Indian Journal of Experimental Biology
Vol. 38, January 2000, pp. 6-17
Review Article
Transgenic strategies for genetic improvement of Basm~ i rice
Rajinder K. Jainl* & Sunita Jain 2
IDepartment of Biotechnology and Molecular Biology, 2Department of Biochemistry, CCS Haryana Agricultural University,
Hisar 125004, India
Transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of Basmati
rice because they enable the introduction of one or more genes into a leading cultivar without affecting its genetic background. During the last ten years, a rapid progress has been made towards the development of transformation methods in
rice. Several transformation methods including Agrobacterium, biolistic, and DNA uptake by protoplasts, have heen employed to produce transgenic rice. An array of useful genes is now available and many of these have already been transferred
in rice to improve the resistance against biotic and abiotic stresses. In Basmati rice, a beginning has already been made regarding the development of tissue culture protocols, transformation methods and production of useful transgenic plants. The
application and future prospects of transformation technology to engineer the resistance against insect pests (stem horer, leaf
folder, brown plant hopper, gall midge), fungal diseases (blast, bakanaelfoot. rot), bacterial di seases (bacterial leaf blight ,
sheath blight), abiotic stresses (salinity and drought) and improved nutritional quality (accumulation of pro vitamin A and
essential amino acids in endosperm) in Basmati rice, have been addressed.
Introgression of specific genes/ traits from various
genetic resources into Basmati rice through conventional breeding based on sexual hybridization, genetic
recombination and selection, has been cumbersome,
time consuming and expensive. Transformation approaches offer an attractive alternative to the conventional techniques to introduce one or more novel useful genes to improve Basmati rice without disrupting
their otherwise desirable genetic make-up. Improvement in the transformation ' technology for rice including indicas, has been remarkable in the past few
years l •2 • In addition to the direct gene-delivery methods (protoplast transformation, biolistic, electroporation), rice can also be transformed efficiently using
the Agrobacterium method. Genes for several important traits are now available that can be transferred in
to Basmati rice to improve its resistance/tolerance
against insect pests, fungal diseases, drought and salinity and to improve its nutritional quality . This review article deals with the application of transgenic
technology for the genetic improvement of Basmati
rice.
Basmati rice breeding: present status,
research priorities and problems
Among the various aromatic rice types available
throughout the world, Basmati rice is the most pre*Fax: 91-1662-33884
E-mail: [email protected]
ferred 3-5 . Basmati rice is characterized by superfine
long slender grain, exquisite aroma, sweet taste, soft
texture, delicate curvature and extra elongation with a
least breadth-wise swelling on cooking. Cooked Basmati rice is non-sticky, and has longer shelf life and is
easily ' digestible. Cooking and eating qualities of
milled rice are mainly due to its specific starch properties; t 6-25 % amylose content, intermediate to low
starch gelatinization temperature, and medium to soft
gel consistency. Basmati rice commends high premium in India as well as in International market. The
value of export of Basmati rice from India has increased from t 53 million US $ in 1986-87 to 337
million $ in 1996-97. This accounts for a 5% of the
total export of agricultural and allied products in
t 996-97. The Basmati rice cultivars have long grains
and a morphologically distinct phenotype that has
been classified as part of the indica subspecies. However, isozyme data have shown that Basmati rice varieties (Group 5) are closer to japonica (Varietal
6
Group 6) than indica (Group I ) rice varieties . Cultivation of Basmati rice is confined to the northwestern
states of India (Haryana, Punjab, Himachal Pradesh ,
Western Uttar Pradesh and Jammu and Kasmir) and
Pakistan.
Basmati rice varieties have a poor plant type (tall
plant stature, light green and non-erect leaves) and are
photoperiod sensitive and poor yielding. Basmati rice
is host to a number of insect pests and diseases3.4.
JAIN & JAIN: TRANSGENIC STRATEGIES FOR GENETIC IMPROVEMENT OF RICE
Blast and bakanae/foot rot are important among the
fungal diseases while stem borer and leaf folder are
major insect pests. Though susceptible, incidence of
bacterial leaf blight and sheath blight has been rare as
these disease results from the use of nitrogenous fertilizers in higher doses, a practice, which is otherwise
detrimental to Basmati crop because of lodging. No
major outbreak of tungro, dwarf and stunt viral diseases have been reported in most of traditional Basmati rice varieties. However, not much data is available to show that Basmati rice varieties have true resistance against these viral diseases. Basmati varieties
are moderately susceptible to sheath rot. Insect pests,
such as yellow stem borer and leaf folder, brown
plant hopper and gall midge invariably affect Basmati
and cause huge yield losses. However, some of the
Basmati rice varieties have been reported to be resistant to white backed plant hopper. Basmati rice collections are found susceptible to all major abiotic
stresses (drought, salinity).
Basmati rice breeding has been difficult due to
complicated nature of quality traits and poor combining ability of Basmati rice varieties. The inheritance of grain quality is very complicated due to
epistasis, maternal and cytoplasmic effects, and triploid nature of endosperm? When Basmati rice varieties are crossed with dwarf, high yielding indica rice
parents, the hybrids show a high degree of sterility
thus preventing many desirable segregants to appear
in the segregating population. The genetics of each of
the Basmati grain quality components (aroma, intermediate amylose content, intermediate gel consistency and high grain elongation) may not be very
complex but to find a desirable segregant possessing
all these components require screening of segregants
at a very large scale. The grain quality traits are also
tightly linked to poor Basmati rice plant type. The
breeding strategy adopted to combine Basmati rice
grain quality and improved plant type and yield potential, has been quite cumbersome. It involved the
crosses between the Basmati rice and dwarf, higher
yielding non-Basmati parents and the selection of
lines with improved plant type having one or two
components of Basmati grain quality. These lines
were then intercrossed to develop lines that have
higher yield potential with all the components of
Basmati grain quality. To give an example, this
breeding approach has been used to develop Pusa
Basmati I (a semi-dwarf, relatively photoperiod insensitive, high yielding Basmati variety) from the
7
Pusa 150 (indica) x Karnallocal (Basmati) crosses .
Application of transgenic technology
in Basmati rice breeding
A rapid progress has been made towards the development of genetic transformation technology in rice.
Initially, rice transformation was carried out by the
direct gene-delivery methods such as DNA uptake
into protoplasts by polyethylene glycol (PEG) treatment or electroporation and microprojectile bombardment. Hiei et af. 8 reported that rice could also be
efficiently transformed
using Agrohacteriummediated gene transfer method . Most success regarding rice transformation has been achieved in case
of japonica rice cultivars where a number of potentially useful genes have been transferred and in some
cases ttansgenic plants have been field tested for a
number of years. Subsequently, a number of indica,
javanica and Basmati rice varieties have also been
transformed. Potentially useful genes has been isolated that may have a function in improving resistance
against various biotic and abiotic stresses that severely affect the plant productivity lJ . Many of these
genes have already been transferred in model plant
species like tobacco, Arahidopsis, or rice and the
transgenic plants have been field-tested for transgenic
trait. Transformation technology can greatly
strengthen Basmati rice breeding programs by transferring these novel genes to improve its resistance
against insect/pests, fungal diseases, salinity, drought,
etc. The other advantage of transformation is that a
useful trait for example resistance to yellow stem
borer, 'can be engineered in Basmati rice without altering its genetic background/quality traits which has
been difficult to achieve through sexual hybridization
involving crosses with the non-Basmati rice varieties .
A. Plant regeneration from cells, tissues and
protoplasts
Before targeting a particular rice variety to ill vitro
genetic transformation, there is a need to develop efficient procedure(s) for regeneration of green, fertile
plants from explant tissues, cells or protoplasts with
minimal somaclonal variation. Indeed, it is the totipotency of plant cells that underlies the efficiency of
most plant transformation systems. Japonica rice varieties have shown to be more responsive to in vitro
culture and plant regeneration than indica rice varieties. As a result, it has been easier to transform japonica rice varieties. However, a lot of research has
8
INDIAN J EXP BIOL, JANUARY 2000
been done to develop tissue culture protocols in indica rice varieties to achieve a workable efficiency of
gene transfer, selection and regeneration of transformants. Most success has been achieved in this regard
by using either the explants such as immature embryos with many regenerable cells or freshly initiated
scutella-derived embryogenic calli . The choices were
made based on the easy and round the year availability of the explant tissues with the option to keep tissue culture regime to the minimum. Detailed consideration of optimization of tissue culture systems useful for rice transformation is beyond the scope of this
review, but the progress made and protocols developed for rice tissue culture, have been addressed in
several reviews and technical manuals l,lo I2.
A number of factors such as the plant genotype and
explant type, composition of the medium including
carbohydrates and growth regulators, presence of
nurse cells, partial desiccation, cryopreservation and
water stress treatments, have been examined in attempts to break the recalcitrance of Basmati type indica rice varietiesl3018. In Basmati rice, immature embryos and embryogenic calli or cell suspensions initiated from mature/ immature seed scutella and microspores, have been used for in vitro genetic manipulation experiments. In general, addition of 2,4-0 to the
medium causes callus induction and proliferation in
rice explants, cells and tissues, whereas plant regeneration occurred in either hormone-free media or media containing a combination of auxin (NAA) and
cytokinin (BAP or kinetin). Shoots are generally
rooted in hormone-free media or an auxin (NAA)
containing medium.
o
Jain et al. 13 reported an improved procedure for the
protoplast culture of several rice varieties including
Pusa Basmati I; the procedure involved plating of
protoplasts isolated from embryogenic cell suspensions on the surface of filter membranes overlaying
agarose-embedded feeder cells of OryZG ridleyi and
Latium muitiflorum. Nurse cultures were essential for
protoplast culture. L. muitiflorum feeder cells induced
a 6-fold higher plating efficiency than feeder cells of
O. ridieyi; the two types of feeder cells when used in
combination showed an additive effect on protoplast
plating efficiency. However, somaclonal variation
and sterility problems marred the application of the
protoplast system for transgenic plant production.
Jain et al. 14 reported maltose to be preferential carbon
source compared to sucrose, glucose, fructose, etc. for
both somatic embryogenesis and plant regeneration
from cell suspension and protoplast-derived calli of
Basmati rice varieties. Jain et ai. 16 reported severalfold increase in frequency of shoot regeneration fol~
lowing water stress treatment (partial desiccation for
24 hr, use of 1.0 % agarose instead of 0.5 % for medium solidification or O.4M mannitol-containing medium) of cell clumps in Pusa Basmati and Basmati
385 .. Best regeneration frequencies (54-98 %) were
obtained when 24 hr-desiccated calli were grown 011
15
regeneration with 1.0 % (w/v) agarose. Jain et al.
reported a simple freeze preservation procedure for
the cryopreservation of embryogenic cells of Pusa
Basmati I and Basmati 385; the cryopreservation
treatment also led to the enrichment of embryogenic
cells. Khanna and Raina l!) reported that ill vitro green
plant regeneration efficiency could be greatly enhanced through modification of nitrate-nitro~en and
ammonium-nitrogen concentrations in the callusing
medium. The highest frequen cy of plant regeneration
(100%) and a maximum number green plants (7 per
callus) were obtained in calli derived from the medium having 35 mM KNO~ and 5 mM (NH 4 h SO.j.
20
Bishnoi et al. reported efficient production of androgenic calli in several Basmati and non-Basmati
indica rice varieties and their FI . hybrids/ F:~ plants
using an improved anther culture medium (modified
RZ21 medium). The calli regenerated green shoots
with higher frequencies suggesting that androgenic
calli can also be used as the starting material for
transformation.
B. Gene transfer methods
Protoplast-based direct gene transfer. biolistic
transformation and Agrobacterium-mediated gene
transfer are the major techniques that are being routinely used for transgenic plant production in rice.
nn
Other methods such as tissue electroporation . , laser mediation 24 , pollen tube pathwa/' , DNA uptake
by imbibition of dry embryos26, silicon carbide fibermediated transformation 27 , etc . have been reported to
produce the transgenic rice plants but have not found
widespread use up to now. Several excellent re.
I 2
.
vIews'
an d Iaboratory manua Is P-.-ox on trans fO
ormatIOn
methods have been published. There are still many
problems that have to be solved in terms of reproducibility . Most of the above mentioned methods
could deliver DNA into the cell, but the subsequent
events in cells, nuclei and organelles are not controlled and the integration of foreign DNA is random.
Gene silencing and interactions between multiple
JAIN & JAIN: TRANSGENIC STRATEGIES FOR GENETIC IMPROVEMENT OF RICE
copies of the same trans gene or different transgenes
result in unexpected expression pattern of foreign
genes 29 -3J _ Several independent transformants with a
specific gene cassette are necessary, to find one
transgenic plant with the desired transgene expression
pattern and level.
Protoplast-mediated trallsjormatiofl-The first approach to transfer foreign DNA into rice cells was the
direct gene transfer into protoplasts. Both polyethylene glycol (PEG) and electroporation methods have
been used for rice transformation . Protoplasts are the
ideal material for gene transfer. While the delivery of
DNA into protoplasts has been quite easy, major
problems relate to the establishment of embryogenic
cell suspension cultures (source of protoplasts) and
regeneration of green, fertile plants from protoplasts.
Protoplast culture and regeneration procedure(s) are
quite labor intensive and time consuming; for example it may take up to six months to establish embryogenic cell suspensions that are ideal for protoplast
isolation. This method is strongly genotype dependent
and has not been worked out for many elite indica
rice varieties. Jain et at. J3 reported that protoplast
culture in two indica rice varieties, Pusa Basmati I,
Jaya, required the use of specific nurse cultures. In
many cases, transgenic rice plants produced through
protoplast transformation were reported to be sterile,
carry extensive somaclonal variation, or multiple
copies of the transgene. Inspite of these limitations,
protoplasts have been successfully used for the production of transgenic plants containing agronomically
useful genes in some responsive japonica and indica
rice varieties 34 .35 (Table I). This method of transformation is no longer a method of choice for rice transformation. However, protoplasts may have an advantage to accomplish the transfer of large segments of
DNA and organelles into a single cell.
Biolistic trallsjormation-Biolistic method employs high-velocity metal particles to deliver biologically active DNA into regenerable plant cells, tissues
and organs. This method depends far less on sophisticated tissue culture procedures and provides a fairly
genotype-independent transformation system bypassing Agrobacterium host specificity and protoplast-related regeneration difficulties. Chances of tissue culture raised somaclonal variation can also be
minimized using the explants exhibiting direct shoot
regeneration with a minimum of callusing phase. The
transformation of several elite indica rice vanetles
became possible due to the extended range of cell
9
tissues/ explants that can be targeted by microprojectiles. In rice, biolistic method has been successfully
used for the transfer of many useful genes in over 40
japoniea, indica and Basmati rice varieties'6 (Table
I). In most cases, embryogenic calli or suspension
cells derived from immature and mature embryos,
were used as 'the target tissue. Mostly, ~us gene has
been used as the reporter gene and hygR and har genes
have been used as selectable marker genes for bioi istic transformation. A number of factors including osmotic pre-conditioning of the target tissues, nature
and size of metallic particles used for DNA delivery,
humidity and temperature, DNA loading on microprojectiles, target cell distance, accelerating force and
depth of penetration, have been reported to influence
the biolistic transformation frequency 10,37. Biolistic
has also been used for organelle transformation especially the plastids which is an attractive target for
crop engineering due to the high level transgene expression,g,39. Besides plasmids, RNA, Y AC (yeast
artificial chromosome) DNA clones, or even E. coil
or Agrobacterillll1 cells have been successfully used
for biolistic transformation 40 . Biolistic method' has
some drawbacks including the high cost of the
equipment and consumables, integration of transgene
in high copy number, transgene rearrangement, and
gene silencing.
Jain et al. 17 • IS reported an improved, reproducible
biolistic procedure for the transformation of embryogenic suspension cells/calli of Pusa Basmati I. The
procedure involved the osmotic pre-conditioning of
cells for 24 hr on a medium supplemented with 0.25M
mannitol prior to bombardment, use of gold particles
for DNA delivery, and use of plant regeneration medium with 1.0% agarose. This procedure consistently
produced over 600 transient transformants and at
least five fertile plants showing integrative transformation per bombarded filter. Minhas et al. 41 reported
the optimum parameters for the biolistic introduction
of GlIS (uidA) reporter gene into embryogenic callus
cultures of Basmati 370 rice variety . Khanna et al.42
reported the biolistic procedure for the transformation
of a Basmati rice cultivar Kamal Local lIsing the
plasmid carrying Gus and bar gene cassettes, five-day
precu1tured mature embryos and glufosinatecontaining liquid medium for selection,
Agrobacterium -mediated trallsjormation-Severalmonocots including rice, are now among the plants
that can be transformed routinely by A, tumejaciens.
10
INDIAN J EXP BIOL, JANUARY 2000
Table I-A list of useful genes transferred in rice
Useful trait! Gene
Transformation method
Rice
subspeices
Reference( s)
Insect and pest control
Bt, S-endotoxin Cry/Arb)
Cry/Arb)
Cry/Arb)
Cry/Arb)
Cry/Arb)
Cry/Arb) & Cry/Arc)
Cry/A (c)
Cry2A
Protoplast electroporation
Biolistic
Biolistic
Biloistic
Biolistic
Agrobacterium
Biolistic
Biolistic
Japonica
Indica
Aromatic Iranian
Indica, Japonica
Japonica
Japonica
Indica
Indica, Basmati
56
57.103
60
5H
104
59
105
61
Potato protease inhbitor II (Pin II)
Cowpea trypsin inhibitor (CpTi)
Corn cystatin (CC)
Oryzacystatin (Oc)
Biolistic
PEG-Protoplast
Protoplast electro po ration
Protoplast transformation
Japonica
Japonica
Japonica
Japonica
62
63
35
64
Snowdrop lectin (GNA)
Protoplast-electroporation
Japonica
67
Biolistic
Biolistic
Japonica
Indica
75
76. 106
Fungal diseases (sheath blight)
Chitinase gene, Chil /
PEG-Protoplast
Indica
34
Fungal diseases (Pyricu/aria oryzae)
Grapevine stilbene synthase gene
PEG-Protoplast
Japonica
70
CodA
Adc
GPAT
Biolistic
Biolistic
Biolistic
Agrobacterium
biolistic
Agrobacterium
Japonica
Japonica
Basmati
Japonica
Japonica
Japonica
81
83
17. IX
39
86
107
Resistance against viral diseases
RNA-2, -3 & capsid genes from BMV
Outer coat protein (S8) gene of RDV
RTSV coat protein genes
RNA-dep. RNA Polymerase ofRYMV
Protoplast electroporation
Biolistic
Biolistic
Biolistic
Japonica
Japonica
Indica, Japonica
African Indica
lOX
95
94
109
Nematode resistance
OC-ltID86
Biolistic
African Indica
65
Nutritional quality
Daffodil phytoene synthase gene
Biolistic
Japonica
96
Dwarfism
Rgpl
Protoplast electroporation
Japonica
9H
Photosynthetic efficiency
PEPC (maize)
Agrobacterium
Japonica
110
Bacterial leaf blight resistance
Xa2/
Salt, drought and cold tolerance
P5CS
HVA/ (LEA3)
Adc, arginine decarboxylase, CodA, choline oxidase gene from a soil bacterium Arthrobacter globiformis; HVA /. Barley late embryogenesis-abundant (LEA 3) protein gene; OC-ltID86, an engineered cysteine proteinase inhibitor (Oryzacystatill-ltID86) gene: P5CS.
t1 1-Pyrroline-5-carboxylate synthetase; Rgpl, a small GTP -binding protein gene from rice; RTSV. rice tungro spherical virus ;
RYMV, rice yellow mottle virus.
JAIN & JAIN: TRANSGENIC STRATEGIES FOR GENETIC IMPROVEMENT OF RICE
II
cultivars including Pusa Basmati I using the matureAgrobacterium-mediated gene transfer system is beseed scutella derived calli and Agrobacterium strain
coming the method of choice in rice due to its low
LBA4404 (pTOK233). Based on the total number of
costs, convenience, high transformation efficiency,
calli co-cultivated, the transformation frequency of
high probability of single copy integration with
independent transgenic Pusa Basmati rice plants was
minimal rearrangements, the transfer of relatively
13.5%. Data from Southern hybridization analysis
large segments of DNA and high fertility of trans8
proved that foreign genes on T-DNA were stably ingenic plants. In 1994, Hiei et al. reported an efficient
tegrated into rice genome at low copy/ site numbers.
Agrobacterium-mediated transformation procedure in
Jain 49 observed a positive correlation between emjaponica rice. Since then a rapid progress has been
bryogenic potential and regeneration capacities of
made towards the development of AgrobacteriumBasmati rice calli and Agrobacterium (strain
mediated transformation protocols in various rice vaLBA4404 with pTOK233)-mediated transformation
rieties including indicas and several useful genes have
frequencies using the modified Hiei et al. x procedure.
been transferred using this approach43 (Table I). In
general, the procedure involved the cocultivation of
Pusa Basmati I calli in comparison to those of Taraori Basmati had higher regeneration potential and
actively dividing, embryogenic cells such as immaconsequently also showed higher transformation freture embryos, isolated shoot apices and calli induced
quency. Regeneration potential also varied with the
from scutella in the presence of acetosyringone,
age of callus cultures. In Pusa Basmati I, six-weekwhich is a potent inducer of the virulence genes. Sevold micro-calli obtained after one subculture of maeral Agrobacterium strains such as LBA4404
ture seeds, had the maximum regeneration potential
(pT0K233), EHAlOI (pIGI2IHm), and EHAlOl
and were found . to be best for Agrobacterium(pSMABuba), have been used for rice transformation.
50
A series of improved binary vectors have been conmediated transformation . We used a novel Agrostructed that are suitable for monocot transformation.
bacterium-mediated transformation system for the
These binary vectors have either bar or hph resistance
introduction of useful insect-resistant and drought
R
genes as selection markers. Hph gene .has been imtolerant genes into Basmati rice. The procedure inproved by the introduction of an intron into its coding
volved the mobilization of pCAMBIA vectors carrying a cloned useful gene cassette into Agrobacterium
so as to abolish its expression in A. tumefaciens, renstrain LBA4404 carrying pSB I plasmid (lacks Tdering the bacterium susceptible to the hygromycin
B44. In addition, binary bacterial artificial chromoDNA but carries virulence genes as in case of
some (BIBAC) vector has been developed that is capTOK233) by tri-parental mating. The resultant
pable of transferring at least 150 kb of foreign DNA
strains have been used successfully for Pusa Basmati
45
46
into plant nuclear genome . . The BIBABC vector
1 transformation 5o .
has the minimal origin of replication for both E. coli
F and A. rhizogenes Ri plasmids and it replicates as a
Potentially useful genes that can improve the
single copy in E. coli as well as A. tumefaciens. The
Basmati rice
large BIBAC T-DNAs in conjunction with the helper
Since the first report of fertile transgenic plant proplasmid that carries additional copies of virulence
duction in 1989, remarkable progress has been made
genes, have been used for high frequency transformatowards the transfer of useful genes in japonica as
tion. The ability to introduce high molecular weight
well as indica rice varieties. A variety of useful genes
DNA into plant chromosomes should accelerate the
conferring resistance against abiotic and biotic
studies on gene identification, genome organization
stresses have been transferred in rice and these have
and genetic engineering of complex polygenic traits.
been summarized in Table I. The Table do not conRashid et al. 47 reported the production of genetitain the exhaustive list of the reports on rice transcally stable transgenic plants of three Basmati rice
formation, but it serves to illustrate the type of genes
varieties (Basmati 370, Basmati 385 and Basmati ' . that can be 'used for the genetic improvement of Bas- , .
6129) essentially using the Hiei et aL 8 transformation
.mati rice. The breeding objectives in Basmati rice
procedure. They used acetosyringone at 50' ~ conthat can be well addressed using the transgenic technology and the traits for which novel genes are availcentration (instead of I 00 ~ for cocultivation.
48
able, are described as under:
Zhang et al. reported A. tumefaciens-mediated transformation of . several commercially important rice
Resistance against insectlpests-The 8-endotoxin
c.
12
INDIAN J EXP BIOL. JANUARY 2000
crystal protein genes from Bacillus thuringiensis -and
plant defensive genes, protease inhibitors and lectins,
are among the insect resistance genes 51 -55 that have
been transferred in japonica and indica (including
Basmatij rice varieties (Table I). The transgenic
plants have been reported to be genetically stable and
display the improved resistance against a variety of
stem borers, leaf-hoppers or leaf folder insects.
B.thuringiensis 8-endotoxin crystal (cry) proteins
are part of a large and still growing family of homologous proteins. About 130 cry genes have been
identified to date. Most Bt crystal proteins are synthesized in a protoxin form and are proteolytically converted into smaller toxic polypeptides in the insect
midgut. These toxins kill insects by binding to midgut
membranes causing lesions. Cry genes have been recently classified on the basis of amino acid homology
into four major classes, cry /, cry2, cry3 and cry4,
which are, respectively, specific for insects belonging
to Lepidotera (caterpillars), Lepidotera and Diptera
(flies and mosquitos), Coleoptera (beetles) and Diptera. In rice, cry genes belonging to first two classes
have been used for transgenic plant production. Fujimoto et al. 56 produced the transgenic japonica rice
plants containing truncated and codon-modified 8endotoxin gene using the protoplast-electroporation
method. The R2 generation transgenic plants were
more resistant to striped stem borer (SSB) and leafhopper than the non-transgenic plants. Wiinn et al. 57
reported increased insecticidal effect on several lepidoteran insect pests (yellow stem borer, striped stem
borer and leaf folder) in the Ro, RI and R2 generation
transgenic IR58 plants expressing the synthetic
cry 1A(b) gene. Datta et al. 58 produced transgenic
plants of several indica and japonica rice varieties
containing cry/Arb) gene driven by either constitutive
(CaMV35S, Actin I) or tissue-specific (PEP carboxylase, pith specific) promoters using the biolistic
and protoplast transformation systems. The transgene,
crylA, driven by different promoters showed a wide
range of expression (low to high) but conferred enhanced resistance to yellow stem borer (YSB). Out of
800 Southern-positive plants that were bio-assayed,
81 transgenic plants showed 100% mortality of insect
larvae of the YSB . Cheng et al. 59 reported the production of over 2600 transgenic j.aponica rice plants
containing the cry/Arb) or cryJA(c) synthetic gene
driven by maize ubiquitin, CaMV35S and Brassica Bp
10 gene promoters. The transgenic Ro plants accumulated the cry/Arb) and cry/Arc) protein at higher
levels (up to 3% of soluble protein). The RI generation transgenic plants were highly toxic to SSB and
YSB with mortalities of 97-100% after 5 days of infestation. Ghareyazie et al. 60 reported the transfer of
the cry/Arb) gene driven by the tissue-specific maize
C 4 PEP carboxylase gene promoter in an Iranian aromatic rice variety Tarom Molaii belonging to group
V. The transgene was expressed in the leaf blades but
was not expressed to a detectable level in dehulled
mature grain . Transgenic plants showed enhanced
resistance against to first-instar larvae of SSB and
YSB. Maqbool et al. 61 reported the production of
transgenic Basmati 370 and M7 plants expressing the
cry2A gene via biolistic method. The gene product
was expressed up to 5% of total leaf protein and the
transgenic plants had higher insecticidal activity
against YSB and rice leaf folder.
Another classes of genes that have been used for
improving insect/pest resistance are the protease inhibitor or lectin genes of plant origin. whose expression have shown anti-metabolic effects against certain
insects. Expression of protease inhibitor genes have
been useful for the control of insects which feed by
chewing plant tissues, such as insects belonging to
Lepidotera and Coleoptera, while that of lectin genes
have been toxic to the sap-sucking insects belonging
to the order Homoptera (brown plant hopper, green
plant hopper). Duan et al. 62 and Xu et al. (,:1 produced
transgenic japonica rice plants containing potato
proteinase inhibitor II (Pinll) and cowpea trypsin inhibitor (CpTi) genes, respectively. The transgenes
were driven by wound-inducible Pinll gene promoter
with rice actin 1 intron and rice actin I promoter, respectively. Transgenic plants had high level accumulation of the PinIII CpTi proteins and their progenies
showed increased resistance against pink stem borer
and/or SSB. Hosoyama et al. 64 and Irie et al. 1 ) produced 'transgenic rice plants containing oryzacystatin
(Dc) and corncystatin (Cc) genes, respectively, which
showed potent inhibitory activity against the cysteine
proteinases that occur in the gut of insect pests. Vain
et al. 65 reported increased nematode resistance in
transgenic plants containing an engineered cysteine
proteinase inhibitor (Oryzacystatin -/t1D86) gene.
These reports clearly indicate that proteinase inhibitor
genes can be used to engineer the resistance against
insect pests and nematodes in rice.
Among the various plant lectins, snowdrop lectin
(Galanthus nivalis agglutinin: GNA) has been reported to be most toxic to the brown plant hopper
JAIN & JAIN: TRANSGENIC STRATEGIES FOR GENETIC IMPROVEMENT OF RICE
(BPH) and non-toxic to mammals 66 • GNA is a tetrameric protein consisting of identical sub-units of 12
kD. Rao et al. 67 reported the production of transgenic
rice plants containing snowdrop lectin gene driven by
either phloem-specific rice sucrose synthase-l gene
promoter (RSsl; Shi et a1. 68 ) or the constitutive maize
ubiquitin-l promoter (ubi). Phloem-specific promoters have been used to specifically express this gene in
phloem tissue of transgenic rice plants conferring resistance against sap-sucking insects. Some of the
transgenic plants had GNA at levels of up to 2.0% of
total protein. Insect bioassays and feeding studies
showed that GNA expressed in the transgenic plants
decreased the insect survival rate, retarded insect development, and had a deterrent effect on BPH feeding.
Resistance against fungal diseases-Rice blast
(Pyricularia grisea, oryzae) and Bakanae/foot rot
(Fusarium moniliforme) are the two major fungal diseases that cause severe yield losses in Basmati rice4 .
In response to fungal attack, plants synthesize an assortment of new proteins commonly known as pathogenesis-related (PR) proteins including chitinases and
~-1 ,3-glucanases34,69. Chitinase preparations alone
and in combination with ~-1,3-glucanases, have been
reported to inhibit fungal growth in vitro. Transgenic
approach based on constitutive expression of a
chitinase gene in transgenic tobacco have been shown
to result in an increased ability to survive in soil infested with fungal pathogens and delayed develop69
ment of disease symptoms . Lin et al. 34 developed
transgenic indica rice plants that expressed chitinase
gene constitutively. The plants with high levels of
chitinase exhibited increased resistance to infection
by the sheath blight pathogen, R. solani.
Stark-Lorenzen et al.70 reported active transcription
of grapevine stilbene-synthase gene in transgenic rice
plants after incubation with the fungus of the rice
blast P. oryzae. Preliminary results indicated an enhanced resistance of the transgenic rice to P. oryzae.
Stilbene synthase in some plant species synthesize a
phytoalexin trans-resveratrol that seems to have a role
in the early protection of plants against fungal pathogens. The effectiveness of stilbene synthase genes in
enhancing resistance to another fungal pathogen ,
Phytophthora infestans, has been demonstrated in
transgenic tomatoes 71 • Tada et al. 72 reported that
transgenic rice plants expressing bar-gene showed
decreased symptoms of rice blast disease caused by
Magnporthe grisea following bialaphos treatment.
13
Resistance against bacterial leaf blight-Basmati
rice is also susceptible to bacterial leaf blight (BLB)
although incidence of bacterial leaf blight has been
low probably due to the non-use of nitrogenous fertilizers for Basmati rice cultivation. A significant
progress has been made towards the genetic engineering for BLB resistance in rice7.l. A dominant gene
for resistance to BLB was transferred from a wild
species, O. longistaminata, to the cultivated variety
"IR24" (Khush et al. 74). This gene designated as
Xa21, confers resistance to all known races of Xanthomonas oryzae pv. oryzae (Xoo) . Wang et aC 5 reported that transgenic
, rice plants expressing Xa21
gene conferred multi-isolate resistance to 29 diverse
isolates from eight countries indicating that a single
cloned gene is sufficient to confer multi-i solate resistance. Tu et aC6 reported higher resistance against
two prevalent races (4 and 6) of Xoo in transgenic
IR72 plants containing Xa21 gene. Resistance against
race 4 was higher due to the pyramiding of transgene
Xa21 and Xa4 (already present in IR72) . The Xa21
encodes a receptor kinase-like protein and it may
have a role in cellular signaling for plant disease resistance.
Salinity and drought tolerance-A number of
transgenic strategies are being used to increase tolerx1
ance to salinity and drought in plants 77 - . These include the late embryogenesis abundant proteins,
overproduction of enzymes responsible for biosynthesis of osmolytes, and detoxification enzymes . This
approach has met with preliminary success and there
are now several reports on transgenic plant production containing stress-tolerant genes. Since expression
of these genes individually confers marginal tolerance
to abiotic stresses, it may be necessary to pyramid
these different genes for higher level of stress tolerance. It may also be desirable to use stress-inducible
promoters instead of constitutive promoters for the
. 0 f stress to Ierant genes gry-.
expreSSIon
Late-embryogenesis abundant protein (LEA) genes
are normally expressed in the seed during maturation
stage that involves desiccation and also in vegetative
tissues during water deficit. LEA genes are induced
by ABA and by osmotic stress resulting from drought,
salinity stress, or cold temperatures. LEA proteins
ha~e long been suggested as important in water retention or protection, ion sequestration, and as molecular chaperones, although their preci se functioning
is not clear. Xu et al. 83 produced the transgen ic japonica rice plants containing barley LEA3 (HVAJ)
14
INDIAN J EXP BIOL, JANUARY 2000
gene driven by a constitutive rice actin I promoter.
The transgenic plants showed the constitutive accumulation of the HV A I protein both in leaves and
roots. The second generation transgenic plants
showed increased tolerance to both water and salt
stresses. Artus et al. 84 reported that constitutive expression of a LEA-related gene Cori5a, in transgenic
Arabidopsis plants enhanced the freezing tolerance of
both chloroplasts and protoplasts. Cor J5a expression
has been suggested to affect the cryostability of the
plasma membrane possibly through the interaction of
corISa polypeptide with lipid bilayers.
During ()smotic stress, plant cells accumulate low
molecular weight osmolytes to prevent water loss and
maintain turgor. Transgenic approach has been used
to engineer biosynthesis pathways of some of these
osmolytes. Transgenic plants containing key genes
encoding enzymes involved in the production of
mannitol, proline, fructans, trehalose, glycine-betaine,
D-ononitol and polyamines have been produced in
model plant species like Arabidopsis, tobacco and
.
.
. 777980 Th
. plants have been
Japomca
nce
. '.
e transgemc
reported to accumulate the corresponding osmolyte at
higher levels and consequently showed a marginal to
significant increase in the dehydration, salinity and/or
cold tolerance. To give a few examples in rice; Zhu et
at. 85 reported that overexpression of ABAIstressinducible promoter driven moth bean pyrroline-5carboxylate synthetase (P5CS) gene resulted in up to
2.S-fold higher proline accumulation under stress
conditions. The second generation transgenic plants
compared to the non-transgenic plants had higher
shoot and root biomass under salt- and water-stress
39
conditions. Sakamoto et al. reported that transgenic
rice plants over-expressing the choline oxidase (codA;
isolated from a soil bacterium Arthrobacter globiformis) gene accumulated glycine betaine at higher levels and were more tolerant to salt and low temperature stresses. The study also showed that such a gene
if targeted to chloroplast was more effective in improving the stress tolerance. Capell et al. 86 produced
the transgenic rice plants over-expressing the oat arginine decarboxylase (ADC) gene. The transgenic
plants showed improved drought tolerance in terms of
chlorophyll loss, however, constitutive expression of
this gene severely affected the development patterns
in vitro.
Much of the injury to plants caused by abiotic
stresses, is associated with oxidative damage at the
cellular level 87 . The production of transgenic plants
with increased capacity for detoxification and scavenging reactive oxygen intermediates (ROIs) could be
an another important strategy to engineer stress tolerance. Several groups have developed transgenic alfalfa and tobacco plants expressing a oxidative stressre Ia ted gene suc h as superoxi.d e d'Ismutase xx
" X<) ,g Iuta90
thione-S-transferase , and iron-binding protein ferritin 91 • The transgenic plants were reported to display
greater tolerance to oxidative damage, salt stress,
water deficit, and/or freezing.
Rec~ntly, Jaglo-Ottosen et ul.'!2 reported that overexpression of a regulator gene, CBF I, whose product
is a transcriptional activator, induced COR gene expression and increased the freezing tolerance in
transgenic Arabidopsis plants. Kasuga et ai.')1 transformed Arabidopsis with a gene encoding DREBIA, a
homologue of CBFI, driveQ by a stress-inducible
rd29A promoter. The overexpression"" of this gene in
transgenic plants activated the expression of many
stress-tolerance genes, rd 17 and rd29A (LEA like
proteins), Kin J, Cor 6.6, Cor 15a, and P5CS; the
transgenic plants showed greater tolerance to drought,
salt and freezing stresses. The transformation of
plants using such regulatory genes could be more rewarding to develop tolerance against complex traits
like drought and salt tolerance.
Viral resistance-Research is also in progress to
transfer genes for resistance against viruses (RTSV,
rice tungro spherical virus; RTBV, rice tungro bacilliform virus; RDV, rice dwarf virus) in rice. Sivamani
et al. 94 produced transgenic indica and japonica rice
plants .containing RTSV coat protein genes. Most of
transgenic plants, as well as their Rio R2 and/or Rl
progeny that contained the target gene, showed the
moderate levels of protection to RTSV infection and
a significant delay of virus replication. Zheng et al.')S
developed the transgenic plants expressing the rice
dwarf virus coat protein gene (S8), which is an important step towards studying the function of RDV
genes and obtaining RDV -resistant rice plants.
Improved nutritional quality-Milled rice lacks
provitamin A and is also deficient in essential amino
acids (cysteine, methionine, lysine). Insufficient dietary provitamin A leads to severe clinical symptoms
(eye disease xeropthalmia leading to blindness, increased susceptibility to other diseases such as diarrhoea, respiratory problems, measles, etc) . Burkhardt
et al. 96 reported accumulation of phytoene. which is a
key intermediate of provitamin A biosynthetic pathway, in endosperm tissue of the transgenic rice plants
JAIN & JAIN : TRANSGENIC STRATEGIES FOR GENETIC IMPROVEMENT OF RICE
expressing the daffodil phytoene synthase gene driven
by the endospenn specific promoter. Further work is
in progress to engineer the metabolic pathways for
the synthesis of provitamin A and essential art}ino
acids so as produce them in sufficient quantities in
rice endosperm97.
Dwarfism-One of the major breeding objective in
basmati rice is to develop short-statured plant type.
98
Recently, Kisaka et al. reported the production of
transgenic japonica rice plants expressing the transgene, rgpJ (encodes for a GTP binding protein).
These transgenic plants had distinct characteristics;
namely, dwarfism, early flowering and high grain
yield, and these characteristics were stable and heritable. The exact physiological function of GTPbinding proteins remains to be worked out. However,
rgpJ gene has been shown to result in the reduction
of apical dominance and high-level synthesis of cyto99
kinins in transgenic tobacc0 . It will be worthwhile
to study the impact of such GTP-binding protein
genes in Basmati rice.
Conclusions and future prospects
Basmati rice breeding has been difficult due to
complicated nature of quality traits and poor combining ability of Basmati rice varieties . Major breeding objectives in Basmati rice includes the development of short-statured, photoperiod-insensitive and
high yielding genotypes with increased resistance
against insect pests (stem borer, leaf folder, brown
plant hopper, gall midge), fungal diseases (blast, bakanae/foot rot), bacterial diseases (bacterial leaf
blight, sheath blight), and abiotic stresses (salinity
and drought). Transgenic approach holds great promise to achieve several of these objectives in Basmati
rice without affecting its desirable genetic background and traits (aroma, grain quality, cooking quality). Rice is already a model monocot plant species
for genetic transfonnation studies and is being routinely transformed by both direct gene transfer (biolistic, DNA uptake by protoplasts) and A. tumefaciens
methods. Valuable genes for improving several important traits are now available and many of these
have already been transferred in rice to improve the
resistance against insect pests, fungal and viral diseases, salinity and drought stresses. Efforts are underway to engineer biosynthetic pathways for provitamin A and certain essential amino acids to improve the nutritional quality of rice.
15
Transgenic research to engineer complex agronomic traits (e.g. yield, drought tolerance, grain quality) and metabolic or regulatory pathways involving
many genes or gene complexes, is likely to gain mo1oo
mentum in future . This is pretty evident from the
progress made towards the genome mapping and gene
tagging research in rice and other plant species, development of tissue specific or stress-inducible promoters, and development of the ARrohacterium vectors for the transfer of fairly large DNA fragments.
The problem of gene silencing can be tackled at least
partially, by preferably using the ARrohacterium
4
method of transfonnation \ selecting the transgenic
plants with only one copy of the transgene.1 l, or by
using the MAR (Matrix Attachment Regions) sequences which has been shown to reduce the position
effect)OI.lo2. Issues such as safety and environmental
concerns, public acceptance and the fees to be paid to
the legal patent owner for the use of a particular technology or product (promoter, transgene, etc .), have to
be taken into account before the application of transgenic technology.
Further research may be necessary to transform
elite, commercially important indica rice varieties
including Basmati rice varieties. A good beginning
has already been made towards the development of
tissue culture and transformation protocols in several
commercially cultivated Basmati rice varieties including Pusa Basmati I and Basmati 370. Transgenic
Basmati 370 plants containing Bt (cry 2A) gene have
been shown to be resistant to yellow stem borer and
leaf folder 61 • With the recent advances in rice molecular and transformation technologies, it should be
feasible to design various transgenic strategies for the
genetic improvement of Basmati rice and to increase
its productivity.
Acknowledgement
Thanks are due to Drs Ray Wu and M Maheswaran
for critically reading of the manuscript and di scussion . We would also like to thank the Rockefeller
Foundation, New York, USA, for providing research
grants for rice biotechnology research at CCS Haryana Agricultural University, Hisar (India) and Cornell, Ithaca, NY (USA).
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