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PLANTS: Improvement of Sorghum through transgenic technology

------------------------------- GENET-news -------------------------------
TITLE:  Improvement of Sorghum through transgenic technology
SOURCE: Information Systems for Biotechnology News Report, USA
AUTHOR: KBRS Visarada and N Sai Kishore
DATE:   01.03.2007

Improvement of Sorghum through transgenic technology
Sorghum is the fifth most important cereal crop in the world. It is
largely grown on marginal soils with residual moisture where other major
cereals cannot be grown due to inadequate water. Sorghum is a
multipurpose crop and the species shows great diversity. For a large
part of Asia and Africa, sorghum's grain is used as food and its stalk
as fodder and feed. In rest of the world, sorghum is considered as
forage crop and also as stock for ethanol production.

Sorghum yield has been substantially increased through conventional
breeding in India. However, resistance to abiotic stresses and biotic
stresses such as shoot fly, stem borer, grain mold, and charcoal rot is
limited due to inadequate genetic resources that can be readily used in
crop improvement programs. Therefore, genetic engineering technology can
assist the production of agronomically desirable crops that exhibit
increased resistance to pests, pathogens, and environmental stress and
enhancement of nutritional qualities.

Sorghum research has received less attention compared to other cereals
for adoption of modern molecular tools, and very few laboratories in the
world are addressing sorghum crop improvement programs through novel
methods. Extensive research has been focused on other cereal crops, and
a number of genes conferring agronomic advantages have been introduced
through Agrobacterium and particle bombardment. In this article, we
present the current status, progress, and prospects in transgenic
sorghum technology and future approaches to increase its economic value,
thereby providing monetary benefits to sorghum farmers.

Sorghum Transformation

The first report of the successful transformation of sorghum appeared as
early as the 1990s. Yet, sorghum is considered to be the most
recalcitrant crop for tissue culture and plant regeneration, thereby for
genetic transformation. Recalcitrance in sorghum tissue culture is
reportedly due to the release of phenolics, lack of regeneration in long
term in vitro cultures, and a high degree of genotype dependence. The
release of phenolics into the culturing medium can be overcome by
frequent subculture and by the addition of polyvinyl pyrrolidone
phosphate (PVPP) in the medium. However, transformation followed by
regeneration remains extremely complicated in sorghum transgenic technology.

Genetic transformation of sorghum has picked up momentum in recent
years, with a greater number of reports published in the last couple of
years. Though different explant sources such as immature inflorescence,
immature embryo, and shoot meristem are reported in sorghum
transformation, successful recovery of transgenic plants through
Agrobacterium mediated and particle bombardment are mainly achieved
using immature embryos. In general, it takes 10 - 12 months for a highly-
responding immature embryo to regenerate into a transgenic plant,
despite much labor-intensive work.

Though both systems of transformation, i.e., Agrobacterium-mediated and
particle bombardment, are successful in sorghum, the most effective
method to date is Agrobacterium based transformation, with a high
transformation efficiency that ranges from 2.1% - 4.5% (Howe et al.,
2006; Gao et al., 2005; and Zhao et al., 2000). Zhao et al. (2000)
reported the first production of transgenic sorghum plants by
Agrobacterium-mediated transformation using immature embryos. They have
tested a number of parameters that include optimization of media,
concentration of bacterial culture, and duration of co-cultivation, for
delivery of t-DNA in immature embryos.

Tadesse et al. (2003) optimized transformation conditions through the
biolistic approach for production of transgenic sorghum plants.
Transgenic sorghum plants were produced using immature zygotic embryos
combined with optimized transformation conditions and a strong monocot
gene promoter. Transformation efficiency was marginally higher (1.3%)
than earlier reports of transgenic sorghum (0.08 - 1%) via
microprojectile bombardment.

Girijashankar et al. (2005) reported successful recovery of transgenic
sorghum plants by particle bombardment of shoot apices and production of
transgenic plants, with a transformation frequency of 1.5%. Though
production of transgenic plants using shoot apices or meristems reduces
the time involved in regenerating transgenic sorghum plants, the
associated limiting factors for their suitability for efficient
production of transgenics are the i) additional skills required for the
isolation of meristems; ii) frequent need for subculture/clippings; and
iii) possibility of production of transgenic chimeras.

Crop improvement traits

Sorghum is chiefly grown in low input conditions; therefore, development
of host plant resistance to biotic and abiotic stresses is a viable
option. Demand for sorghum as a health food is gaining importance, and
thus, incorporation of certain value added traits is advantageous to the
food industry.

Worldwide, sorghum producers face a major threat to their crops from
insect pests, and the most destructive pests are the lepidopteran stem
borer (Chilo partellus) and the dipterans, midge (Stenodiplosis
sorghicola) and shoot fly (Atherigona soccata). Building resistance
through conventional breeding is limited due to a lack of reliable
resistance sources. Insecticidal crystal proteins (CRY) from Bacillus
thuringiensis are very effective against the lepidopterans and
dipterans. Bt and other genes with insecticidal activities are being
evaluated for eventual use in transforming crops and reducing losses due
to these pests.

Girijashankar et al. (2005) produced transgenic sorghum plants carrying
a synthetic gene, Bt cry1Ac, under the control of a wound inducible
promoter from a maize protease inhibitor gene (mpi). They reported low
levels of Bt protein of 1 - 8 ng per gram of fresh leaf tissue. A
moderate level of tolerance was reported, which in turn conferred
partial protection against neonate larvae of the spotted stem borer
(Chilo partellus).

Padmaja produced transgenic plants carrying a synthetic gene Bt cry1B
under the control of the constitutive promoter maize ubiquitin (ubi) in
the parental lines of Indian hybrids via particle bombardment. Some of
the events are promising in insect bioassays with 80% larval mortality
compared to non-transformed control plants (Padmaja, personal communication).

The agronomically important gene chi II, encoding rice chitinase under
the constitutive CaMV 35S promoter, has been transferred to sorghum for
resistance to stalk rot (Fusarium thapsinum) by Zhu et al. (1998) and
Krishnaveni et al. (2001).

Trials are also underway to engineer sorghum to withstand abiotic stress
conditions, such as drought and salinity. Efforts are in progress to
transfer genes mtlD, p5CSf129A, and codA to Indian sorghum genotypes for
biosynthesis of osmoprotectants. Expression of these genes leads to
accumulation of osmolytes, resulting in tolerance to various abiotic
stresses (Maheshwari et al., personal communication). Overexpression of
the gene for mannitol-1-phosphate dehydrogenase (mtld) for biosynthesis
of mannitol enhances tolerance to water deficit stress, primarily
through an osmotic adjustment that improves growth of transgenic plants
under water stress and salinity. The p5CSf129A gene codes for
pyrroline-5-carboxylate synthase, which catalyses the first two steps of
proline biosynthesis in plants. codA codes for choline oxidase, which
converts choline into glycine betaine.

Sorghum grain is loaded with starch and is relatively poor in protein
and lipid. Tadesse and Jacob (2003) introduced the dhdps-raec1 mutated
gene, which encodes an insensitive form of dihydropicolinate synthase,
the key regulatory enzyme of the lysine pathway. Overexpression of the
gene produces sorghum lines with elevated lysine content. Enrichment of
the essential amino acid lysine in sorghum grain improves nutritional
quality. Efforts are also underway to transfer the high molecular weight
(HMW) wheat glutenin gene 1Ax1 into sorghum to alter dough quality to
meet demands from the bakery industry (SV Rao, personal communication).

Biosafety concerns

There are no transgenic sorghum crops under commercial cultivation to
date. The most important issue related to biosafety concerns in sorghum
is pollen-mediated gene flow to the wild species Sorghum halepense
(Johnsongrass), a wild weedy relative, reported to occur naturally at
frequencies of 2.5% at a distance of 13m (Schmidt and Bothma, 2006). The
concern is that transfer of the herbicide tolerance gene to S. halepense
through gene flow would make control of the weed unattainable. Godwin
(2005) reported that hybridization of S. halepense (2n=40) and
cultivated sorghum, S. bicolor (2n=20), would produce unviable
triploids. Transgenic technology in sorghum is at a juvenile stage.
Recently Gao et al. (2005) established transgenic technology using the
positive selectable marker gene pmi (phosphate mannose isomerase), which
is biosafe and found widely in other crops.

Future prospects

Despite the use of other monocot promoters such as rice actin (act-1)
and maize ubiquitin (ubi1), use of native promoters in sorghum may be
explored if it helps to increase the levels of transgene expression. In
the eukaryotic genome, DNA elements called scaffold/matrix attachment
regions (MARs) are primarily involved in structural and functional
organization. They are thought to influence gene expression, and
evidence from other transgenic crops reveals that these sequences, when
flanking the transgene, result in enhanced expression of the integrated
gene(s). Research in making potentially well-defined synthetic MARs and
improving stable transgene expression in sorghum are areas of immediate
attention. Construction of a detailed genetic map of sorghum is
underway, and it offers a wealth of genomic tools to the sorghum
scientific community with great potential to improve sorghum (Bedell et
al, 2005). Financial assistance from non-governmental organizations like
the Bill and Melinda Gates Foundation, Andhra Pradesh-Netherlands
Biotechnology Programme (APNLB), and CGIAR grants, in addition to public
sector support, to improve sorghum nutritional quality with enhanced
levels of vitamins, minerals, and protein, and also to withstand biotic
and abiotic stresses will improve sorghum transgenic technology and lead
to increased quality and productivity in the coming years.


Bedell JA, et al. (2005) Sorghum genome sequencing by methylation
filtration. PLoS Biol 3,e13
Gao Z, et at. (2005) Agrobacterium tumefaciens-mediated sorghum
transformation using a mannose selection system. Plant Biotechnology
Journal 3, 591-599
Girijashankar V, et at. (2005) Development of transgenic sorghum for
insect resistance against the spotted stem borer (Chilo partellus) Plant
Cell Rep 24, 513-522
Godwin ID (2005) Sorghum genetic engineering: Current status and
prospectus. Pages 1-8 in Sorghum tissue culture and transformation. N.
Seetharama and Ian Godwin, eds. Oxford & IBH Publishing Co. Pvt. Ltd,
New Delhi
Howe A, et at. (2006) Rapid and reproducible Agrobacterium-mediated
transformation of sorghum. Plant Cell Reports 25, 751-758
Krishnaveni S, et at. (2001) Transgenic sorghum plants constitutively
expressing a rice chitinase gene show improved resistance to stalk rot.
Journal of Genetics and Breeding 55, 151-158
Tadesse Y, et at. (2003) Optimization of transformation conditions and
production of transgenic sorghum (Sorghum bicolor) via microparticle
bombardment. Plant Cell Tissue Organ Cult 75, 1-18
Zhao Z, et at. (2000) Agrobacterium-mediated sorghum transformation.
Plant Mol Biol 44, 789-798
Zhu H, et at. (1998) Biolistic transformation of sorghum using a rice
chitinase gene. Journal of Genetics and Breeding 52, 243-252

KBRS Visarada, N Sai Kishore
NRC for Sorghum (ICAR)
Rajendranagar, Hyderabad-500 030
AP, India

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