Transgenic Rice Plants Essay, Research Paper

For centuries, rice has been

one of the most important staple crops for the world and it now currently feeds

more than two billion people, mostly living in developing countries. Rice

is the major food source of Japan and China and it enjoys a long history of

use in both cultures. In 1994, worldwide rice production peaked at 530 million

metric tons. Yet, more than 200 million tons of rice are lost each year to

biotic stresses such as disease and insect infestation. This extreme loss

of crop is estimated to cost at least several billion dollars per year and

heavy losses often leave third world countries desperate for their staple food.

Therefore, measures must be taken to decrease the amount of crop loss and

increase yields that could be used to feed the populations of the world. One

method to increase rice crop yields is the institution of transgenic rice plants

that express insect resistance genes. The two major ways to accomplish insect

resistance in rice are the introduction of the potato proteinas

e inhibitor

II gene or the introduction of the Bacillus thuringiensis toxin gene into the

plant’s genome. Other experimental methods of instituting insect resistance

include the use of the arcelin gene, the snowdrop

lectin/GNA (galanthus nivallis

agglutinin) protein, and phloem specific promoters and finally the SBTI gene.

The introduction of the potato proteinase inhibitor II gene, or PINII,

marks the first time that useful genes were successfully transferred from a

dicotyledonus plant to a monocotyledonous plant. Whenever the plant is wounded

by insects, the PINII gene produces a protein that interferes with the insect’s

digestive processes. These protein inhibitors can be detrimental to the growth

and development of a wide range of insects that attack rice plants and result

in insects eating less of the plant material. Proteinase inhibitors are of

particular interest because they are part of the rice plant’s natural defense

system against insects. They are also beneficial because they are inactivated

by cooking and therefore pose no environmental or health hazards to the human

consumption of PINII treated rice.

In order to produce fertile transgenic

rice plants, plasmid pTW was used, coupled with the pin 2 promoter and the

inserted rice actin intron, act 1. The combination of the pin 2 promoter and

act 1 intron has been shown to produce a high level, wound inducible expression

of foreign genes in transgenic plants. This was useful for delivering the

protein inhibitor to insects which eat plant material. The selectable marker

in this trial was the bacterial phosphinothricin acetyl transferase gene (bar)

which was linked to the cauliflower mosaic virus (CaMV) 35S promoter. Next

the plasmid pTW was injected into cell cultures of Japonica rice using the

BiolisticTM particle delivery system. The BiolisticTM

system proceeds as

follows:

Immature embryos and embryonic calli of six rice materials were

bombarded with

tungsten particles coated with DNA of two plasmids containing

the appropriate

genes.

The plant materials showed high frequency

of expression of genes when stained

with X-Gluc. The number of blue

or transgenic units was approximately 1,000.

After one week, the transgenic

cells were transferred onto selection medium

containing hygromycin

B. After two weeks, fresh cell cultures could be

seen on bombarded

tissue. Some cultures were white and some cultures were blue.

Isolated cell

cultures were further selected on hygromycin resistance. However,

no

control plant survived.

Then twenty plates of cells were bombarded with

the PINII gene, from which over two hundred plants were regenerated and grown

in a greenhouse. After their growth, they were tested for PINII gene using

DNA blot hybridization and 73% of the plants were found to be transgenic.

DNA blot hybridization is the process by which DNA from each sample was digested

by a suitable restriction endonuclease, separated on an aragose gel, transferred

to a nylon membrane, and then finally hybridized with the 1.5 kb DNA fragment

with pin 2 coding and 3′ regions as the probe. The results also indicate that

the PINII gene was inherited by offspring of the original transgenic line,

that the PINII levels were higher among many of the offspring and that when

PINII levels rose in wounded leaves, the PINII levels in unwounded leaves also

rose. However, the PINII gene is not 100% effective in eliminating insects

because it does not produce an insect toxin, just a proteinase inhibitor.

Yet, greater insect resistance can be achiev

ed by adding genes to produce

the Bacillus thuringiensis or BT toxin.

Bacillus thuringiensis is an entomocidal

spore-forming soil bacterium that offers a way of controlling stem boring insects.

Stem borers such as the pink and striped varieties are difficult to control

because the larvae enter the stem of the plant shortly after hatching and continue

to develop inside the plant, away from the toxins of sprayed insecticides.

Therefore, the stable institution of the BT gene into the rice plant’s genome

would provide a method of reaching stem borers with toxins that are expressed

in the plant tissues themselves.

Bacillus thuringiensis is comprised of

so-called cry genes that encode insect specific endotoxins. Recently some

lower varieties of rice, such as Japonica, have been successfully transformed

with cry genes, but the real challenge lies in transforming Indica rice, an

elite breeding rice that composes almost 80% of the world’s rice production.

In order to transform Indica rice, the synthetic cry IA gene must be used

because it is the only cry gene to produce enough of the BT protein. Next,

the synthetic cry IA gene under the control of the CaMV 35S promoter is attached

to a CaMV cassette for hygromycin selection of transformed tissues. Following

the linkage of the cry IA and the CaMV 35S cassette, the DNA is delivered to

the embryonic cells by particle bombardment with a particle inflow gun. More

specific transformation includes the following:

Immature Indica rice embryos

were isolated for ten to sixteen days after pollination from other greenhouse

plants and were plated on a solid MS medium containing sucrose (3%) and cefotaxime.

After twenty four hours, embryos were transferred to a thin layer of highly

osmotic medium containing a higher percentage of sucrose (10%), were incubated,

and then were bombarded with plasmid pSBHI and gold particles by the particle

inflow gun. After bombardment, the thin layer of 10% sucrose was placed on

the layer of 3% sucrose. This sandwich technique allowed continuous adaptation

of the target tissue to the osmotic conditions, which was shown to be optimal

for callus induction. After twenty four hours, the 10% sucrose layer was removed

and the embryos were cultured on the 3% sucrose layer. After one week, they

were transferred to a 3% sucrose medium that was selected for hygromycin B

resistance. After a further three to four weeks, regenerated plants were transferred

to soil and placed in the greenhouse under

appropriate conditions. The results

of this process were eleven transgenic plants out of a total of thirty six.

Transgeneicy of the rice plants was confirmed by similar banding patterns

in Southern blotting. The presence of the BT protein was also demonstrated

in Western blot analysis, where a protein with the expected size of sixty-five

kilobases was found in all plants tested. Interestingly enough, the BT protein

levels were higher in older plants than in younger plants, possibly questioning

the role of inheritance of BT gene. Yet, inheritance was determined by using

DNA blot hybridization, which revealed a segregation ratio of 3:1. This indicates

the integration of all copies of transgene at a single locus.

To assess

the mortality rate among different insects, both petri dish assays and whole

plant assays were performed. In petri dish assays, mortality rates were as

follows:

European corn borer = 85-95%

Yellow stem borer = 100%

Striped stem

borer = 100%

Cnaphalocrocis medinalis (leaffolder) = 67%

Marasmia patnalis

(leaffolder) = 55%

In whole plant assays, no surviving insects were found

on any BT expressing plants, although insects still survived on the control

plants or non expressing BT plants.

In addition to this recent insertion

of the BT gene into Indica rice, a similar procedure was conducted on Shuahggei

36, a variety of Indica rice. Transgeneicy of Shuahggei 36 was achieved by

taking plasmid P41ORH, which contained the coding region of the BT gene with

the marker CaMV 35S-HPI-NOS plus 1.0 kb of DNA fragment, and inserting it into

the pollen tube pathway. More specifically, the plasmid DNA was applied at

the cut ends of rice florets from one to four hours after pollination. Next

the seeds that were harvested were germinated under hygromycin B resistance.

However only 3% of the plants survived hygromycin resistance. After this,

the seedlings from the second generation were again segregated for hygromycin

resistance. From these seeds, seventy plant lines were screened for transgeneicy

and fifteen displayed the BT protein. These results and the inheritance of

the BT gene into offspring were confirmed by Southern blotting. Nevertheless,

the question remains whether the BT gene was really

integrated into the genome

or whether it was expressed only as a plasmid.

The use of the arcelin gene

is another experimental method of creating transgenic rice plants. The arcelin

gene is a translationally enhanced Bacillus thuringiensis toxin construct that

is effective on the rice water weevil. The rice water weevil or RWW is the

major pest of the Texan rice crop. Previously, the RWW was combated by granular

carbofuran, an insecticide that kills the RWW but has deleterious effects on

water fowl that live in the crop area. So environmentalists have forced the

cessation of the use of granular carbofuran and therefore, new methods have

to be developed. One of the major genes that confer resistance to the RWW

is the arcelin gene. Arcelin is a lectin that was originally discovered in

the seeds of bean cultivators that showed resistance to the Mexican bean weevil.

Next, researchers isolated a genomic clone encoding arcelin from the bean

seed and then placed it under regulation of a rice actin promoter. Then the

clone with the rice promoter was introduced into rice protoplast

s. Transgeneicy

and inheritance was then confirmed by genomic DNA blots and immunochemical

blots. In two separate experiments, six transgenic rice plants were subjected

to RWW infestation under controlled conditions. The results of the first experiment

are that similar numbers of RWW larvae were recovered from each set of six

plants, but the size of those from arcelin expressing plants were significantly

smaller. In the second experiment, although many normal larvae were recovered

from control plants, only three small larvae came from arcelin expressing plants.

This would indicate the benefits of inserting the arcelin gene into rice plants

for RWW resistance.

Another experimental method of creating transgenic rice

plants that are insect resistant includes the use of snowdrop lectin or galanthus

nivallis agglutinin (GNA). Snowdrop lectin helps to control the sporadically

serious pest the brown planthopper (BPH), which has developed a resistance

to many pesticides. Luckily for the environment, snowdrop lectin provides

high levels of toxicity to BPH but not to other animals. BPH is a member of

the order Homoptera and feeds by sucking the phloem sap from the stems of rice

plants. The major problem with combating BPH is that rice plants can not be

engineered for BT toxin resistance against this pest because BT toxins that

effect Homopterans have not yet been discovered or reported. Therefore, other

types of genes had to be manipulated in order to produce insect resistance

against BPH. The best plant protein that provides resistance to BPHs turns

out to be snowdrop lectin, and this was first confirmed by artificial diet

bioassays. To create the transgenic rice

plants, embryonic cell suspension

cultures were initiated from mature embryos from two Japonica rice varieties,

Taipei 309 and Zhonghua 8. Next, the protoplasts isolated from these cell

suspension cultures were transformed by using the plasmid pSCGUSR, containing

the nos-npt II gene as a selectable marker. Plasmid uptake was then induced

by the PEG process and geneticin was used as a selection agent. Geneticin

was added to the protoplast-derived colonies during the four and eight cell

stages. From this, more than fifty putative transgenic plants have been regenerated

from one thousand resistant colonies.

Another way of combating the brown

planthopper is by producing phloem-specific promoters. These promoters are

necessary because phloem is the exact site of feeding for the BPH. Although

the CaMV promoter is active in phloem tissue, the possibility exists to institute

a promoter from a gene that is specifically expressed only in phloem. This

would be advantageous if there are other parts of the plant that may be negatively

affected by the promoter and in this scenario, they would be unaffected. Recently,

a phloem specific promoter has been obtained from the rice sucrose synthase

gene RSs 1. RSs 1 promoter was used to drive the snowdrop lectin or GNA protein.

The results were confirmed by the use of immunological assays and they indicated

that not only is the gene being expressed in the phloem tissues, but that the

protein product has been successfully transported to phloem sap.

Unfortunately,

RSs 1 is heavily expressed in the seeds of rice plants, so an alternative promoter

called PP2 is currently under study. So far, PP2 has been purified and partially

sequenced. Also, a full cDNA library has been created for the gene and it

has been used to probe a genomic library to obtain the corresponding gene.

The promoter region form the PP2 gene is now being assayed.

One final

method of creating insect resistance in rice plants is the use of the SBTI

gene. SBTI gene is a trypsin inhibitor that acts against pests such as the

yellow stem borer and the gall midge. Greater insect resistance can be created

by introducing the Kunitz soybean trypsin inhibitor (SBTI) gene into varieties

of Indica rice plants. First, a PCP product corresponding to the protein was

isolated by oligonucleotide primers. Then, the resulting fragment was cloned,

sequenced and expressed in E. coli cell cultures. The results were a recombinant

SBTI gene that effectively fought off gall midges and yellow stem borers.

Presently, the SBTI gene is being cloned into vectors and is being used to

transform other types of embryos using the particle gun technique.

In

conclusion, through the use of new technologies such as the introduction of

potato proteinase inhibitor II gene, the establishment of the Bacillus thuringiensis

toxin gene and the experimental methods of using the arcelin gene, the snowdrop

lectin/GNA (galanthus nivallis agglutinin) protein, and phloem specific promoters

and finally the SBTI gene, rice plants have become almost completely resistant

to insects that used to destroy much of the crop. This has been an important

step in biotechnology because the improvement of rice plants is a major concern

that could potentially effect almost all of the populations of the world.

Biotechnology has become an increasingly accepted method of solving some of

the major problems in agriculture, medicine, and industry. Potentially, with

the advancements of many techniques, almost whenever people eat, drink, take

medicine, or go to work, they will be touched in some way by the many complicated

processes of biotechnology, that are striving to make our world a better place to exist in.