunduaga et al., 1988). Proline and glutamine have stimulatory effects on callus induction among the organic N compounds tested (Shetty and Asano, 1991). Embryogenic turfgrass callus is characteristically friable, somewhat organized, and generally white to light yellow in color. Because of a low frequency of occurrence (Vasil and Vasil, 1994) and the heterogeneous nature of embryogenic cultures, selection during subculture is effective in enriching embryogenic cultures (Krishnaraj and Vasil, 1995).

Embryogenic cell suspensions provide the only source of totipotent cells to isolate protoplasts from grass species (Vasil, 1988; Potrykus, 1990), in addition to being the direct targets for genetic transformation. Regenerable cell suspensions are established by selectively transferring embryogenic calli to liquid medium. Finely dispersed and fast-growing cell suspensions are maintained in liquid media with high levels of auxins and subcultured every 3 to 7 d. Most turfgrass embryos only develop to either a globular or early scutellar stage in the liquid medium, as is also the case for most other gramineous species (Vasil, 1985, 1987). Cell suspensions need to be plated onto semi-solid media for further embryo development and plant regeneration. The only exception was found in orchardgrass, where embryos can develop to reach a germinable stage in a single regime of liquid medium (Gray et al., 1984; Gray and Conger, 1985; Conger et al., 1989). Successful establishment of embryogenic cell suspensions and subsequent plant regeneration have been reported in several major turfgrass species, including creeping bentgrass (Terakawa et al., 1992; Hartman et al., 1994; Lee et al., 1995), tall fescue (Festuca arundinacea Schreber) (Rajoelina et al., 1990; Takamizo et al., 1990; Dalton, 1988a,b, 1993; Wang et al., 1992, 1995; Ha et al., 1992; Dalton et al., 1995), red fescue (Spangenberg et al., 1994; Wang et al., 1995), meadow fescue (Wang et al., 1993a), redtop grass (Agrostis alba L.) (Asano and Sugiura, 1990); Kentucky bluegrass (Nielsen et al., 1993; Nielsen and Knudsen, 1993), Italian ryegrass (Rajoelina et al., 1990; Dalton, 1988b, 1993; Wang et al., 1993b, 1995), perennial ryegrass (Dalton, 1988a,b; Zaghmout and Torello, 1992; Wang et al., 1993b, 1995; Olesen et al., 1995a), and the hybrid ryegrass (L. x boucheanum) (Wang et al., 1993b) (Table 1). In most of these studies, embryogenic cell suspensions were used for protoplast isolation and subsequent plant regeneration. One major problem in cell suspension cultures is the rapid decline of regenerability with time. To retain high levels of green-plant regeneration frequency, cryopreservation of suspension cultures in liquid N has been successfully introduced and optimized for perennial ryegrass, ltalian ryegrass, tall fescue, and red fescue (Wang et al., 1993b; Spangenberg et al., 1994; Wang et al., 1995).

Protoplasts are useful for multiple manipulations in turfgrass biotechnology. Plants have been recovered from protoplasts of several turfgrass species (Table 1). Although having many advantages for in vitro manipulations, protoplasts are still considered the most difficult tissue-culture explants from which to recover plantlets. Embryogenic callus-derived, well-dispersed, finely grained, and fast-growing cell suspensions are required as the donor material to isolate competent protoplasts. The age of the donor-cell suspensions is very critical to the competency of protoplasts (Takamizo et al., 1990; Nielsen et al., 1993). To retain morphogenic potential of donor suspensions, addition of amino acids, such as L-proline, and an osmoticum, such as sorbitol, was beneficial (Asano and Sugiura, 1990). Using high quality enzymes for cell-wall hydrolyzation, improves protoplast isolation. In addition to the basic requirements for protoplast culture in terms of the need for nutrients, growth regulators, and osmotic pressure, the medium volume per cell ratio (plating density) and other environmental factors need to be adjusted for specific genotypes. Agarose embedding of protoplasts (bead-type culture) was used to promote plant regeneration from protoplasts (Asano and Sugiura, 1990; Takamizo et al., 1990; Wang et al., 1992, 1995). Some special treatments have been used in turfgrass species for a better regeneration response. The feeder layer system, in which protoplasts are cocultured with a layer of physically separated "nurse" or "feeder cells", has been found effective for increasing plating efficiency of protoplasts and is necessary for the formation of protoplast-derived callus and plant regeneration in creeping bentgrass (Lee et al., 1995), tall fescue, red fescue and meadow fescue (Wang et al., 1995), and Italian ryegrass, perennial ryegrass, and L. x bouceanum (Wang et al., 1995; Olesen et al., 1995a). This method was frequently used for other turfgrass species (Takamizo et al., 1990; Ha et al., 1992; Wang et al., 1992). Proline in proliferation medium enhanced regeneration of Kentucky bluegrass from protoplasts (Takamizo et al., 1990). Despite the remarkable success that has been achieved in turfgrass species, protoplast culture still remains difficult in practice because of the intensive labor involved, significant genotype-dependence in culture establishment, and relatively short longevity of the donor-cell suspensions, problems of albinism, low frequency in plant regeneration from protoplasts, and difficulties in selection after genetic manipulations. Manipulations of culture conditions provide strong potential to overcome the recalcitrance caused by epigenetic effects, as indicated by Halperin (1986).

Shoot Apex Culture

The shoot apex is a small, distalmost portion of the shoot, typically including such regions as the apical meristem, three to six lateral primordia, and a subapical region (Medford, 1992). Studies of some grass species have found that the development of their shoot apices can be reprogrammed by in vitro culture. This developmental plasticity was even obvious in the early studies when fertile plants were recovered directly from shoot apices of maize (Raman et al., 1980), Italian ryegrass, perennial ryegrass, meadow fescue, tall fescue, and red fescue (Dale, 1975, 1977a,b). The more recent studies found that in vitro culture regimes could affect shoot apex development in more profound ways. Somatic embryos can be induced to develop from isolated sorghum shoot apices (Bhaskaran et al., 1988). By manipulating plant growth regulators in the medium, sorghum shoot apices can give rise to not only single or multiple shoots but also embryogenic, shooty, or rooty callus (Bhaskaran et al., 1992). Other studies also revealed the versatile developmental patterns of sorghum shoot apices (Zhong et al., 1998), and that of shoot apices of maize (Zhong et al., 1992), rice (Christou and Ford, 1995), and oat (Avena sativa L.) (Zhang et al., 1996) under several different culture regimes.

In turfgrass, shoot apex cultures were established for virus eradication and for long-term aseptic storage of germplasm (Dale, 1975, 1977a). Kinetin and 2,4-D (0.01-0.2 mg [[.-1]) in the culture medium were beneficial for plant regeneration from shoot apices (Dale, 1977b). The sizes of shoot apices excised were critical to virus elimination, survival rate in the culture, and regeneration behavior (Dale, 1975,1977b). An interesting finding was that some larger shoot apices produced an inflorescence instead of maintaining vegetative growth (Dale, 1975, 1977b). Besides vegetative shoot apices, floral apices of perennial and Italian ryegrasses were cultured for the production of functional pollen (Perez-Vicente et al., 1993). In this study, floral apices were excised from immature inflorescences and cultured in MS liquid medium supplemented with kinetin and 2,4-D to facilitate differentiation of spikelets. The differentiated spikelets completed full development on a hormonefree MS solidified medium.

The shoot apical explant, derived from either vegetative or floral apex, could provide an alternative target for turfgrass transformation. In studies of turfgrass and cereal crops, plant regeneration from shoot apices was readily achieved and nearly genotype-independent (Sautter et al., 1995; Park et al., 1996). Plant regeneration from shoot apices can also be manipulated to avoid an intervening callus phase and adventitious regeneration, which increase the risk of undesirable somaclonal variation (Karp, 1994; Park et al., 1996). The competence of shoot apices was demonstrated for biolisticsmediated transformation of maize (Lowe et al., 1995; Zhong et al., 1996), rice (Christou and Ford, 1995), and for Agrobacterium-mediated transformation of rice (Park et al., 1996). Transient gene expression was also observed in meristem tissues of vegetative and floral apices of perennial ryegrass (Perez-Vicente et al., 1993) and wheat (Triticum aestivum L.) (Sautter et al., 1995).

Because shoot apex (of shoot meristems)-targeted transformation offen has led to chimeric sectors in primary transformants (R0) (Christou and Ford, 1995; Sautter et al., 1995; Lowe et al., 1995; Park et al., 1996; Zhong et al., 1996), transgenic progeny can only be obtained from those transgenic sectors that happen to be germline cells in the shoot apices. In order to produce a high frequency of nonchimeric R1 transformants, follow-up manipulations on the sectored R0 transformants are offen needed. The approach developed by Lowe et al. (1995) and Zhong et al. (1996) for maize should be seen as a solid step in this respect. By multiplication of shoot apices on the selection medium, nontransgenic sectors were reduced in size or even eliminated as transgenic sectors enlarged and became predominant in each shoot apex. After these manipulations, transgenic sectors had a greater likelihood of contributing to the germline.

Anther Culture

Haploids recovered from anthers have become an integrated part of modern breeding practices (Wenzel et al., 1995). The culture procedure is comprised of two steps: induction of microspore callus and subsequent regeneration. The induction medium contains an auxin, such as 2,4-D, indole-3-acetic acid (IAA), or naphthalene acetic acid (NAA), and cytokinin, such as kinetin; while the regeneration medium contains cytokinins, such as 6-BA. The later spikes with most of the microspores in the mid- or late-uninucleate stage, are used to initiate the culture. Haploid plantlets have been regenerated from anther cultures of tall fescue (Kasperbauer et al., 1980); meadow fescue (Rose et al., 1987); Italian ryegrass (Boppenmeier et al., 1989); perennial ryegrass (Stanis and Butenko, 1984; Halberg et al., 1990; Opsahl-Ferstad et al., 1994a,b; Olesen et al., 1995b; Madsen et al., 1995), and the intergenera hybrid (L. multiflorum x Festuca pratensis) (Rose et al., 1987). Genotype-dependence and inheritance of anther-culture response have been investigated (Opsahl-Ferstad et al., 1994a; Madsen et al., 1995). The genotype of perennial ryegrass determined the degree of correlation between different characters of androgenetic response, such as embryo-like structures per 100 anthers, and green plants per 100 anthers (Opsahl-Ferstad et al., 1994a). The high genotype-dependent anther response of ryegrass can be transferred via back crossing to produce superior genotypes capable of anther culture (Halberg et al., 1990: Madsen et al., 1995). Strong genotype-dependence makes it necessary to modify the chemical composition of the culture medium and other in vitro conditions to fit the specific genotypes for improved androgenetic responses (Opsahl-Ferstad et al., 1994a). The total N content, ratio of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], solid vs. liquid medium, and cold pretreatment affected the fate of anther culture in perennial ryegrasses (Opsahl-Ferstad et al., 1994b).

Effects of Genotype on In Vitro Regenerability

The genotypic effect is a significant factor controlling the response of explants to in vitro culture conditions. For example, the competence for direct embryogenesis in orchardgrass was found to be genotype dependent (Hanning and Conger, 1982; Conger et al., 1983). The genotype regenerability dependency may be more pronounced in cell suspensions and protoplasts than in callus cultures (van Heeswijck et al., 1994), but the opposite situation was also reported (Olesen et al., 1995a). Genotype and culture response were correlated, including the correlation value of callus induction-plant regeneration in tall rescue (Takamizo et al., 1990), meadow rescue (Wang et al., 1993a), and perennial ryegrass (Opsahl-Ferstad et al., 1994a; Olesen et al., 1995a). Genotypes with high regeneration percentages and high frequencies of green-plant recovery also showed superior suspension culture regeneration both in longevity and ability (Wang et al., 1995; Olesen et al., 1995a). Cultures derived from single genotypes may help to reduce inconsistent in vitro behaviors (Takamizo et al., 1990; Wang et al., 1995; Yamamoto and Engelke, 1997). This practice is more important for cool-season turf species than warm-season species because most of the cool-season turfgrass species are genetically heterogeneous within cultivars or population (Huff, 1997). Single-genotype-derived cultures improved the efficiency of in vitro manipulations in heterogeneous populations (Takamizo et al., 1990; Wang et al., 1995), because researchers can selectively use individual culture lines that have the best response to in vitro manipulations. Single-genotype-derived cultures from vegetative tissues of cross-pollinated or vegetatively propagated cultivars facilitate postculture selection (Yamamoto and Engelke, 1997). This system could also help to evaluate and optimize other culture condition factors.

Genotype selection within cultivars, especially cultivars with broad genetic backgrounds, for better response to in vitro cultures, would probably be effective for several turfgrass species, since androgenetic response in perennial ryegrass is probably determined by relatively few loci (Opsahl-Ferstad et al., 1994a). Transfer of culture response via back-crossing is a possible approach for nonculture responsive breeding material. Androgenesis superior to that of their parents was attained in hybrid clones of perennial ryegrass (Halberg et al., 1990; Madsen et al., 1995).


Genetic transformation can accelerate crop improvement. Transgenic plants of orchardgrass were first regenerated from protoplasts (Horn et al., 1988). A recent review by Lee (1996) listed six cool-season species of turfgrass transformed by a variety of transformation systems. The transgenes include the [Beta]-glucuronidase (GUS) gene, the phosphinothricin acetyltransferase (bar) gene, and the hygromycin phosphotransferase gene. Progress in turfgrass transformation has been made in exploring and optimizing transformation systems that have been used for other grass species and dicots.

Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation has been widely used for many dicot plants as a convenient, highly efficient method for DNA transfer. A great benefit of Agrobacterium-mediated transformation is its capability to transform intact, regenerable plant tissues and organs (Horsch et al., 1985). Gene integration patterns are more predictable in Agrobacterium-mediated transformation than other gene transfer methods (Birch and Franks, 1991; Smith and Hood, 1995). Transformants from Agrobacteriaum-mediated transformation often contain genomic insertions by exact and single- or low-copy number of transgene cassettes (DeBlock, 1993). Single-copy insertions facilitate gene expression because they are less prone to co-suppression or gene-silencing effects that are usually found in multicopy insertion events (Vasil, 1995). Another benefit of Agrobacterium-mediated DNA delivery is the minimal exposure of explant material to tissue culture conditions that induce genetic instability (Karp, 1994). Although grass and other monocots have been generally considered outside the host range of A. tumefaciens, Agrobacterium-mediated transformation has been used successfully in a few grass species including maize (Gould et al., 1991; Ishida et al., 1996), rice (Chan et al., 1992, 1993; Liu et al., 1992: Park et al., 1996), and wheat (Deng et al., 1988). An examination of successful monocot transformation points to several factors that may be critical to A. tumefaciens-mediated monocot transformation. These factors include the use of meristematic tissue for cocultivation with A. tumefaciens, the addition of phenolic compounds to the cocultivation media, and the use of the compatible strains of A. tumefaciens (see review by Smith and Hood, 1995). Agrobacterium-mediated transformation has not been reported in turfgrass. More efforts are warranted before this method becomes a routine transformation approach for grass species.

Transformation by Direct DNA Delivery DNA Uptake by Protoplasts

Direct DNA delivery into protoplasts is one of the methods by which successful transformation has been achieved in turfgrasses. With the physical barriers of the cell walls removed, protoplasts can be induced to uptake DNA from the culture by osmotic treatment, e.g., polyethylene glycol, or electric shock (electroporation). Protoplast cultures have served as genotype-independent targets for free DNA uptake. By protoplast-mediated gene transfer, transgenic plants were obtained in tall rescue (Wang et al., 1992; Ha et al., 1992; Dalton et al., 1995), red fescue (Spangenberg et al., 1994), orchardgrass (Horn et al., 1988), creeping bentgrass (Lee et al., 1995; Sugiura et al., 1997), and redtop (Asano et al., 1991; Asano and Ugaki, 1994). Although protoplast-mediated transformation has proven successful in these turfgrass species, plant regeneration from protoplasts is still difficult to achieve (Park et al., 1996) due to such uncontrollable parameters as genotype-dependent competence for regeneration (Potrykus, 1990; Hinchee et al., 1994; Spangenberg et al., 1995a). Other constraints of this process include infertility problems, undesired integration of multiple and rearranged transgene copies, and somaclonal variation resulting from the extensive cytogenetic disturbance in protoplast culture (Karp, 1994; Spangenberg et al., 1995a).

Biolistic Bombardment

Microbiolistic delivery systems for turfgrass genetic engineering have achieved success. Invented by Sanford and others (Sanford et al., 1987; Sanford, 1988), biolistics delivers DNA molecules carried by tiny particles of Au or W into intact cells. Physical force generated from compressed He or gun powder to penetrate cell walls for DNA delivery circumvents the host range limitations of Agrobacterium vectors and also eliminates the need for plant regeneration from protoplasts. The biolistic DNA delivery method is versatile and culture-system independent, compared with protoplast DNA uptake methods. Several different types of explant material have been used successfully in turfgrasses to recover transgenic plants after biolistic bombardment. Transformation by biolistic delivery has been reported with embryogenic calli of creeping bentgrass (Zhong et al., 1993; Lee et al., 1995; Liu et al., 1998; Warkentin et al., 1997), and with cell suspensions of tall fescue and red fescue (Spangenberg et al., 1995a), creeping bentgrass (Hartman et al., 1994), and perennial ryegrass (Spangenberg et al., 1995b). Transgenic orchardgrass plantlets have been regenerated directly from leaf mesophyll cells after biolistic bombardment (McDaniel et al., 1997). Biolistic gene delivery also provides a unique way for direct transformation of organelle genomes and rapid assessment of transient expression of genetic constructs in plants (Birch and Franks, 1991).

Although biolistic DNA delivery methods were suggested to be universal for every species (Birch and Franks, 1991), they have the shortcomings analogous to those in other direct gene transfer methods. The complex gene integration patterns that often result from this method may have significant implications for developing stable transgenic plants.


The production of transgenic plants from microinjected cells has only been successful in tobacco (Nicotiana tabacum L.) and rapeseed (Brassica napus L.) (Neuhaus et al., 1987). The microinjection apparatus consists of three basic components: a micropipette made of glass, an injection system applying injection driving force to the micropipette, and a d controlling the microscopic motions of the injection pipette (Mathias, 1987). Since it can deliver a DNA solution into the nuclei of the target cells, microinjection has the potential to transform any plant species when the proper target cells are available (Hinchee et al., 1994). The potential target cells for whole-plant transformation by microinjection were listed by Mathias (1987). Among these cells, protoplasts, suspension culture cells, and callus cells are the most readily available turfgrass in vitro cultures and therefore have the potential to become the target cells in microinjection-mediated transformation. Nevertheless, the feasibility of using microinjection for whole-plant transformation is restricted by the technical difficulties. The operation of microinjection is laborious and requires the difficult process of regenerating plantlets from isolated single cells or small cell clusters because large tissues cannot be resolved clearly enough for accurate needle placement (Hinchee et al., 1994). This task is made even more demanding when protoplasts are used as the target cells in transformation, since the plating efficiency and regeneration efficiency from protoplasts also play a significant role in determining transformation efficiency (Hinchee et al., 1994). Compared with other gene delivery systems, microinjection is not as well proven for creating transgenic plants. Turfgrass transformation via microinjection has not been reported.

Silicon Carbide Fiber-Mediated Transformation

Silicon carbide fiber-mediated transformation for plant cells was developed by Kaeppler et al. (1990). This method involves vortexing the cell or tissue culture with plasmid DNA and CSi fibers averaging 0.6 **m in diameter and 10 to 80 **m in length, which facilitate DNA delivery into nuclei by serving as microinjection needles (Kaeppler et al., 1990, 1992). Progress in CSi-mediated DNA delivery has been slow. The scarce number of studies include earlier reports on cell suspensions of maize, tobacco (Kaeppler et al., 1990), redtop grass (Asano et al., 1991), and a more recent one using mature embryos of wheat (Serik et al., 1996), in which only GUS transient expression was detected in the Csi-DNA-treated cultured cells. Stable transformation of plant cells was first reported by Kaeppler et al. (1992) in maize. The study by Frame et al. (1994) was the first and the only one so far, in which fertile, transgenic maize plants were recovered from plant cells transformed via CSi-mediated gene delivery. The transformation frequency was estimated as [10.sup.-6] transformants per cell treated (Kaeppler et al., 1992), lower than with biolistic bombardment. Nevertheless, it might be premature to judge this method solely on the transformation frequency data generated from one or two studies, since the transformation frequency of maize cells using other methods varied significantly from case to case with differences up to two magnitudes, as shown by Kaeppler et al. (1992). The observed reduction in cell viability as the result of CSi treatment (Kaeppler et al., 1992) is a potential drawback of this method. Advantages of CSi fiber-mediated transformation over biolistic bombardment include very low equipment cost, relative ease of the procedure (Kaeppler et al., 1992), and better control of the DNA quantity delivered (Thompson et al., 1995). While these features are attractive to plant researchers, the potential of this method needs to be further explored in order to obtain transgenic plants in other species. Regenerable targets of gene delivery should be extended to include tissues and organs other than suspension cells.

Selection for Transgenic Plants

Cells containing gene(s) of interest are selected and regenerated into individual plants. A selectable marker gene in transgene constructs is usually necessary to confer the preferential growth of transformed cells in the presence of the corresponding selective agent. Except for the case in which creeping bentgrass was transformed by Zhong et al. (1993) using the GUS gene as the screening marker, all other transformation events in turfgrass species have relied on using selectable markers for identifying transgenic plants. Antibiotic markers, such as hygromycin resistance, and herbicide markers, such as phosphinothricin resistance, have been successfully transferred and expressed in transgenic turfgrasses as selectable markers (see the list of selectable markers in the review by Lee, 1996). Selection schemes used for turfgrasses vary widely among different genotypes and culture types in terms of concentrations of selective agents and the timing of application because of the differing sensitivity of cultures to the selective agents. Since selection efficiency can be measured as a function of the recovery rate of transgenic cells and the nontransgenic "escapes", the minimal lethal dosage of the selective agents to nontransgenic cells must be empirically determined. Selection by herbicides usually needs to be delayed for a certain period of time after gene transfer in order to allow the transformed cells to recover from physical damage (especially in electroporation and particle bombardment) and to produce sufficient quantities of the resistance enzyme for protection. Either a fixed concentration or stepwise increase in concentration of selective agents has been used frequently in the selection schemes for turfgrass transformation. The finding that continuous vs. discontinuous selection schemes are correlated with the copy number of integrated transgenes in tall rescue (Dalton et al., 1995) might be important in efforts to minimize down-regulating or "gene-silencing" effects caused by multiple integrations.

Selectable markers are essential to genetic transformation. However, fewer selectable markers can be used for monocot species than for dicot plants (Zhou et al., 1995). Given the fact that the presence of a selectable marker gene in plant genomes makes it impossible to carry out the subsequent gene transfers with the same marker, it would be beneficial to develop strategies to remove selectable markers from the transgenic plants. Transformation by co-transferring unlinked transgenes has been achieved with high efficiencies in several crops, including maize (Schocher et al., 1986; Gordon-Kamm et al., 1990), rice (Chair et al., 1996), barley (Wan and Lemaux, 1994), rapeseed (Herve et al., 1993), and tobacco (Carter and Maliga, 1995). This method was originally used to speed up transformation because it eliminates the need to prepare a transgene construct harboring both the desired gene and the selectable marker (Schocher et al., 1986). It also provides a simple approach to recycling selectable markers. If there is no physical linkage between the selectable marker and the desired transgene, removal of the selectable-marker locus will be readily achieved by sexual segregation. However, the cotransferred genes could become linked in the genomes of the transformants if they are coprecipirated and transferred by using the biolistic method (Gordon-Kamm et al., 1990; Spencer et al., 1990). Another strategy for removing selectable marker genes involves using site-specific recombination systems obtained from microorganisms, such as budding yeast (Saccharomyces cerevisia) (Cregg and Madden, 1989), fission yeast (Schizosaccharomyces pombe) (Lyznik et al., 1993), and bacteriophage P1 (Dale and Ow, 1991; Sauer, 1994). Each of these systems consists of a pair of inverted repeat DNA sequences as the recognition site and a specific recombinase gene. The specific enzymatic activity of this recombinase results in the excision of the inverted repeats and the sequence between them. Plasmid constructs for gene transfer can be made to contain the selectable marker flanked by the inverted repeats and used to transform plant cells. As the established recombination target, the selectable marker can be excised from the plant genome by the corresponding site-specific recombinase activity, which can be introduced through either transfer of the recombinase gene or direct application of purified recombinase enzyme (Dale and Ow, 1991). Biolistic bombardment systems may be suitable for these manipulations.

Recycling of selectable marker genes should be included in transformation practices in turfgrass species. Although the strategy of using recombination systems has not yet been tested in turfgrass, there is strong reason to be optimistic about its success, since gene removal has already been achieved in tobacco by the Cre/lox recombination system (Dale and Ow, 1991), and the yeast FLP/FRT recombination system has already been tested to be functional in maize and rice protoplasts (Lyznik et al., 1993).

After the fertile transgenic plants have been regenerated, selfing is usually a necessary step to obtain homozygous seed progeny from the heterozygous parents at the transgene loci. Self-fertilized turfgrass species provide a more convenient system to achieve this task than the outcrossing species, in which self-infertility limits inbreeding, and apomictic species, in which either self-fertilization or cross-fertilization is limited.


Endophytes are organisms that are contained or grow entirely within plants and spend all or nearly all of their life cycles in their hosts (Siegel, 1987; Breen, 1993a,b). Darnel (Lolium temulentum L.) was the first grass species found to have endophytic mycelium in seeds (Vogl, 1898). Currently, fungal endophytes have been recognized as widely distributed in virtually all large genera, tribes, and subfamilies of the Gramineae family (Bacon and De Battista, 1991; Clay, 1993). To estimate the level of endophyte infection in the grass family in natural settings (Clay, 1994) or to control these endophytic fungi in the field is difficult (Siegel, 1987).

Among grass endophytes, true endophytes (endophytes sensu stricto, Schardl, 1994), are currently recognized as most relevant in grass breeding. This type of endophyte never produces external reproductive stages on the host plants and thus is less destructive and even relatively symptomless to the hosts compared with those that produce external mycelium or spores. In fact, symbiotic associations between true endophytes and grass hosts could be characterized as stable and mutualistic; the endophytes are transmitted asexually via host seeds. The genera Acremonium and Epichloe in the tribe Balansiae of Clavicipitaceae are the most important true endophytes and have been the focus of study because of the immediate applications to turfgrass development (Hill, 1994). Nevertheless, the use of true endophytes in turfgrass improvement is greatly limited by the fact that these two genera are not found in warm-season turfgrass species (Richardson et al., 1997).

Altered Performance of Endophyte-Infected Grasses

The effects of endophytic fungi on forage and turf grasses and on grazing animals have been a focus in studies of grass-endophyte associations (Porter, 1994). Endophyte-infected pasture grasses cost livestock producers hundreds of millions of dollars annually in lost production due to fescue toxicosis and ryegrass staggers (Siegel, 1987); however, grass endophytes play an important role in enhancing survival of host plants suffering from environmental stresses. Endophyte-conferred protection against insects (Prestidge, 1982; Funk et al., 1983; Ahmad et al., 1986; Johnson-Cicalese and White, 1990; Mathias et al., 1990; Breen, 1993a,b) and drought and heat (Arachevaleta et al., 1989) has been well documented, though endophyte-enhanced drought tolerance was not supported by other reports, such as White et al. (1992). Grass-endophyte interactions have had significant impacts on turfgrass evolution and breeding. Many current turf cultivars were derived from endophyte-infected plants, while breeders were originally unaware of the presence and benefits of endophytes (Funk et al., 1994).

Turfgrass Breeding Using Endophytes Strategies

Grass endophytes present new opportunities for turfgrass improvement. The close mutualistic associations with host plants make it possible to use true endophytes to manipulate the performance turfgrass plants. Endophyte strategies include several approaches to exploit endophytes as breeding agents.

The direct use of naturally endophyte-infected germplasm in breeding programs provides a convenient way to improve turfgrasses. Germplasm derived from old low-maintenance turfs of the USA and some naturalized turfs are believed to have established stable mutualisms with endophytes and other beneficial microorganisms as the result of long-term natural selection. They have become well adapted to local edaphic, climatic, and pest environments (Funk et al., 1994). Endophyte-infected turfgrass germplasm exhibits enhanced resistance to a list of insect pests (see reviews of Siegel, 1987; Clement et al., 1994; and Rowan and Latch, 1994) including sod webworm (Crambus spp.) (Funk et al., 1983), chinch bugs (Blissus leucopterus hirtus Montandon) (Funk et al., 1985; Mathias, 1988; Siegel, 1987), the Argentine stem weevil (Listronotis bonariensis Kuschel) (Prestidge, 1982; Stewart, 1985), bluegrass billbugs (Sphenophorus parvulus Gyllenhal) (Ahmad et al., 1986), and fall armyworms [Spodoptera frugiperda (J.E. Smith)] (Hardy et al., 1985, 1986; Breen, 1993a). Endophyte-enhanced persistence under abiotic stresses such as drought and resistance to disease such as brown patch (Rhizoctonia solani Kuhn) and dollar spot (Sclerotinia homoeocarpa F.T. Bennett) are also valuable in turfgrass germplasm (Funk et al., 1994). The widespread availability of A cremonium endophytes in elite turfgrass germplasm makes it highly effective to develop new cultivars with endophyte-enhanced performance (Funk et al., 1994). Many commercially released turfgrass cultivars feature high levels of Acremonium endophytes (Funk et al., 1983). Perennial ryegrass and tall fescues are the most successful examples of the use of endophyte-enhanced germplasm. However, the limited diversity endophytes associated with turfgrass germplasm should be a concern in future breeding programs (Funk et al., 1994).

Considerable efforts have also been directed at incorporating beneficial endophytes into genetically improved turfgrass cultivars. Hybridization and direct inoculation are the current approaches to establish novel endophyte-host combinations. Several commercially released fine fescues were developed (Funk et al., 1994). Identification of the desirable endophytes and a better understanding of the physiology of the interactions between the endophytes and their turfgrass hosts are needed to ensure success in other turf species.

Manipulating the performance of grass hosts via genetic engineering of the associated endophytes represents an alternative approach to transform grasses (Murray et al., 1992). Surrogate transformation has been successful by transforming Acremonium spp. (Murray et al., 1992; Tsai et al., 1992), and the reintroduction of transformed Acremonium into perennial ryegrass (Murray et al., 1992). Progress in transforming filamentous fungi (reviewed by Fincham, 1987) using protoplasts along with polyethylene glycol [[Alpha]-hydro-t[Omega]-hydroxypoly(oxy-l,2-ethanediyl)] treatment and osmotic conditioning provides the basic protocol for grass endophyte transformation. Transformation frequencies for Acremonium spp. were achieved up to 700 to 800 transformants [micro]g-1 plasmid DNA (Murray et al., 1992), depending on such parameters as regeneration medium (Oliver et al., 1987; Murray et al., 1992), osmoticum techniques, and stares of vector DNA (linear or circular) (Murray et al., 1992). Electroporation was used by Tsai et al. (1992) to facilitate DNA transfer because it requires few postmanipulations and might facilitate homologous integration or gene conversion (Schardl, 1994). Surrogate transformation makes it possible to manipulate grass hosts by introducing heterologous genes into endophyte genomes. Most known molecules responsible for endophyte-enhanced plant protections are the end products of biosynthetic pathways (Schardl, 1994). Examples are peramine, which confers protection to the hosts from damage by Argentine stem weevil (Rowan et al., 1986), and lolitrems, the cause of ryegrass staggers syndrome (Gallagher et al., 1981, 1982, 1984; Weedon and Mantle, 1987). Identification of the genes responsible for ratelimiting steps would be very critical in attempts to manipulate endophytes. Progress has been made with other filamentous fungi (Schardl, 1994). Dimethylallyltryptophan synthase (DMAT synthase), which is involved in ergoline alkaloid biosynthesis pathways, was isolated from Clavicepspurpurea Fr. (Tul.) (Shibuya et al., 1990). Because of possible shared homologies due to the evolutionary relatedness in endophytes, genetic studies of turfgrass endophytes would certainly benefit from findings in other filamentous fungi. By using stare-of-the art molecular techniques as listed by Schardl (1994), various gene manipulations might be readily performed for grass endophytes once the relevant genes have been cloned and characterized.

In addition to improving turf and forage grasses, endophyte strategies could be employed to implement molecular farming to produce an array of chemicals such as natural plant regulators, insecticides, natural herbicides, and medicinal agents (Porter, 1994).

Concerns and Problems Associated with Using Endophytes

Compatibility between grass hosts and endophytes is an important issue for plant breeders. A greater knowledge of the interactions that determine grass-endophyte compatibility would help to identify suitable ways to manipulate the endophyte host range. Endophytes with broader host ranges might prove to be valuable because surrogate transformation of them would cover a diversity of host plants; host plants that are compatible with multiple strains of endophytes would have access to great endophytic variability. Since more diversity means more options, the value of a broad host range would be even more significant when pest insects and microorganism pathogens overcome the protection conferred by individual endophytes. In practice, experimental inoculations are the only way to examine compatibility, which is usually measured by longevity of persistence in host plants, reciprocal infection between hosts, and transmission capability.

While several attempts have succeeded in bringing about novel combinations of endophyte-grass associations (Koga et al., 1993; Siegel et al., 1990), evidence indicates that Acremonium endophytes have a high degree of specificity for their original hosts (Leuchtmann, 1994). In addition to genotype-dependent compatibility, artificial endophytic infections also use young seedlings (Latch and Christensen, 1985; Johnson et al., 1986; Leuchtmann and Clay, 1988) or undifferentiated tissues, such as callus cultures (Johnson et al., 1986), somatic embryos (Kearney et al., 1991), and plantlets derived from meristems (O'Sullivan and Latch, 1993), which seem predisposed to infection (Leuchtmann, 1994). Even for those compatible combinations, multiple inoculations are often needed to establish infections that have low efficiencies (Latch and Christensen, 1985; Leuchtmann and Clay, 1989). Observations have been made regarding the mechanisms of compatibility. Leuchtmann (1994) proposed correlations between the presence of enzymes capable of degrading the middle lamella of cell walls, such as esterase and galactosidase, and grass-endophyte incompatibility and suggested that the capability of endophytes to overcome physical barriers and utilize the nutritional supply within the apoplast of the host tissue might be important in compatibility interactions. On the other hand, activation of nonspecific defense systems of hosts marked by elevated levels of pathogenesis-related proteins (PR-proteins) such as chitinase (Roberts et al., 1992) and changed permeability of the extracellular matrix (Fineran et al., 1983) might also be involved in incompatibility reactions. Host range of endophytes, as determined by compatibility interactions, would, to some extent, contribute to defining how much can be achieved in grass biotechnology using endophyte strategies.

Although animal toxicosis is of little concern in grasses used exclusively as turf, the presence of compounds in turfgrasses, such as ergot alkaloids and indole diterpenes, that are harmful to mammalian herbivores still present animal toxicoses because seed production of turfgrasses is often combined with grazing of pastures or stubble after harvest (Rowan and Latch, 1994). For some grasses used both as turf and forage grasses, such as perennial ryegrass and tall fescues, improvements in persistence, pest resistance, and stress tolerance without deleterious effects on livestock have been a breeding goal (Funk et al., 1994). Genetic engineering of endophytes is a promising approach to manipulate endophytes to minimize animal toxicoses while improving endophyte-enhanced benefits.


Turfgrasses have a great impact on people's lives in the United States. The cash value of the turfgrass industry had grown from 4 billion dollars in 1974 to 45 billion dollars in 1994 and will approach 90 billion dollars by the end of this century (Duble, 1996). However, the turfgrass industry faces great challenges, including water shortage, rising costs of energy, limited labor resources, and environment-oriented restrictions on the use of fertilizers, herbicides, fungicides, and pesticides (Duble, 1996). Biotechnology could play an important role in providing technical solutions to these problems. Previous progress has proven that the necessary elements of applying biotechnology to turfgrasses have become available, and in practice, it is feasible to use biotechnology to solve certain problems in turfgrasses (van Heeswijck et al., 1994). With genetic engineering as the central element (Boulter, 1995), biotechnology provides a set of powerful tools in the development of desirable turfgrass cultivars, including cultivars tolerant to drought and salts for water conservation, cultivars with resistance to microbial diseases and insect pests, and herbicide-resistant plants that can be used to simultaneously control both weeds and turfgrass diseases after being sprayed with glufosinate herbicides, which were found to have an inhibition effect on fungal diseases (Liu et al., 1998). Other desirable tasks are to develop turfgrass cultivars with low maintenance requirements such as dwarf or rosette turfgrass (Xu et al., 1995). Nevertheless, the potential of biotechnology in turfgrass improvement largely depends on how well plant regeneration, signal transduction, and gene expression is understood. Traditionally, funding by government and large industry for turfgrass research has been and will continue to be limited. The progress in turfgrass biotechnology will greatly benefit from achievements in other grass species. Examples of achievements in other grass species include the development of somaclonal variants that tolerate environmental extremes, such as salt-tolerant rice (Bong et al., 1996), chilling-tolerant rice (Bertin et al., 1996), drought-tolerant rice (Adkins et al., 1995), frost-tolerant wheat (Sutka, 1994), drought-tolerant wheat (Hsissou and Bouharmont, 1994), dwarf wheat (Guenzi et al., 1992), glyphosate-tolerant barley (Escorial et al., 1996), drought- and acid- soil-tolerant sorghum (Waskom et al., 1990), eyespot disease [Pseudocercosporella herpotrichoides (Fron) Deighton]-resistant sugarcane (Saccharum officinarum L.) (Ramos et al., 1996), Fusarium-resistant wheat (Ahmed et al., 1991), leaf rust (Puccinia recondita Roberge ex Desmaz.)-resistant wheat (Oberthur et al., 1993), and downy mildew [Sclerophthora macrospora (Sacc.) Thirum, Shaw, and Naras.]-resistant millet [Pennisetum glaucum (L.) R. Br.] (Nagarathna et al., 1993). It may also be possible to produce somaclonal variations of turfgrasses that can tolerate shade. Other examples include using isolated and characterized genes from other plant species that can potentially produce rosette or dwarf turfgrasses (Xu et al., 1995), and genes from bacteria (Tarczynski et al., 1993) or from plants (Kavi Kishor et al., 1995) for the development of salt- and drought-tolerant turfgrass cultivars. Development of value-added turfgrasses may not be a dream. Should bioecologists ever become able to transfer genes from N-fixation plants into cereal crops, turfgrass cultivars that fix N via Rhizobia or other sources are feasible and will further enhance their environmental compatibility.


We thank Dr. Heng Zhong, Dr. Joseph Saunders, and Mr. Donald Warkentin for their constructive suggestions in writing this manuscript. Mr. Benli Chai's graduate assistantship is provided by funding from United States Golf Association.


Adkins, S.W., R. Kunanuvatchaidach, and I.D. Godwin. 1995. Somaclonal variation in rice: drought tolerance and other agronomic characters. Aus. J. Bot. 43:201-209.

Ahloowalia, B.S. 1975. Regeneration of ryegrass plants in tissue culture. Crop Sci. 15:449-452.

Ahmad, S., S. Govindarajan, J.M. Johnson-Cicalese, W.K. Dickson, and C.R. Funk. 1986. Endophytes-enhanced resistance in perennial ryegrass to the bluegrass billbug, Sphenophorus parvulus. Entomol. Exp. Appl. 39:183-190.

Ahmed, K.Z., A. Mesterhazy, and F. Sagi. 1991. In vitro techniques for selecting wheat for Fusarium resistance. Euphytica 47:251-257.

Ahn, B.J., F.H. Huang, and J.W. King. 1985. Plant regeneration through somatic embryogenesis in common bermudagrass tissue culture. Crop Sci. 25:1107-1109.

Ahn, B.J., F.H. Huang, and J.W. King. 1987. Regeneration of bermudagrass cultivars and evidence of somatic embryogenesis. Crop Sci. 27:594-597.

Akkaya, M.S., A.A. Bhagwat, and P.B. Gregan. 1992. Length polymorphisms of simple sequence repeat DNA in soybean. Genetics 132:1131-1139.

Al-Khayri, J.M., F.H. Huang, L.F. Thompson, and J.W. King. 1989. In vitro plant regeneration of zoysiagrass. Arkansas Farm Res. 38(21:11.

Arachevaleta, M., C.W. Bacon, C.S. Hoveland, and D.E. Radcliffe. 1989. Effect of the tall fescue endophyte on plant response to environmental stress. Agron. J. 81:83-90.

Artunduaga, I.R., C.M. Taliaferro, and B.B. Johnson. 1988. Effects of auxin concentration on embryogenic callus induction from cultured young infloresccnces of old world blue stems (Bothriochloa spp.) and bermudagrasses (Cynodon spp.). Plant Cell Tissue Organ Cult. 12:13-19.

Artunduaga, I.R., C.M. Taliaferro, and B.B. Johnson. 1989. Induction and growth of callus from immature inflorescences of "Zebra" bermudagrass as affected by casein hydrolysate and 2,4-D concentration. In Vitro Cell. Dev. Biol. 25(8):753-756.

Asano, Y., Y. Otsuki, and M. Ugaki. 1991. Electroporation-mediated and silicon carbide fiber-mediated DNA delivery in Agrostis alba L. (redtop). Plant Sci. 79:247-252.

Asano, Y., and K. Sugiura. 1990. Plant regeneration from suspension culture-derived protoplasts of Agrostis alba L. (redtop). Plant Sci. 72:267-273.

Asano, Y., and M. Ugaki. 1994. Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts. Plant Cell Rep. 13:243-246.

Asay, K.H., and D.A. Sleper. 1989. Contributions from breeding forage and turf grasses -- An overview, p. 1-3. In D.A. Sleper et al. (ed.) Contributions from breeding forage and turfgrasses. CSSA Spec. Publ. 15. CSSA, Madison, WI.

Bacon, C., and J. De Battista. 1991. Endophytic fungi of grasses, p. 231-256. In D.K. Arora et al. (ed.) Soil and plants. Handbook of applied mycology. Vol. 1. Marcel Dekker, New York.

Beard, J.B. 1973. Turfgrass: Science and culture. Prentice-Hall, Englewood Cliffs, N.J.

Becker, J., and M. Heun. 1995. Barley microsatellites: Allele variation mapping. Plant Molec. Biol. 27:835-845.

Becker, J., P. Vos, M. Kuiper, F. Salamini, and M. Heun. 1995. Combined mapping of AFLP and RFLP markers in barley. Molec. Gen. Genet. 249:65-73.

Beckmann, J.S., and M. Soller. 1986. Restriction fragment length polymorphisms and genetic improvement of agricultural species. Euphytica 35:111-124.

Bertin, P., J.M. Kinet, and J. Bouharmont. t996. Heritable chilling tolerance improvement in rice through somaclonal variation and cell selection. Aus. J. Bot. 44:91-105.

Bhaskaran, S., A.J. Neumann, and R.H. Smith. 1988. Origin of somatic embryos from cultured shoot tips of Sorghum bicolor (L.) Moench. In Vitro Cell. Dev. Biol. 24:947-95(I.

Bhaskaran, S., M. Rigoldi, and R.H. Smith. 1992. Developemental potential of sorghum shoot apices in culture. J. Plant Physiol. 140:481-484.

Birch, R.G., and T. Franks. 1991. Development and optimization of microprojectile systems for plant genetic transformation. Aus. J. Plant Physiol. 18:453469.

Blanche, F.C., J.V. Krans, and G.E. Coats. 1986. Improvement in callus growth and plantlet formation in creeping bentgrass. Crop Sci. 26:1245-1248.

Bong, B.B., S. Tabita, and T. Senboku. 1996. Variation in salt tolerance of rice plants regenerated from salt-selective calli of a susceptible variety. Intl. Rice Res. Newsl. 21:38-39.

Boppenmeier, J., S. Zuechner, and W.B. Forough. 1989. Haploid production from barley yellow dwarf virus resistant clones of Lolium. Plant Breed. 103:216-220.

Botstein, D., R.L. White, M. Skolnick, and R.W. Davis. 1980. Construction of a genetic linkage map in human using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32:314-331.

Boulter, D. 1995. Plant biotechnology: Facts and public perception. Phytochemistry 40:1-9.

Bovo, O.A., and L.A. Mroginski. 1986. Tissue culture in Paspalum (Gramineae): Plant regeneration from cultured inflorescences. J. Plant Physiol. 124:481-492.

Boyd, L.A., and P.J. Dale. 1986. Callus production and plant regeneration from mature embryos of Poa pratensis L. Plant Breed. 97: 246-254.

Breen, J.P. 1993a. Enhanced resistance to fall armyworm (Lepidoptera: Noctudiae) in Acremonium endophyte-infected turfgrasses. J. Econ. Entomol. 86:621-629.

Breen, J.P. 1993b. Enhanced resistance to three species of aphids (Homoptera: Aphididae) in Acremonium endophyte-infected turfgrasses. J. Econ. Entomol. 86:1279-1286.

Brown, S.M., M.S. Hopkins, S.E. Mitchell, M.L. Senior, T.Y. Wang, R.R. Duncan, F. Gonzalez, and S. Kresovich. 1996. Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench.]. Theor. Appl. Genet. 93:190-198.

Brown, S.M., S.E. Mitcheil, C.A. Jester, Z.W. Liu, S. Kresovich, and R.R. Duncan. 1997. DNA typing (profiling) of seashore paspalum (Paspalum vaginatum Swartz) ecotypes and cultivars, p. 39-51. In M.B. Sticklen and M.P. Kenna (ed.) Turfgrass biotechnology -Cell and molecular genetic approaches to turfgrass improvement. Ann Arbor Press, MI.

Caetano-Anolles, G., B.J. Bassam, and P.M. Gresshoff. 1991a. DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Bio/Technology 9:553-556.

Caetano-Anolles, G., B.J. Bassam, and P.M. Gresshoff. 1991b. DNA amplification fingerprinting: A strategy for genome analysis. Plant Molec. Biol. Rep. 9:294-307.

Caetano-Anolles, G., I.M. Callahan, P.E. Williams, K.R. Weaver, P.M. Gresshoff. 1995. DNA amplification fingerprinting analysis of bermudagrass (Cynodon) genetic relationships between species and interspecific crosses. Theor. Appl. Genet. 9:228-235.

Callahan, L.M., K.R. Weaver, G. Caetano-Anolles, B.J. Bassam, and P.M. Gresshoff. 1993. DNA fingerprinting of turfgrasses, p. 761-767 In R.N. Carrow et al. (ed.) Int. Turf. Soc. Res. J. Vol. 7. Interec Publishing Corp., Overland Park, KS.

Carrer, H., and P. Maliga. 1995. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene. Bio/Technology. 13:791-794.

Chair, H., T. Legavre, and E. Guiderdoni. 1996. Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.). Plant Cell Rep. 15:766-770.

Chan, M-T., T-M. Lee, and H-H. Chang. 1992. Transformation of indica rice (Orvza sativa L.) by Agrobacterium tumefaciens. Plant Cell Physiol. 33:577-583.

Chan, M.T., H.H. Chang, S.L. Ho, W.F. Tong, and S.M. Yu. 1993. Agrobacterium-mediated production of transgenic rice plants expressing a chimeric [Alpha]-amylase promoter/I3-glucuronidase gene. Plant Molec. Biol. 22:491-506.

Christou, P., and T.L. Ford. 1995. The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants. Transgenic Res. 4:44-51.

Clark, K.W., A. Hussain, K. Bamford, and W. Bushuk. 1989. Identification of cultivars of Agrostis species by polyacrylamide gel electrophoresis of seed proteins, p. 121-125. In H. Takatoh (ed.) Proc. Int. Turf. Res. Conf., 6th, Tokyo. 31 July-5 Aug. 1989. Int. Turfgrass Soc. and Jpns. Soc. Turfgrass Sci., Tokyo.

Clay, K. 1993. The ecology and evolution of endophytes. Agric. Ecosyst. Environ. 44:39-64.

Clay, K. 1994. The potential role of endophytes in ecosystems, p. 73-86. in C.W. Bacon and J.F. White (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Clement, S.L., W.J. Kaiser, and H. Eichenseer. 1994. Acremonium endophytes in germplasms of major grasses and their utilization for insect resistance, p. 185-199. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Condit, R.C., and S.P. Hubbell. 1991. Abundance and DNA sequence of two-base repeat regions in tropical tree genomes. Genome 34:66-71.

Conger, B.V., and J.V. Carabia. 1978. Callus induction and plantlet regeneration in orchardgrass. Crop Sci. 18:157-159.

Conger, B.V., G.E. Hanning, D.J. Gray, and J.K. McDanniel. 1983. Direct embryogenesis from mesophyll cells of orchardgrass. Science 221:850-851.

Conger, B.V., and R.E. McDonnell. 1983. Plantlet formation from cultured inflorescence of Dactylis glomerata L. Plant Cell Tissue Organ Cult. 2:191-197.

Conger, B.V., J.C. Hovanesian, R.N. Trigiano, and D.J. Gray. 1989. Somatic embryo ontogeny in suspension cultures of orchardgrass. Crop Sci. 29:448452.

Creemers-Molenaar, T., J.P.M. Loeffe, and M.A.C.M. Zaal. 1988. Isolation, culture and regeneration of Lolium perenne and Lolium multiflorum protoplasts. Curr. Plant Sci. Biotech. Agric. 7:53-54.

Cregg, J.M., and K.R. Madden. 1989. Use of site-specific recombination to regenerate selectable markers. Molec. & Gen. Genet. 219:320-323.

Dale, E.C., and D.W. Ow. 1991. Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA 88:10558-10562.

Dale, P.J. 1975. Meristem tip culture in Lolium multiflorum. J. Exp. Bot. 26:731-736.

Dale, P.J. 1977a. The elimination of ryegrass mosaic virus from Lolium multiflorum by meristem tip culture. Ann. Appl. Biol. 85:93-96.

Dale, P.J. 1977b. Meristem tip culture in Lolium, Festuca, Phleum and Dactylis. Plant Sci. Lett. 9:333-338.

Dale, P.J. 1980. Embryoids from cultured immature embryos of Lolium multiflorum. Z. Pflanzenphysiol. 100:73-77.

Dalton, S.J. 1988a. Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb. (tall fescue) and Lolium perenne L. (perennial ryegrass). J. Plant Physiol. 132:170-175.

Dalton, S.J. 1988b. Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb., Lolium perenne L., and L. multiflorum Lam. Plant Cell Tissue Organ Cult. 12:137-140.

Dalton, S.J. 1993. Regeneration of plants from protoplasts of Lolium (Ryegrasses) and Festuca (Fescues). p. 46-68. In Y.P.S. Bajaj (ed.) Plant protoplasts and genetic engineering. Springer-Verlag, Berlin.

Dalton, S.J., A.J.E. Bettany, E. Timms, and P. Morris. 1995. The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb.). Plant Sci. 108:63-70.

Dalton, S.J., and P.J. Dale. 1985. The application of in vitro tiller induction in Lolium multiflorum. Euphytica 34:897-904.

DeBlock, M. 1993. The cell biology of plant transformation: Culture state, problems, prospects and the implications for the plant breeding. Euphytica. 71:1-14.

Deng, W., X. Lin, and Q. Shao. 1988. Transformation of some cereal crops with Agrobacterium tumefaciens. Genetic Manipulation Crops Newsl. 4:1-2.

Duble, R.L. 1996. Turfgrasses: Their management and use in the southern zone. 2nd ed. Texas A&M Univ. Press, College Station.

Edwards, K., C. Johnstone, and C. Thompson. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nuclear Acids Res. 19:1349.

Escorial, M.C., H. Sixto, J.M. Garcia-Baudin, and M.C. Chueca. 1996. In vitro culture selection increases glyphosate tolerance in barley. Plant Cell Tissue Organ Cult. 46:179-186.

Fincham, J.R.S. 1987. Transformation in fungi. Microbiol. Rev. 53: 148-170.

Fineran, B.A., I.C. Harvey, and M. Ingerfeld. 1983. Unusual crystalloids and aggregates of tubules in the Lolium endophytes of ryegrass leaf sheaths. Protoplasma 117:17-23.

Frame, B.R., P.R. Drayton, S.V. Bagnall, C.J. Lewnau, and W.P. Bullock. 1994. Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant J. 6:941948.

Funk, C.R., C. F. Belanger, and J.A. Murphy. 1994. Role of endophytes in grasses used for turf and soil conservation, p. 201-209. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Funk, C.R., P.M. Halisky, S. Ahmad, and R.H. Hurley. 1985. How endophytes modify turfgrass performance and response to insect pests in turfgrass breeding and evaluation trials, p. 137. In F. Lemair (ed.) Proc. Int. Turf. Res. Conf., 5th, Avignon, France. 1-5 July 1985. Int. Turfgrass Soc. and INRA, Paris.

Funk, C.R., P.M. Halisky, M.C. Johnson, M.R. Seigel, A.V. Stewart, S. Ahmad, R.H. Hurley, and I.C. Harvey. 1983. An endophytic fungus and resistance to sod webworms: Association in Lolium perennne. Bio/Technology 1:189-191.

Gallagher, R.T., A.G. Campbell, A.D. Harwkes, P.T. Holland, D.A. McGaveston, E.A. Pansier, and I.C. Harvey. 1982. Ryegrass staggers: The presence of lolitrem neurotoxins in perennial ryegrass seed. NZ Ver. J. 30:183-184.

Gallagher, R.T., A.D. Hawkes, P.S. Steyn, and R. Vleggaar. 1984. Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: Structure elucidation of lolitrem B. J. Chem. Soc. Chem. Commun. 1984:614-616.

Gallagher, R.T., E.P. White, and P.H. Mortimer. 1981. Ryegrass staggers: Isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. NZ Vet. J. 29:189-190.

Gordon-Kamm, W.J., T.M. Spencer, M.L. Mangano, T.R. Adams, R.J. Daines, W.G. Start, J.V. O'Brien, S.A. Chambers, W.R. Adams, and N.G. Willetts. 1990. Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603-618.

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

Gray, D.J., B.V. Conger, and G.E. Hanning. 1984. Somatic embryogenesis in suspension and suspension-derived callus culture of Dactylis glomerata. Protoplasma 122:196-202.

Gray, D.J., and B.V. Conger. 1985. Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae). Plant Cell Tissue Organ Cult. 4:123-133.

Guenzi, A.C., D.W. Mornhinweg, and B.B. Johnson. 1992. Genetic analysis of a grass dwarf mutation induced by wheat callus culture. Theor. Appl. Genet. 84:952-957.

Ha, S.B., F.S. Wu, and T.K. Thorne. 1992. Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. Plant Cell Rep. 11:601-604.

Halberg, N., A. Olesen, I.K.D. Tuvesson, and S.B. Andersen. 1990. Genotypes of perennial ryegrass (Lolium perenne L.) with high anther-culture response through hybridization. Plant Breed. 105: 89-94.

Halperin, W. 1986. Attainment and retention of morphogenetic capacity in vitro, p. 3-47. In I.K. Vasil (ed.) Cell culture and somatic cell genetics of plants. Vol. 3. Academic Press, Orlando, FL.

Hanning, G.E., and B.V. Conger. 1982. Embryoid and plantlet formation from leaf segments of Dactylis glomerata L. Theor. Appl. Genet. 63:155-159.

Hanning, G.E., and B.V. Conger. 1986. Factors influencing somatic embryogenic from cultured leaf segments of Dactylis glomerata. J. Plant Physiol. 123:23-29.

Hardy, T.N., K. Clay, and A.M. Hammod, Jr. 1985. Fall armyworm (Lepidoptera: Noctuidae): A laboratory bioassay and larval preference study for the fungal endophyte. J. Econ. Entomol. 78:571-575.

Hardy, T.N., K. Clay, and A.M. Hammod, Jr. 1986. Leaf age and related factors affecting endophyte-mediated resistance to fall armyworm (Lepidoptera: Noctuidae) in tall fescue. Environ. Entomol. 15:1083-1089.

Hartman, C.L., L. Lee, R.P. Day, and N.E. Turner. 1994. Herbicide resistant turfgrass (Agrostis palustris Huds.) by biolistic transformation. Bio/Technology 12:919-923.

Herve, C., D. Rouan, P. Guerche, M.H. Montane, and P. Yot. 1993. Molecular analysis of transgenic rapeseed plants obtained by direct transfer of two separate plasmids containing, respectively, the cauliflower mosaic virus coat protein gene and a selectable marker gene. Plant Sci. 91:181-193.

Hill, N.S. 1994. Ecological relationships of Balansiae-infected graminoids, p. 59-72. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Hinchee, M.A.W., D.R. Corbin, C.L. Armstrong, J.E. Fry, S.S. Sato, D.L. DeBoer, W.L. Petersen, T.A. Armstrong, D.V. Connor-Ward, J.G. Layton, and R.B. Horsch. 1994. Plant transformation, p. 231270. In I.K. Vasil and Trevor A. Thorpe (ed.) Plant cell and tissue culture. Kluwer Academic Publ., Hingham, MA.

Horn, M.E., R.D. Shillito, B.V. Conger, and C.T. Harms. 1988. Transgenic plants of orchardgrass (Dactylis glomerata L.) from protoplasts. Plant Cell Rep. 7:469-472.

Horsch, R.B., J. Fry, N. Hoffmann, M. Wallroth, D. Eichholtz, S.G. Rogers, R.T. Fraley. 1985. A simple and general methods for transferring genes into plants. Science 227:1229-1231.

Hsissou, D., and J. Bouharmont. 1994. In vitro selection and characterization of drought tolerance plants of durum wheat (Triticum durum Desf. ). Agronomie (Paris) 14:65-70.

Huff, D. 1997. RAPD characterization of heterogeneous perennial ryegrass cultivars. Crop Sci. 37:557-564.

Huff, D.R., and J.M. Bara. 1993. Determining genetic origins of aberrant progeny from facultative apomictic Kentucky bluegrass using a combination of flow cytometry and silver-stained RAPD marker. Theor. Appl. Genet. 87:201-208.

Huff, D.R., R. Peakall, and P.E. Smouse. 1993. RAPD variation within and among natural populations of outcrossing buffalograss [Buchloe dactyloides (Nutt.) Engelm.]. Theor. Appl. Genet. 86: 927-934.

Ishida, Y., H. Saito, S. Ohta, Y. Hiei, T. Komari, and T. Kumashiro. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology 14: 745-750.

Jackson, J.A., and P.J. Dale. 1988. Callus induction, plant regeneration and assessment of cytological variation in regenerated plants of Lolium multiflorum L. J. Plant Physiol. 132:351-355.

Johnson, M.C., L.P. Bush, M.R. Siegel. 1986. Infection of tall fescue with Acremonium coenophilaum by means of callus culture. Plant Dis. 70:380-382.

Johnson-Cicalcse, J.M., and R.H. White. 1990. Effect of Acremonium endophytes on four species of billbug found on New Jersey turfgrasses. J. Am. Soc. Hortic. Soc. 115:602-604.

Kaeppler, H.F., W. Gu, D.A. Somers, H.W. Rines, and A.F. Cockburn. 1990. Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Rep. 9:415-418.

Kaeppler, H.F., D.A. Somers, H.W. Rines, and A.F. Cockburn. 1992. Silicon carbide fiber-mediated stable transformation of plant cells. Theor. Appl. Genet. 84:56(7)-566.

Karp, A. 1994. Origins, causes and uses of variation in plant tissue culture, p. 139-151. In I.K. Vasil and Trevor A. Thorpe (ed.) Plant cell and tissue culture. Kluwer Academic Publ., Hingham, MA.

Kasperbauer, M.J., R.C. Buckner, and W.D. Springer. 1980. Haploid plants by anther-panicle culture of tall fescue. Crop Sci. 20:103-107.

Katayama, H., and O. Yaasunari. 1993. Structure alternations of the chloroplast genome found in grasses are not common in monocots. Curr. Genet. 23:160-165.

Kavi Kishor, P.B., Z. Hong, G.H. Miao, C.A. Hu, and D.P. Verma. 1995. Overexpression of [[Delta].sup.1]-pyrroline-5-caroxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 108:1387-1394.

Kearney, J.F., W.A. Parrott, and N.S. Hill. 1991. Infection of somatic embryos of tall rescue with Acremonium coenophialum. Crop Sci. 31:979-984.

Kidd, G. 1993. Why do agbiotech firms neglect turf grasses? Bio/ Technology 11:268.

Koga, H., M.H. Christensen, and R.J. Bennett. 1993. Cellular interactions of some grass/Acremonium endophyte associations. Mycolog. Res. 97:1237-1244.

Krans, J.V. 1981. Cell culture of turfgrasses, p. 27-33. In R.W. Sheard (ed.) Proc. Int. Turf. Res. Conf., 4th, Guelph, Ontario, Canada. 19-23 July 1980. lnt. Turfgrass Soc. and Univ. Guelph, Ontario, Canada.

Krans, J.V. 1989. Biotechnology advances in turfgrass, p. 11-15. In H. Takatoh (ed.) Proc. Int. Turfgrass Res. Conf., 6th, Tokyo. 31 July-5 August. Int. Turfgrass Soc. and Jpns. Soc. Turfgrass Sci.

Krans, J.V., and F.C.B. Blanche. 1985. Tissue culture of centipedegrass, p. 159-164. In F. Lemaire (ed.) Proc. Int. Turfgrass Res. Conf., 5th, Avignon, France. 1-5 July 1985. Int. Turfgrass Soc. and INRA, Paris.

Krans, J.V., V.T. Henning, and K.C. Torres. 1982. Callus induction, maintenance and plantlets regeneration in creeping bentgrass. Crop Sci. 22:1193-1197.

Krishnaraj, S., and I. Vasil. 1995. Somatic embryogenesis in herbaceous monocots, p. 417-470. In T.A. Thorpe (ed.) In vitro embryogenesis in plants. Kluwer Academic Publ., Hingham, MA.

Kuo, Y., and M.A.L. Smith. 1993. Plant regeneration from St. Augustine-grass immature embryo derived callus. Crop Sci. 33: 1394-1396.

Latch, G.C.M., and M.J. Christensen. 1985. Artificial infections of grasses with endophytes. Ann. Appl. Biol. 107:17-24.

Lee, L. 1996. Turfgrass biotechnology. Plant Sci. 115:1-8.

Lee, L., C. Hartman, C. Laramore, N. Turner, and P. Day. 1995. Herbicide-resistant creeping bentgrass. USGA Green Section Record 33:16-18.

Leuchtmann, A. 1994. lsozyme characterization, persistence, and compatibility of fungi and grass mutualists, p. 21-29. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Leuchtmann, A., and K. Clay. 1988. Experimental infection of host grasses and sedges with Atkinsonella hypoxylon and Balansia cyperi. Mycologia 80:291-297.

Leuchtmann, A., and K. Clay. 1989. Experimental evidence for genetic variation in compatibility between the fungus Atkinsonella hypoxylon and its three host grasses. Evolution 43:825-834. 1989.

Liu, C.A., H. Zhong, J. Vargas, D. Penner, and M. Sticklen. 1998. Prevention of fungal diseases in transgenic, bialaphos--and glufosinate-resistant creeping bentgrass (Agrostis palustris L.). Weed Sci. 46:139-146.

Liu, C.N., X.Q. LI, and S.B. Gelvin. 1992. Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobaterium tumefaciens. Plant Molec. Biol. 20: 1037-1992.

Liu, Z.W., R.L. Jarret, S. Kresovich, and R.R. Duncan. 1995. Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz). Theor. Appl. Genet. 91:47-52.

Liu, Z.W., R.M. Biyashev, and M.A. Saghai Maroof. 1996. Development of simple sequence repeat DNA markers and their integration into a barley linkage map. Theor. Appl. Genet. 93:869-876.

Lowe, K., B. Bowen, G. Hoerster, M. Ross, D. Bond, D. Pierce, and B. Gordon-Kamm. 1995. Germline transformation of maize following manipulation of chimeric shoot meristems. Bio/Technology 13:677-682.

Lowe, K.W., and B.V. Conger. 1979. Root and shoot formation from callus cultures of tall fescue. Crop Sci. 19:397-400.

Lyznik, L.A., J.C. Mitchell, L. Hirayama, and T.K. Hodges. 1993. Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Res. 21:969-975.

Mackill, D.J., Z. Zhang, E.D. Redona, and P.M. Colowit. 1996. Level of polymorphism and genetic mapping of AFLP markers in rice. Genome 39:969-977.

Madsen, S., A, Olesen., B. Dennis, and S.B. Andersen. 1995. Inheritance of anther-culture response in perennial ryegrass (Lolium perenne L.). Plant Breed. 114:165-168.

Mathias, J.K. 1988. The relationship of endophytic fungi in perennial ryegrass and resistance to the hairy chinch bug and sod webworm, Ph.D. diss., Univ. of Maryland, College Park.

Mathias, J.K., R.H. Ratcliffe, and J.L. Hellman. 1990. Association of an endophytic fungus in perennial ryegrass and resistance to the hairy chinch bug (Hemiptera: Lygaeidae). J. Econ. Entomol. 83: 1640-1646.

Mathias, R.J. 1987. Plant microinjection techniques. Genet. Eng.: Princ. Meth. 9:199-227.

Maughan, P.J., M.S. Saghai Maroof, and G.R. Buss. 1996. Amplified fragment length polymorphism (AFLP) in soybean: Species diversity, inheritance, and near-isogenic line analysis. Theor. Appl. Genet. 93:392-401.

McDaniel, J.K., B.V. Conger, and E.T. Graham. 1982. A histological study of tissue proliferation, embryogenesis, and organogenesis from tissue cultures of Dactylis glomerata L. Protoplasma. 110: 121-128.

McDaniel, J.K., P.D. Denchev, D.D. Songstad, and B.V. Conger. 1997. Direct transformation of orchardgrass leaf cells by microprojectile bombardment. In Vitro Cell. Dev. Bio. 33:49a (P-1019).

McDonnell, R.E., and B.V. Conger. 1984. Callus induction and plantlet formation from mature embryo explants of Kentucky bluegrass. Crop Sci. 24:573-578.

Medford, J.I. 1992. Vegetative apical meristems. The Plant Cell 4:1029-1039.

Meksem, K., D. Leister, J. Peleman, M. Zabeau, F. Salamini, and C. Gebhardt. 1995. A high resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers. Molec. Gen. Genet. 249:74-81.

Moberg, E.L. 1972. Turfgrass cultivar identification by peroxidase isoenzyme composition. Ph.D. diss., Penn. State Univ., University Park. (Diss. Abstr. 33: 4620B).

Morgante, M., and A.M. Olivieri. 1993. PCR-amplified microsatellites as markers in plant genetics. Plant J. 3:175-182.

Morgante, M., J.A. Rafalski, P. Biddle, S. Tingey, and A.M. Olivieri. 1994. Genetic mapping and variability of seven soybean simple sequence repeat loci. Genome 37:763-769.

Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-493.

Murray, F.R., G.C.M. Latch, and D.B. Scott. 1992. Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte. Molec. Gen. Genet. 233:l-9.

Nagarathna, K.C., S.A. Shetty, G. Harinarayana, and H.S. Shetty. 1993. Selection for downy mildew resistance from the regenerants of pearl millet. Plant Sci. 90:53-60.

Neuhaus, G., G. Spangenberg, O. Mittelsten Scheid, and H.G. Schweiger. 1987. Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor. Appl. Genet. 75:30-36.

Nielsen, K.A., and E. Knudsen. 1993. Regeneration of green plants from embryogenic suspension culture of Kentucky bluegrass (Poa pratensis L.). J. Plant Physiol. 141:589-595.

Nielsen, K.A., E. Larsen, and E. Knudsen. 1993. Regeneration of protoplast-derived green plants of Kentucky bluegrass (Poa pratensis L.). Plant Cell Rep. 12:537-540.

Oberthur, L.E., S.A. Harrison, T.P. Croughan, and D.L. Long. 1993. Inheritance of improved leaf rust resistance in somaclones of wheat. Crop Sci. 33:444-448.

Ohmura, T., M. Yaneshita, S. Kaneko, Y. Ogihara, and T. Sasakuma. 1993. Turfgrass species and cultivars identification by RFLP analysis of chloroplast and nuclear DNA. p. 754-760. In R.N. Carrow et at. (ed.) Int. Turf. Soc. Res. J. Vol. 7. Interec Publishing Corp., Overland Park, KS.

Olesen, A., M. Storgaard, M. Folling, S. Madsen, and S.B. Andersen. 1995a. Protoplast, callus and suspension cultures of perennial ryegrass -- Effect of genotype and culture system, p. 69-74. In M. Terzi et al. (ed.) Current issues in plant molecular and cellular biology. Kluwer Academic Publ., Hingham, MA.

Olesen, A., M. Storgaard, S. Madsen, and S.B. Andersen. 1995b. Somatic in vitro culture response of Lolium perenne L.: Genetic effects and correlations with anther culture. Euphytica 86:199-209.

Oliver, R.P., I.N. Roberts, R. Harling, L. Kenyon, P.J. Punt, M.A. Dingemanse, and C.A.M.J.J. van den Hondel. 1987. Transformation of Fulvia fulea, a fungal pathogen of tomato, to hygromycin B resistance. Curt. Genet. 12:231-233.

Opsahl-Ferstad, H.G., A. Bjornstad, and O.A. Rognli. 1994a. Genetic control of androgenetic response in Lolium perenne L. Theor. Appl. Genet. 89:133-138.

Opsahl-Ferstad, H.G., A. Bjornstad, and O.A. Rognli. 1994b. Influence of medium and cold pretreatment on androgenetic response in Lolium perenne L. Plant Cell Rep. 13:594-600.

Orford, S.J., N. Steele-Scott, and J.N. Timmis. 1995. A hybervariable middle repetitive DNA sequence from citrus. Theor. Appi. Genet. 91:1248-1252.

O'Sullivan, B.D., and G.C.M. Latch. 1993. Infection of plantlets, derived from ryegrass tall fescue meristems, with Acremonium endophytes, p. 16. In D.E. Hume et al. (ed.) Proc. 2nd Int. Symp. Acremonium/Grass Interactions. AgResearch, Grasslands Research Center, Palmerston North, New Zealand.

Palmer, J.D., R.K. Jansen, H.J. Michaels, M.W. Chase, and J.R. Manhart. 1988. Chloroplast DNA variation and plant phylogeny. Ann. Missouri Bot. Garden 75:1180-1206.

Park, S.H., S.R.M. Pinson, and R.H. Smith. 1996. T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices. Plant Molec. Bio. 32:1135-1148.

Perez-Vicente, R., L. Petris, M. Osusky, I. Potrykus. 1992. Spangenberg, G. Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: Applications in the analysis of Festulolium hybrids. Theor. Appl. Genet. 84:145-154.

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

Porter, J.K. 1994. Chemical constituents of grass endophytes, p. 103123. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Potrykus I., C.T. Harms, and H. Lorz. 1979. Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor. Appl. Genet. 54:209-214.

Potrykus, I. 1990. Gene transfer to plants: An assessment. Bio/Technology 8:535-542.

Powell, W., M. Morgante, C. Andre, M. Hanafey, and J. Vogel. 1996. The comparison of RFLP, RAPD, AFLP, and SSR (microsatellite) markers for germplasm analysis. Molec. Breed. 2:225-238.

Prabhu, R.R., and P.M. Gresshoff. 1994. Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean. Plant Molec. Biol. 26:105-116.

Prestidge, R.A. 1982. An association of the Lolium endophyte with ryegrass resistance to Argentine stem weevil. In M.J. Hartley (ed.) Proc. NZ Weed & Pest Control Conf., 35th. Hamilton, New Zealand. 9-19 Aug. 1982. NX Weed & Pest Control Soc. and Ministry of Agric. and Fish., Palmerston North, New Zealand.

Rajoelina, S.R., G. Alibert, and C. Planchon. 1990. Continuous plant regeneration from established embryogenic cell suspension culture of Italian ryegrass and tall rescue. Plant Breed. 104:265-271.

Raman, K., D.B. Walden, and R.I. Greyson. 1980. Propagation of Zea mays L. by shoot tip culture: A feasible study. Ann. Bot. 45:183-189.

Ramos, L.M., R.H. Maribona, A. Ruiz, S. Korneva, E. Canales, T.D. Dinkova, F. Izquierdo, O. Coto, and D. Rizo. 1996. Somaclonal variation as a source of resistance to eyespot disease of sugarcane. Plant Breed. 115:37-42.

Richardson, M.D., J.F. White, Jr., and F.C. Belanger. 1997. The use of endophytes to improve turfgrass performance, p. 97-111. In M.B. Sticklen and M.P. Kenna (ed.) Turfgrass biotechnology -Cell and molecular genetic approaches to turfgrass improvement. Ann Arbor Press, MI.

Riordan, T.P., S. Fei, and P.G. Johnson. 1997. Plant breeding, plant regeneration, and flow cytometry in buffalograss, p. 183-193. In M.B. Sticklen and M.P. Kenna (ed.) Turfgrass biotechnology -Cell and molecular genetic approaches to turfgrass improvement. Ann Arbor Press, MI.

Roberts, G.A., S.M. Marek, T.L. Niblack, A.L. Karr. 1992. Parasitic Meloidogyne and mutualistic Acremonium increase chitinase in tall fescue. J. Chem. Ecology 18:1107-1116.

Rongwen, J., M.S. Akkaya, A.A. Bhagwat, U. Lavi, and P.B. Cregan. 1995. The use of microsatellite DNA markers for soybean genotype identification. Theor. Appi. Genet. 90:43-48.

Rose, J.B., J.M. Dunwe!l, and N. Sunderland. 1987. Anther culture of Lolium temulentum, Festuca pretensis and Lolium x Festuca hybrids. II. Anther and pollen development in vivo and in vitro. Ann. Bot. 60:213-214.

Rowan, D.D., M.B. Hunt, and D.L. Gaynor. 1986. Pereramine, a novel insect feeding deterrent from ryegrass infected with the endophyte Acremonium loliae. J. Chem. Soc. Commun. 1986:935-936.

Rowan, D.D., and G.C.M. Latch. 1994. Utilization of endophyte-infected perennial ryegrasses for increased insect resistance, p. 169-183. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Saghai-Maroof, M.A., R.M. Biyashev, G.P. Yang, Q. Zhang, and R.W. Allard. 1994. Extrordinarily polymorphic microsatellite DNA in barley: Species diversity, chromosamal location, and population dynamics. Proc. Natl. Acad. Sci. USA 91:5466-5470.

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

Sanford, J.C., T.M. Klein, E.D. Wolf, and N. Allen. 1987. Delivery of substances into cells and tissues using a particle bombardment process. Part. Sci. Technol. 5:27-37.

Sauer, B. 1994. Recycling selectable markers in yeast. Biotechniques 16:1086-1088.

Sauer, K.M., S.H. Hulbert, and N.A. Tisserat. 1993. Identification of Ophiosphaerella herpotricha by cloned DNA probes. Phytopathology 83:97-102.

Sautter, C., N. Leduc, R. Bilang, V.A. Iglesias, A. Gisel, X. Wen, and I. Potrykus. 1995. Shoot apical meristems as a target for gene transfer by microbiolistics. Euphytica 85:45-51.

Schardl, C. 1994. Molecular and genetic methodologies and transformation of grass endophytes, p. 151-165. In C.W. Bacon and J.F. White, Jr. (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL.

Schocher, R.J., R.D. Shillito, M.W. Saul, J. Paszkowski, and I. Potrykus. 1986. Co-transformation of unlinked foreign genes into plants direct gene transfer. Bio/Technology. 4:1093-1096.

Senior, M.L., and M. Heun. 1993. Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer. Genome 36:884-889.

Serik, O., I. Ainur, K. Murat, M. Tetsuo, and I. Masaki. 1996. Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum aestivum L.) mature embryo. Plant Cell Rep. 16:133-136.

Shetty, K., and Y. Asano. 1991. The influence of organic nitrogen sources on the induction of embryogenic callus in Agrostis alba L. J. Plant. Physiol. 139:82-85.

Shibuya, M., H.M. Chou, M. Fountoulakis, S. Hassam, S.U. Klm, K. Kobayashi, H. Otsuka, E. Rogalska, J.M. Cassady, and H.G. Floss. 1990. Stereochemistry of the isoprenylation of tryptophan catalyzed by 4-(g,g-dimethlally)tryptophan synthase from Claviceps, the 1st pathway-specific enzyme in ergot alkaloid biosynthesis. J. Amer. Chem. Soc. 112:297-304.

Siegel, M.R. 1987. Fungal endophytes of grasses. Ann. Rev. Phytopathol. 25:293-315.

Siegel, M.R., G.C.M. Latch, L.P. Bush, F.F. Fannin, D.D. Rowan, B.A. Tapper, C.W. Bacon, and M.C. Johnson. 1990. Fungal endophyte-infected grasses: alkaloid accumulation and aphid response. J. Chem. Ecol. 16:3301-3315.

Smith, R.H., and E.E Hood. 1995. Agrobacterium tumefaciens transformation of monocots. Crop Sci. 35:301-309.

Spangenberg, G., Z.Y. Wang, J. Nagel, and I. Potrykus. 1994. Protoplast culture and generation of transgenic plants in red rescue (Festuca ruba L.). Plant Sci. 97:83-94.

Spangenberg, G., Z.Y. Wang, X.L. Wu, J. Nagel, V.A. Iglesias, and I. Potrykus. 1995a. Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra ) plants from microprojectile bombardment of embryogenic suspension cells. J. Plant Physiol. 145:693-701.

Spangenberg, G., Z.Y. Wang, J. Nagel, X.L. Wu, and I. Potrykus. 1995b. Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci. 108:209-217.

Spencer, T.M., W.J. Gordon-Kamm, R.J. Daines, W.G. Start, and P.G. Lemaux. 1990. Bialaphos selection of stable transformants from maize cell culture. Theor. Appl. Genet. 79:625-631.

Stanis, V.A., and R.G. Butenko. 1984. Developing viable haploid plants in anther culture of ryegrass. Dokl. Biol. Sci. 275:249-251.

Stewart, A.V. 1985. Perennial ryegrass seedling resistance to Argentine stem weevil. NZ. J. Agric. Res. 28:403407.

Sugiura K., C. lnokuma, N. Imaizumi, and C. Cho. 1997. Transgenic creeping bentgrass (Agrostis palustris Huds.) plants regenerated from protoplasts. J. Turf. Manag. 2:43-53.

Sutka, J. 1994. Genetic control of frost tolerance in wheat (Triticum aestivum L.) Euphohytica. 77:277-282.

Sweeney, P.M., and T.K. Danneberger. 1994. Random amplified polymorphic DNA in perennial ryegrass: A comparison of bulk samples vs. individuals. HortScience 29:624-626.

Takamizo, T., K.I. Suginobu, and R. Ohsugi. 1990. Plant regeneration from suspension culture derived protoplasts of tall fescue (Festuca arundinacea Schreb.) of a single genotype. Plant Sci. 72:125-131.

Tanksley, S.D., N.D. Young, A.H. Paterson, and M.W. Bonierbale. 1989. RFLP mapping in plant breeding: New tools for an old science. Bio/Technology 7:257-265.

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

Tautz, D. 1989. Hypervariability of simple sequences as a general source for polymorphic DNA marker. Nucleic Acids Res. 17: 6463-6471.

Terakawa, T., T. Sato, M. Koike. 1992. Plant regeneration from protoplasts isolated from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris Huds.). Plant Cell Rep. 11:457-461.

Thompson, J.A., P.R. Drayton, B.R. Frame, K. Wang, and J.M. Dunwell. 1995. Maize transformation utilizing silicon carbide whiskers: A review. Euphytica 85:75-80.

Tisserat, N.A., S.H. Hulbert, and A. Nus. 1991. Identification of Leptosphaeria korrae by cloned DNA probes. Phytopathology 81' 917-921.

Tisserat, N.A., S.H. Hulbert, and K.M Sauer. 1994. Selective amplification of rDNA internal transcribed spacer regions to detect Ophiosphaerella korrae and O. herpotricha. Phytopathology 84:478-482.

Torello, W.A., A.G. Symington, and R. Rufner. 1984. Callus initiation, plant regeneration, and evidence of somatic embryogenesis in red rescue. Crop Sci. 24:1037-1040.

Trigiano, R.N., D.J. Gray, B.V. Conger, and J.K. McDaniel. 1989. Origin of direct somatic embryos from cultured leaf segments of Dactylis glomerata (orchardgrass). Bot. Gaz. (Chicago) 150:72-77. Tsai, H.F., M.R. Siegel, and C.L. Schardl. 1992. Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea. Curr. Genet. 22:399.406.

Valles, M.P., Z.Y. Wang, P. Montavon, I. Potrykus, and G. Spangenberg. 1993. Analysis of genetic stability of plant regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis L.). Plant Cell Rep. 12:101-106.

van der Valk, P., M.A.C.M. Zaal, and J. Creemers-Molenaar. 1989. Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass). Plant Cell Rep. 7:644-647.

van Heeswijck, R., J. Hutchinson, G. McDonald, and J. Woodward. 1994. The role of biotechnology in perennial grass improvement for temperate pastures. NZ. J. Agric. Res. 37:427-438.

Vasil, I.K. 1985. Somatic embryogenesis and its consequences in the Gramineae. p. 31-47. In R.R. Henke et al. (ed.) Tissue culture in forestry and agriculture. Plenum Publ., New York.

Vasil, I.K. 1987. Developing cell and tissue culture systems for the improvement of cereal and grass crops. J. Plant Physiol. 128: 193-218.

Vasil, I.K. 1988. Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology 6:397-402.

Vasil, I.K., and V. Vasil. t994. In vitro culture of cereals and grasses. p. 293-312. In I.K. Vasil and T.A. Thorpe (ed.) Plant cell and tissue culture. Kluwer Academic Publ., Hingham, MA.

Vasil, I.K. 1995. Cellular and molecular genetic improvement of cereals. p. 5-8. In M. Terzi et al. (ed.) Current issues in plant molecular and cellular biology. Kluwer Academic Publ., Hingham, MA.

Villamil, C.B, R.W. Duell, D.E. Fairbrothers, and J. Sadowski. 1982. Isoelectric focusing of esterases for fine fescue identification. Crop Sci. 22:786-793.

Vogl, A.E. 1898. Mehl und die anderen Mehlprodukte der Cerealien und Leguminosen. Nahrungsm. Unters. Hyg. Warenk. 12:25-29.

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

Wang, Z.Y., G. Legris, M.P. Valles, I. Potrykus, and G. Spangenberg. 1995. Plant regeneration from suspension and protoplast culture in the temperate grasses Festuca and Lolium. p. 81-86. In M. Terzi et al. (ed.) Current issues in plant molecular and cellular biology. Kluwer Academic Publ., Hingham, MA.

Wang, Z.Y., J. Nagel, I. Potrykus, and G. Spangenberg. 1993a. Fertile plant regeneration from protoplasts of meadow rescue (Festuca pratensis Huds.). Plant Cell Rep. 12:95-100.

Wang, Z.Y., J. Nagel, I. Potrykus, and G. Spangenberg. 1993b. Plants from cell suspension-derived protoplasts in Lolium species. Plant Sci. 94:179-193.

Wang, Z.Y., T. Takamizo, V.A. Iglesias, M. Osusky, J. Nagel, I. Potrykus, and G. Spangenberg. 1992. Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts. Bio/Technology 10:691-696.

Warkentin, D., B. Chai, R.K. Hajela, H. Zhong, and M.B. Sticklen. 1997. Development of transgenic creeping bentgrass (Agrostis palustris Huds.) for fungal disease resistance, p. 153-161. In M.B. Sticklen and M.P. Kenna (ed.) Turfgrass biotechnology -- Cell and molecular genetic approaches to turfgrass improvement. Ann Arbor Press, MI.

Warnke, S.E., D.S. Douches, and B.E. Branham. 1997. Relationships among creeping bentgrass (Agrostispalustris Huds.) cultivars based on isozyme polymorphisms. Crop Sci. 37:203-207.

Warnke, S.E., D.S. Douches, and B.E. Branham. 1998. Isozyme analysis supports allotetroploid inheritance in tetraploid creeping bentgrass (Agrostis palustris Huds.). Crop Sci. 38:817-822.

Waskom, R.M., D.R. Miller, G.E. Hanning, R.R. Duncan, R.L. Voigt, and M.W. Nabors. 1990. Field variation of tissue culture derived sorghum for increased tolerance to acid soils and drought stress. Can. J. Plant Sci. 70:997-1004.

Weeden, N.F., and A.C. Emmo 1985. Isozyme characterization of Kentucky bluegrass cultivars. Can. J. Plant Sci. 65:985-994.

Weedon, C.M., and P.G. Mantle. 1987. Paxilline biosynthesis by Acremonium loliae: A step towards defining the origin of lolitrem neurotoxins. Phytochemistry 26:969-971.

Wehner, J.D., J.M. Duich, and T.L. Watschke. 1976. Separation of Kentucky bluegrass cultivars using peroxidase isoenzyme banding patterns. Crop Sci. 16:476-479.

Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213-7218.

Wenzel, G., U. Frei, A. Jahoor, and A. Graner. 1995. Haploids -An integral part of applied and basic research, p. 127-135. In M. Tcrzi et al. (ed.) Current issues in plant molecular and cellular biology. Kluwer Academic PUN., Hingham, MA.

White, D.W.R. 1988. Use of cell and molecular genetic manipulation to improve pasture plants, p. 67-72. In R.E. Burgess (ed.) Proc. NZ Grass. Assoc. Conf., 59th. Matamata, New Zealand. 3-5 Nov. 1987. NZ Grass. Assoc., Palmerston North, New Zealand.

White, R.H., M.C. Engelke, S.J. Morton, J.M. Johnson-Cicalese, and B.A. Ruemmele. 1992. Acrernonium endophyte effects on tall fescue drought tolerance. Crop Sci. 32:1392-1396.

Wilkinson, J.F., and J.B. Beard. 1972. Electrophoretic identification of Agrostis palustris and Poa pratensis cultivars. Crop Sci. 12:833-834.

Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535.

Wu, K.S., and S. Tanksley. 1993. Abundance, polymorphism and genetic mapping of microsatellites in rice. Molec. Gen. Genet. 241: 225-235.

Wu, L., A.H. Harivandi, J.A. Harding, and W.B. Davis. 1984. Identification of Kentucky bluegrass cultivars with esterase and phosoglucomutase isocnzyme markers. Crop Sci. 24:763-768.

Wu, L., and R. Jampates. 1986. Chromosome number and isoenzyme variation in Kentucky bluegrass cultivars and plants regeneration from tissue culture. Cytologia 51:125-132.

Wu, L., and H. Lin. 1994. Identifying buffalograss (Buchloe dactyloides (Nutt.) Engelm.) cultivar breeding lines using random amplified polymorphic DNA (RAPD) markers. J. Am. Soc. Hort. Sci. 119: 126-130.

Wyman, A.R., and R. White. 1980. A highly polymorphic locus in human DNA. Proc. Natl. Acad. Sci. USA. 77:6754-6758.

Xu, Y.L., L. Li, K. Wu, A.J.M. Peters, D.A. Gage, and J.A.D. Zeevaart. 1995. Thc GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression. Proc. Natl. Acad. Sci. USA. 92:6640-6644.

Yamamoto, I., and M.C. Engelke. 1997. How can we utilize in vitro culture for direct improvement of existing turfgrass cultivars, p. 165-172. In M.B. Sticklen and M.P. Kenna (ed.) Turfgrass biotechnology -- Cell and molecular genetic approaches to turfgrass improvement. Ann Arbor Press, M1.

Yaneshita, M., R. Nagasawa, S. Kaneko, Y. Ogihara, and T. Sasakuma. 1993b. Genetic characterization of zoysiagrasses by RFLP analysis of nuclear DNA. p. 786-792. In R.N. Carrow et al. (ed.) Int. Turf. Soc. Res. J. Vol. 7. Interec Publ. Corp., Overland Park, KS.

Yaneshita, M., T. Ohmura, T. Sasakuma, and Y. Ogihara. 1993a. Phylogenetic relationships of turfgrasses as revealed by restriction fragment analysis of chloroplast DNA. Theor. Appl. Genet. 87: 129-135.

Yong, G.C., M.W. Blair, P. Olivier, and S.R. McCouch. 1996. Cloning and mapping of variety-specific rice genomic DNA sequences: Amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels. Genome 39:373-378.

Zabeau, M., and P. Vos. 1993. Selective restriction fragment amplification: A general method for DNA fingerprinting. European Patent Application no. 0534858 Al.

Zaghmout, O.M.F., and W.A. Torello. 1988. Enhanced regeneration in long-term callus culture of red rescue by pretreatment with activated charcol. HortScience 23:615-616.

Zaghmout, O.M.F., and W.A. Torello. 1992. Plant regeneration from callus and protoplasts of perennial ryegrass (Lolium perenne L.). J. Plant Physiol. 140:101-105.

Zhang, S., H. Zhong, M.B. Sticklen. 1996. Production of multiple shoots from shoot apical meristems of oat (Arena sativa L.). J. Plant Physiol. 148:667-671.

Zhao, X., and G. Kochert. 1992. Characterization and genetic mapping of microsatelliates in rice. Molec. Gen. Genet. 241:225-235.

Zhao, X., and G. Kochert. 1993. Phylogenetic distribution and genetic mapping of a (GGC), microsatellite from rice (Oryza sativa L.) Plant Molec. Biol. 21:607-614.

Zhong, H., M.G. Bolyard, C. Srinivasan, and M.B. Sticklen. 1993. Transgenic plants of turfgrass (Agrostis palustris Huds) from microprojectile bombardment of embryogenic callus. Plant Cell Rep. 13:1-6.

Zhong, H., C. Srinivasan, and M.B. Sticklen. 1991. Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds). Plant Cell Rep. 10:453-456.

Zhong, H., C. Srinivasan, and M.B. Sticklen. 1992. In-vitro morphogenesis of corn (Zea mays L.) II. Planta 187:490497.

Zhong, H., B. Sun, D. Warkentin, S. Zhang, R. Wu, T. Wu, and M.B. Sticklen. 1996. The competence of maize shoot meristems for integrative transformation and inherited expression of transgenes. Plant Physiol. 110:1097-1107.

Zhong, H., W. Wang, and M.B. Sticklen. 1998. In vitro morphogenesis of Sorghum bicolor(l.) Moench: Efficient plant regeneration from shoot apices. J. Plant Physiol. (in press).

Zhou, H., J.W. Arrowsmith, M.E. Fromm, C.M. Hironaka, M.L. Taylor, D. Rodriguez, M.E. Pajeau, S.M. Brown, C.G. Santhino, and J.E. Fry. 1995. Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Rep. 15: 159-163.

Benli Chai and Mariam B. Sticklen, 202 Pesticide Research Center, Dep. of Crop and Soil Sciences, Michigan State Univ., East Lansing, MI 48824. Received 5 Aug. 1997. *Corresponding author (sticklel@

Abbreviations: AFLP, Amplified Fragment Length Polymorphism; cpDNA, chloroplast DNA; DAF, DNA amplification fingerprinting; PCR, Polymerase Chain Reaction; RAPD, Random Amplified Polymorphic DNA; RFLP, Restriction Fragment Length Polymorphisms; SSR, Simple Sequence Repeat; UPGMA, Unweighted Pair-Group Method of Analysis.
COPYRIGHT 1998 Crop Science Society of America
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 1998 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Chai, Benli; Sticklen, Mariam B.
Publication:Crop Science
Date:Sep 1, 1998
Previous Article:The NIa-proteinase of different plant potyviruses provides specific resistance to viral infection.
Next Article:Genetic variation among tomato accessions from primary and secondary centers of diversity.

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters