2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2177/11/11_chapter...
Post on 14-Jul-2020
6 Views
Preview:
TRANSCRIPT
2. REVIEW OF LITERATURE
2.1 Sorghum is an important crop plant of mankind
Sorghum is an important tropical cereal food, feed and fodder
crop. Botanically, sorghum belongs to the Genus Sorghum and Family
Gramineae. There are several types of sorghum including grain
sorghum, grass sorghum (for pasture and hay), sweet sorghum (for
syrup) and broom corn. Among known species the genus, Sorghum
bicolor (L) Moench is important. Other names of this species are
Sorghum vulgare pers and Andropogon sorghum (L) Brot. Common
names of sorghum in different countries are sorghum (United States,
Australia), Durra (Africa), Jowar (India) and Bachanta (Ethiopia).
It is an important grain and forage crop of semiarid regions due
to its high adaptability and suitability to rain-fed low input
agriculture. It has substantial popularity amongst farmers due to its
greater adaptability and various forms of utilization like green fodder,
stover, silage and hay to suit the diverse needs of farming system,
besides its grain.
2.2 Cyanogenesis is a major problem in sorghum
The value of sorghum fodder has increased over the years
compared to that of grain. But, one of the major factors limiting the
utilization of sorghum fodder is the production of cyanogenic (HCN-
producing) glycoside dhurrin that lowers the nutritive value of fodder
due to its toxic effects on the feeding livestock (Kojima et al., 1979).
6
Dhurrin is problematic when the digestive enzymes of grazing cattle
hydrolyse the compound into hydrocyanic acid (HCN).
Leaves and stems of all sorghum species contain hydrocyanic
acid or prussic acid (HCN) glycoside dhurrin. Some other plants also
produce HCN but in lesser amounts whereas in sorghum it is
produced in large quantities (above tolerance threshold) which are
hazardous to animal species. The dhurrin is hydrolyzed in the rumen
liberating the toxic HCN. HCN or hydrocyanic acid, can build up to
toxic levels (200 g/g dry weight is the threshold limit, McBee et al.,
1980) in the leaves of forage sorghum. Hydrocyanic acid can rapidly
make cattle ill and doses as little as 0.5 g are sufficient to kill a cow.
HCN causes death of animals by interfering with the ability of red
corpuscles in the blood to transfer oxygen.
Muthuswamy et al. (1976) estimated the HCN content of CSH 5
sorghum hybrid at different growth stages. They reported that HCN
content was more at the early stage of crop and it decreased at
maturity stage. They found that the HCN content was high 18 days
after sowing and decreased gradually upto 53 days. (Table 2.1).
However, HCN content in root did not come down substantially with
time. According to this study, HCN was more in shoot tissue (leaves
and stem) and less in root portion and it was also time dependent.
7
Table 2.1: HCN content of CSH 5 sorghum hybrid (Muthuswamy et
al., 1976)
Days from
sowing
HCN content (ppm; fresh weight basis)
Shoot Root
18 650 375
20 600 425
23 575 500
27 300 575
30 200 575
34 150 500
40 75 325
45 43 400
49 7 350
53 15 300
Chaturvedi et al. (1994) estimated HCN at flowering and grain
maturity stage in seven sorghum hybrids and their eight parents,
nine varieties, four high lysine grain sorghum lines and two check
varieties. Samples were collected at two growth stages; the first growth
stage (65 days after showing) represented the green crop at the
flowering stage and the second growth stage (100 days after sowing)
represented the crop at the grain maturity stage. The HCN content
was analyzed as per the method of Watten-Barger et al. (1968). The
genotypes differed significantly in their HCN content at both the
growth stages. Overall, P 721 (shrivelled grain), IS 84, BP 53, Aispuri,
Chitta Jonna and 296B had significantly lower HCN content than
other genotypes. The HCN content decreased significantly from 65
days after sowing to grain maturity stage only in 10 genotypes viz.,
CSH 11, CSH 13R, CK 60, 2077B, 2219B, IS 84, Swarna, Aispuri, P
8
721(plump grain) and N 914 (plump grain). Wheeler et al. (1990) also
reported decrease in HCN content with plant age in three genotypes of
sorghum.
Mc Bee et al., (1980) also found genotypic difference in HCN
content in grain sorghum genotypes. Mc Bee et al., (1980) used 200
ppm HCN on dry weight basis as the threshold for classifying sorghum
genotypes safe or unsafe. According to this classification, Chaturvedi
et al., (1994) grouped 20 genotypes CSH 5 (225 ppm), CSH 6 (278
ppm), CSH 9 (300 ppm), CSH 10 (260 ppm), CSH 11 (458 ppm), CSH
13R (247 ppm), CK 60 (277 ppm), 2077B (214 ppm), 2219B (210
ppm), 296B (326 ppm), CS 3541B (218 ppm), MR 750B (279 ppm),
RS29 (393ppm), Swarna (460ppm), SPV 462 (391ppm), SPV 473 (340
ppm), SPV 346 (372 ppm), SPV 351 (400 ppm), SSV 84 (606 ppm) and
P721 (271 ppm) as “unsafe” for feeding to cattle at the flowering stage.
In five genotypes i.e, CSH 9 (208 ppm), RS 29 (213 ppm), SPV 462
(224 ppm), SPV 346 (230 ppm) and SPV 351 (215 ppm), HCN levels
were found unsafe at grain maturity stage.
Wu Xian-rong et al., (1989) also studied change in HCN content
during the growth of seedlings. They measured the HCN potential
(HCN-p) of 148 sorghum and Sudan-grass (Sorghum sudanense)
varieties during seedling growth. The results showed that most of the
varieties had their HCN potential more than 1000 ppm (94.59%).
Among them, 33.11% belonged to 1400–1600 ppm, 22.97% to 1200–
1400 ppm, 17.57% to 1000–1200 ppm, and 14.86% to 1600–1800
ppm. The varieties in which HCN-p was less than 1000 ppm or higher
9
than 1800 ppm were a smaller proportion (11.44%). The varieties with
the lowest HCN-p were Xinliang 80 (672 ppm), Sudancao (753 ppm),
Huangke Sudancao (856 ppm), Limuji (860 ppm), and MI03 (876
ppm). Those with the highest HCN-p were Yuanxin lA (1967 ppm),
Shisanjie (1904 ppm), Mi- bangz (Da Lai) (1900 ppm), 7503 A (1889
ppm), and Mijia Honggaoliang (1883 ppm). They also reported that
sudangrass had the lowest HCN-p (about 700 ppm) and sweet
sorghum had higher HCN potential (about 1500 ppm). With the
seedling growth, HCN potential reached its highest value in 4-days-old
seedling. The first leaf had the highest HCN content, the second leaf
and sheath had lower and root had the lowest HCN content.
2.2.1 Cyanogenesis pathway in Sorghum
Cyanogenic glycosides are a related group of amino acid derived
natural products that also have oxime as intermediates (Moller and
Seigler, 1999). Studies of biosynthetic pathway of the tyrosine derived
cyanogenic glycoside dhurrin in sorghum (Sorghum bicolor [L.]
Moench) has shown that tyrosine is converted to p-
hydroxyphenylacetaldoxime by two multifunctional cytochrome P450
enzymes (CYP79A1 and CYP71E1), each encoded by a single gene
(Koch et al., 1995). CYP79A1, catalyses two consecutive N-
hydroxylation reactions followed by a dehydration and decarboxylation
reaction (Sibbesen et al., 1995) this reaction is conversion of tyrosine
to p-hydroxyphenylacetaldoxime (Koch et al., 1995; Sibbesen et al.,
1995). The oxime is then converted by another cytochrome P450,
CYP71E1 to the aglycon p-hydroxymandelonitrile which occurs by
10
dehydration to the nitrile followed by a C-hydroxylation (Bak et al.,
1998; Kahn et al., 1997). This p-hydroxymandelonitrile is
subsequently converted into dhurrin by UDPG-glycosyltransferase
which adds a glucose moiety to stabilize p-hydroxymandelonitrile
(Jones et al., 1999; Kahn et al., 1997). The UDP-glucosep-
hydroxymandelonitrile-O-glucosyl transferase was isolated from
etiolated seedlings of S. bicolor, cloned and characterized by Jones et
al., (1999). Through sequencing studies, the open reading frame of
cytochrome P450tyr gene were sequenced and found that this gene
encodes a protein with a molecular mass of 61,887 Da. This gene
encoded protein is multifunctional N-hydroxylase that is hemethiolate
protein cytochrome P450tyr.
The CYP79A1 enzyme has a high specificity for tyrosine as its
substrate (Kahn et al., 1999). CYP71E1 has a broader substrate
Fig-2.1 Dhurrin biosynthesis
and break down pathway in
sorghum
The CYP79A1 enzyme convert s
tyrosine to oxime which is
responsible for hydrogen cyanide
production (Koch et al., 1995;
Sibbesen et al., 1995)
11
specificity than CYP79A1 and can metabolize the oxime derived from
phenylalanine, valine and isoleucine in addition to those derived from
tyrosine (Kahn et al., 1999). Expression of CYP79A1 and CYP79E1
together in Arabidopsis led to the accumulation of tyrosine derived
cyanogenic metabolites that are not normally found in Arabidopsis
and also of p-hydroxybenzylglucosinolate (Bak et al., 1999).
2.2.2 Cyanogenesis metabolism involved in sorghum
The ability of plant to synthesize cyanogenic glycoside is known
as “cyanogenesis”. In cyanogenesis, the sugar moiety is cleaved from
the cyanogenic glycoside in a process catalyzed by one or more β
glycosidases. The resulting cyanohydrin is relatively unstable and
degrades (Conn, 1981). Autotoxicity is prevented by spatial separation
either at the subcellular or tissue level of the degradative enzymes and
the cyanogenic glycosides (Sanders et al., 1977; Frehner and Conn,
1987; Puolton, 1988; Swain et al., 1992)
Living plant contains both cyanogenic glycoside dhurrin and
enzyme β- glycosidase in separate cells. When plant tissues are
damaged by freezing, chopping or chewing, enzymes can come in
contact with cyanogenic glycoside and produce HCN. Upon tissue
disruption, cyanogenic glycoside degradation is initiated by cleavage of
carbohydrate moiety by one or more β- glycosidases, yielding the
corresponding cyanohydrin. This intermediate may decompose either
spontaneously or enzymatically in the presence of an α-hydroxynitrile
lyase to release HCN and an aldehyde or ketone. Bacterial action in
12
the rumen of cattle and sheep can also release prussic acid from
glycoside. Prussic acid production is apparently more likely to occur
in ruminants because both chewing and rumen bacteria release
cyanide. Hydrochloric acid in the stomach of horses and swine
destroys plant enzyme that release the toxin.
The level of cyanogenic glycoside produced is dependent upon
the age and variety of the plant, as well as environmental factors
(Cooper-Driver and Swain, 1976; Woodhead and Bernays 1977). High
amount of dhurrin accumulate in the tissue of the sorghum plant
during early growth phase as well as when rapid growth occurs
following stress like drought and frost. This potential is genetically
regulated, so that different cultivars can have very different HCN
potential values under similar circumstances.
Cyanohydric acid is extremely toxic to a wide spectrum of
organisms, due to its ability of linking with metal (Fe++, Mn++, Cu++)
that are functional group of many enzymes, inhibiting process like the
reduction of oxygen in the cytochrome respiratory chain, electron
transport in the photosynthesis, and the activity of enzymes like
catalase, oxidase (Cheeke, 1995; Mc Mahan et al., 1995). Once
cyanide is absorbed, it is readily transported throughout the body and
is very toxic to all animals. In cells, cyanide reacts with cytochrome
oxidase (an enzyme involved in the electron transport system that
enables cells to use the oxygen) to form a stable, inactive complex. As
a result the ion inhibits the release of oxygen from the hemoglobin of
13
blood to the individual cells. Without oxygen, cellular respiration
ceases and cells die rapidly due to hypoxia.
The association between cyanogenic plants and poisoning in
humans and domestic animals is well known. There are many well-
documented examples of death and serious illness from consumption
of cyanogenic plants in humans (Cock, 1982; Cardoso et al., 1998;
Banea-Mayambu et al., 2000), cattle (Robinson, 1930; Cooper-Driver
et al., 1977; Crush and Caradus, 1995), goats (Webber et al., 1985),
and other grazing animals (Harborne, 1982; Saucy et al., 1999).
2.3 Need for low HCN potential sorghum through genetic engineering
In this background, enhancement of fodder quality and
utilization in sorghum is possible by reducing the cyanogenic
potential. One of the feasible approaches available for this purpose is
the reduction of dhurrin production by down-regulating the enzymes
involved in its synthesis. In this direction, CYP79A1 gene is the
candidate of choice as it is the first enzyme involved in the pathway
and leads to no accumulation of secondary products. Such a down-
regulation of CYP79A1 gene expression is possible by anti-sense DNA
transformation approach, where the CYP79A1 is inserted (by
transformation) in sorghum genome in anti-sense orientation. This
approach is most feasible and would be highly effective, as has been
achieved in cassava (Siritunga and Sayre, 2003) who obtained cassava
plants that showed 94% reduction in cyanogen production by using
anti-sense transgenes of CYP79D1 and CYP79D2 genes that regulate
14
the cyanogens pathway in cassava. Moreover, the effectiveness
CYP79A1 and CYP71E genes of sorghum in inducing cyanogen
production had been demonstrated in Arabidopsis and tobacco (Bak et
al., 2000).
2.3.1 Antisense technology
When mRNA forms a duplex with a complimentary anti-sense
RNA sequence, translation is blocked. Antisense RNAs were initially
recognized in bacteria as naturally occurring mechanisms for
regulation of gene expression (Simons et al., 1983) which
subsequently led to the design of artificial antisense control strategies
(Green et al., 1986; Simons, 1988). Even though the first engineered
experiments in this area were done in mouse cells expressing
complementary RNAs against thymidine kinase (Izant et al., 1984), it
was in plants that the first multicellular eukaryotic organism was
transformed with a foreign antisense gene (Rothstein et al., 1987). The
technology of antisense has been exploited to a much greater degree
in prokaryotic and mammalian than in plant systems. As the
literature grows with more and more successes since the first report of
inhibition of the chloramphenicol acetyltransferase marker gene in
carrot protoplasts (Ecker et al., 1986) and that of the chalcone
synthase endogenous gene in transgenic petunia plants (Van der Krol
et al., 1988), antisense strategies were increasingly utilized in plant
systems as a means of down regulating specific genes of interest.
15
2.3.2 Antisense technology has been successfully applied in
plants
The FLAVR SAVRTM tomato was developed through the use of
antisense RNA to regulate the expression of the enzyme
polygalacturonase (PG) in ripening tomato fruit (Kramer et al., 1992).
This enzyme was one of the most abundant proteins in ripe tomato
fruit and has long been thought to be responsible for softening in ripe
tomatoes. The use of an antisense strategy to reduce the expression of
the polygalacturonase (PG) gene in tomatoes causes decreased pectin
solublization in the ripening fruit which in fresh market tomatoes
results in ripe fruit that remain intact for extended periods of time
(Kramer et al., 1992). The FLAVR SAVRTM tomato was the first
genetically engineered whole food to be sold in commerce. In terms of
a commercially viable product, the technology allows for the
production of fresh market tomatoes which can be vine-ripened for
enhanced flavor and have a longer shelf life yet still survive the
traditional distribution system intact.
André D‟Aoust (1999) developed transgenic tomato plants with
reduced sucrose synthase (SuSy) activity in fruit by expressing an
antisense RNA fragment of the TOMSSF gene under the control of the
cauliflower mosaic virus 35S promoter. Inhibition was only slight in
the endosperm and was undetectable in the embryo, shoot, petiole
and leaf tissues. The inhibition of sucrose synthase (SuSy) activity in
the flowers was perhaps because the TOMSSF cDNA was isolated from
tomato pistil mRNAs and is therefore expressed in this tissue as well
16
as in the fruit (Wang et al., 1993). The only effect on the carbohydrate
content of young fruit was a slight reduction in starch accumulation.
The in vitro sucrose import capacity of fruits was not reduced by
sucrose synthase inhibition at 23 days after anthesis, and the rate of
starch synthesized from the imported sucrose was not lessened even
when sucrose synthase activity was decreased by 98%. Reduced fruit
set, leading to markedly less fruit per plant at maturity, was observed
for the plants with the least sucrose synthase activity.
Wong et al., (2001) identified chilling-inducible ACC synthase
gene (CS-ACS1) gene from Citrus sinensis. The CS-ACS1 gene was
constructed in an inverted orientation and placed under the control of
the double 35S promoter. The antisense CS-ACS1 transgene was
introduced into Carrizo citrange, C. sinensis (L.) Osbeck and Poncirus
trifoliate by Agrobacterium-mediated gene transfer. The transgenic
citrus lines that produce higher level (over expression) of antisense
ACS RNA were found to repress the increase of ACC content following
the chilling treatment. This work was the first example of controlling
the ethylene biosynthesis in citrus plants through the genetic
engineering approach.
He et al. (2003) reported the effectiveness of expressing antisense
sorghum O-methyltransferase gene (omt) to down-regulate maize OMT
and reduce lignin. Lignin is a complex, aromatic polymer that limits
plant cell wall degradation by ruminants and reduces the nutritional
value of forages. Genetic engineering using antisense strategy offered
the potential to modulate enzymes in the lignin biosynthetic pathway
17
to reduce lignin, thereby improving forage quality and animal
performance. Constructs contained a sorghum omt coding region in
the antisense orientation driven by the maize ubiquitin-1 (Ubi)
promoter (with the first intron and exon) along with bar, that confers
glufosinate herbicide resistance, driven by the CaMV 35S promoter.
Twenty-eight T0 plants regenerated from 17 herbicide-resistant callus
lines from 13 independent bombardments expressed the brown midrib
(low lignin) phenotype. O-methyltransferase activity was significantly
lower in T1 transgenics compared with controls, with some plants
showing a 60% reduction. Those T1 transgenics with down-regulated
OMT averaged 20% less lignin in stems and 12% less lignin in leaves
compared with controls. On a whole-plant basis, lignin was reduced
by an average of 17% with the greatest reduction being 31%.
Digestibility was significantly improved in transgenic plants by 2% in
leaves and 7% in stems. Mean whole-plant digestibility increased from
72 to 76%.
In Petunia plant BoACS1 (broccoli ACC synthase) and BoACO1
(broccoli ACC oxidase) coding sequences of enzymes involved in
biosynthesis of ethylene in broccoli plants. Li-Chun Huang et al.
(2007) with the help of antisense technology transformed the
antisense BoACS1 gene and antisense BoACO1 gene in Petunia plant.
The integration of these genes with an antisense orientation was
verified by PCR analyses of kanamycin-resistant regenerants. The
expression of transgenes and endogenous genes was further
confirmed by RT-PCR analysis. Production of ethylene in shoot tissues
18
was reduced among most transgenic plants. Flowers of transformants,
especially excised flowers, generally remained fresh longer than those
of untransformed controls. The delayed flower senescence was more
pronounced with the antisense BoACO1 than the antisense BoACS1.
Transgenic tissues were, nevertheless, still responsive to ethylene.
They concluded that the antisense BoACO1 gene from Brassica
oleracea is able to reduce ethylene biosynthesis and delay flower
senescence of Petunia hybrida more efficiently than the antisense
BoACS1 gene.
Research investigation aimed at down-regulation of cyanogenic
potential by anti-sense approach has been done in cassava plant.
Cassava is one of the major root starch crops grown in the tropics.
Like sorghum cassava contains potentially toxic levels of cyanogenic
glycoside linamarin in roots, synthesized from valine. As in sorghum,
two cytochrome P450 enzymes (CYP79D1 and CYP79D2) catalyze the
first dedicated step in linamarin synthesis (McMahan et al., 1995;
Andersen et al., 2000; Siritunga and Sayre 2003). Siritunga and Sayre
(2003) developed transgenic cassava through reduced levels of
CYP79D1 and CYP79D2 encoded enzyme, which led to the inhibition
of cyanogen production by antisense technology.
2.4 Genetic transformation studies in plants with special
reference to sorghum
Efficient plant transformation system depends upon the
availability of levels of plant regeneration protocol. Besides an
established tissue culture and regeneration system, following factors
19
are also critical for successful transformation: (i) suitable explant, (ii)
method of DNA delivery into cell, (iii) target gene in a suitable vector
with reporter (to confirm alien gene introduction into cell or tissue)
and selectable (to select only those cells into which foreign DNA has
been incorporated) marker genes, and (iv) efficient testing methods for
the confirmation of transformed phenotype. It is equally important to
ensure consistent inheritance of transgene in the progeny and lack of
gene silencing or pleotropism to commercialize a transgenic plant. In
this regard, herbicide resistance is the most common phenotype
obtained by transformation in cereals, followed by abiotic and biotic
stress resistance (Metz et al., 1998). Genetic transformation with
specific genes conferring disease and insect resistance provides an
efficient tool to complement traditional breeding (Harshavardhan et
al., 2002).
2.4.1 Genetic transformation studies in sorghum
Gene transfer to crop plants can be achieved using several
methods such as direct DNA uptake, Agrobacterium-mediated DNA
transfer (Christou, 1995; Zhao et al., 2000) and particle bombardment
(Casas et al., 1993; Zhu et al., 1998). Due to greater difficulties in
Agrobacterium-mediated gene transfer, biolistic approach has been
used extensively for the genetic transformation of the monocot species
(Christou, 1995). Transformation systems have been described for all
major cereals (Bajaj, 2000) including maize, oat, rice, wheat and
barley. In contrast, in the past one-decade, very little attention is
20
given in developing transgenics of semi-arid crops like sorghum which
can benefit millions of needy farmers and consumers in the developing
world (Sharma et al., 2001; Reddy and Seetharama, 2002). The
protocols for producing transgenic sorghum are still being
standardized using various methods of DNA transfer (Gurel et al.,
2009).
Hagio et al (1991) reported first time success in obtaining stable
cellular transformation in sorghum using the biolistic technique. They
reported that sorghum cells that were transformed by biolistics with
genes conferring resistance to hygromycin (hph gene) and kanamycin
(nptII) exhibited resistance to these antibiotics. This report encouraged
the deployment of biolistic technique for monocot plant
transformation in general and sorghum transformation in particular.
Battraw and Hall (1991) used protoplasts for incorporating nptII
and uidA genes through electroporation. They studied transient
expression of reporter genes using different factors such as
linearization of the plasmid and effect of co-bombardment with two
different gene constructs. However, their analyses of gene integration
using PCR and Southern blots confined only putatively transformed
calli, and no transgenic plants were regenerated.
The first set of sorghum transgenics developed through
microprojectile bombardment route were from immature embryo
explants (Casas et al., 1993) and calli from immature inflorescence
(Casas et al., 1997). The culture time to develop transgenics was seven
months. Nevertheless, the transformation frequency was relatively low
21
(0.2%). However, they had used the sorghum genotype PI 898012
which has poor agronomic traits.
In sorghum immature embryos, the parameters involved in the
DNA delivery process were optimized using the maize R and C1 gene
that code anthocyanin transcription factors. (Casas et al., 1993) The
expression frequency of transient UidA (GUS) was less than 20 stained
foci per embryo. These result suggested that in sorghum transgene
expression levels was lower than that in maize because of genotype
and acceleration pressure effects or their interactions, besides the
inherent characteristics of sorghum scutellar tissue. Kononowicz et al.
(1995) also reported that response of sorghum explants for biolistics
and regeneration were genotype-specific while they used immature
embryo and immature inflorescence explants.
Zhu et al., (1998) reported incorporation of rice chitinase gene
and bar gene into sorghum conferring resistance to fungi and
herbicide (Basta), respectively by micro projectile bombardment.
In sorghum, Zhao et al. (2000) reported the first set of
transgenics developed by Agrobacterium-mediated transformation.
They used two sorghum lines - a public line P898012 and a
commercial line PH I391 for Agrobacterium –mediated transformation
of sorghum. Several workers (Casas et al., 1993; Kaeppler and
Pedersen, 1997; Carvalho et al., 2004) found that coconut water
adjuvant was helpful for generating embryogenic superior calli from
the sorghum accession P 898012, at a greater frequency.
22
Zhao et al. (2000) investigated a number of factors that can
improve sorghum transformation by Agrobacterium, such as medium
composition, the inclusion of polyvinylpyrrolidone and frequent
subculture in selection medium. They also reported that mean
frequency of transformation was 2.1%. Inclusion of 100 µm
acetosyringone in the co-cultivation media to induce Agrobacterium vir
operon increased the transformation frequency.
The results of Jeoung et al. (2002) while comparing the
transformation of reporter genes, gfp and gus, for Agrobacterium and
biolistic transformation, recorded that gfp gene was superior to gus
gene for transgene expression in transiently transformed materials in
both methods of transformation. Using GFP as the screenable marker,
they optimized the sorghum transformation with respect to the
conditions for transformation, type of explants, promoters, and
inbreds. Similarly, Able et al., (2001) utilized the gfp gene to optimize
sorghum transformation and regeneration via particle bombardment.
They also optimized the conditions for biolistic transformation, such
as the distance between the rupture disk and the target tissue, helium
inlet aperture and pressure of helium gas used for accelerating the
micro-projectiles, and the age of tungsten and spermidine solution
used in these experiments.
Tadesse et al. (2003) optimized transformation parameters in
sorghum via biolistics. For optimization, they used four types of
explants of sorghum based on transient GUS expression. They tested
physical parameters including acceleration pressure, target distance,
23
gap width and microprojectile travel distance. The sorghum explants
studied were immature and mature embryos, shoot tips and
embryogenic calli. In addition, the activity of four heterologous
promoters was determined both by histochemical staining and
determining enzymatic activity to assay GUS expression in immature
embryos and shoot tips. The highest GUS expression was attributed to
the promoter Ubi1, followed by Act1D, Adh1 and CaMV35S promoters,
in the decreasing order. The optimized bombardment conditions were
applied for selecting phosphinothricin- or genticin-resistant in vitro
cultures in order to generate transgenic plants.
Carvalho et al. (2004) proposed that efficiency of sorghum
transformation using Agrobacterium is influenced by the sensitivity of
the explants to agro-infection, the growth conditions of the explants,
donor plant and the composition of co-cultivation medium. They
reported that major problem during the development of protocol was
necrosis of explants after co-cultivation, to which sorghum immature
embryos were particularly sensitive. Agro-infection led to death of
many explants by necrosis, limiting the transformation efficiency.
Therefore, Carvalho et al. (2004) observed that specific attention may
be given to concentration of Agrobacterium in the inoculum, selection
of the explants and the genotype. They enumerated the benefits of
adding coconut water to the medium in enhancing callus recovery,
reducing the pigment production and improving callus growth.
Though P 898012 was very responsive to coconut water, other
genotypes may require different preparations of coconut water as
24
many genotypes showed no response to coconut water (Kaeppler and
Pederson, 1997).
Gray et al. (2004) obtained insect resistance plant by
microprojectile bombardment of shoot meristems with cry1Ab and
cry1Ac. Devi et al. (2004) obtained for drought tolerance by
bombarding shoot meristems isolated from germinating seedlings with
HVA1 gene.
For grain sorghum transformation, Gao et al. (2005a) used a
visual marker gene (gfp) and a target gene (tlp). Three genotypes (two
inbreds, Tx 430 and C 401, and a commercial hybrid, Pioneer 8505)
were used. They obtained a large number (1011, in total) of fertile
transgenics from 61 independent callus lines, which were produced
from 2463 zygotic immature embryos via Agrobacterium-mediated
transformation. The tlp target gene codes thaumatin-like protein that
enhances resistance to fungal diseases and drought. Both gfp and tlp
genes were regulated by the maize ubi1 promoter in the binary vector
pPZP201. The mean transformation efficiency was 2.5%, which was
greater than that reported earlier by Zhao et al. (2000).
Gao et al. (2005b) employed a dual marker plasmid containing
the selectable marker gene, manA and the reporter gene, gfp (both
regulated by Ubi1 promoter), to transform sorghum immature
embryos utilizing the Agrobacterium-mediated transformation method.
They observed that mannose selection did not result in necrosis and
mannose had lesser negative effect on regeneration of transgenics.
25
Gao et al. (2005 a&b) did not observe gene silencing for either the gfp
gene or the tlp gene in T0 and T1 generations.
Howe et al., (2006) conducted stable transformation
experiments in sorghum using immature embryos of Tx 430 and C2-
97 genotypes. They used the Agrobacterium tumefaciens C58 strain
that harbours nptII as a selectable marker. Transformation frequency
was approximately 1% for both genotypes.
Nguyen et al. (2007) developed an improved regeneration
protocol suitable for sorghum transformation. The improvements
focused on limiting the production of phenolic compounds and the
use of suitable culture vessels for each developmental stage in plant
regeneration from immature embryo derived calli. Inclusion of
activated charcoal in callusing medium resulted in reduced
development of black pigment, however it also inhibited the callus
formation from immature embryo explants. A one-day 4°C treatment
of immature seeds significantly improved the callus formation from
immature embryos and reduced the need for frequent subculture.
Agrobacterium-mediated transformation using the improved
regeneration protocol and the hygromycin phosphotransferase gene as
selectable marker resulted in the recovery of 15 transgenic plants
from 300 initial immature embryos with transformation efficiency of
5%.
Gurel et al. (2009) reported that Agrobacterium transformation
is affected by several parameters. Agrobacterium infections were tested
to optimize transformation frequencies of sorghum. They tried the
26
following treatments to improve Agrobacterium transformation
efficiency.
(1) Using different temperatures and centrifugation conditions to
pre-treat immature embryos prior to Agrobacterium infection,
(2) Altering cooling temperatures following heat treatment of
immature embryos
(3) Varying temperatures during and after centrifugation
(4) Pre-treating panicles in cold prior to immature embryo isolation.
The effects of different treatments on frequencies of transient and
stable transformation were determined by monitoring GFP expression
during callus formation and mannose selection and by conducting
PCR, DNA hybridization and western analyses of regenerated shoots.
According to Nguyen et al. (2007), a one day, 4°C pre-treatment of
immature seeds significantly improved callus formation from
immature embryo of an African red sorghum cultivar and reduced the
need for frequent sub culturing due to reduction of phenolics. But
according to Gurel et al. (2009), 1-day pre-treatments at 4°C of two US
sorghum lines, P 898012 (Type II) and Tx 430 (Type I), did not
significantly increase frequency of immature embryo survival or callus
induction; in fact with Tx 430 the frequency decreased significantly
after one day of pre-treatment. Prolonged (five days) cold pre-
treatment of panicle prior to isolation of immature embryo
significantly enhanced the frequencies of immature embryos from
both genotypes that survived culturing, produced callus, blackened
and expressed GFP. Cold pre-treatment, however, did reduce phenolic
27
production, most likely due to effects of low temperature on reducing
key enzyme activities (polyphenol oxidases and peroxidases) that are
involved in phenolic compound synthesis (Dicko et al. 2006). Using
different heating times at 43°C prior to infection showed 3 min was
optimal. Centrifuging immature embryos with no heat or heating at
various temperatures decreased frequencies of all tissue responses;
however, both heat and centrifugation increased de-differentiation of
tissue. The most optimal treatment, 43°C for 3 min, cooling at 25°C
and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3%
stable transformation frequency. Transformation frequencies greater
than 7% were routinely observed using similar treatments over five
months of testing.
Emani et al., (2002) provided the evidence for methylation-based
transgene silencing in sorghum. By use of the cytidine analog, 5-
azacytidine (azaC), the methylation-mediated transgene silencing in
sorghum could be reversed. Methylation-mediated transgene silencing
is known in dicots (Matzke et al., 1995), wheat (Demeke et al., 1999)
and rice (Kumpatla et al., 1997; Kohli et al., 1999; Fu et al., 2000).
Emani et al. (2002) concluded that methylation-based silencing may
be more frequent in sorghum and it was probably responsible for
transgene inactivation in earlier reports by sorghum workers. The
summary of attempts by earlier researchers to genetically transform
sorghum is presented in Table 2.2.
Table 2.2: History of genetic transformation in sorghum
S.No
Transformation method
Explant/ culture system
Gene of interest
Promoter Selection agent/ conc.used
Remarks Reference
1
Electroporation
Protoplasts
Cat
CaMV35S/ Copia promoter of drosophila
Chloramphenicol
Efficient gene expression under both promoters
Ou-Lee et al. (1986)
2 Electroporation Cell suspension and protoplasts
nptII CaMV35S Kanamycin, 100 mg/l
Stable transformation Battraw and Hall(1991)
3 PDS- 1000/He (Bio-Rad)
Cell suspension
nptII,hpt, uidA
adh1/ CaMV35S Kanamycin/ hygromycin
Stable transformation
Hagio et al. (1991)
4 PDS- 1000/He (Bio-Rad)
Immature embryo
bar,uidA CaMV35S Bialophos, 3 mg/l Plants regenerated at low frequency
Casas et al. (1993)
5 PDS-1000/He Immature embryo/inflorescence callus
bar,uidA
Bialophos, Plants regenerated at low frequency
Kononowicz et al.(1995)
6 PIG(Particle-Inflow Gun)
Immature embryo/inflorescence derived callus
bar CaMV 35S/Act1 Biolophos/ 2 mg/l
Single plant regenerated
Rathus et al. (1996)
7 PDS-1000/He Immature inflorescence
bar,uidA
CaMV35S
Biolophos
Plants regenerated at low frequency
Casas et al. (1997)
29
S.No
Transformation method
Explant/ culture system
Gene of interest
Promoter Selection agent/ conc.used
Remarks Reference
8 PDS-1000/He Immature embryo
bar/ chitinase 1
CaMV35 S Basta/ 1-2 mg/l
Gene integration confirmed
Zhu et al. (1998)
9 PIG Immature embryo
bar CaMV35S /Act1
Basta/1-2 mg/l Casein hydrolysate used for increasing
regeneration frequency
Rathus and Godwin
(2000)
10 Agrobacterium mediated
Immature embryo callus
bar Ubi1 PPT/5 mg/l 2.1% transformation frequency reported
Zhao et al. (2000)
11 Particle bombardment
Immature embryo callus
uidA, bar,gfp
Act1, Ubi1, CaMV35S.
bialophos/2 mg /l
Ubi>Act1>CaMV35S Able et al. (2001)
12 Particle bombardment
Immature embryo calli
uid Ubi1, Act1, Adh1,CaMV 35S
Nil CaMV35S>Ubi 1>Act1>Adh1
Hill- Ambroz et al. (2001)
13 Particle bombardment
Immature embryo calli
uidA, GFP
Ubi, Act1, Adh1, CaMV35S
Observing GFP expression
Ubi>CaMV35S >Act1>Adh1
Jeoung et al. (2002)
14 Particle bombardment
Immature embryo calli
Uid,bar
Act I, UbiI PPT/5 mg/l
Methylation based transgene silencing
Emani et al. (2002)
15 Particle Bombardment
Immature embryo and shoot tips
Uid A bar and neo
Ubi I, ActI, Adh1,CaMV35S
Observed GFP expression
Ubi> CaMV35S>Act>AdhI
Tadesse et al. (2003)
16 PDS- 1000/He Immature embryo/shoot tips
npt II/dhdps-raec 1
Act1/Adh1/ CaMV35S, Ubi1
Kanamycin/ For more lysine content
Obtained 13 plants. Southern reported
Tadesse and Jacobs (2004)
30
S.No
Transformation method
Explant/ culture system
Gene of interest
Promoter Selection agent/ conc.used
Remarks Reference
17 PDS- 1000/He
Shoot meristems from germinating seedlings
bar/HVA I CaMV35S Glufosinate/10 mg/l
For drought tolerance; Southern confirmed
Devi et al. (2004)
18 PIG
Shoot meristems isolated from germinating seedlings
bar/cryIAb,cryIB
CaMV35S/Act 1 Basta /2 mg/l For insect resistance; No Southerns
Gray et al. (2004)
19 Agrobacterium mediated
Immature embryo derived callus
bar/ T1p, rice chitinase G11
Ubi 1 Bialophos,3 mg /l
Southern Jeoung et al. (2004)
20 Agrobacterium mediated
Immature embryo callus
Gfp/bar/ tlp, rice G11
CaMV35S
Hygromycin
Southern Carvalho et al. ( 2004)
21 Particle bombardment
Shoot tips
Uid,bar, cryIAc
MpiCI
Basta /2 mg/l
PCR, Southern and ELISA
Girijashanker et al. (2005)
22 Agrobacterium mediated
Immature embryo callus
Gfp,tlp
UbiI
No marker
Southern for tlp gene
Gao et al. (2005a)
23 Agrobacterium mediated
Immature embryo callus
Gfp,ManA
Ubi I
Mannose sugar added in medium
Southern and Western
Gao et al. (2005b)
24 Agrobacterium mediated
Immature embryo callus
Npt II, uidA
Nil Gentamycin or Paromycin
Nil Howe et al. (2006)
25 Agrobacterium mediated
Immature embryo callus
hpt
Nil Hygromycin
Southern Nguyen et al. (2007)
31
S.No
Transformation method
Explant/ culture system
Gene of interest
Promoter Selection agent/ conc.used
Remarks Reference
26 Mild ultra sonication
Pollen
Npt II, uid A Nil Nil Southern and PCR Wang et al. (2007)
27 Agrobacterium mediated
Immature embryo callus
Gfp, ManA
Ubi I
Mannose
PCR, Western, and Southern
Gurel et al. (2009)
28 Agrobacterium mediated co-transformation
Immature embryo callus
bar,sorghum lysyl tRNA synthetase
CaMV35S maize ZeinC Z19 B I
PPT
PCR and Southern
Lu et al. (2009)
Abbreviations: cat- Chloramphenicol acetyl transferase, npt II- neomycin phosphotransferase; bar- bailophos resistance; gfp-
green fluorescence protein; hpt- hygromycin phosphotransferase; act 1- rice actin promoter; ubi – maize ubiquitin1 promoter; adh
1- alcohol dehydrogenase promoter; CaMV35S – Cauliflower mosaic virus 35S promoter; PPT- phosphinothricin
2.5 Tissue culture and in vitro regeneration studies
Explants derived from meristematic tissues at early stages of
development are most amenable to tissue culture conditions
(Puddephat et al., 1996). In cereals, immature embryo and immature
inflorescences have been widely used as explants for successful plant
regeneration (Bregiter et al., 1989 & 1991). However, these explants
are seasonal and available during certain time period only. Mature
tissues such as seed embryo and hypocotyls are readily accessible
year round sources of cereal explants (Conger et al., 1982); also the
shoots tips and shoot apices isolated from germinating seedlings
(Zhong et al., 1998).
2.5.1 Pathways of in vitro plant regeneration
In vitro plant regeneration can follow either of the following two
pathways: (i) Organogenesis involving the development of auxillary
buds following inhibition of apical dominance, or de novo organization
of shoot meristems in callus cultures, and (ii) Somatic embryogenesis.
In the latter case, regenerates arise from single cells either directly or
follow the formation of a mass of proembryonic cells. Earlier reports in
Gramineae described only shoot morphogenesis (Green, 1978).
However, now extensive evidence is available for the regeneration of
plants via somatic embryogenesis. Further it is suggested that, there
exists a common pathway of regeneration in Gramineae tissue
cultures (Vasil, 1987). In recent studies, it has been shown that
33
minimum in vitro culture period would minimize somaclonal
variations (Seetharama et al., 2000).
2.5.2 Role of genotype on plant regeneration
The relationship between plant genotype and differential in vitro
response is well known in cereals (Green, 1978). Further, there are
many instances, both within the Gramineae and in other
angiosperms, where in plant regeneration was obtained in almost all
the genotypes tested (Bajaj, 2000). These results strongly suggest
that, the physiological state and the developmental stage of the
explant are critical. The same is true for in vitro response in sorghum.
In almost all the cases reported, employment of MS or LS basal
medium with 2-4-D with or without kinetin resulted in successful
morphogenic response (Table 2.2). Use of a variety of explants such as
immature inflorescences, immature embryos, and use of shoot tips or
apices have reported regeneration frequencies ranging from 0-100%
across the genotypes tested.
2.5.3 Role of growth regulators in in vitro plant regeneration
Plant growth regulators (PGR) controlled the morphogenic
competency, pathway and speed of regeneration from isolated shoot
meristems (Harshavardhan et al., 2002; Zhong et al., 1998). The
auxin: cytokinin ratio in the control of regeneration was well
documented by Skoog and Miller (1957). Polisetty et al., (1997)
reported that eliminating the apical dominance of main buds by
physical means or through the use of higher cytokinin concentration
34
in the culture medium, a large number of shootlets could be raised in
vitro.
Two types of cytokinin-like plant growth regulators are available;
one is phenyl urea (Thidiazuron, TDZ) and the other is a naturally
occurring purine-based derivative N6-benzylaminopurine (BAP). TDZ
efficiently stimulated cytokinin-dependent shoot regeneration. Two
hypotheses regarding the mechanism of action of TDZ are that (i) TDZ
could directly promote growth due to its own biological activity in a
way similar to that of cytokinins and (ii) it might affect the
accumulation of endogenous cytokinins (by reducing the rate of
degradation) or increase the synthesis of endogenous cytokinins
(Huetteman and Preece, 1993).
The addition of TDZ in the induction medium was effective for
multiple bud formation from bulged meristems and promoted the
induction of direct somatic embryos on shoot apices of sorghum
(Harshavardhan et al., 2002). It was reported that TDZ changed the
regeneration mode of shoots from organogenesis pathway to
embryogenesis type in tobacco leaf disc cultures (Gill and Saxena,
1993). TDZ-mediated somatic embryogenesis induction was also
reported in woody species (Fiola et al., 1990).
Higher levels of 2,4-D (1-2.5 mg/l) coupled with low levels of BAP
(0.05-0.5 mg/l) or kinetin were required to promote the formation and
proliferation of embryogenic callus from shoot apices of sorghum in
the studies of Bhaskaran et al. (1988), Bhaskaran and Smith (1990),
Lusardi and Lupotto (1990) and Nahdi and deWet (1995).
35
Zhong et al. (1998) reported that low levels of BAP induced the
formation of auxillary buds while high levels (2-4 mg/l) of BAP
stimulated the differentiation of adventitious buds from shoot apices
of sorghum. The addition of a low level (0.5 mg/l) of 2, 4-D in the BAP
containing media, not only triggered the higher frequency of
adventitious shoot formation, but also resulted in efficient
embryogenesis directly from the shoot apical domes of cultured
sorghum shoot apices.
The use of MS medium supplemented with (2-4 mg/l) of BAP and
(0.5 mg/l) of 2, 4-D was always accompanied with certain degree of
callus formation (Harshavardhan et al., 2002). However, replacement
of 2, 4-D with NAA resulted in the effective induction of somatic
embryos without any callus formation. Induction of callus formation
in rice and sorghum was reported earlier with higher concentrations of
2,4-D i.e., 3 mg/l (Abe and Futsuhara, 1985) and 2 mg/l (George and
Eapen, 1988). Seetharama et al. (2000) reported the induction of
friable embryogenic calli and somatic embryos from shoot tips
cultures by culturing on Linsmaier and Skoog (LS) medium
supplemented with 2,4-D (2.0 mg/l) and kinetin (0.1 mg/l) indicating
the role of 2,4-D in induction of indirect somatic embryogenesis.
The embryogenic calli from scutella of immature embryos was
induced in 10-15 days after the embryos were plated on MS medium
with 0.25 mg/l of zeatin and 0.5 mg/l of 2, 4-D and that the induction
was poor in medium with 2,4-D alone (Sairam et al. (2000). It was also
seen that age-dependent variation in in vitro responses were linked to
36
differences in endogenous auxin levels (Cassels et al., 1982) or
endogenous cytokinin levels (Josphina et al., 1990). Harshavardhan et
al. (2002) reported that isolated meristems from seven-day-old
germinating seedlings were optimum for sorghum in vitro plant
regeneration.
2.5.4. Genetic variability in plants regenerated in vitro
Larkin and Scowcroft (1981) termed the variation in tissue
culture derived plants as 'somaclonal variation'. A variety of nuclear
and cytoplasmic factors like point mutations, chromosomal
rearrangements, recombination, DNA methylation and transposable
elements, are responsible to its origin, and this is influenced by
genotype, explant type, culture medium, and age of the donor plant
(Jain, 2001). A majority of these variations are epigenetic in nature
(Micke, 1999). In case of sorghum, somaclonal variation for leaf
morphology and growth habit was reported, by Gamborg et al. (1977).
Similarly, Bhaskaran et al. (1983) obtained sodium chloride tolerant
callus from mature seeds. Sorghum variety GAC, tolerant to
aluminum in acid saturated soils was developed by Duncan et al.
(1991) and Waskom et al. (1990) reported increased tolerance to acidic
soils and drought stress at field level. Maralappanavar et al. (2000)
studied variation in both qualitative and quantitative characters like
chlorophyll variation, altered phyllotaxy, branching phenotype, ear
head weight and total grain weight in two sorghum cultivars, M 35-1
and A1. Out of a wide variety of molecular methods available for
37
analysis of somaclonal variation in plants, RFLP and RAPD's are
increasingly applied in the recent period (Hashmi et al., 1997; Henry,
1998; Jain, 2001).
The positive aspect of such variation is its potential in crop
improvement, if properly incorporated into the existing plant breeding
programmes (Mythili et al., 1997). But from genetic transformation
point of view, such a variation is unwanted. Therefore, a system which
has no room for generation of somaclonal variation is the most
suitable candidate (Birch, 1997).
2.6. Successful transformation strategies
After tissue culture and regeneration system, the following
factors are responsible for successful transformation (i) suitable gene
in a suitable vector that contain reporter and selectable marker genes;
(ii) DNA delivery into target cell; and (iii) efficient method of testing to
confirm transformation events. It is equally important to ensure
consistent expression and inheritance of the transgene in the progeny.
According to Seetharama et al. (2002), lack of pleiotropic effects, and
consideration of bio-safety issues are important before useful
transgenic can be commercialized.
2.6.1 Choice of the explants used for transformation
Pre-cultured immature embryos or isolated scutella with competent
cells for somatic embryogenesis have been proven to be excellent
targets for genetic transformation of cereals (Bommineni et al., 1997).
The first report of successful sorghum transformation came from
38
Casas et al. (1993) using immature embryos. They were able to
regenerate two plants, out of 600 bombarded embryos. To date, this
has been the most tried explant in sorghum transformation (Casas et
al., 1993; Rathus et al., 1996; Zhu et al., 1998; Zhao et al., 2000;
Rathus and Godwin, 2000; Jeoung et al., 2002).
Apart from the above-mentioned explants, Tadesse and Jacobs
(2004) have used shoot tips as explants. Of late, shoot meristems and
apices dissected out from germinating seedlings are being used as
explants (Gray et al., 2004; Devi et al., 2001). It is convenient to
obtain shoot apices from germinating seeds rather than to wait until
young panicles are formed (Seetharama et al., 2000). Protocols for
efficient and reproducible plant regeneration system have been
reported from shoot apices of germinating seedlings of sorghum
(Zhong et al., 1998; Seetharama et al., 2000; Harshavardhan et al.,
2002).
2.6.2 Choice of plant promoters
Transgene expression efficiency is dependent on the promoter
regulating it, which also depends on the plant species that is being
examined (Able et al., 2001). Promoter heterologous for sorghum such
as CaMV35S promoter, rice actin promoter and maize Ubiquitin
promoter have been used in cereals (Bajaj, 2000). Actin1 promoter
and ubiquitin1 promoter have showed naturally high constitutive
activity (McElroy and Brettell, 1994). In sorghum, all these promoters
have been tested (cited in Table 2.2) for their strength as gene
39
regulators. Hagio et al. (1991) have observed that CaMV35S and
maize Adh1 were poor in expressing transgenes in sorghum. For high
levels of gene expression, either recombinant genes having two copies
of promoter (Casas et al., 1993) or with added enhancers and introns
have been used (Bajaj, 2000).The promoter constructs of ubiquitin1
and actin1 contain one native intron in the transcription unit, which
is perhaps responsible for elevated mRNA abundance and enhanced
gene expression in the transformed cells in cereals (Callis et al., 1987;
Luehen and Walbot, 1991). In sorghum, Able et al. (2001) probed the
expression (transient GUS assay based) of ubiquitin1, actin1 and
CaMV35S promoters and found that a significantly higher expression
was obtained with ubiquitin promoter, compared to Actin1 and
CaMV35S promoters. Tadesse et al. (2003), determined the strength of
four promoters in sorghum, which was found to be in the order as
ubi1, followed by Act1D, adh1 and CaMV35S. Jeoung et al. (2002)
found the order of promoter strength as measured by green
fluorescent protein (GFP) expression in calli was highest in ubi1
compared to CaMV35S. The order of promoter strength for GUS
expression was highest in ubi1 followed by CaMV35S, Act1 and Adh1.
2.6.3 Selectable marker for transformation
In genetic transformation experiments, identification of
transformed cells and culling out non-transformed ones is often
facilitated by the use of selectable markers which selectively allow the
growth of transformants, in medium containing the specific selection
40
agents. The most common selection markers are those that confer
resistance to antibiotics or herbicides (Casas et al., 1995). For
dicotyledons (tobacco and carrot reports of Hardegger and Sturm,
1998) the antibiotic kanamycin, and the neomycin
phosphotransferase II (npt II) gene isolated from E. coli (Bevan et al.,
1983) were highly useful. However, nptII gene based selection has
proven to be less effective for monocot transformation, as monocot
tissues are not affected by the antibiotic kanamycin (Dekeyser et al.,
1989). Selection in earlier sorghum experiments [Battraw and Hall,
1991; 100 mg/l and Hagio et al., 1991; 75-100 mg/l kanamycin] was
not satisfactory due to the natural resistance of sorghum cell cultures
to kanamycin. Therefore, the possibility of using alternate selection
agents was explored. More promising results were obtained with E.
coli hygromycin phosphotransferase (hpt) gene (Gritz and Davies,
1983). This was effectively used to select transformed tissues in maize
(Walters et al., 1992) and rice (Li et al., 1993). Hagio et al. (1991) used
1-2 mg/l hygromycin for selection of transformed sorghum explants.
Hygromycin is highly photosensitive and therefore it cannot be used
for selection during plant regeneration in cases where explants (like
shoot meristems) need light for growth. So far, the most successful
and popular selection agent in transformation of sorghum has been
the phosphinothricin (PPT), the resistance to which is conferred by the
bar gene of Streptomyces hygroscopicus. The bar gene encodes the
enzyme phosphinothricin acetyltransferase (PAT) that provides
resistance to the herbicide phosphinothricin. PPT has been used for
41
selection to obtain transgenic plants in cereals including sorghum
(Casas et al., 1993; Rathus et al., 1996; Zhu et al., 1998; and Zhao et
al., 2000, in sorghum). The concentration of selection agent (with bar
gene) used were - 8-10 mg/l of glufosinate or 2-3 mg/l of bialophos or
2-5 mg/l phosphinothricin (PPT).
2.6.4 Method of gene transfer
Plant transformation is performed using a wide range of tools
such as Agrobacterium Ti plasmid vectors, microprojectile
bombardment, microinjection and chemical (PEG) treatment of
protoplasts. Though all methods have advantages that are unique to
each of them, transformation using Agrobacterium and microprojectile
bombardment are currently the most extensively used methods
(Veluthambi et al., 2003). Owing to the difficulty in Agrobacterium
mediated gene transfer, biolistic approach has been used extensively
for the genetic transformation of the monocot species (Christou,
1995).
2.6.4.1 Microprojectile bombardment
Particle bombardment is an efficient method of genetic
transformation of cereals, where in, biological molecules are driven at
high velocity into the target tissue. It offers advantages like
introduction of multiple genes, the simplicity of introducing genes,
and transformation in those plants, where agro-infection is difficult.
This process was initiated by J. C. Sanford and T.M. Klein at Cornell
University in 80's. Their device included a barrel into which a
gunpowder charge was fitted, which accelerated the coated tungsten
42
powder placed at the tip of the micro-projectile (Sanford, 1988). The
commercial version of this device after a series of structural
modifications was changed to a safer compressed helium system
(Sanford et al., 1991).
The first set of successful applications of this process included
bombardment of DNA and RNA into epidermal cells of onion (Sanford
et al., 1987) and (Klein et al., 1987) few other reports also appeared in
the same year (Klein et al., 1988a, 1988b; Christou et al., 1988).
These experiments mainly focused on transient expression, and once
the method became routine, the utilization extended to genetic
transformation of plants for which the existing methods of
transformation like electroporation and agro-infection were considered
difficult.
Currently, a number of instruments based on various
accelerating mechanisms are in use. These include the original gun
powder device (Sanford et al., 1987), an apparatus based on electric
discharge (McCabe & Christou, 1993), a micro-targetting apparatus
(Sautter et al., 1991), a pneumatic instrument (Iida et al., 1991), an
instrument based on flowing helium (Finer et al., 1992; Takeuchi et
al., 1992), and an improved version of both the original gun powder
device utilizing compressed helium (Sanford et al., 1991). Hand held
version of the original Biolistic R device and the Accell device are also
in use. Biolistic transformation has allowed the recovery of transgenic
fertile plants in many cereal food crops such as rice (Chen et al.,
43
1998), maize (Brettschneider et al., 1997), wheat (Bommineni et al.,
1997) oat and barley (Zhang et al., 1999) and sorghum (Casas et al.,
1993 and Zhu et al., 1998).
2.6.4.1.1 Parameters that affect DNA delivery
Production of transgenic plants by particle bombardment can be
divided into two processes: (i) That of introduction of DNA into cells
with minimum tissue damage, and (ii) Regeneration from transformed
cells. Bombardment pressure, flight distance, amount of particles and
DNA used per shot, and the number of shots per target.
Transformation is also affected by donor plant variables like,
temperature, photoperiod and humidity, nature of explants (McCabe
and Christou, 1993; Smith et al., 2001). Optimization of physical and
biological parameters can increase the efficiency of these processes
(Birch and Bower, 1994). Increased transient expression and stable
transformation efficiencies resulted from treatment of the target
tissues with osmoticum (Vain et al. 1993). Generally, gold or tungsten
particles are used as micro-carriers in particle bombardment. Size of
the micro-carrier in the range of (0.5-1.0 m) used for bombardment
has an effect on transformation efficiency, as observed in wheat
transformation (Altpeter et al., 1996). Of the two, tungsten particles
are less expensive, but are more heterogeneous in size compared to
gold. But the disadvantage of using tungsten is that, it can
catalytically degrade DNA over a period of time, and may be toxic to
some cell types (Russel et al., 1992).
44
2.6.4.1.2 Post-bombardment selection strategies
The key to establishment of a successful transformation
strategy lies in adoption of an effective and foolproof selection
strategy. In general, post-bombardment selection should be just
enough to allow the survival of transformed tissues, without
hampering the regeneration process. This is determined by the type of
marker gene used and the type of explant transformed. Different doses
of the selection agent can be used to limit the number of non-
transformed cells that survive due to cross-protection by the
transformed cells. This optimal concentration for selection, in turn
depends on the species (Somers et al., 1992), which has to be
evaluated experimentally, while taking into consideration the effect of
post-bombardment tissue damage on selection process (Taylor and
Vasil., 1991). Therefore, an important benchmark for using such
selection strategy lies in the prior establishment of kill curves for the
particular marker gene to be used. For sorghum, Battraw and Hall
(1991) recorded 100 mg/l kanamycin in the culture medium to be
sufficient to ensure selection of transformed protoplasts, while Haigo
et al. (1991) have used, as high as 500 mg/l kanamycin for effective
selection for suspension cultures. They have also bombarded hpt gene
that provides resistance to hygromycin, and used 50 mg/l in the
selection medium. Yet another marker gene, that has wide usage is
the bar gene sourced from Streptomyces hygroscopicus, encoding the
enzyme phosphinothricin acetyltransferase (PAT). This enzyme confers
resistance to the herbicide Basta (or glufosinate, bialophos and
45
phosphinothricin) (Casas et al., 1993; Zhu et al., 1998; Zhao et al.,
2000). For screening the transgenic tissues transformed using bar
gene, the concentrations of selection agents used were 8-10 mg/l of
glufosinate or 2-3 mg/l of bialophos or 2-5 mg/l of phosphinothricin.
Casas et al. (1993) employed bialophos at 1-3 mg/l concentration.
Strategy followed here was the imposition of selection immediately
after bombardment on 1 mg/l or after two weeks of incubation on 3
mg/l, and maintenance of cultures further at this concentration
during the rest of the culture period. At times, the above chemical
selection proved too rigorous leading to loss of regenerative capacity of
the transformed tissue as observed in barley (Stiff et al., 1995) and
banana (Sagi et al., 1995).
2.6.4.2 Agrobacterium-mediated genetic transformation
Agrobacterium mediated transformation of plants is believed to
be more practical. Unlike the biolistic method, complex equipment is
not involved. However, for quite some time in the early history of
Agrobacterium-mediated transformation, monocotyledons were
considered not suited for transformation using Agrobacterium since
they are outside the host range of Agrobacterium. But, with better
understanding of the biology of agro-infection and the availability of
suitable promoters (Wilmink et al., 1995) and selectable markers,
Agrobacterium transformation in monocotyledons became a success
(Smith and Hood, 1995). Since then, many cereals including rice
(Chan et al., 1993), wheat (Chen et al., 1997), maize (Zhao et al.,
46
1998) and barley (Tingay et al., 1997) have been transformed by this
method.
Zhao et al., (2000) presented the first report of genetic
transformation of sorghum through Agrobacterium- mediated
bar gene delivery and they obtained overall transformation
frequency of 2.1%. The latest work by Lu et al. (2009) deals
with the development of marker-free transgenic sorghum
plants harbouring tRNA synthetase gene for enhanced lysine
content in sorghum seed.
These reports have established the feasibility of Agrobacterium-
mediated method, for transforming sorghum. However, several
variations and modifications would be needed to increase the
efficiency of transformation before this method can be handled on a
routine basis.
2.7 Molecular mechanisms of transgene integration
The analysis of molecular characteristics of transgenic loci would
provide invaluable information on the mechanism of the integration of
transgenes in to the host genome. This information may pave way for
improving the transformation techniques for recalcitrant plant
species. Morikawa et al. (2002) attempted to study the mechanism of
transgene integration into a host genome. They suspected that MARs
(nuclear matrix attachment regions) located in a transgenic locus
increases the transformation frequency, contribute to DNA
rearrangement and to the revolution of plant genomes. The transgene
47
structure and organization in cereals, as hypothesized by Kohli et al.
(1998), involves a two-phase integration mechanism. At the first level,
fragments of transgene DNA may join together end-to-end, to form
contiguous transgenes. Next, such clusters integrate (with the host
genome) at breaks in the genome, which occur naturally in all cells.
Such breaks may occur randomly, but transgene integration occurs in
easily accessible regions of the host cell chromatin. The first molecule
which gets integrated attracts the integration of additional transgene
molecules to the same site. This would eventually lead to the
formation of larger individual transgene clusters which may be
separated by shorter regions of host genomic DNA (Kohli et al., 1998).
2.8 Cyanogenesis in crop plants
Cyanogenesis, the ability of plants and other living organisms to
release hydrogen cyanide, has been recognized in over 3000 species of
higher plants distributed throughout 110 different families of ferns,
gymnosperms, and both monocotyledonous and dicotyledonous
angiosperms (Conn, 1981). However, only in approximately 300 plant
species, the source of HCN has been identified. In certain
Sapindaceous seeds, HCN may arise during cyanolipid hydrolysis. All
higher plants probably form low levels of HCN as a co-product of
ethylene biosynthesis (Kende, 1993). This might explain why even
'acyanogenic' plants contain significant levels of the cyanide
detoxifying enzyme f3-cyanoalanine synthase. The level of cyanogenic
glycosides produced is dependent upon the age and variety of the
plant, as well as environmental factors (Cooper-Driver and Swain,
48
1976; Woodhead and Bernays, 1977). It is usual to find cyanogenic
and acyanogenic plants within the same species, where the function of
cyanogenesis is revealed through their phenotypic characteristics.
Cyanogenesis may not necessarily be used for plant survival; it may
take part in metabolic and excretory processes but there certainly is a
characteristic of value for these species (Harborne, 1972; Cooper-
Driver and Swain, 1976; Woodhead and Bernays, 1977; Tokarnia et
al., 1994).
The major food sources of cyanogenic glucoside include bitter
almonds, cassava root, sorghum and lima beans (Shibamoto and
Bjeldanes, 1993). Toxicity of cyanogenic glucosides is due to the
liberation of hydrogen cyanide (Table 2.3). Hydrogen cyanide is
released from cyanogenic glucosides in chewed or chopped plants or
following ingestion by an enzymatic process involving two enzymes
(Bokanga et al., 1994). Cyanide release from cyanogenic glucosides
occurs readily in the laboratory by acid or base hydrolysis.
Table 2.3: Food sources of cyanogenic glycosides and amount of
hydrogen cyanide (HCN) produced (Shibamoto and Bjeldanes, 1993)
Plant part Amount of HCN (100mg/100g)
Glycoside
Bitter almonds 250 Amygdalin
Cassava root 53 Linamarin
Sorghum (whole plant) 250 Dhurrin
Lima beans 10-312 Linamarin
49
2.9 Method of HCN estimation
Cyanogenesis, or the emanation of hydrogen cyanide (HCN), has
long been recognized as an effective means of deterring predation
(Ellis et al., 1977; Conn, 1981; Netted, 1988; Scrapper and Shore,
1999; Magadha‟s et al., 2000). Plants, in particular, are capable of
yielding HCN (Jones, 1999; Vetter 2000) when their tissues are
crushed during maceration by chewing herbivores (Vetter, 2000). In
some tropical environments where insect pressure is high, as many as
4% of woody plants are cyanogenic and they concentrate HCN
precursors in reproductive parts (Thomsen and Brier, 1997). Many
methods have been developed for determination of the total cyanogen
(total cyanide) content of cassava (Cooke, 1978., Bradbury et al.,
1991,1994), sorghum (Haskins et al., 1988), flax (Palmer, et al., 1980;
Oomph, et al.,1992), giant taro (Bradbury et al., 1995; Netted, 1975)
and bamboo (Schwarzmaier, 1976,1977). The picrate method
(Adsersen, et al., 1988) and the Feigl-Anger spot test (Van Wyck,
1989) have been used to survey for cyanogenesis in wide range of
plants.
A general method was developed for determination of the total
cyanide content of all cyanogenic plants and foods by Rezaul Haque et
al. (2002). Ten cyanogenic substrates (cassava, flax seed, sorghum
and giant taro leaves, stones of peach, plum, nectarine and apricot,
apple seeds and bamboo shoot) were chosen, as well as various model
compounds, and the total cyanide contents determined by the acid
hydrolysis and picrate kit methods. The hydrolysis of cyanoglucosides
50
in 2 M sulfuric acid at 100°C in a glass stoppered test tube causes
some loss of HCN which is corrected for by extrapolation to zero time.
However, using model compounds including replicate analyses on
amygdalin, the picrate method was found to be more accurate and
reproducible than the acid hydrolysis method. For eleven different
samples of flax seed and flax seed meal, the total cyanide content was
140–370 ppm. Bamboo shoots contained up to 1600 ppm total
cyanide in the tip reducing to 110 ppm in the base. The total cyanide
content of sorghum leaves was 740 ppm one week after germination
but reduced to 60 ppm three weeks later. The acid hydrolysis method
is generally applicable to all plants, but is much more difficult to use
and is less accurate and reproducible than picrate method, which is
the method of choice for plants of importance for human food (Rezaul
Haque et al., 2002).
2.10 Molecular characterization of antisense transgenics
Molecular analyses such as polymerase chain reaction (PCR)
and Southern hybridization help in detecting the presence and
integration of the transgenes in host genome. By using the transgene-
specific primers that amplify (by PCR) the target transgene sequence
in the transformed plants, a large number of putative transgenic
plants can be rapidly analyzed in a relatively short period (Bajaj,
2000).
The antisense transgenic Arabidopsis plants with an AtProDH
cDNA encoding praline dehydrogenase (ProDH), which catalyzes
proline degradation, provided evidence for a key role of ProDH in
51
proline degradation in Arabidopsis (Nanjo et al., 1999). The membrane
was probed with 32P-labeled AtProDH antisense RNA, which detects
1.8 kb of At- ProDH mRNA. The AtProDH antisense RNA probe was
used for Northern hybridization.
He et al. (2003) reported the effectiveness of expressing
antisense sorghum O-methyl transferase gene (omt) to down-regulate
maize OMT and reduce lignin. Constructs contained a sorghum omt
coding region in the antisense orientation driven by the maize
ubiquitin-1 (Ubi) promoter (with the first intron and exon) along with
bar, that confers glufosinate herbicide resistance, driven by the CaMV
35S promoter. PCR analysis was performed with the help of bar and
Ubi promoter sequence.
Cyanogen-free transgenic cassava was also developed by
antisense technology (Siritunga and Sayre 2003). They characterized
the antisense transgenics with the help of PCR. The DNA primers
specific for the CYP79D1 gene were designed to amplify the region
between the Cab1 promoter/CYP79D1 junction and the
CYP79D1/NOS terminator junction. A diagnostic 700 bp CYP79D1
fragment was amplified in each of the five transformants and its
identity was confirmed by DNA sequence analysis.
BoACS1 (broccoli ACC synthase) and BoACO1 (broccoli ACC
oxidase) coding sequences of enzymes were involved in biosynthesis of
ethylene in broccoli plants. Li-Chun Huang et al. (2007) transfer the
antisense BoACS1 gene and antisense BoACO1 gene in petunia. The
integration of these genes with an antisense orientation was verified
52
by PCR analyses of kanamycin-resistant regenerants. The expression
of transgenes and endogenous genes was further confirmed by RT-
PCR analysis.
top related