Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-014-0653-7
REVIEW
In vitro regeneration, somatic hybridization and genetic
transformation studies: an appraisal on biotechnological
interventions in grasses
C. C. Giri • M. Praveena
Received: 7 June 2014 / Accepted: 25 October 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract The grass family Poaceae is one of the largest
groups of monocots comprising approximately 650–900
genera and 10,000 species. Grasses include plants with
wider environmental adaptability sustaining extremes of
cold, heat, drought, and dominant in its distribution on
variety of landscapes world over. Conventional approaches
have contributed substantially in the past for the genetic
improvement of grasses. However, the necessity of
improvement for quality traits, nutritional value and as
biofuel source has sought biotechnological interventions in
grasses. Regeneration of grasses is a prerequisite for
genetic manipulation in vitro. Last one decade, has seen
accumulation of large amount of literature in grass on
in vitro regeneration. Further, against the background of
the limitations for transferring polygenic traits coupled
with prevalent innate sexual incompatibilities, somatic
hybridization through protoplast fusion may be a possible
option for gene transfer within grasses and from grasses to
lead cereals. In addition, grasses gain additional impor-
tance by realizing novel nuclear- cytoplasm-genome
combinations from somatic fusion products. There are no
detailed recent reviews available depicting overall research
progress with major emphasis on in vitro plant regenera-
tion, somatic hybridization and genetic transformation in
grasses. In the present review, attempts is made towards
making a inclusive survey illustrating research findings on
in vitro regeneration, somatic hybridization and genetic
transformation studies covering last one decade. Mention
of a few new and novel developments for grass genetic
C. C. Giri (&)  M. Praveena
Centre for Plant Molecular Biology (CPMB), Osmania
University, Hyderabad 500007, India
e-mail: giriccin@yahoo.co.in
improvement using biotechnological approaches is dis-
cussed in this review.
Keywords Assessment  Genetic transformation 
Grasses  In vitro regeneration  Poaceae  Quality traits 
Somatic hybridization
Introduction
Grasses embraces all cereal crops, cultivated forage, and
are tougher contrast to any other flowering plants. Very few
of them genuinely been exploited for genetic improvement
as crops or turf. The most intensively used forage and lawn
grasses include fescues, ryegrasses, bentgrasses, blue
grasses, bromegrasses, orchardgrass, bermudagrass and
many others. Besides its contribution to livestock and feed
for wildlife, grasses play an important role in soil conser-
vation and environmental protection. Further, some of the
grasses, are specifically grown for turf or amenity purposes
on sports fields, golf courses, parks, lawns, and roadsides.
Conventional approaches has contributed for the
improvement of grasses in the past, however; confronted
with several inherent bottlenecks. Because, grass geno-
types; vary immensely in their reproductive performances,
incompatibility associations, chromosome numbers and
modes of pollination. Due to these impediments, biotech-
nological interventions find its genesis and significance. In
the past, methods concerning in vitro regeneration, somatic
hybridization and gene transfer techniques were evaluated
involving grasses.
In vitro plant regeneration protocol is a prerequisite for
propagation of grasses and manipulation of genomes
through somatic hybridization and genetic transformation
(Han et al. 2009; Zhang et al. 2010; Li et al. 2010a; Seo
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et al. 2011; Song et al. 2012; King et al. 2014; Ran et al.
2014). Success of plant regeneration in grasses depends on
the control of morphogenesis in vitro. This is influenced by
a number of factors e.g. origin of explants, nutritional
components, plant growth regulators and culture environ-
ment (Barampuram et al. 2009; Głowacka et al. 2010; Chai
et al. 2011; Praveena and Giri 2012; Chen et al. 2012;
Zhang et al. 2012; Zhao et al. 2013; Carloni et al. 2014).
Somatic hybridization through protoplast fusion can be a
relevant tool in genetic improvement of grasses (Mostageer
and Elshihy 2003; Cui et al. 2009; Xiang et al. 2010).
Somatic hybridization might be of importance in grass
improvement, however; it is reported in a relatively few
grass species (Liu et al. 1999; Xiang et al. 1999; Zhou et al.
2001a, b; Xia et al. 2003; Li et al. 2004a, b; Cheng and Xia
2004; Ge et al. 2006a; Cai et al. 2007; Cui et al. 2009;
Xiang et al. 2010). There have been several recent reports
on grass genetic transformation (Long et al. 2011; Liu et al.
2012; Song et al. 2012; Ramamoorthy and Kumar 2012;
Patel et al. 2013; Hwang et al. 2014). In the past some
reviews on biotechnological interventions in grasses has
been reported (Wang et al. 2001a; Wang and Ge 2006).
However, there are no recent detailed reviews presented
with emphasis on plant regeneration, somatic hybridiza-
tion, and gene transfer studies in grasses. The present
communication summarizes an inclusive study trends on
in vitro regeneration, somatic hybridization, genetic
transformation and some of the novel approaches for
improvement of grasses covering last decade.
In vitro plant regeneration
Plant regeneration protocols have been developed for a
wide range of forage and other grasses including cereals
(Giri and Vijaya Laxmi 2000; Barampuram et al. 2009;
Chai et al. 2011; Praveena and Giri 2012; Zhang et al.
2012; Kim et al. 2012). Plant regeneration in grasses
commonly influenced by a number of factors such as
explants origin, genotype, different basal media, media
supplements, media additives, carbohydrate sources, type
and concentrations of plant growth regulators (Table 1).
Explants such as nodes, immature embryos, mature
embryos, immature inflorescence, mature seeds, and shoot
meristems are frequently used (Table 1; Fig. 1).
MS (Murashige and Skoog 1962) based media are pre-
dominantly used in grass tissue culture (Fig. 2) compared
to others such as LS (Linsmaier and Skoog 1965) and B5
(Gamborg et al. 1968). The plant growth regulators such as
auxins (2,4-D, dicamba, NAA, IAA, TIBA, picloram and
GA3), cytokinins (BA, KN, zeatin) were used for in vitro
callus induction and plant regeneration in grasses. 2,4-D is
the most frequently supplemented auxin for grass callus
induction and maintenance. Other auxins such as dicamba,
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Plant Cell Tiss Organ Cult
picloram, NAA and cytokinin (e.g. BA, KN and others),
was also used for callus induction (Figs. 3, 4). Either total
removal or using reduced concentrations of auxins was
necessary during plant regeneration process. However,
addition of cytokinins such as KN, BA to the culture media
was must for plant regeneration depending on the grass
species (Figs. 5, 6). Distinct types of callus identified in
grass callus cultures based on their morphology, cellular
structure, and capacity to regenerate plants. More impor-
tantly, certain specific type of callus is competent to
regenerate plants, while others are not capable of forming
any organized structures regardless of culture media or
hormone employed (Praveena and Giri 2012).
Somatic hybridization
Somatic hybridization via protoplast fusion brings together
the genomes of two species and exploited to transfer mono or
polygenic traits (Liu et al. 2005). It also creates novel
genotypes by combining cytoplasmic genomes of different
species or cultivars. Many interspecific, intergeneric, inter-
tribal, and even interfamilial somatic hybrid plants generated
through this approach. A countable number of reports are
available on somatic hybrids involving grasses (Table 2).
Amongst different protoplast fusion methods used, except
for few electrofusion attempts, chemical (PEG) mediated
fusion were predominant (Table 2). Somatic fusion products
include both symmetric and asymmetric somatic hybrids
(Liu et al. 2005). To mention a few, protoplast fusion was
reported between rice and with Zizania latifolia (Liu et al.
1999), and Porteresia coarctata (Jelodar et al. 1999).
Asymmetric somatic hybridization was exploited for
transfer of bacterial blight resistance trait from wild Oryza
meyeriana L. to Oryza sativa L. ssp. Japonica (Yan et al.
2004). Protoplast fusion resulting in asymmetric hybrids was
also reported involving genus Triticum, which includes
Agropyron elongatum (Xia et al. 2003), Avena sativa (Xiang
et al. 2003a, b) and Lolium multiflorum (Ge et al. 2006a).
Very few symmetrical hybrids reported, for instance Hord-
eum vulgare L. (Kisaka et al. 1997) and Triticum aestivum
(Xia et al. 2003). Various screening methods were employed
for selection of somatic hybrids (Table 2). Symmetric
hybridization may present new genetic variability and is a
positive outcome of this method.
Genetic transformation studies
Genetic transformation of plants is an indispensable tool
for transfer of genes from an exotic and endemic source.
Choice of explants competent for transformation is a cru-
cial factor. Starting explants such as proliferating callus,
embryogenic callus, embryogenic cell suspension cultures,
isolated protoplasts, embryogenic regenerable green callus,
 Table 1 Plant tissue culture studies depicting explants source, media composition, and plant regeneration responses in grasses
Plant species/cultivar Explants source Media composition Culture Regeneration response References
response
Media ? additives PGRs Media PGRs R

Agrostis stolonifera L., S MS ? 500 mg/l CH, 3 % 6.6 mg/l dicamba, 0.5 mg/l BA EC – – – Luo et al. (2004)
cv. Penn-A-4 sucrose, 2 g/l phytagel
Agrostis palustris S MS ? 500 mg/l CH 6.63 mg/l dicamba, EC MS 1.0 mg/l KN or BA Y Wang et al. (2002a)
Huds. 0.5–2.0 mg/l BA
Plant Cell Tiss Organ Cult

Agrostis canina L. MS ? 500 mg/l CH 6.63 mg/l dicamba, 1.0 mg/l KN

0.5–2.0 mg/l BA
Agrostis capillaries L. MS ? 500 mg/l CH 6.63 mg/l dicamba, 1.0 mg/l BA, 100 mg/l MI
0.5–2.0 mg/l BA
Bouteloua eriopda AM MS 0.99 mg/l 2,4-D, 1.99 mg/l BA, C MS – Y Osuna and Barrow
(Torr.) Torr. 40 g/l adenine (2004)
AM-C MS 0.99 mg/l dicamba SE MS – Y
Bouteloua gracilis (H. ST MS 2.0 mg/l 2,4-D, 0.25/0.5 mg/l EC MS – Y Aguado-Santacruz
B. K.) Lag. ex Steud BA ‘ S MS et al. (2001)
1.0 mg/l 2,4-D, BA 0.5 mg/l MS 1.0 mg/l GA3 Y
Brachiaria brizantha NS LS 1.0 mg/l NAA, 3.0 mg/l KN O – – Y Cabral et al. (2011)
S MS ? 100 mg/l CH, 3.0 mg/l BA, 0.3 mg/l NAA O – – Y
14.6 g/l glutamine
S, SB, L MS ? 100/300 mg/l CH 2.0/3.0/4.0 mg/l 2,4-D, 0.2 mg/ SE MS ?100/300 mg/l 0.1 mg/l NAA, 0.4/4.0 mg/l Y
l BA MI KN/0.5 mg/l NAA, 1.0 mg/l
BA, 2.5 mg/l KN
Brachypodium ImE MS ? 3 % sucrose 1.0 mg/l 2,4-D C MS 0.1 mg/l BA Y Hammami et al. (2011)
distachyon
Cenchrus ciliaris L. S, ST, ImI MS 3.0 mg/l 2,4-D, 0.5 mg/l BA EC MS 3.0 mg/l BA Y Yadav et al. (2009)
0.25 mg/l 2,4-D, 1.0/5.0 mg/l SE MS 3.0 mg/l BA Y
BA
Cenchrus ciliaris L. ST MS 3.0 mg/l TDZ MSI MS 3.0 mg/l GA3 Y Kumar and Bhat(2012)
Cenchrus ciliaris L. A MS ? 3 % sucrose 6.0 mg/l 2,4-D C MS 05 mg/l NAA, 1.0 mg/l BA Y Carloni et al. (2014)
Chloris gayana Kunth AM 0.3 % gellan gum 0.1 mg /l 2,4-D, 2.0 mg/l BAP MSI MS – Y Gondo et al. (2007)
2.0 mg/l 2,4-D EC MS 0.01 mg/l NAA, 2.0 mg/l BAP Y
Cynodon dactylon cv. ImI, NS MS ?1.16 g/l proline 1/1.5 mg/l 2,4-D, 0.01 mg/l BA EC MS 0.1 mg/l 2,4-D, 0.5/4.0 mg/l Y Zhang et al. (2007)
(TifEagle, TifSport, BA
Tift97-4)
Tetraploid genotypes
(Tift93-132, Tift93-
135, Tift93-156,
Tift93-157)
Cynodon NS NB ? B5 vit., 0.5 mg/l 6.6 mg/l dicamba, 0.6 mg/l BA EC NB ? B5 vit., 0.6 mg/l BA Y Lu et al. (2006)
dactylon 9 C. CH, 0.5 g/l glutamine, 0.5 mg/l CH,
transvaalensis cv. 0.5 g/l, proline, 0.1 g/l 0.5 g/l glutamine,
Tifeagle MI, 3 % sucrose 0.1 g/l MI, 3 %
sucrose

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 Table 1 continued
Plant species/cultivar Explants source Media composition Culture Regeneration response References
response
Media ? additives PGRs Media PGRs R

123
Cynodon dactylon [L.] S MS ? 3 % sucrose 13.26 mg/l 2,4-D C MS 1.68 mg/l BA Y Salehi and Khosh-Khui
Pers, Festuca rubra (2005)
L. var. rubra, L.
perenne L. Poa
pratensis L.
Cynodon dactylon [L.] ImI MS ? 3.0 % sucrose, 0.99 mg/l 2,4-D, 0.009/ EC MS ? 3.0 % 0.99 mg/l BAP, 0.279 mg/l Y Li and Qu (2004)
Pers 3.0 g/l phytagel 0.202 mg/l BAP, 0.5 mg/l sucrose, 3.0 g/l NAA, 0.45 mg/l GA3
ABA phytagel
Dichanthium ST MS ? 3 % Maltose, B5 vit. 2.0 mg/l 2,4-D C MS ? 3 % maltose 1 mg/l BA, 1 mg/l NAA Y Dalton et al. (2003)
annulatum
Eragrostis curvula L MS ? 2 % sucrose 2 mg/l 2,4-D, 0.01 mg/l BA C MS ? 6 % Sucrose 2 mg/l 2,4-D, 0.01 mg/l BA Y Ncanana et al. (2005)
S MS ? 2 % sucrose 2 mg/l 2,4-D, 0.01 mg/l BA C MS ? 3 % sucrose 2 mg/l 2,4-D, 0.01 mg/l BA or Y
–
Eragrostis tef P MS ? 0.3 % gelrite 2.03 mg/l 2,4-D EC MS – Y Gugsa et al. (2006)
Eragrostis tef DZ-01- S MS ? 1 mg/l thiamine 2.0 mg/l 2,4-D, 4.0 mg/l TIBA, C – – –
354 HCl, 250 mg/l MI, 3 % 5.0 mg/l dicamba
sucrose
Eremochloa S MS 4.5 mg/l 2,4-D, 1.0 mg/l BA EC MS 2 mg/l BA, 1 mg/l NAA Y Liu et al. (2008)
ophiuroides [Munro]
Hack.
ST MS 2.0 mg/l 2,4-D EC – – –
Eulaliopsis binata S MS 3.0 mg/l 2,4-D EC MS 0.2 mg/l NAA, 6.0 mg/l KN Y Ma et al. (2013)
F. arundinacea Schreb. S MS ? 9.9 mg/l thiamine, 7.0 mg/l 2,4-D, 0.05 mg/l KN EC MSH ?0.5 g/l CH, 2.0 mg/l KN, 0.5 mg/l NAA Y Zhang et al. (2006)
4.5 mg/l nicotinic acid, 2 % sucrose
9.5 mg/l pyridoxine,
1.0 g/l CH
Leptochloa fusca (L.). ImI MS 2.0 mg/l dicamba 0.25 mg/l C MS 2.0 mg/l NAA, 0.5 mg/l BA Y Praveena and Giri
BA (2012)
Leymus chinensis Trin. ImI N6 1.99 mg/l 2,4-D C N6 1.0 mg/l KN, 1.0 mg/l BA Y Liu et al. (2004)
Leymus chinensis S, SB MS ? 5.0 mg/l L-glutamic 2.0 mg/l 2,4-D EC MS ?2.0 g/l CH 0.2 mg/l NAA, 2.0 mg/l KN Y Sun and Hong (2010)
(Trin.) acid
L. multiflorum S MS ? 500 mg/l proline, 5.0 mg/l 2,4-D, BA 0.5 mg/l EC MS ? 500 mg/l 1.0 mg/l 2,4-D, 5.0 mg/l BA Y Lee et al. (2009)
1 g/l CH, 3 % sucrose, proline, 1 g/l CH,
7 mg/l AgNO3, 2 mg/l 3 % sucrose,
CuSO4 7 mg/l AgNO3,
2 mg/l CuSO4,
1 mg/l thiamine-
HCl
L. perenne L. S MS 5 mg/l 2,4-D, 5 mg/l BA EC MS 3 mg/l BA, 1 mg/l NAA, 1 mg/ Y Liu et al. (2006)
l KN
M. sinensis 9 M. ImI MS 5 mg/l 2,4-D, 0.1 mg/l BA EC MS 2.0 mg/l BA Y Głowacka et al. (2010)
giganteus
Plant Cell Tiss Organ Cult
 Table 1 continued
Plant species/cultivar Explants source Media composition Culture Regeneration response References
response
Media ? additives PGRs Media PGRs R

Miscanthus 9 ImI MS 0.5 mg/l 2,4-D, 0.5 mg/l BA EC MS 5.0 mg/l BA, 1.0 mg/l 2,4-D, Y Kim et al. (2012)
giganteus 1.0 g/l proline, 300 mg/l CH
Miscanthus sinensis ST MS ? 3 % sucrose 1.0 mg/l BA, 2.0 mg/lKN, MSI – ‘ MS ? 0.3 mg/l NAA Y Zhang et al. (2012)
0.05 mg /l NAA
Plant Cell Tiss Organ Cult

E HB ? 0.1 mg/l thiamine 2.0 mg/l 2,4-D, 0.5 mg/l BA EC HB 2.0 mg/l BA, 0.05 mg/l IBA, Y
and 0.05 mg/l NAA
Miscanthus ImI MS 4 mg/l 2,4-D C MS 4.0 mg/l BA, 1.0 mg/l NAA Y Zhao et al. (2013)
sacchariflorus ssp.
lutarioriparius
Panicum spp. S MS ? 0.30 % gelrite 4.0 mg/l 2,4-D C MS 1.0 mg/l KN, 0.4 % gelrite Y Seo et al. (2008)
Panicum spp. S MS ? 3.0 % sucrose, 4.0 mg/l 2,4-D C MS ? 3.0 % 1.0 mg/l TDZ Y Seo et al. (2010)
3.00 % gelrite maltose
ImE, ST MS ? 3.00 % sucrose, 10.0 mg/l 2,4-D C MS ? 3.0 % 1.0 mg/l TDZ Y
3.00 % gelrite maltose
Paspalum notatum S MS ? 3.00 % sucrose 6.63 mg/l dicamba, 1.12 mg/l EC MS 1.12 mg/l BA, 0.34 mg/l GA3 Y Grando et al. (2002)
Flügge cv. Tifton 9 BA
Paspalum simplex ME, ES MS ? 0.4 mg/l thiamine- 2.0 mg/l 2,4-D, 0.05 mg/l KN C 0.4 mg/l thiamine- 0.5 mg/l NAA, 2.0 mg/l BA Y Molinari et al. (2003)
HC, L, RT HCl, 100 mg/l MI, 3.0 % HCl, 100 mg/l
sucrose MI, 3.0 %
sucrose
Paspalum vaginatum ImI MS 3 mg/l dicamba, 1 mg/l BA EC MS – Y Neibaur et al. (2008)
Swartz
Phragmites australis ImI 1 mg/l 2,4-D EC – – Y Lauzer et al. (2000)
(Cav.) Trin.
MS ? 100 mg/l MI 1 mg/l 2,4-D EC MS –
MS ? 3 % sucrose 2 mg/l 2,4-D
Phragmites communis NS MS ? 200 mg/l MI, 2.0 mg/l 2,4-D C MS 2.997 mg/l BA, 0.999 mg/l Y Yang et al. (2003)
Trin. 4.00 % sucrose NAA, 0.1 mg/l 2,4-D,
0.1 mg/l NAA
0.99 mg/l 2,4-D EC MS
0.099 mg/l 2,4-D SE MS
Poa pratensis S MS 2.0 mg/l 2,4-D, 0.2 mg/l BA C SH Y Zong et al. (2010)
Psathyrostachys juncea ME MS ? 3.00 % sucrose 5.0 mg/l 2,4-D EC MS ? 3.0 % 0.2 mg/l KN Y Wang et al. (2002b)
(Fisch.) Nevski sucrose
AA ? 5 mg/1 2,4-D ECS
?2.0 %, sucrose, 3.0 %
sorbitol
Schizachyrium EC MS ? 10 % CW 1.5 mg/l 2,4-D ECS, SE MS ? 500 mg/l 3.0 mg/l 2,4-D, 1.0 mg/l BA, Y Chakbavarty et al.
scoparium (Michx.) 0.2 mg/l BAP, 0.5 mg/l zeatin, CH, 10 % CW 1.0 mg/l GA3, 0.5 mg/l NAA (2001)
Nash var. 0.2 mg/l GA3
stoloniferum (Nash)
J. Wipff

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 Table 1 continued
Plant species/cultivar Explants source Media composition Culture Regeneration response References
response
Media ? additives PGRs Media PGRs R

123
Stenotaphrum ImE, SB MS 1.0 mg/l 2,4-D, 0.5 mg/l BA EC MS 1.0 mg/l BA, 0.2 mg/l NAA, Y Li et al. (2006)
secundatum (Walt.) 0.5 mg/l GA3
Kuntze
Thinopyrum, ME, ImE, ImI MS ? 3.0 % sucrose 4.99 mg/l 2,4-D EC MS ? 3.0 % 0.19 mg/l Y Wang et al. (2003)
Agropyron and sucrose
Pascopyrum spp. AA medium 3.0 % sucrose, 1.5 mg/l 2,4-D ESC KN
3.00 % sorbitol
Tripsacum dactyloides AM MS ? 400 mg /l thiamine 5 mg/l 2,4-D EC MS 100 mg/l MI, 1.0 mg/l KN, 10 mg/l BA Y Sairam et al. (2002)
(L.) HCl 100 mg/l glycine
Zoysia japonica Steud. S N6 ? MS ? 500 mg/l 3.0 mg/l 2,4-D, 0.01 mg/l BA EC MS 0.3 mg/l NAA, 0.5 mg/l KN, Y Liu et al. (2009)
proline, 500 mg/l CH, 1.0 mg/l BA
500 mg/l MI, 5 mg/l
thiamine–HCl, 40 g/l
sucrose, 10 g/l glucose
Zoysia matrella L. ImI MS 0.99/l/1.498/1.98 mg/l 2,4-D, C – – – Dhandapani et al.
Merr 0.099 mg/l BA (2008)
Zoysia. matrella [L.] NS MS ? 1.15 g/l proline, 2 mg/l 2,4-D 0.02 mg/l KN/BA EC ‘ MS ? 2 % 0.1 mg/l BA/KN Y Chai et al. (2011)
Merr. 40 g/l sucrose, 3 g/l sucrose, 3.0 g/l
phytagel phytagel
Zoysia matrella [L.] NS MS ? 1.15 g/l proline, 4 % 2.0 mg/l 2,4-D, 0.02 mg/l BA EC ‘ MS ? 2.0 % 0.1 mg/l BA Y Chen et al. (2012)
Merr sucrose, 3 g/l phytagel sucrose, 3.0 g/l
phytagel
Zoysia sinica Hance S MS ? 3.00 % sucrose 2.0 mg/l 2,4-D, 0.05/0.2 mg/l EC MS ? 3.0 % 1 mg/l BA, 0.2 mg/l NAA Y Li et al. (2006)
BA sucrose

AA Muller and Grafe amino acid medium (Müller and Grafe 1978), ABA abscisic acid, AM apical meristem/shoot meristem, AM-C apical meristem derived callus, BA 6-benzylaminopurine, B5 Gamborg’s medium
(Gamborg et al. 1968), C callus, CH casein hydrolysate, CW coconut water, Dicamba 3,6-dichloro-2-methoxybenzoic acid, EC embryogenic callus, ECS embryogenic suspension culture, GA3 gibberellic acid, HB
Holley and Baker medium (Holley and Baker 1963), IBA indole-3-butyric acid, ImE immature embryo, ImI Immature inflorescence, KN kinetin, L young leaf/leaves/leaf blade, LS Linsmaier and Skoog, ME mature
embryo, MI myo-inositol, MS Murashige and Skoog medium (Murashige and Skoog 1962), MSH modified Schenk and Hilderbrandt medium (Schenk and Hildebrandt 1972), MSI multiple shoot induction/adventitious
shoot buds, NB N6 macro ? B5 micro elements ? EDTA Fe, NS stem nodes/nodal segment, N6 medium (Chu 1978), NAA a-naphthaleneacetic acid, PGRs plant growth regulator, P pistil, R regeneration, S mature
seeds/seeds, SB shoot base/leaf base, SE somatic embryo, ST shoot apices/shoot tips, TDZ thidiazuron, TIBA 2,3,5-tri iodobenzoic acid, Vit vitamin, 2,4-D 2,4-dichlorophenoxy acetic acid, – devoid of hormones
Plant Cell Tiss Organ Cult
Plant Cell Tiss Organ Cult
Fig. 1 Random depiction of data on the frequency of the explants use
for development of in vitro culture and plant and plant regeneration in
grasses. ImI immature inflorescence, ME mature embryo, ImE
immature embryo, NS nodal segment, ST stolons, MISLNS
miscellaneous
Fig. 2 Random portrayal of data on the frequency of the media used
for development of in vitro culture and plant regeneration in grasses.
MS Murashige and Skoog medium, MS ? B5 MS ? Gamborgs
medium, LS Linsmaier and Skoog medium, N6 Chu medium, MISLNS
miscellaneous
Fig. 3 Random representation of data on the frequency of plant
growth regulators used for callus induction in grasses. 2,4-D 2,4-
dichlorophenoxyacetic acid, DICAMBA 3,6-dichloro-o-anisic acid,
NAA a-napthaleneacetic acid, MISLNS miscellaneous
Fig. 4 Random representation of data on the frequency of cytokinin
and others used for callus induction in grasses. BA N6-benzyladenine,
KN kinetin, ABA abscisic acid, MISLNS miscellaneous
Fig. 5 Random version of data on the frequency of plant growth
regulators (auxins) used for plant regeneration in grasses. 2,4-D 2,4-
dichlorophenoxyacetic acid, DICAMBA 3,6-dichloro-o-anisic acid,
PIC picloram, NAA a-napthaleneacetic acid, GA3 gibberellic acid,
MISLNS miscellaneous
Fig. 6 Random account of data on the frequency of plant growth
regulators (cytokinins) used for plant regenration in grasses. BA N6-
benzyladenine, KN kinetin, TDZ thidiazuron, ZTN zeatin
and stolon nodes were frequently used for genetic trans-
formation studies in grasses (Table 3). However, explants
such as embryogenic callus, embryogenic cell suspension
cultures and isolated protoplasts were used most of the
transformation experiments. Assessment of research
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Table 2 Somatic hybridization studies in grasses illustrating species involved, fusion method, type of fusion products and characterization of
somatic hybrids
Plant species Fusion Type of somatic Characterization technique/ References
method fusion products method

T. aestivum L. cv, Jinan 177 ? A. sativa L. cv. Mapur PEG ASH FISH, SSR Xiang et al. (2010)
A. elongatum (tall wheatgrass) ? T. aestivum cv. PEG ASH GISH, AFLP Cui et al. (2009)
Jinan177 (bread wheat)
F. arundinacea Schreb. ? F. arundinacea Schreb. PEG ASH RAPD, FISH, RFLP, IA Cai et al. (2007)
T. aestivum L. ? L. multiflorum Lam. EF CYB RFLP, IA Ge et al. (2006a)
T. aestivum L. ? L. multiflorum Lam. EF ASH, SH RFLP, GISH Cheng et al. (2006)
T. aestivum L. ? Haynaldia villosa Schur. PEG ASH GISH, RFLP Zhou and Xia
(2005)
T. aestivum L. ? Psathyrostachys juncea PEG SSH RAPD, GISH, IA Li et al. (2004a)
T. aestivum L. cv. Jinan 177 (cha9 and PEG ASH RAPD, CA, IA Li and Xia (2004)
176) ? Psathyrostachys juncea (Fisch.) Nevski
T. aestivum L. ? Leymus chinensis (Trin.) Tzvel. PEG ASH RAPD, RFLP, PA, CA, IA, Chen et al. (2004a)
5S rDNA SSA
T. aestivum L. cv. Jinan 177 ? L. multiflorum Lam. EF ASH RAPD, RFLP, SSR, CA, IA Cheng and Xia
(2004)
T. aestivum L. ? Thinopyrum ponticum PEG ASH CA Chen et al. (2004b)
T. aestivum L. ? A. elongatum (Host) Nevishi PEG ASH RAPD, RFLP, IA, CA, Cheng et al. (2004)
5S-rDNA SSA
O. meyeriana L. ? O. sativa L. ssp. japonica PEG ASH RAPD Yan et al. (2004)
T. aestivum ? A. sativa L. PEG SSH GISH, IA Xiang et al.
(2003a)
T. aestivum ? A. sativa L. PEG ASH GISH, CA, IA, 5S-rDNA Xiang et al.
SSA (2003b)
O. sativa ? Phragmites communis EF SSH RAPD Mostageer and
Elshihy (2003)
T. aestivum L. ? A. elongatum PEG SSH RAPD, GISH, IA Xia et al. (2003)
T. aestivum L. ? Zea mays L. PEG ASH PA, IA, RFLP, SSR, GISH Xu et al. (2003)
T. aestivum L. ? Zea mays L. PEG ASH CA, IA, 5S-rDNA SSA Zhi et al. (2002)
T. aestivum L. c.v. Jinan 177 ? Haynaldia villosa PEG ASH, SSH GISH Zhou et al. (2001a)
T. aestivum L. c.v. Jinan 177 ? Haynaldia villosa PEG SSH PCR, GISH, RFLP, CA Zhou et al. (2001b)
T. aestivum L. c.v. Jinan 177 ? Aeleuropus littorulis PEG ASH RAPD, CA, IA Wei et al.(2001)
sinensis
T. aestivum L. ? A. elongatum PEG SSH RAPD, CA, IA Xia et al. (1999)
O. sativa L. line RCPL 1–2C ? O. sativa L. line V20A EF CYB RFLP Bhattacharjee et al.
(1999)
O. sativa L. ? Porteresia coarctata EF SSH, ASH GISH, RAPD Jelodar et al.
(1999)
O. sativa L. ? Z. latifolia (Griseb). Turez.Ex Stapf EF ASH SA Liu et al. (1999)
T. aestivum ? Bromus inermis Leyss PEG ASH RAPD, IA Xiang et al. (1999)
AFLP amplified fragment length polymorphism, ASH asymmetric somatic hybrid, CA cytological/chromosome analysis, CYB cybrid, EF
electrofusion, GISH genomic in situ hybridization, IA isozyme analysis, ISH in situ hybridization, PA phytotype analysis, PCR polymerase chain
reaction, PEG polyethylene glycol, RAPD random amplified polymorphic DNA, RFLF restriction fragment length polymorphism, SA southern
analysis, SSA space sequence analysis, SSH symmetric somatic hybrid, SSR simple sequence repeats

progress has found that both Agrobacterium based and
particle bombardment genetic transformation is the main
method used for developing transgenics in grasses (Fig. 7).
Microprojectile particle bombardment for gene delivery to
plant cells and tissues has led to the production of trans-
genic plants from different recalcitrant species bypassing
protoplast culture and Agrobacterium host specificity.
Embryogenic callus cultures used as ideal targets for par-
ticle bombardment mediated transformation (Gao et al.
2008; Long et al. 2011). Agrobacterium-mediated genetic
transformation for developing transgenics in grasses has
been used (Wang et al. 2009; Lee et al. 2010; Li et al.

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Table 3 Genetic transformation studies in grasses depicting explants source, transformation method, genes transferred and production of
transgenic plants
Plant species Nature Transformation Genes transferred Transgenic References
of method plants
tissue

Agrostis palustris L., cv. Penncross EC Ag VuNCED1, hph Y Aswath et al. (2005)
Agrostis palustris Huds EC PB gfp Y Jayaraj et al. (2008)
Agrostis stolonifera L. EC PB PAP-Y, PAP-C or Y Dai et al. (2003)
PAPII, hph
Agrostis stolonifera L., cv. Penn-A-4 EC Ag gusA, hph, bar Y Luo et al. (2004)
Agrostis stolonifera L. EC Ag tlpD34, bar Y Fu et al. (2005)
Agrostis stolonifera L. EC Ag barley hva1, bar Y Fu et al. (2007)
Agrostis stolonifera L. EC Ag Lc, or Pl, gusA, bar Y Han et al. (2009)
Agrostis stolonifera L., cv. Penn A-4 EC Ag AVP1, bar Y Li et al. (2010a)
Agrostis stolonifera L., cv. Penn A-4 EC Ag Pen 4-1, bar Y Zhou et al (2011)
Agrostis tenuis Sibth. Fl. Oxen C Ag gusA, hph Y Chai et al. (2004)
Bouteloua gracilis (H.B.K.) Lag. ex Steud. CEC PB rol A Y Aguado-Santacruz et al.
(2009)
Brachypodium distachyon EC PB gusA, bar Y Christiansen et al. (2005)
Brachypodium distachyon (Brachypodium) C Ag hph, gusA Y Vogel and Hill (2008)
Bromus inermis Leyss NECS Ag gfp, gusA – Nakamura and Ishikawa
(2006)
Cynodon dactylon (L.) Pers. C PB hph, gusA, bar Y Li and Qu (2004)
Cynodon dactylon (L.) Pers. EC Ag cry1Ac, nptII Y Salehi et al. (2005)
Cynodon dactylon 9 C. transvaalensis EC Ag bar, gusA Y Hu et al. (2005a)
Cynodon dactylon C Ag hph Y Li et al. (2005)
ECS
Cynodon spp. Agrostis stolonifera NS Ag hph, gusA Y Wang and Ge (2005a)
Dactylis glomerata L. RGT PB hph, gus A, bar Y Cho et al. (2001)
Dichanthium annulatum (Forssk) C PB gusA, hph Y Dalton et al. (2003)
E. breviaristatus C Ag ppip, hph Y Li et al. (2007)
Eragrostis curvula C PB Hsp 12 Y Ncanana et al. (2005)
gusA
Eremochloa ophiuroides [Munro] Hack EC Ag hph, gusA Y Liu et al. (2012)
Eulaliopsis binata S Ag hpt, gfp Y Ma et al. (2013)
Festuca arundinaceae schreb ECS PB SFA8, mhpt Y Wang et al. (2001b)
F. arundinacea (Schreb.) ECS PB cad, hph Y Chen et al. (2003)
Festuca arundinaceae EC PB ipt, bar Y Hu et al. (2005b)
Festuca arundinaceae EC Ag hph, gusA, gfp Y Wang and Ge (2005b)
Festuca arundinaceae EC Ag cbf1 Y Wu et al. (2006)
F. arundinacea Schreb. cv. Kentucky-31 C Ag CuZnSOD, APX Y Lee et al. (2007)
F. arundinacea Schreb. C Ag AtNHX1 Y Zhao et al. (2007)
F. arundinacea Schreb. Ag AGLU1, SI, Pi9 Dong et al. (2007)
F. arundinacea Schreb. (Tall fescue) ECS PB faeA, sgfp Y Buanafina et al. (2008)
F. arundinacea Schreb. EC Ag OsALS (dm) Y Sato et al. (2009)
F. arundinacea Ag 2-Cys Prx Y Kim et al. (2010)
F. arundinacea Schreb EC PB bar, hph, nptII Y Long et al. (2011)
Festuca pratensis Huds. EC Ag bar, gusA Y Gao et al. (2009)
Festulolium plants (L. perenne L. 9 Festuca C Ag hph, gusA Y Guo et al. (2009)
pratensis Huds.)
Leymus chinensis (Trin.) Tzvel. C PB PAT Y Shu et al. (2005)
Leymus chinensis (Trin.) C Ag TaLEA3 Y Wang et al. (2009)

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Table 3 continued
Plant species Nature Transformation Genes transferred Transgenic References
of method plants
tissue

L. multiflorum Lam ECS PB sacB, hph Y Ye et al. (2001)

L. multiflorum Lam ECS PB SEE1, IPT, gusA, hph Y Li et al. (2004b)
L. multiflorum Lam ECS PB Lol p1, Lol p2, gusA, Y Petrovska et al. (2004)
L. perenne bar
L. multiflorum Lam C PB hph Y Takahashi et al. (2005)
Cht 2
RCC 2
L. multiflorum C Ag gusA, hph Y Lee et al. (2010)
L. perenne L. C PB RgMV-CP, nptII Y Xu et al. (2001)
L. perenne L. EC PB wft1, wft2, bar Y Hisano et al. (2004)
L. perenne L. cv. Friend PPTS EP Exogenous substance – Unno and Yamamoto
Fructan (2005)
L. perenne L. EC Ag OsNHX1, bar Y Wu et al. (2005)
L. perenne C PB arg E, hph Y Chen et al. (2005)
L. perenne L. EC Ag gusA, hph Y Sato and Takamizo
(2006)
L. perenne L. EC Ag hph Y Bajaj et al. (2006)
gusA
L. perenne L. EC Ag gusA, hph, cbf3 Y Li et al. (2011)
L. perenne L. C Ag cry8Ca2 or cry8Ga, bar Y Wu et al. (2012)
L. perenne L. EC Ag bar, gfp Y Patel et al. (2013)
Lolium rigidum SE-C Ag hph, gusA Y Ran et al. (2014)
Lolium temulentum L. EC Ag hph, gusA Y Ge et al. (2007)
Miscanthus. sinensis EC Ag hph, egfp Y Hwang et al. (2014)
P. meyerianum Nees. C Ag GFP:GUS, hpt Y Seo et al. (2011)
Panicum virgatum L. EC PB bar, sgfp Y Richards et al. (2001)
Panicum virgatum SE, EC Ag bar, gusA Y Somleva et al. (2002)
Panicum virgatum L. EC Ag hph, gusA Y Xi et al. (2009)
Panicum virgatum L. SLG Ag npt II, gusA, bar Y Song et al. (2012)
EC
Panicum virgatum L. EC PB pporRFP, hph Y King et al. (2014)
Panicum virgatum L. cv. Alamo C Ag sgfp Y Ramamoorthy and Kumar
(2012)
Paspalum notatum EC, C PB bar, gusA Y Gondo et al. (2005)
Paspalum notatum Flugge EC PB gfp Y Himuro et al. (2006)
Paspalum notatum Flugge cv. Argentine) EC PB nptII, bar MCs Y Sandhu and Alpeter
(2008)
Poa pratensis L. EC PB hph, bar, gusA Y Gao et al. (2006)
Poa pratensis L. AM Ag betA, als Y Zhang et al. (2010)
Psathyrostachys juncea ECS PB hph, gusA Y Wang et al. (2004)
Zoysia japonica Steud. NS Ag hph, gusA, gfp Y Ge et al. (2006b)
Zoysia japonica Steud. C Ag bar, gusA Y Toyama et al. (2003)
Zoysia sinica Hance EC Ag CBF1/DREB1b, bar Y Li et al. (2006)

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Plant Cell Tiss Organ Cult

Table 3 continued
Plant species Nature Transformation Genes transferred Transgenic References
of method plants
tissue

Zoysia tenuifolia Willd. ex Trin C Ag hph, gusA Y Li et al. (2010b)

Ag agrobacterium-mediated, AGLU1 the alfalfa b-1,3-glucanase, als gene for herbicide resistance, APX gene encoding ascorbate peroxidase, AM
apical meristem, argE gene encoding N-acetyl ornithinase, AtNHX1 Arabidopsis vacuolar Na?/H? antiporter, AVP1 the Arabidopsis vacuolar
H?-pyrophosphatase, bar/pat phosphinothricin acetyltransferase, Barley hva1 LEA protein from barley, betA gene encoding choline dehy-
drogenase, C callus, cad cinnamyl alcohol dehydrogenase, cbf1 C-repeat (CRT) binding factor 1, CBF1/DREB1b C-repeat (CRT)/dehydration
responsive element binding factor, cbf3 C-repeat (CRT) binding factor 3, CEC chlorophyllous embryogenic callus, Cht-2, RCC2 rice chitinase
gene, cry1Ac Bacillus thuringenesis Berliner gene encoding endotoxin, CuZnSOD gene encoding CuZn superoxide dismutase, EC embryogenic
callus, ECS embyogenic cell suspension, egfp enhanced green fluorescence protein gene, EP electroporation, faeA ferulic acid esterase gene, gfp
green fluorescent protein, gusA b-glucuronidasehph/hpt hygromycin phosphotransferase, Hsp 12 heat shock protein 12, IPT cytokinin biosyn-
thesis gene, ipt isopentenyl transferase gene, Lc leaf color, Pl purple leaf transcription factor genes, Lol p1 and Lol p2 main allergenic
determinants in rye grass pollen, MC minimal cassettes, mhpt modified hygromycin phosphotransferase, NECS non embryogenic cell suspension,
nptII neomycin phosphotransferase II, NS stolon nodes, OsALS (dm) a two-point mutated rice ALS gene, OsNHX1 rice Na?/H? antiporter gene,
PAP-Y, PAP-C or PAPII poke weed antiviral proteins, PB particle bombardment, Pen 4-1 the antimicrobial peptide—penaeidin4-1 from the
shrimp Litopenaeus setiferus, Ppip Pseudomonas pseudoalcaligenes insecticidal protein gene, pporRFP red flourescence protein (RFP) reporter
gene, PPTS protoplasts, Pi9 the rice gene, RGT regenerative green tissue, RgMV-CP rye grass mosaic virus coat protein gene, rol A gene from
Agrobacterium rhizogenes for hairy embryo, S seeds, sacB Bacillus subtilis levansucrose, SEE1 maize cysteine protease gene, SE somatic
embryo, SE-C somatic embryo derived callus, SFA8 sulfur-rich sunflower albumin gene, sgfp synthetic green fluorescent protein, SI a truncated
frog dermaseptin gene, SLG seedling, TaLEA3 T. aestivum LEA gene, tlpD34 rice thaumatin-like protein, VuNCED1 9-cis-epoxycarotenoid
dioxygenase gene, wft1 and wft2 wheat fructosyltransferase genes, 2-Cys Prx 2-Cys peroxiredoxins, Y yes

Fig. 7 Random depiction of data on the frequency of explant types

uses for genetic transformation studies in grasses. PPTS protoplasts, Fig. 8 Random account of data on the frequency of genetic
ECS embryogenic cell suspension, ECAL embryogenic cell suspen- transformation methods used in grasses. PB particle bombardment,
sion, ECAL embryogenic callus, CAL callus, MISLNS miscellaneous ElecP electroporation, PEG polythene glycol mediated, Ag-Med
Agrobacterium-mediated, MISLNS miscellaneous

2010b; Ramamoorthy and Kumar 2012; Hwang et al. 2014;

Ran et al. 2014; King et al. 2014). Many transgenic plants
were produced in the grasses in the last decade using diverse
methods of gene transfer (Table 3; Fig. 8). Several chimeric
and agronomically useful genes were transferred and
transgenic grasses have been developed in grasses (Fig. 9).
Some of the selected and specific paradigms on genetically
modified transgenic grasses are summarized as follows.
Generation of potentially useful transgenic grasses
Improvement of grasses for agronomically useful traits
In grasses, herbicide resistance may be useful for control-
ling unwanted weeds where economically important
grasses are the target crop for cultivation. A random rep-
resentation of genetic transformation studies and transgenic
grasses with agronomic and useful traits are documented
(Fig. 9). The particle inflow gun-mediated transformation
for bialaphos (L-PPT) resistance of diploid bahiagrass was
demonstrated and green transgenic plants were recovered
(Gondo et al. 2005). Shu et al. (2005) reported generation
of transgenic Chinese leymus resistant to herbicide basta.
The transgenic-resistant plants actively produced OsALS
(dm) protein under herbicide treatment (Sato et al. 2009).
Environmental stresses such as low temperature,
drought and salinity are the major adverse factors that limit
crop productivity worldwide. Exposure to abiotic stresses
such as drought, cold, heat, pollutants, and heavy metals,

123

Fig. 9 Random representation of data on the frequency of genetic
transformation studies in grasses. bar Bialaphos resistance, hph
hygromycin phospho transferase, nptII neomycin phospho transferase,
gus glucuronidase, gfp green florescent protein, ast abiotic stress
tolerance, bst biotic stress tolerance, qt quality traits
can give rise to excess accumulation of reactive oxygen
species (ROS) in plant cells. Lee et al. (2007) generated
transgenic tall fescue expressing the CuZnSOD and APX
genes in chloroplasts conferred enhanced tolerance to
methyl viologen (MV), and protection against heavy metal-
induced oxidative damage. Recently, transgenic Kentucky
blue grass cultivar was produced by the introduction of
betA gene from Escherichia coli (Zhang et al. 2010).
Drought is a major abiotic stress limiting the growth of
plants and productivity. Commonly LEA protein families
are involved in plant abiotic stress tolerance. The trans-
genic lines showed enhanced growth ability under drought
stress with increased relative water content, leaf water
potential and average growth rate compared to untrans-
formed control plants (Wang et al. 2009). Agrobacterium-
mediated transformation of bentgrass with VuNCED1 gene
showed a threefold and fourfold increase in biomass pro-
duction compared with wild type and increase in tolerance
to drought and salt concentration (Aswath et al. 2005).
Amongst the various abiotic stresses, salinity stress is
equally damaging compared to drought and submergence
and ultimately reduces production considerably. Trans-
genic grasses were also developed involving Na?/H?
antiporter gene. Over expression of the Na?/H? antiporter
gene (OsNHX1) in transgenic perennial ryegrass has been
demonstrated which noticeably improved salt tolerance
(Wu et al. 2005). Transgenic grasses possibly might be
used for cultivation of target grass species in seashore or
brackish water affected land and protect soil from wearing
away.
Transcription factors play an important role in molecular
biology studies for drought and freezing stress tolerance.
Transgenic grasses conferring tolerance to freezing and
low temperature was developed (Table 3). Recently,
transgenic L. perenne plants that constitutively expressed
the Arabidopsis DREB1A/CBF3 gene has been developed
123
Plant Cell Tiss Organ Cult
and found to enhance freezing tolerance (Li et al. 2011). A
recent report demonstrates that pretreatment with hydrogen
peroxide (H2O2) at appropriate concentration may improve
the tolerance of warm-season Zoysia grasses to chilling
stress through regulation of anti-oxidative systems,
including enhancement of anti-oxidative enzymes regula-
tion and alteration of levels of antioxidants (Wang et al.
2010). Heat stresses cause a decline in forage and turf grass
quality that has been associated with reductions in root
growth, leaf water potential, cell membrane stability,
photosynthetic rate, photochemical efficiency, and carbo-
hydrate accumulation (Jiang and Haung 2000). Transgenic
grasses conferring tolerance to high temperature was
developed (Table 3). Recently, transgenic plants were
generated by overexpression of an Arabidopsis 2-Cys Prx
in transgenic tall fescue (Festuca arundinacea) plants by
means of Agrobacterium-mediated genetic transformation
to confer tolerance against heat and MV stress (Kim et al.
2010). Fructan metabolism in grasses is considered to be
important in assimilate partitioning and in the plant’s
response to environmental stresses such as drought and
cold. Transgenic ryegrass plants with manipulated fructan
metabolism through the transgenic expression of a bacterial
levansucrase gene (chimeric sac B) were generated (Ye
et al. 2001). The isolation of fructan biosynthetic genes and
their expression in grasses would enable us to study the
regulation and role of fructan metabolism, the physiologi-
cal consequences of its manipulation, and clarify their
functional role in abiotic stress.
Commonly, fungal diseases are serious problems for all
crops, including lawn and recreational grasses. Transgenic
creeping bentgrass plants expressed ribosome- inactivating
proteins from pokeweed. They are termed pokeweed anti-
viral proteins (PAP) and they are the reduced toxicity
mutants, PAP-C and PAP-Y. These mutants are native
form of PAPII. Under greenhouse conditions, PAP-Y
transformed plants appeared to have normal phenotype
(Dai et al. 2003). The other rice Pi9 gene was transferred
into two elite cultivars, ‘Coronado’ and ‘Matador’, of tall
fescue through Agrobacterium-mediated transformation
and conferred resistance to gray leaf spot and brown patch
disease (Dong et al. 2007). Insect pest and weeds are two
major problems for grasses. Damage caused by the insects
such as grasshoppers plague has become major destruction
to the maintenance of pasture. The introduction of insec-
ticidal gene ppip into Elymus breviaristatus by Agrobac-
terium-mediated method and the effectiveness of the ppip
gene in transgenic plants were evaluated (Li et al. 2007). A
recent report demonstrated that Scarabaeid larvae and
herbicide resistant transgenic perennial ryegrass (Lolium
perenne L.) was obtained by transforming it with cry
(cry8Ca2 or cry8Ga) and bar genes, respectively; via the
Agrobacterium-mediated method (Wu et al. 2012).
Plant Cell Tiss Organ Cult
Improvement of grasses for quality traits
Economically important cool-season turf grasses with fine
texture, dense growth are suitable for extensive use on
putting greens and fairways of golf courses in temperate
climates (Warnke 2003). In order to stimulate interest in
such grasses as an ornamental species, researchers focused
on the flavonoid/anthocyanin pathway of plants (Tanaka
et al. 2008). Transgenic purple colored creeping bentgrass
plants that carried the flavonoid/anthocyanin pathway
regulatory genes, Lc and Pl were developed by means of
Agrobacterium-mediated transformation (Han et al. 2009).
Grasses conferring delayed senescence was developed with
a putative senescence-enhanced promoter and a regulator
of senescence e.g. maize SEE1 50 -flanking sequence
(PSEE1) by genetic modification of ryegrass (Li et al.
2004b). Leaves displayed a stay-green phenotype thus,
confirming PSEE1 functions as a senescence-enhanced
promoter with the potential to auto regulate senescence.
The sulphur-containing (S-) amino acids, methionine
and cysteine, are among the most limiting essential amino
acids in ruminant animal nutrition and wool growth in
sheep. Transgenic tall fescue (F. arundinacea Schreb.)
plants have been generated that express SFA8 gene
encoding rumen-stable protein rich in sulphur-containing
amino acids and accumulated up to 0.2 % of the total
soluble protein in individual transgenic plants (Wang et al.
2001b). Grasses are also exploited for the production of
biopharmaceuticals and food supplements for commercial
use through biotechnological interventions. Coenzyme Q
(CoQ or ubiquinone) has strong antioxidative activity and
plays an important role in energy production. The length of
the side chain of CoQ is determined by polyprenyl
diphosphate synthase, an enzyme that synthesizes a given
length of isoprenoid chain (Kawamukai 2009). Recently,
ddsA gene has been transferred to Panicum meyerianum, an
important warm-season forage grass and biomass crop via
an efficient Agrobacterium-mediated transformation
method and successfully generated a CoQ10-producing
warm-season grass (Seo et al. 2011).
Lignification of forage tissues limits the amount of
digestible energy available to livestock, resulting in an
incomplete utilization of cellulose and hemicelluloses by
ruminant animals (Casler et al. 2002). cDNA sequences
encoding, cinnamyl alcohol dehydrogenase (CAD) were
cloned from the widely grown monocotyledonous forage
species tall fescue (F. arundinacea Schreb.). Transgenic
tall fescue plants were developed carrying either sense or
antisense CAD gene constructs. The CAD down-regu-
lated transgenic lines had significantly decreased lignin
content (Chen et al. 2003). World energy demand is
expected to increase more than 50 % by 2025 (Ersahin
et al. 2011). Plant-based biofuels are being considered as
a viable alternative to fossil fuels while reducing
greenhouse gas emissions through the fixation of atmo-
spheric carbon into useful plant biomass (Ceotto 2009).
Currently, corn, rapeseed, soybean, and sorghum are the
primary bioenergy feedstocks in the USA (Schmer et al.
2008). Several candidate species of Miscanthus consid-
ered for sustainable production of biomass for generating
renewable energy (Głowacka et al. 2010). Miscan-
thus 9 giganteus found to be an especially valuable
resource and is the only species being commercially
cultivated for biomass production (Chung and Kim
2012). Agrobacterium-mediated transformation system
for M. sinensis successfully produced herbicide-resistant
transgenics by introducing bar gene in Miscanthus plants
a source for biofuel and generates optimism for genetic
manipulation of this important grass species (Hwang
et al. 2014).
Conclusion
Amongst grasses, forage and turf grasses are major target
for grassland agriculture. Grasses have tremendous eco-
logical and economic value, as well as unique biological
significance of scientific interest. There has been tremen-
dous advancement on biotechnological interventions in
grasses. Grasses are among the most recalcitrant crop
species for genetic manipulation in vitro. Establishment of
an efficient tissue culture system to regenerate plants
in vitro is prime requisite for genetic manipulation at the
cellular level. Strategies to regenerate plants from tissue
culture of cereals and grasses have evolved steadily during
the past decade. Successes achieved on in vitro plant
regeneration, commonly obtained from young inflores-
cence, immature embryos; shoot-buds have opened up new
opportunities for the application of in vitro techniques to
improve grasses. In the last decade, several protocols
developed for in vitro plant regeneration, and genetic
transformation of grasses. The demand for genetically
improved grass cultivars with agronomic and quality traits
is increasing owing to its immeasurable value for livestock
wildlife feed and environmental protection and recreation.
In the midst of biotechnological interventions, somatic
hybridization can be an option towards adding extra
genetic variability to grasses. There have been reports on
somatic hybridization within grasses, between grass and
cereals. An assessment in the success trend of studies on
somatic hybridization is positive, however; mostly con-
fronted with unfinished success. However, somatic
hybridization approach remains a vital strategy in hand to
manipulate grasses at genomic and cytoplasm (plastom-
e ? chondriome) level for overall genetic improvement of
grasses.
123

The current targets for biotechnological intervention of
grasses include abiotic/biotic stress tolerance, expression of
foreign genes of industrial/economic relevance, and
improvement of forage pasture etc. The most common
problems experienced by grasses are abiotic stresses.
Abiotic stresses such as drought, salinity, cold, and freez-
ing affect the growth of both forage and turf grasses.
Transgenic expression of genes, regulatory genes/tran-
scription factors involved in stress responses has great
potential for improving environmental stress tolerance in
grasses. An improvement of disease resistance in grasses
by genetic engineering should be relatively straightfor-
ward. Many strategies for engineering disease resistance in
transgenic plants in general and grasses in particular were
developed over the last decade.
In the past decade, a number of findings on transfor-
mation techniques and its applications in grasses have been
reported. It is found that Agrobacterium mediated trans-
formation is used in most cases followed by particle
bombardment technique. Recent developments in grasses
can have multidirectional benefits both in fundamental
understanding of transformation requirements and ultimate
applied value. An armory of disease resistant genes such as
chitinases, glucanases, plant defensins, phytoalexins, ribo-
some inactivating proteins, viral coat proteins, etc. are
involved in genetic modification of plants in general and
can be well extrapolated to grasses. In addition, certain
forage species are currently engineered to make biophar-
maceuticals and bioindustrial products. Further, the bene-
fits range from modification of senescence, accumulation
of pigments in grass for decoration of lawns. Further,
manipulation of fructans and improvement in sulphur
containing amino acids in grasses can be beneficial for
abiotic stress tolerance and nutritional quality of animal
protein. These successes demonstrate the potential of these
techniques in the improvement of forage and turf grass
species.
Keeping in view the inherent difficulties in developing
efficient protocols for plant regeneration, development of
model system for forage and turf grasses, genetic trans-
formation and somatic hybridization, recent progress might
be adequate to functionally evaluate and validate the can-
didate genes of interest. With recent research developments
in molecular and biotechnological approaches, the genetic
improvement of grasses can revive the missing ground and
the future may see grass species are tailored for special
agronomic and quality characteristics of significance for
human welfare.
Acknowledgments Authors would like to thank Department of
Science and Technology (DST), New Delhi for financial support. Ms.
M. Praveena thanks DST, New Delhi and DBT-OU-ISLARE for the
award of Research Fellowships.
123
Plant Cell Tiss Organ Cult
References
Aguado-Santacruz GA, Cabrera-Ponce JL, Olalde-Portugal V, San-
chez-Gonzalez MAR, Marouez-Guzman J, Herrera-Estrella L
(2001) Tissue culture and plant regeneration of blue grama grass,
Bouteloua gracilis (H.B.K.) Lag. Ex Steud. In Vitro Cell Dev
Biol Plant 37:182–189
Aguado-Santacruz GA, Rascón-Cruz Q, Moreno-Gómez B, Guevara-
González RG, Guevara-Olvera L, Jiménez-Bremont JF, Arévalo-
Gallegos S, Garcı́a-Moya E (2009) Genetic transformation of
blue grama grass with the rolA gene from Agrobacterium
rhizogenes: regeneration of transgenic plants involves a ‘‘hairy
embryo’’ stage. In Vitro Cell Dev Biol Plant 45:681–692
Aswath CR, Kim SH, Mo SY, Kim DH (2005) Transgenic plants of
creeping bent grass harboring the stress inducible gene, 9-cis-
epoxycarotenoid dioxygenase, are highly tolerant to drought and
NaCl stress. Plant Growth Regul 47:129–139
Bajaj S, Ran Y, Phillips J, Kularajathevan G, Pal S, Cohen D,
Elborough K, Puthigae S (2006) A high throughput Agrobacte-
rium tumefaciens-mediated transformation method for functional
genomics of perennial ryegrass (Lolium perenne L.). Plant Cell
Rep 25:651–659
Barampuram S, Chung BY, Lee SS, An BC, Lee EM, Cho JY (2009)
Development of an embryogenic callus induction method for
centipede grass (Eremochloa ophiuroides Munro) and subse-
quent plant regeneration. In Vitro Cell Dev Biol Plant
45:155–161
Bhattacharjee B, Sane AP, Gupta HS (1999) Transfer of wild abortive
cytoplasmic male sterility through protoplast fusion in rice. Mol
Breed 5:319–327
Buanafina MM, Langdon T, Hauck B, Dalton S, Morris P (2008)
Expression of a fungal ferulic acid esterase increases cell wall
digestibility of tall fescue (Festuca arundinace). Plant Biotech-
nol J 6:264–280
Cabral GB, Carneiro VTC, Lacerda AL, do Valle CB, Martinelli AP,
Dusi DMA (2011) Somatic embryogenesis and organogenesis in
apomictic and sexual Brachiaria brizantha. Plant Cell Tissue
Organ Cult 107:271–282
Cai Y, Xiang F, Zhi D, Liu H, Xia G (2007) Genotyping of somatic
hybrids between Festuca arundinacea Schreb. and Triticum
aestivum L. Plant Cell Rep 26:1809–1819
Carloni E, Ribotta A, Colomba EL, Griffa S, Quiroga M, Tommasino
E, Grunberg K (2014) Somatic embryogenesis from in vitro
anther culture of apomictic buffel grass genotypes and analysis
of regenerated plants using flow cytometry. Plant Cell Tissue
Organ Cult 117:311–322
Casler MD, Buxton DR, Vogel KP (2002) Genetic modification of
lignin concentration affects fitness of perennial herbaceous
plants. Theor Appl Genet 104:127–131
Ceotto E (2009) Grasslands for bioenergy production: a review. In:
Lichtfouse E, Navarrete M, Debaeke P, Veronique S, Alberola C
(eds) Sustainable agriculture. Springer, New York, pp 141–151
Chai ML, Senthil KK, Kim DH (2004) Transgenic plants of colonial
bentgrass from embryogenic callus via Agrobacterium-mediated
transformation. Plant Cell Tissue Organ Cult 77:165–171
Chai M, Jia Y, Chen S, Gao Z, Wang H, Liu L, Wang P, Hou D
(2011) Callus induction, plant regeneration, and long-term
maintenance of embryogenic cultures in Zoysia matrella [L.]
Merr. Plant Cell Tissue Org Cult 104:187–192
Chakbavarty T, Norcini JG, Aldrich JH, Kalmbacher RS (2001) Plant
regeneration of creeping bluestem (Schizachyrium scoparium
(Michx.) Nash var. Stoloniferum (Nash) J. Wipff) via somatic
embryogenesis. In Vitro Cell Dev Biol Plant 37:550–554
Chen L, Auh CK, Dowling P, Bell J, Chen F, Hopkins A, Dixon RA,
Wang ZY (2003) Improved forage digestibility of tall fescue
Plant Cell Tiss Organ Cult
(Festuca arundinacea) by transgenic down-regulation of cin-
namyl alcohol dehydrogenase. Plant Biotechnol J 1:437–449
Chen SY, Xia GM, Quan TY, Xiang FN, Jin Y, Chen HM (2004a)
Introgression of salt-tolerance from somatic hybrids between
common wheat and Thinopyrum ponticum. Plant Sci
167:773–779
Chen XL, Xia GM, Chen HM (2004b) Nuclear and cytoplasmic
genome analysis of somatic hybrid of Triticum aestivum L. and
Leymus chinensis (Trin.) Tzvel]. Sci China C Life Sci
30:379–386
Chen X, Yang WQ, Sivamani E, Bruneau AH, Wang BH, Qu RD
(2005) Selective elimination of perennial ryegrass by activation
of a pro-herbicide through engineering E. coli argE gene. Mol
Breed 15:339–347
Chen S, Chai M, Jia Y, Gao Z, Zhang L, Gu M, Lin W, Wang L
(2012) In vitro selection of glyphosate-tolerant variants from
long-term callus cultures of Zoysia matrella [L.] Merr. Plant Cell
Tissue Organ Cult 111:199–207
Cheng A, Xia G (2004) Somatic hybridization between common
wheat and Italian ryegrass. Plant Sci 166:1219–1226
Cheng AX, Xia GM, Zhi DY, Chen HM (2004) Intermediate fertile
Triticum aestivum (?) Agropyron elongatum somatic hybrids are
generated by low doses of UV irradiation. Cell Res 14:86–91
Cheng A, Cui H, Xia G (2006) Construction of a primary RH panel of
Italian ryegrass genome via UV-induced protoplast fusion. Plant
Biol 8:673–679
Cho MJ, Choi HW, Lemaux PG (2001) Transformed T0 orchardgrass
(Dactylis glomerata L.) plants produced from highly regenera-
tive tissues derived from mature seeds. Plant Cell Rep
20:318–324
Christiansen P, Andersen CH, Didion T, Folling M, Nielsen KK
(2005) A rapid and efficient transformation protocol for the grass
Brachypodium distachyon. Plant Cell Rep 23:751–758
Chu CC (1978) The N6 medium and its applications to anther culture
of cereal crops. In: Proceedings of symposium on plant tissue
culture, Science Press, Beijing, pp 43–50
Chung JH, Kim DS (2012) Miscanthus as a potential bioenergy crop
in East Asia. J Crop Sci Biotechnol 15:65–77
Cui H, Yu Z, Deng J, Gao X, Sun Y, Xia G (2009) Introgression of
bread wheat chromatin into tall wheatgrass via somatic hybrid-
ization. Planta 229:323–330
Dai WD, Bonos S, Guo Z, Meyer WA, Day PR, Belanger FC (2003)
Expression of pokeweed antiviral proteins in creeping bentgrass.
Plant Cell Rep 21:497–502
Dalton SJ, Bettany AJE, Bhat V, Gupta MG, Bailey K, Timms E,
Morris P (2003) Genetic transformation of Dichanthium annul-
atum (Forssk)—an apomictic tropical forage grass. Plant Cell
Rep 21:974–980
Dhandapani M, Hong SB, Aswath CR, Kim DH (2008) Regeneration
of zoysia grass (Zoysia matrella L. Merr.) cv. Konhee from
young inflorescences and stem nodes. In Vitro Cell Dev Biol
Plant 44:8–13
Dong S, Tredway LP, Shew HD, Wang GL, Sivamani E, Qu R (2007)
Resistance of transgenic tall fescue to two major fungal diseases.
Plant Sci 173:501–509
Ersahin ME, Gomec CY, Dereli RK, Arikan O, Ozturk I (2011)
Biomethane production as an alternative bioenergy source from
codigesters treating municipal sludge and organic fraction of
municipal solid wastes. J Biomed Biotechnol 2011:1–8
Fu D, Tisseratb NA, Xiao Y, Settle D, Muthukrishnan S, Liang GH
(2005) Overexpression of rice TLPD34 enhances dollar-spot
resistance in transgenic bentgrass. Plant Sci 168:671–680
Fu D, Huang B, Xiao Y, Muthukrishnan S, Liang GH (2007)
Overexpression of barley hva1 gene in creeping bentgrass for
improving drought tolerance. Plant Cell Rep 26:467–477
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of
suspension culture of soybean root cells. Exp Cell Res
50:151–158
Gao C, Jiang L, Folling M, Han L, Nielsen KK (2006) Generation of large
numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants
following biolistic gene transfer. Plant Cell Rep 25:19–25
Gao C, Long D, Lenk I, Nielsen KK (2008) Comparative analysis of
transgenic tall fescue (Festuca arundinacea Schreb.) plants
obtained by Agrobacterium-mediated transformation and particle
bombardment. Plant Cell Rep 27:1601–1609
Gao C, Liu J, Nielsen KK (2009) Agrobacterium-mediated transfor-
mation of meadow fescue (Festuca pratensis Huds.). Plant Cell
Rep 28:1431–1437
Ge TM, Lin XH, Qin FL, Yu SW, Yu YJ (2006a) Protoplast
electrofusion between common wheat (Triticum aestivum L.)
and Italian ryegrass (Lolium multiflorum Lam.) and regeneration
of mature cybrids. In Vitro Cell Dev Biol Plant 42:179–187
Ge Y, Norton T, Wang ZY (2006b) Transgenic zoysiagrass (Zoysia
japonica) plants obtained by Agrobacterium-mediated transfor-
mation. Plant Cell Rep 25:792–798
Ge Y, Cheng X, Hopkins A, Wang ZY (2007) Generation of
transgenic Lolium temulentum plants by Agrobacterium tum-
efaciens-mediated transformation. Plant Cell Rep 26:783–789
Giri CC, Vijaya Laxmi G (2000) Production of transgenic rice with
agronomically useful genes: an assessment. Biotechnol Adv
18:653–683
Głowacka K, Jezowski S, Kaczmarek Z (2010) The effects of
genotype, inflorescence developmental stage and induction
medium on callus induction and plant regeneration in two
Miscanthus species. Plant Cell Tissue Organ Cult 102:79–86
Gondo T, Tsuruta SI, Akashi R, Kawamura O, Hoffmann F (2005)
Green, herbicide-resistant plants by particle inflow gun-mediated
gene transfer to diploid bahiagrass (Paspalum notatum). J Plant
Physiol 162:1367–1375
Gondo T, Matsumoto J, Yamakawa K, Tsuruta SI, Ebina M, Akashi R
(2007) Somatic embryogenesis and multiple-shoot formation
from seed-derived shoot apical meristems of rhodesgrass (Chl-
oris gayana Kunth). Grassl Sci 53:138–142
Grando MF, Franklin CI, Shatters RG Jr (2002) Optimizing embryo-
genic callus production and plant regeneration from ‘Tifton 9’
bahiagrass seed explants for genetic manipulation. Plant Cell
Tissue Organ Cult 71:213–222
Gugsa L, Sarial AK, Lorz H, Kumlehn J (2006) Gynogenic plant
regeneration from unpollinated flower explants of Eragrostis tef
(Zuccagni) Trotter. Plant Cell Rep 25:1287–1293
Guo Y-D, Hisano H, Shimamoto Y, Yamada T (2009) Transformation
of androgenic-derived Festulolium plants (Lolium perenne
L. 9 Festuca pratensis Huds.) by Agrobacterium tumefaciens.
Plant Cell Tissue Organ Cult 96:219–227
Hammami R, Cuadrado A, Friero E, Jouve N, Soler C, González JM
(2011) Callus induction and plant regeneration from immature
embryos of Brachypodium distachyon with different chromo-
some numbers. Biol Plant 55:797–800
Han YJ, Kim YM, Lee JY, Kim SJ, Cho KC, Chandrasekhar T, Song
PS, Woo YM, Kim JI (2009) Production of purple-colored
creeping bentgrass using maize transcription factor genes Pl and
Lc through Agrobacterium-mediated transformation. Plant Cell
Rep 28:397–406
Himuro Y, Gondo T, Yamakawa K, Akashi R (2006) Genetic
transformation of bahiagrass (Paspalum notatum Flugge) by
visually screening cells expressing green fluorescent protein.
Grassl Sci 55:216–220
Hisano H, Kanazawa A, Kawakami A, Yoshida M, Shimamoto Y,
Yamada T (2004) Transgenic perennial ryegrass plants express-
ing wheat fructosyltransferase genes accumulate increased
123

amounts of fructan and acquire increased tolerance on a cellular
level to freezing. Plant Sci 167:861–868
Holley WD, Baker R (1963) Carnation Production. W.C. Brown,
Dubuque, p 142
Hu F, Zhang L, Wang X, Ding J, Wu D (2005a) Agrobacterium-
mediated transformed transgenic triploid bermudagrass (Cyn-
odon dactylon 9 C. transvaalensis) plants are highly resistant to
the glufosinate herbicide Liberty. Plant Cell Tissue Organ Cult
83:13–19
Hu Y, Jia W, Wang J, Zhang Y, Yang L, Lin Z (2005b) Transgenic
tall fescue containing the Agrobacterium tumefaciens ipt gene
shows enhanced cold tolerance. Plant Cell Rep 23:705–709
Hwang OJ, Cho MA, Han YJ, Kim YM, Lim SH, Kim DS, Hwang I,
Kim JI (2014) Agrobacterium-mediated genetic transformation
of Miscanthus sinensis. Plant Cell Tissue Organ Cult 117:51–63
Jayaraj J, Liang GH, Muthukrishnan S, Punja ZK (2008) Generation
of low copy number and stably expressing transgenic creeping
bentgrass plants using minimal gene cassette bombardment. Biol
Plant 52:215–221
Jelodar NB, Blackhall NW, Hartman TPV, Brar DS, Khush G, Davey
MR, Cocking EC, Powar JB (1999) Intergeneric somatic hybrids
of rice [Oryza sativa L. (?) Porteresia coarctata (Rozb.)
tateoka]. Theor Appl Genet 99:570–577
Jiang Y, Haung B (2000) Effects of drought or heat stress alone and in
combination on Kentucky bluegrass. Crop Sci 40:1358–1362
Kawamukai M (2009) Biosynthesis and bioproduction of coenzyme
Q10 by yeasts and other organisms. Biotechnol Appl Biochem
53:217–226
Kim KH, Alam I, Lee KW, Sharmin SA, Kwak SS, Lee SY, Lee BH
(2010) Enhanced tolerance of transgenic tall fescue plants
overexpressing 2-Cys peroxiredoxin against methyl viologen and
heat stresses. Biotechnol Lett 32:571–576
Kim S, Da K, Mei C (2012) An efficient system for high-quality
large-scale micropropagation of Miscanthus 9 giganteus plants.
In Vitro Cell Dev Biol Plant 48:613–619
King ZR, Bray AL, LaFayette PR, Parrott WA (2014) Biolistic
transformation of elite genotypes of switchgrass (Panicum
virgatum L.). Plant Cell Rep 33:313–322
Kisaka H, Kisaka M, Kanno A, Kameya T (1997) Production and
analysis of plants that are somatic hybrids of barley (Hordeum
vulgare L.) and carrot (Daucus carota L.). Theor Appl Genet
94:221–226
Kumar S, Bhat V (2012) High-frequency direct plant regeneration via
multiple shoot induction in the apomictic forage grass Cenchrus
ciliaris L. In Vitro Cell Dev Biol Plant 48:241–248
Lauzer D, Dallaire S, Vincent G (2000) In vitro propagation of reed
grass by somatic embryogenesis. Plant Cell Tissue Organ Cult
60:229–234
Lee SH, Ahsana N, Lee KW, Kim DH, Lee DG, Kwak SS, Kwon SY,
Kim TH, Lee BH (2007) Simultaneous overexpression of both
CuZn superoxide dismutase and ascorbate peroxidase in trans-
genic tall fescue plants confers increased tolerance to a wide
range of abiotic stresses. J Plant Physiol 164:1626–1638
Lee KW, Choi GJ, Kim KY, Ji HC, Park HS, Yoon SH, Lee SH
(2009) High frequency plant regeneration from mature seed
derived callus of Italian ryegrass (Lolium multiflorum) cultivars.
Afr J Biotechnol 8:6828–6833
Lee KW, Choi GJ, Kim KY, Yoon SH, Ji HC, Park HS, Lim YC, Lee
SH (2010) Genotypic variation of Agrobacterium-mediated
transformation of Italian ryegrass. Electron J Biotechnol 13:1–10
Li L, Qu R (2004) Development of highly regenerable callus lines and
biolistic transformation of turf-type common bermudagrass
[Cynodon dactylon (L.) Pers.]. Plant Cell Rep 22:403–407
Li CL, Xia GM (2004) Asymmetric somatic hybridization between
mixed wheat and Psathyrostachys juncea. Sci China C Life Sci
20:610–614
123
Plant Cell Tiss Organ Cult
Li C, Xia G, Xiang F, Zhou C, Cheng A (2004a) Regeneration of
asymmetric somatic hybrid plants from the fusion of two types
of wheat with Russian wildrye. Plant Cell Rep 23:461–467
Li Q, Robson PRH, Bettany AJE, Donnison IS, Thomas H, Scott IM
(2004b) Modification of senescence in ryegrass transformed with
IPT under the control of a monocot senescence-enhanced
promoter. Plant Cell Rep 22:816–821
Li L, Li R, Fei S, Qu R (2005) Agrobacterium-mediated transforma-
tion of common bermudagrass (Cynodon dactylon). Plant Cell
Tissue Organ Cult 83:223–229
Li RF, Wei JH, Wang HZ, He J, Sun ZY (2006) Development of
highly regenerable callus lines and Agrobacterium-mediated
transformation of Chinese lawngrass (Zoysia sinica Hance) with
a cold inducible transcription factor, CBF1. Plant Cell Tissue
Organ Cult 85:297–305
Li D, Sun Q, Huang M, Zhang J, Bai S, Zheng L, Zhao J, Qiu D, Li L,
Yang Z, You M, Liu G, Zhang Y, Zhang C, Li S (2007)
Agrobacterium–mediated genetic transformation of Elymus
breviaristatus with Pseudomonas pseudoalcaligenes insecticidal
protein gene. Plant Cell Tissue Organ Cult 89:159–168
Li M, Li H, Hu X, Pan X, Wu G (2010a) An Agrobacterium
tumefaciens mediated transformation system using callus of
Zoysia tenuifolia Willd. ex Trin. Plant Cell Tissue Organ Cult
102:321–327
Li Z, Baldwin CM, Hu Q, Liu H, Luo H (2010b) Heterologous
expression of Arabidopsis H?-pyrophosphatase enhances salt
tolerance in transgenic creeping bentgrass (Agrostis stolonifera
L.). Plant Cell Environ 33:272–289
Li X, Cheng X, Liu J, Zeng H, Han L, Tang W (2011) Heterologous
expression of the Arabidopsis DREB1A/CBF3 gene enhances
drought and freezing tolerance in transgenic Lolium perenne
plants. Plant Biotechnol Rep 5:61–69
Linsmaier EM, Skoog F (1965) Organic growth factor requirement
for tobacco tissue cultures. Physiol Plant 18:100–127
Liu B, Liu ZL, Li XW (1999) Production of a highly asymmetric
somatic hybrid between rice and Zizania latifolia (Griseb):
evidence for inter-genomic exchange. Theor Appl Genet
98:1099–1103
Liu GS, Liu JS, Qi DM, Chu CC, Li HJ (2004) Factors affecting plant
regeneration from tissue cultures of Chinese leymus (Leymus
chinensis). Plant Cell Tissue Organ Cult 76:175–178
Liu J, Xu X, Deng X (2005) Intergeneric somatic hybridization and its
application to crop genetic improvement. Plant Cell Tissue
Organ Cult 82:19–44
Liu P, Zhang ZX, Yuan JG, Xi JB, Du XL, Yang ZY (2006) Callus
Induction and plant regeneration in eleven perennial ryegrass
cultivars. Biotechnol Biotechnol Equip 20:30–37
Liu M, Yang J, Lu S, Guo Z, Lin X, Wu H (2008) Somatic
embryogenesis and plant regeneration in centipedegrass (Erem-
ochloa ophiuroides [Munro] Hack). In Vitro Cell Dev Biol Plant
44:100–104
Liu L, Fan X, Zhang J, Yan M, Bao M (2009) Long-term cultured
callus and the effect factor of high-frequency plantlet regener-
ation and somatic embryogenesis maintenance in Zoysia japon-
ica. In Vitro Cell Dev Biol Plant 45:673–680
Liu M, Lu S, Liu L, Tan J, Guo Z (2012) Agrobacterium-mediated
transformation of centipedegrass (Eremochloa ophiuroides
[Munro] Hack.). Plant Cell Tissue Organ Cult 109:557–563
Long D, Wu X, Yang Z, Lenk I, Nielsen KK, Gao C (2011)
Comparison of three selectable marker genes for transformation
of tall fescue (Festuca arundinacea Schreb.) plants by particle
bombardment. In Vitro Cell Dev Biol Plant 47:658–666
Lu S, Wang Z, Peng X, Guo Z, Zhang G, Han L (2006) An efficient
callus suspension culture system for triploid bermudagrass
(Cynodon transvaalensis 9 C. dactylon) and somaclonal varia-
tions. Plant Cell Tissue Organ Cult 87:77–84
Plant Cell Tiss Organ Cult
Luo H, Hu Q, Nelson K, Longo C, Kausch AP, Chandlee JM, Wipff
JK, Fricker CR (2004) Agrobacterium tumefaciens-mediated
creeping bentgrass (Agrostis stolonifera L.) transformation using
phosphinothricin selection results in a high frequency of single-
copy transgene integration. Plant Cell Rep 22:645–652
Ma K, Hu CG, Xu B, Yao JL (2013) Regeneration and Agrobacte-
rium-mediated transformation of the apomictic species Eulali-
opsis binata. Appl Biochem Biotechnol 171:543–552
Molinari L, Busti A, Calderini O, Arcioni S, Pupilli F (2003) Plant
regeneration from callus of apomictic and sexual lines of
Paspalum simplex and RFLP analysis of regenerated plants.
Plant Cell Rep 21:1040–1046
Mostageer A, Elshihy OM (2003) Establishment of a salt tolerant
somatic hybrid through protoplast fusion between rice and ditch
reed. Arab J Biotech 6:1–12
Müller AJ, Grafe R (1978) Isolation and characterization of cell lines
of Nicotiana tabacum lacking nitrate reductase. Mol Gen Genet
161:67–76
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bio assays for tobacco tissue cultures. Physiol Plant 15:473–497
Nakamura T, Ishikawa M (2006) Transformation of suspension
cultures of bromegrass (Bromus inermis) by Agrobacterium
tumefaciens. Plant Cell Tissue Organ Cult 84:293–299
Ncanana S, Brandt W, Lindsey G, Farrant J (2005) Development of
plant regeneration and transformation protocols for the desicca-
tion-sensitive weeping lovegrass Eragrostis curvula. Plant Cell
Rep 24:335–340
Neibaur I, Gallo M, Altpeter F (2008) The effect of auxin type and
cytokinin concentration on callus induction and plant regener-
ation frequency from immature inflorescence segments of
seashore paspalum (Paspalum vaginatum Swartz). In Vitro Cell
Dev Biol Plant 44:480–486
Osuna P, Barrow JR (2004) Regeneration of black grama (Bouteloua
eriopoda torr. torr) plants via somatic embryogenesis. In Vitro
Cell Dev Biol Plant 40:299–302
Patel M, Dewey RE, Qu R (2013) Enhancing Agrobacterium
tumefaciens-mediated transformation efficiency of perennial
ryegrass and rice using heat and high maltose treatments during
bacterial infection. Plant Cell Tissue Organ Cult 114:19–29
Petrovska N, Wu X, Donato R, Wang Z, Ong EK, Jones E, Forster J,
Emmerling M, Sidoli A, O’Hehir R, Spangenberg G (2004)
Transgenic ryegrasses (Lolium spp.) with down-regulation of
main pollen allergens. Mol Breed 14:489–501
Praveena M, Giri CC (2012) Plant regeneration from immature
inflorescence derived callus cultures of salt tolerant kallar grass
(Leptochloa fusca L.). Physiol Mol Biol Plants 18:345–356
Ramamoorthy R, Kumar PP (2012) A simplified protocol for genetic
transformation of switchgrass (Panicum virgatum L.). Plant Cell
Rep 31:1923–1931
Ran Y, Patron N, Yu Q, Georges S, Mason J, Spangenberg G (2014)
Agrobacterium-mediated transformation of Lolium rigidum
Gaud. Plant Cell Tissue Organ Cult 118:67–75
Richards HA, Rudas VA, Sun H, McDaniel JK, Tomaszewski Z,
Conger BV (2001) Construction of a GFP-BAR plasmid and its
use for switchgrass transformation. Plant Cell Rep 20:48–54
Sairam RV, Wilber C, Franklin J, Smith B, Bazil J, Hassel R,
Whaling D, Frutiger K, Blakey CA, Vierling R, Goldman SL
(2002) High frequency callus induction and plant regeneration in
Tripsacum dactyloides (L.). In Vitro Cell Dev Biol Plant
38:435–440
Salehi H, Khosh-Khui M (2005) Effects of genotype and plant growth
regulator on callus induction and plant regeneration in four
important turfgrass genera: a comparative study. In Vitro Cell
Dev Biol Plant 41:157–161
Salehi H, Seddighi Z, Kravchenko AN, Sticklen MB (2005)
Expression of cry1Ac in ‘Arizona Common’ Common
bermudagrass via Agrobacterium-mediated transformation and
control of black cutworm. J Am Soc Hortic Sci 130:619–623
Sandhu S, Alpeter F (2008) Co-integration, co-expression and
inheritance of unlinked minimal transgene expression cassettes
in an apomictic turf and forage grass (Paspalum notatum
Flugge). Plant Cell Rep 27:1755–1765
Sato H, Takamizo T (2006) Agrobacterium tumefaciens-mediated
transformation of forage-type perennial ryegrass (Lolium per-
enne L.). Grassl Sci 52:95–98
Sato H, Takamizo T, Shimizu T, Kawai K, Kaku K (2009) Conferred
resistance to an acetolactate synthase-inhibiting herbicide in
transgenic tall fescue (Festuca arundinacea Schreb.). Hort-
Science 44:1254–1257
Schenk RV, Hildebrandt AC (1972) Medium and techniques for
induction and growth monocotyledonous and dicotyledonous
plant cell cultures. Can J Bot 50:199–204
Schmer MR, Vogel KP, Mitchell RB, Perrin RK (2008) Net energy of
cellulosic ethanol from switchgrass. Proc Natl Acad Sci USA
105:464–469
Seo MS, Takahara M, Ebina M, Takamizo T (2008) Evaluation of
tissue culture response from mature seeds of Panicum spp.
Grassl Sci 54:125–130
Seo MS, Takahara M, Takamizo T (2010) Optimization of culture
conditions for plant regeneration of Panicum spp. through
somatic embryogenesis. Grassl Sci 56:6–12
Seo MS, Takahashi S, Kadowaki KI, Kawamukai M, Takahara M,
Takamizo T (2011) Expression of CoQ10-producing ddsA
transgene by efficient Agrobacterium-mediated transformation in
Panicum meyerianum. Plant Cell Tissue Organ Cult 107:325–332
Shu QY, Liu GS, Xu SX, Li XF, Li HJ (2005) Genetic transformation
of Leymus chinensis with the PAT gene through microprojectile
bombardment to improve resistance to the herbicide Basta. Plant
Cell Rep 24:36–44
Somleva MN, Tomaszewski Z, Conger BV (2002) Agrobacterium-
mediated genetic transformation of switchgrass. Crop Sci
42:2080–2087
Song G, Walworth A, Hancock JF (2012) Factors influencing
Agrobacterium-mediated transformation of switchgrass culti-
vars. Plant Cell Tissue Organ Cult 108:445–453
Sun YL, Hong SK (2010) Effects of plant growth regulators and L-
glutamic acid on shoot organogenesis in the halophyte Leymus
chinensis (Trin.). Plant Cell Tissue Organ Cult 100:317–328
Takahashi W, Fujimori M, Miura Y, Komatsu T, Nishizawa Y, Hibi
T, Takamizo T (2005) Increased resistance to crown rust disease
in transgenic Italian ryegrass (Lolium multiflorum Lam.)
expressing the rice chitinase gene. Plant Cell Rep 23:811–818
Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant
pigments: anthocyanins, betalains and carotenoids. Plant J
54:733–749
Toyama K, Bae CH, Kang JG, Lim YP, Adachi T, Riu KZ, Song PS,
Lee HY (2003) Production of herbicide-tolerant zoysiagrass by
Agrobacterium-mediated transformation. Mol Cells 16:19–27
Unno H, Yamamoto S (2005) Introduction of exogenous substance
into protoplasts of perennial ryegrass by electroporation. Grassl
Sci 51:165–168
Vogel J, Hill T (2008) High-efficiency Agrobacterium-mediated
transformation of Brachypodium distachyon inbred line Bd21-3.
Plant Cell Rep 27:471–478
Wang ZY, Ge Y (2005a) Rapid and efficient production of transgenic
bermudagrass and creeping bentgrass bypassing the callus
formation phase. Funct Plant Biol 32:769–777
Wang ZY, Ge Y (2005b) Agrobacterium-mediated high efficiency
transformation of tall fescue (Festuca arundinacea). J Plant
Physiol 162:103–113
Wang ZY, Ge Y (2006) Recent advances in genetic transformation of
forage and turf grasses. In Vitro Cell Dev Biol Plant 42:1–18
123

Wang Z, Hopkins A, Mian R (2001a) Forage and turf grass
biotechnology. Crit Rev Plant Sci 20:573–619
Wang ZY, Ye XD, Nagel J, Potrykus I, Spangenberg G (2001b)
Expression of a sulphur-rich sunflower albumin gene in trans-
genic tall fescue (Festuca arundinacea Schreb.) plants. Plant
Cell Rep 20:213–219
Wang Y, Ruemmele BA, Chandlee JM, Sullivan WM, Knapp JE,
Kausch AP (2002a) Embryogenic callus induction and plant
regeneration media for bentgrasses and annual bluegrass.
In Vitro Cell Dev Biol Plant 38:460–467
Wang Z, Lehmann D, Bell J, Hopkins A (2002b) Development of an
efficient plant regeneration system for Russian wildrye (Psathy-
rostachys juncea). Plant Cell Rep 20:797–801
Wang ZY, Bell J, Hopkins A (2003) Establishment of a plant
regeneration system for wheatgrasses (Thinopyrum, Agropyron
and Pascopyrum). Plant Cell Tissue Organ Cult 73:265–273
Wang ZY, Bell J, Lehmann D (2004) Transgenic Russian wildrye
(Psathyrostachys juncea) plants obtained by biolistic transforma-
tion of embryogenic suspension cells. Plant Cell Rep 22:903–909
Wang L, Li X, Chen S, Liu G (2009) Enhanced drought tolerance in
transgenic Leymus chinensis plants with constitutively expressed
wheat TaLEA3. Biotechnol Lett 31:313–319
Wang Y, Li J, Wang J, Li Z (2010) Exogenous H2O2 improves the
chilling tolerance of manila grass and mascarenegrass by activat-
ing the antioxidative system. Plant Growth Regul 61:195–204
Warnke S (2003) Creeping bentgrass (Agrostis stolonifera L.). In:
Casler MD, Duncan RR (eds) Turfgrass biology, genetics, and
breeding. Wiley, Hoboken, pp 175–185
Wei Y, Guangmin X, Daying Z, Huimin C (2001) Transfer of salt tolerance
from Aeleuropus littorulis sinensis to wheat (Triticum aestivum L.)
via asymmetric somatic hybridization. Plant Sci 161:259–266
Wu YY, Chen QJ, Chen M, Chen J, Wang XC (2005) Salt-tolerant
transgenic perennial ryegrass (Lolium perenne L.) obtained by
Agrobacterium tumefaciens-mediated transformation of the
vacuolar Na?/H? antiporter gene. Plant Sci 169:65–73
Wu GT, Chen JQ, Hu ZH, Lang CX, Chen XY, Wang FL, Jin W, Xia
YW (2006) Production of transgenic tall fescue plants with
enhanced stress tolerances by Agrobacterium tumefaciens-med-
iated transformation. Agric Sci China 5:330–338
Wu JX, Zhang ZG, Zhang Q, Lang ZH, Sun XH (2012) Scarabaeid
larvae- and herbicide-resistant transgenic perennial ryegrass
(Lolium perenne L.) obtained by Agrobacterium tumefaciens-
mediated transformation of cry8Ca2, cry8Ga and bar genes.
J Integr Agric 11:53–61
Xi Y, Fu C, Ge Y, Nandakumar R, Hisano H, Bouton J, Wang ZY
(2009) Agrobacterium-mediated transformation of switchgrass
and inheritance of the transgenes. Bioenergy Res 2:275–283
Xia GM, Xiang FN, Zhou AF, Wang H, He SX, Chen HM (1999)
Fertile hybrid plant regeneration from somatic hybridization
between Triticum aestivum and Agropyron elongatum. Acta Bot
Sin 41:349–352
Xia G, Xiang F, Zhou A, Wang H, Chen H (2003) Asymmetric somatic
hybridization between wheat (Triticum aestivum L.) and Agropy-
ron elongatum (Host) Nevishi. Theor Appl Genet 107:299–305
Xiang FN, Xia GM, Zhou AF, Chen HM, Huang Y, Zhai XL (1999)
Asymmetric somatic hybridization between wheat (Triticum
aestivum) and Bromus inermis. Acta Bot Sin 41:458–462
Xiang F, Xia G, Chen H (2003a) Effect of UV dosage on somatic
hybridization between common wheat (Triticum aestivum L.)
and Avena sativa L. Plant Sci 164:697–707
Xiang F, Xia G, Chen H (2003b) Asymmetric somatic hybridization
between wheat (Triticum aestivum) and Avena sativa L. Sci
China C Life Sci 46:243–252
Xiang F, Wang J, Xu C, Xia G (2010) The chromosome content and
genotype of two wheat cell lines and of their somatic fusion
product with oat. Planta 231:1201–1210
123
Plant Cell Tiss Organ Cult
Xu J, Schubert J, Altpeter F (2001) Dissection of RNA-mediated
ryegrass mosaic virus resistance in fertile transgenic perennial
ryegrass (Lolium perenne L.). Plant J 26:265–274
Xu CH, Xia GM, Zhi DY, Xiang FN, Chen HM (2003) Integration of
maize nuclear and mitochondrial DNA into the wheat genome
through somatic hybridization. Plant Sci 165:1001–1008
Yadav CB, Jha P, Mahalakshmi C, Anjaiah V, Bhat V (2009) Somatic
embryogenesis and regeneration of Cenchrus ciliaris genotypes
from immature embryo explants. Biol Plant 53:603–609
Yan CQ, Quian KX, Yan QS, Zhang XQ, Xue GP, Huangfu WG, Wu
YF, Zhao YZ, Xue ZY, Huang J, Xu GZ, Wu P (2004) Use of
asymmetric somatic hybridization for transfer of the bacterial
blight resistance trait from Oryza meyeriana L. to O. sativa L.
ssp. Japonica. Plant Cell Rep 22:569–575
Yang YG, Guo YM, Guo Y, Guo ZC, Lin JX (2003) Regeneration and
large-scale propagation of Phragmites communis through somatic
embryogenesis. Plant Cell Tissue Organ Cult 75:287–290
Ye XD, Wu XL, Zhao H, Frehner M, Nösberger J, Potrykus I,
Spangenberg G (2001) Altered fructan accumulation in trans-
genic Lolium multiflorum plants expressing a Bacillus subtilis
sacB gene. Plant Cell Rep 20:205–212
Zhang WJ, Dong JL, Liang BG, Jin YS, Wang T (2006) Highly
efficient embryogenesis and plant regeneration of tall fescue
(Festuca arundinacea Schreb.) from mature seed-derived calli.
In Vitro Cell Dev Biol Plant 42:114–118
Zhang S, Hanna W, Ozias-Akins P (2007) Comparison of callus
induction and plant regeneration from different explants in
triploid and tetraploid turf-type bermudagrasses. Plant Cell
Tissue Organ Cult 90:71–78
Zhang K, Wang J, Hu X, Yang A, Zhang J (2010) Agrobacterium-
mediated transformation of shoot apices of Kentucky bluegrass
(Poa pratensis L.) and production of transgenic plants carrying a
betA gene. Plant Cell Tissue Organ Cult 102:135–143
Zhang QX, Sun Y, Hu HK, Chen B, Hong CT, Guo HP, Pan YH,
Zheng BS (2012) Micropropagation and plant regeneration from
embryogenic callus of Miscanthus sinensis. In Vitro Cell Dev
Biol Plant 48:50–57
Zhao J, Zhi D, Xue Z, Liu H, Xia G (2007) Enhanced salt tolerance of
transgenic progeny of tall fescue (Festuca arundinacea)
expressing a vacuolar Na?/H? antiporter gene from Arabi-
dopsis. J Plant Physiol 164:1377–1383
Zhao L, Hu H, Zhan H, Diao Y, Jin S, Zhou F, Hu Z (2013) Plant
regeneration from the embryogenic calli of five major Miscan-
thus species, the non-food biomass crops. In Vitro Cell Dev Biol
Plant 49:383–387
Zhi D, Xiang F, Chen X, Xia G, Chen H (2002) Production of plants
from somatic hybridization between common wheat and maize
(Zea mays L.). Sci China C Life Sci 37:80–83
Zhou A, Xia G (2005) Introgression of the Haynaldia villosa genome
into gamma-ray-induced asymmetric somatic hybrids of wheat.
Plant Cell Rep 24:289–296
Zhou A, Xia G, Chen H, Hu H (2001a) Comparative study of
symmetric and asymmetric somatic hybridization between
common wheat and Haynaldia villosa. Sci China C Life Sci
44:294–304
Zhou A, Xia G, Zhang X, Chen H, Hu H (2001b) Analysis of
chromosomal and organellar DNA of somatic hybrids between
Triticum aestivum and Haynaldia villosa Schur. Mol Genet
Genomics 265:387–393
Zhou M, Hu Q, Li Z, Li D, Chen CF, Luo H (2011) Expression of a
novel antimicrobial peptide penaeidin4-1 in creeping bentgrass
(Agrostis stolonifera L.) enhances plant fungal disease resis-
tance. PLoS One 6:1–12
Zong L, Ding LM, Xue X, Wang T (2010) Regeneration of green
plants from seed-derived callus cultures of Poa. Afr J Biotechnol
9:3091–3098