2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2177/11/11_chapter...

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).

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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.

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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

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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

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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

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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)

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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

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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

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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

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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.

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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

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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

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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.

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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,

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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

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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



Gene of interest


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


uidA, bar,gfp

Act1, Ubi1, CaMV35S.

bialophos/2 mg /l

Ubi>Act1>CaMV35S Able et al. (2001)


Immature embryo calli

uid Ubi1, Act1, Adh1,CaMV 35S

Nil CaMV35S>Ubi 1>Act1>Adh1

Hill- Ambroz et al. (2001)



uidA, GFP

Ubi, Act1, Adh1, CaMV35S

Observing GFP expression

Ubi>CaMV35S >Act1>Adh1

Jeoung et al. (2002)



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



Gene of interest


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)


Immature embryo derived callus

bar/ T1p, rice chitinase G11

Ubi 1 Bialophos,3 mg /l

Southern Jeoung et al. (2004)



Gfp/bar/ tlp, rice G11

CaMV35S

Hygromycin

Southern Carvalho et al. ( 2004)


Shoot tips

Uid,bar, cryIAc

MpiCI

Basta /2 mg/l

PCR, Southern and ELISA

Girijashanker et al. (2005)



Gfp,tlp

UbiI

No marker

Southern for tlp gene

Gao et al. (2005a)



Gfp,ManA

Ubi I

Mannose sugar added in medium

Southern and Western

Gao et al. (2005b)



Npt II, uidA

Nil Gentamycin or Paromycin

Nil Howe et al. (2006)



hpt

Nil Hygromycin

Southern Nguyen et al. (2007)

31

S.No



Gene of interest


Remarks Reference

26 Mild ultra sonication

Pollen

Npt II, uid A Nil Nil Southern and PCR Wang et al. (2007)



Gfp, ManA

Ubi I

Mannose

PCR, Western, and Southern

Gurel et al. (2009)

28 Agrobacterium mediated co-transformation


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.

2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2177/11/11_chapter...

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