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REVIEW ARTICLES
Nuclear control and mitochondrial transcript
processing with relevance to cytoplasmic male
sterility in higher plants
H. K. Srivastava
Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015, India
Recent advances in the molecular biology of plant
mitochondria have yielded some newer insights. A
common, basic set of genes is encoded in all mitochondrial genomes, although their sizes differ
greatly, with plant mitochondrial DNAs being by far
the most complex. Mitochondrial genome mutation
encodes cytoplasmic male sterility (cms) which in
turn leads to stamen sterility or pollen abortion in
several higher plants. Cms is the resultant of an incompatibility between the nucleus and mitochondrial
genomes such that male pollen is aborted or not
properly formed. Cms system in agriculturally important crops has been used to produce high-yielding
and heterotic hybrid seeds, because it eliminates the
need for labour intensive and expensive hand emasculation. Hybrid seeds (or varieties) hold great potential
for improving crop economic yields, even when the
average yields are much higher in many of the traditional food and feed crops. An important feature of
mutation(s) responsible for cms is the discovery of
chimeric genes or chimeric locus and different open
reading frames joined together, or placed in proximal
locations for cotranscriptions with other standard mitochondrial genes. Twenty-nine mitochondrial cms
related genes in over twelve so far studied higher plant
species have been characterized together with their
specific DNA coding sequences. The recent development of in vitro systems for transcription initiation and
RNA processing has yielded intriguing details of transcriptional and post-transcriptional regulation of plant
mitochondrial cms gene. The nuclear gene Rf or Fr
(restorer of fertility) prevents the expression of male
sterility gene in the cms system in rice (B-atp 6) and
maize (mitochondrial aldehyde dehydrogenase enzyme) by influencing RNA processing and sequential
post-transcriptional editing. The latest findings on the
subject support the notion that ‘intergenomic interaction’, i.e. specific nuclear-mitochondrial gene(s) cotranscription is operational as the plausible molecular
mechanism for the manifestation of maternally and
cytoplasmically inherited cms genetical trait in crop
plants.
THREE genetic systems, one nuclear and two cytoplasmic or extranuclear, mitochondria and chloroplasts are
essential for expression and development of various
quantitative and qualitative traits in higher plants. Plant
mitochondria have been found to cover many facets of
genetics and molecular biology 1. The first evidence for
the presence of genes outside the nucleus was provided
by non-Mendelian inheritance in plants2. Non-Mendelian
inheritance and somatic segregation are taken to indicate
the existence of functional genes that reside outside the
realm of the nucleus and do not undergo hereditary type
conventional segregation and transmission on the meiotic
and mitotic spindles to distribute genomic replicas to
gametes or to daughter cells during microsporogenesis.
The interpretation for the frequent occurrence of novel
heritable genetic or even nonheritable epigenetic variations has been debated among geneticists and breeders for
almost two decades. Exciting new findings have emerged
in terms of plant organelle (mitochondria and chloroplast)
gene expression3,4 in relation to the manifestation
of cytoplasmic male sterility (cms) and heterosis, after the
author’s earlier reviews on the related subject exhibited
some kind of nuclear control of mitochondrial and chloroplast development and function in higher plants which now
provide convincing evidence for a plausible DNA transfer
(mobile genes) between the mitochondria, chloroplast and
nucleus5–7, i.e. intergenomic interaction. Cms provides a
convenient and proprietary means to produce hybrid seeds.
Srivastava5 propounded a theory of ‘intergenomic interaction’ to explain genetical phenomenon of heterosis or hybrid vigour based on experimental evidences then available,
that mitochondria may interact structurally, functionally
and biosynthetically with other cellular-genetical components like nuclear and chloroplast genes so as to endow an
overall superiority to heterotic hybrids in many measurable
attributes, including crop grain yields. Much of the interest
in cms plants stems from their use, or potential use, in producing heterotic hybrid seeds. Presently, hybrid seeds of
some crops are produced with the use of cms inbred (predominantly homozygous and homogeneous) lines, while
production of others depends upon tedious hand emasculation. The eukaryotic cell nucleur DNA content
e-mail: [email protected]
176
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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(× 10–14 g) is immensely greater (4.0–7.0) compared to
chloroplast DNA (0.3–0.9) and mitochondrial DNA
(0.1–0.3) 5. Mitochondria contain their own DNA
(mt DNA) and the transcriptional and translational machinery necessary for protein synthesis. Mitochondria,
however, are not fully autonomous, and their biogenesis
and function depend on coordinated expression of both
nuclear and organellar genomes and genes. mtDNA is
particularly maternally or uniparentally inherited. Plant
mitochondria also possess an alternative route for electron transport system or cyanide-resistant respiration 8.
Many independent mutations that disrupt pollen development or abnormal aborted pollen production have
been observed in a wide variety of higher plant species.
In tomato, for example, nuclear encoded male sterility
mutations have been mapped to over 40 different loci.
Many other male sterile mutations, however, do not
exhibit Mendelian inheritance. Such cytoplasmic inherited mutations have been so far reported in over 140
higher plant species 9 and the possibility of discovery of
novel plant cms gene(s) in other plant species exists
through further intensive research. Despite identification of several mitochondrial genes that specify cms
trait, its precise molecular basis or molecular mechanism is not understood in any plant species. An updated
account of current knowledge concerning the nature of the
nuclear–mitochondrial interaction resulting in plant mitochondrial gene mutation(s) encoding economically and
commercially relevant cms trait in several important food
and other crop species is provided in this article.
Plant mitochondrial genomes organization
The in vivo structure of mitochondrial genome organization has been adequately covered 1,10,11 . Earlier studies
have elucidated the derivation of circular structures
from physical mapping of overlapping restriction fragments; from these maps circular genome structures are
predicted for the majority of mitochondrial genomes in
eukaryotic cells 12 . A more comprehensive coverage of
mitochondrial genome organization, evolution, cms, and
analysis of mtDNA in somatic hybrids has been provided 4,13,14 . Several general features of higher plant mitochondrial genome organization have emerged: (i) The
genomes are larger than those from animals and fungi.
(ii) Restriction endonuclease mapping (using DNA
markers such as RFLPs, RAPDs and AFLPs) indicates
that plant mitochondrial genomes are usually organized
as multiple circular molecules, with conversion of circle
types mediated by recombination between repeated sequences. (iii) Mitochondrial genomes from closely related species are highly conserved in the primary
sequence, but vary greatly in linear gene order. (iv) The
plant mitochondrial genome, like that of other eukaryotes, encodes only a small percentage of proteins located
within the mitochondria, indicating that over the course
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
of evolution most of the genes that resided in the original endosymbiont (earlier as prokaryotes) have migrated
to the nuclear genome. (v) In addition to high molecular
weight mtDNA, plasmid-like molecules are present in the
mitochondria. (vi) Chloroplast DNA and nuclear DNA
sequences are often found inserted in the mtDNA.
Higher plants exhibit an extremely wide range of
variation in mtDNA size with a minimum of ~ 100 kb.
Higher plant mitochondrial genomes, which vary in size
from 200 kb to 2500 kb are much larger. Analyses of
Cucurbit mtDNAs demonstrated a seven-fold range in
genome size within the family, from 330 kb in watermelon to 2500 kb in muskmelon. Despite the large size
differences, there is no correlation of mitochondrial
genome size with repeated DNA sequences, mitochondrial volume or the number of translational products.
The function of additional DNA has not been precisely
enumerated, but it seems likely that much of it either is
noncoded or is subjected to some kind of shuffling
mechanism between ‘intron’ and ‘exon’ sequences. The
size of the genome makes it difficult to isolate the
mtDNA intact, but restriction mapping (RFLPs, RAPDs
and AFLPs) in several crop plants suggests that each
has a single sequence organized as a circle. Within this
circle there are short homologous sequences. Recombination between these elements generates smaller, subgenomic circular molecules that coexist with the
complete ‘master’ genome or ‘principal’ genome explaining the apparent complexity of plant mtDNAs. The
mixing of cytoplasms in higher plants promotes mitochondrial fusion and recombination between varying
mitochondrial genomes 5,6 .
Unlike animal and fungal mtDNAs, which are usually
organized in a single circular molecule, plant mtDNA
often consist of an assortment of genomic and subgenomic circular molecules of different sizes and frequencies 1. The cause of this variability was first explained in
Brassica campestris. Its mtDNA is primarily composed
of a single large 218 kb circle called the ‘master’ chromosome plus smaller circles of 135 kb and 83 kb.
Within the master chromosome are two 2 kb direct repeats separated by 83 kb on one side and 135 kb on the
other. Recombination between the two repeats generates
the smaller 135 kb and 83 kb circles. Moreover, recombination between the two smaller molecules can also regenerate the ‘master’ chromosome. The 570 kb maize
genome has six major pairs of repeats, which may
participate in recombination to produce a large array of
circular molecules of various sizes and frequencies. The
relative proportions of the different circles apparently
depend on the recombinational frequencies among the
different pairs of repeats and whether the various circles
are capable of autonomous replication.
Moreover, genomic rearrangements of the atpA gene
characteristic of cms-S (USDA) and cms-T (Texas)
maize are found at low levels in N (normal) mitochon177
REVIEW ARTICLES
dria 15,16 . It is thought that these ‘substoichiometric’
atpA types are part of a larger circular or linear molecule present in low copy number relative to the rest of
the mitochondrial genome in the cell. The substoichiometric molecules called ‘sublimons’ may have
originated from infrequent recombinational events between very short regions of homology in the mitochondrial genome. Thus, the products of rare or unique
recombinational events are retained in the genome at
low levels and these sublimons may then be expected to
exhibit rapid molecular evolution, because mutational
events are more quickly fixed by chance in a small
population of molecules. Occasional amplification of
sublimons could cause sudden genomic organization,
possibly leading to the evolution of cms 1,17 . It has been
unequivocally demonstrated that mitochondrial genomes of many higher plants contain subprinciple genome DNAs and/or double-stranded RNAs which have
been termed minilinear and minicircular 18 for convenience in distinguishing them from the higher molecular
weight principal genome.
Tobacco tissue culture cells yield a large percentage
of small circular DNAs, which are derived from the
principal genome. Of the smaller circular DNAs, the
smallest ones (10.1, 20.2 and 30.3 kb) merge to reach
the size of some of the larger minicircular and minilinear DNAs, found in other species 19 . It is interesting that
the coding sequences of cytochrome c oxidase subunit
II have been shown to be located on the small circles of
Oenothera 11,20,21 . The small, circular DNA molecules of
animal cells of about 16 kb have been visualized by
electron microscopy and can be isolated as supercoiled
molecule populations by CsCl density gradient centrifugation 22 . The in vivo structure of the larger mitochondrial DNAs of yeast and plants, however, may be
different from the structures predicted by frequent mapping. Pulse field gel electrophoresis exhibits one fraction of molecules to be locked into highly complex
arrangements that migrate with an apparent size of more
than one megabase 22 . Most plant mtDNAs behave like
linear molecules ranging from 50 to 100 kb in a heterogeneous population visible as smear in the gels 10 . Linear
DNA molecules have thus been proposed as the major
in vivo form of mitochondrial genomes in higher
plants 10 . Effects of DNA anchoring at membrane integral proteins and specific mechanisms of replication
such as those leading to catenated network of trypanosome mtDNA 23,24 may be involved in determining the
variation in linear and circular structures in the
mitochondria.
Mitochondrial gene(s) and their transcription
Extranuclear genetic systems are a fundamental feature
of higher plant cells. The cytoplasmic organelles pos178
sess their own DNA. The mitochondrial genomes contribute a limited (10%) but essential number of gene
products to the biogenesis of mitochondria, that are
competent to carry out oxidative phosphorylation and
energetically important cellular respiratory functions.
Mitochondria from all organisms provide ATP as the
principal energy source for the cell and deliver numerous substrates via specific carriers for biosynthetic reactions in the cytoplasm. Many basic features of the
mitochondrial structure and function, developed at an
early stage of evolution, have been highly conserved
between animals and plants despite a billion years of
divergent evolution. The assembly and function of respiratory-competent mitochondrion in eukaryotes result
from a collaboration and coordination between gene
products derived from mitochondrial and nuclear genomes. The inventory of nuclear and mitochondrially
coded proteins required to assemble a functional mitochondrion shows clearly that nuclear and mitochondrial
genomes interact in at least two ways. First, both nuclear and mitochondrial genes contribute to mitochondrial protein function. Second, both nuclear and
mitochondrial genomes interact to affect the synthesis
and assembly of mitochondrial proteins 5,6 . Communication from the mitochondrion to the nucleus probably
involves metabolic signals and one or more signal
transduction pathways that function across the inner
mitochondrial membrane.
Nuclear background influences extensive editing of
plant mitochondrial transcription in particular genomic
regions. The complete nucleotide sequences of several
animal mitochondrial genomes have been published 25–27
and those of certain fungi are nearing completion 28 .
These analyses have made possible the location of the
coding regions (mitochondrial transcript) and construction of detailed mtDNA restriction maps 12,19,29–33 . The
picture as regards the number of genes encoded in the
higher plant mtDNA has become clearer. Isolated plant
mitochondria synthesize some 20 to 50 polypeptides 34
which are presumed to be encoded by the mtDNA. It
has been observed that higher plants also encode 26S
rRNA (ref. 34) and in maize mitochondria coxI, coxII,
cob, atp and 5S, 18S and 26S rRNA genes have been
identified on the circular 570 kb restriction map 35 . The
protein coding genes are obviously not clustered,
whereas the 5S and 18S rRNA genes and the ATPase
gene are transcribed from the same mtDNA strand,
while the 26S rRNA, coxI, coxII and cob genes are transcribed from the opposite strand 36,37 . Approximately
one-tenth of the mitochondrial proteins by weight are
specified by the limited number of genes on the mitochondrial genome. Thus mitochondrial genomes contribute a limited but essential number of gene products
to the biogenesis of mitochondria, that are fully competent to carry out respiratory functions such as electron
transport and oxidative phosphorylation. It is obvious
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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then that a significant number of nuclear genes must be
involved in the synthesis of mitochondrial components,
expression of the mitochondrial genome, and control of
mitochondrial biogenesis. Nuclear background alters
transcription of atpA in the ‘ogura’ cytoplasm of
radish 38 and the cms gene (T-urf 13) in the T cytoplasm
of maize 39 . The T-urf 13 cms associated sequence is
cotranscribed with the open reading frame orf 221. Specific inbred lines of maize revealed some marked influence of nuclear background on the pattern of
transcription and mitochondrial function 40 . Rocheford et
al. 41 demonstrated that the transcript changes were not
only a function of the mitochondrial genomic environment encompassing the T-urf 13/orf 221 region but also
of dominant nuclear gene action (following Mendelian
hereditary transmission principles) influencing the pattern of post-transcriptional processing of the transcripts.
Mitochondrial transcription
The common functions of the mitochondria in plants,
animals and fungi are reflected by similarities in their
genomes 42 . The recent development of in vitro systems
for transcription initiation and tRNA processing has
yielded intriguing details of transcriptional and posttranscriptional regulation in plant mitochondria. Most
of the coding regions are separated by several kilobases
of non-coding regions 20,43–45 . This organization implies
that most of the essential coding information is expressed as monocistronic transcripts from individual
promoters, for example, atp6, atp9, cytob, coxI, coxII,
and coxIII genes in most plants. When transcripts are
analysed by Northern blot hybridization, the mRNAs
are found to be generally much larger than the actual
coding regions of the genes and to include extended
non-coding 5′and 3′transcribed regions of several hundred nucleotides. The frequent genomic recombinations
in plant mitochondrial genomes can place genes that are
far apart in one genome into close proximity in another
species.
Genes located close together almost invariably exhibit co-transcription 11,46,47 , such as the 18S-5S-RNA
genes or nad3-rps12 transcription units. The transcribed
spacers between, for example, the 18S-5S rRNA genes
vary among plant species, ranging from just over 100 nt
to more than 500 nt; sequences apparently can become
integrated or lost by intra-or intermolecular recombination 48 . Larger trancription units would most likely be
found upon further detailed investigation of higher
plants. The stably transcribed fraction of higher plant
mitochondrial genomes has been estimated to be about
one-third of the genomic complexity, for example in
Brassicaceae and in Cucurbitaceae 49 . This RNA population is equivalent to the entire sequence information
common to all plant mitochondrial genomes. The reCURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
maining 70% of the plant mitochondrial genome appears to be non-essential sequence information derived
partly from chloroplast DNA (ct DNA), partly from
nuclear sequences and from the duplication of mitochondrial sequences degenerated to various degrees.
The presence of numerous transcription units implies a
corresponding multitude of individual promoters of
transcription and the presence of several promoters per
genome has been confirmed 4. For some genes, multiple
promoters have been identified which are actively engaged in transcription and may be used for differential
regulation of the respective gene activity. The coxII
gene in maize, for example, is transcribed from a
closely stacked set of promoter sequences, and the atp9
gene in this plant is preceded by at least six active promoters spaced over several hundred nucleotides upstream of the coding region 4,47,50 . The transcripts arising
from such multiple promoters have different sizes and
thus complicate transcription patterns of individual
genes even in monocistronic units 4. Transcription patterns of individual genes are further complicated by the
presence of several gene copies in a given genome. In
Northern blots, detectable transcripts of different sizes
may arise from duplicated genes with different 5′or 3′
regions adjacent to the respective open reading frame
that leads to variable extensions at either end. Common
to mitochondria in animals, fungi and plants appears to
be the positioning of the conserved sequence block and
the first transcribed nucleotide. The conserved sequence
motif generally appears to include the first transcribed
nucleotide of all mRNAs in yeast and the first two nucleotides in plant mitochondria 50 . Mitochondrial activity, although required in all plant tissues, is capable of
adopting to specific requirements by regulated gene
expression 4. Investigation of the factors governing the
quality and quantity of distinct RNAs will define the
extent of interorganelle regulatory interface in mitochondrial gene expression.
In vitro transcription systems have been established
and now permit the identification of sequences necessary and sufficient for efficient transcription initiation 4,51,52 . Post-translational control having some
important role in the regulation of plant mitochondrial
genome expression has been considered earlier 53,54 . Not
much is known about the mechanisms that control gene
expression at post-translational 3′and 5′ends of
mRNAs 4. Like chloroplast transcripts, plant mitochondrial mRNAs tend to contain inverted repeat sequences
in their 3′
-region that can fold into stem–loop structures 55–58 . The steady-state level of mRNA from the
‘ogura’ cms locus of Brassica cybrids being determined
post-transcriptionally by its 3′region has recently been
demonstrated by Bellaoui et al. 12 . Their result strongly
suggests that the steady-state level of mRNA from the
orf 138 locus is determined post-transcriptionally, most
likely by its 3′
-region. In the presence of rapeseed mito179
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chondrial lysate, synthetic RNAs corresponding to the
3′
-region of the Nco 2.7/13 F transcript are, as expected, less stable than RNAs corresponding to the 3′
regions of the Nco 2.5/13S and Bam 4.8/18S transcripts
(see Figure 1). Future research should focus on better
understanding of the role of these 3′regions and their
post-transcriptional regulation in relation to mitochondrial gene expression in general and cms gene in particular.
a
Mitochondrial transcript processing
Transcript processing is a widespread phenomenon in
plant mitochondria though not yet well understood
mechanistically. It was first deduced by the observation
that unusually complex transcripts in certain regions of
the plant mitochondrial genome were produced not only
by multiple transcription start and stop sites, but also by
internal transcript processing 44 . Although the role of
processing in gene regulation is not yet clear, nuclear
regulation of transcript processing could be an important means of mitochondrial gene suppression 4. This
inference is based on the observation that three different
nuclear Rf (restorer of fertility as dominant gene) loci
directly influence transcript processing within the respective mitochondrial cms-associated regions. In cmsT maize, perhaps the most well-studied example of cms,
Figure 2. a, Structure of the maize (Zea mays) cms-associated chimeric gene. Portion of urf13 derived from a normal atp 6 gene and a
normal 26S rRNA gene. Atp6 and 26S rRNA genes are present elsewhere in the maize cms-T mtDNA, but the urf25 gene downstream of
urf13 is the only copy present in maize cms-T (refs 29, 31). Chloroplast DNA sequences derived from an arginine tRNA gene are found
at the 3′terminus of the urf25 gene from both a normal fertile maize
line and cms-T (ref. 32); b, Transcription termini in the maize cms
associated chimeric gene. Arrows indicate the location of mapped
termini. The arrow labelled T–Rf 1 indicates a transcript that is increased in abundance in lines containing Rf 1 allele 30 . (Figure adapted
from Hanson 21 ).
Figure 1. Restriction maps of the three configurations Nco 2.5/13S,
Nco 2.7/13F and Bam 4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) Brassica cybrids, respectively. The sequences
of the Nco 2.7/13F and Bam 4.8/18S fragments which are common
with the Nco 2.5/13S fragment are indicated by a thick black line.
White box: trn fM gene coding for initiator methionine rRNA; light
box with plain line; orf138 gene; dark box: orfB gene; striped box:
atpA gene. In vivo accumulated transcripts from each configuration
are indicated by the bent arrows. (Adapted from Bellaoui et al. 12 ).
fertility restoration (Rf) requires dominant alleles of two
unlinked nuclear loci, Rf 1 and Rf 2 (ref. 59). Compelling
evidence demonstrates that the product of Rf 1, essential
though not sufficient to restore fertility, directly influences transcript processing of the T-urf 13 mitochondrial region (see Figure 2). Processing of T-urf 13
transcripts appears to be directly associated with a
marked reduction in the expression of the encoded
13 kDa T-URF 13 polypeptide 20 . Although the function
of Rf 2 gene in fertility restoration is not yet understood
at the molecular genetic level, it has been speculated
that the Rf 2 gene product may play a role in the detoxification of a pollen-specific product that interacts with
the T-URF 13 protein to cause premature tapetal breakdown leading to premature or aborted pollen grains 60 . In
Sorghum line IS 1112 C, cms is associated with expression of the open reading frame orf 107 (ref. 61). Again
fertility restoration is associated with altered processing
of orf 107 transcripts and the concomitant reduction in
the accumulation of a 12 kDa polypeptide presumed to
be the gene product 61 . Of particular interest is the observation that the transcript processing sites described
in both cms-T maize and cms sorghum share sequence
features 62 , implying that sequence motifs exist within
plant mitochondrial genes that may act as targets for
nuclear-directed gene modulation.
180
b
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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Cms in the B. napus (oilseed rape) polima cytoplasm
is associated with aberrant expression of a region encoding the atp6 gene cotranscribed with a downstream
chimeric sequence, orf 224 (ref. 63). Fertility restoration conditioned by either of two dominant single nuclear loci, results in a transcript processing event that
generates predominantly monocistronic atp6 transcripts 63 . Generation of monocistronic atp6 transcripts
cosegregates with a single dominant fertility restorer
locus, Rfp. Most intriguing, however, is the observation
that an alternate recessive allele at this locus (rfp 1) or a
second locus tightly linked to rfp 1, influences transcript
processing of two other mitochondrial genes not associated with male sterility, nad4 and a cell-like gene that
may be involved in mitochondrial cytochrome c biogenesis 63 . At all four processing sites under Rfp 1 or rfp 1
control, there is UUGUGG or UUGUUG sequence motif located very near the processing site.
RNA editing alters gene products
Although only 0.13% of the codons in the entire maize
chloroplast genome is altered, plant mitochondrial protein coding genes typically have 3–15% of the codons
affected by RNA editing 64 . The discovery of RNA editing has changed the way plant organelle genes must be
analysed. Before 1991, sequencing of genomic DNA
was thought to be sufficient to characterize the coding
regions of most plant mitochondrial genes. Earlier literature did predict protein sequences from genomic
DNA sequences 4. However, some anomalies were
noted; often plant mitochondrial proteins predicted from
genomic sequences differed from mitochondrial proteins in other organisms. As a result, it was proposed
that like yeast mitochondrial genes, the genetic code in
plant mitochondria differed from the universal code.
Because the CGG codon (which codes arginine) was
present where homologous genes in other organisms
exhibited ‘tryptophan’, codons such as TGG, CGG were
at first proposed to encode tryptophan in the plant mitochondrial genetic code. Now it is known that the genetic
code in plant mitochondria does not differ from the
standard code but that some CGG codons actually specify UGG as a result of C to U RNA editing. When editing occurs, the encoded amino acid is likely to be
altered because the codon position of the edited C is not
random. In chloroplasts, the second codon position is
the primary target of editing 65 , in mitochondria, the first
and second positions of codons are more often edited
than the third position 66,67 . Plant organelle proteins are
more similar to their homologous counterparts in other
organisms than was originally realized 4. Protein sequencing of two mitochondrial gene products (wheat
ATP 9 and potato NAD 9) has confirmed the presence
of the amino acid predicted from edited codons 68 .
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
Editing of RNA may shorten the predicted open reading frame to the length expected from analysis of homologous genes. The stop codons UAA, UAG and UGA
could be created by editing the C-containing codons.
Mitochondrial atp6, atp9 and rsp10 genes from several
plant species have ‘edit encoded’ stop codons, which
slightly shorten the open reading frame 69,70 . A bizarre
example of an edit-encoded stop codon occurs in the
petunia rpl16 gene. When 15 cDNAs were sequenced,
all were found to carry an edit-encoded stop codon early
in the open reading frame predicted from the genomic
sequence 71 . Editing evidently has converted the mitochondrial rpl16 gene (mt-rpl16) into a pseudogene. Presumably a functional rpl16 gene may be present in the
nucleus. Most plant chloroplast and plant mitochondrial
genes carry genomically encoded AUG start codons,
unlike human sleeping sickness blood stream protozoa
trypanosome 72 mitochondrial genes, in which insertion
of U often created the start codon. Nevertheless, ACG
to AUG editing creates the start codons of transcripts of
the wheat mt-nad1 gene, the potato mt-coxI gene, the
maize chloroplast rpl12 gene (cp-rpl12) and the tobacco
cp-psbL and cp-ndhD genes 73,74 .
Transcripts of different genes vary in extent of
editing
Mitochondrial genes can be divided into three classes
with respect to their transcript editing. All transcripts of
some mitochondrial genes appear to be fully edited;
direct cDNA sequencing as well as sequencing of multiple, independently derived cDNA clones, indicate that
all editing sites have Ts replacing Cs. Other genes exhibit partially edited precursor transcripts, but the mature transcript population is fully edited 75,76 . The third
class of genes exhibits a low proportion of fully edited
transcripts; most transcripts are partially edited 77,78 .
When a gene exhibits partially edited transcripts, many
individual cDNAs must be sequenced to make sure that
every editing site is detected. The existence of partially
edited transcipts, and the finding that spliced transcripts
are more highly edited than unspliced transcripts, have
led to the view that RNA editing in plant mitochondria
is a post-transcriptional process 12,33 . Fewer data are
available concerning the extent of editing of chloroplast
transcripts, but partially edited transcripts of a few
genes have been reported 4,12,33 . Editing can occur before
splicing in both organelles 4,79 . Transcripts of almost all
mitochondrial protein-coding genes contain at least one
editing site (Table 1; also see Figures 1–4). Mitochondrial editing events have 5′and 3′non-translated regions, introns, ribosomal RNAs and transfer RNAs, but
in general, coding regions are more highly edited than
non-coding regions. Not only does the extent of editing
of different mitochondrial genes’ transcripts vary within
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the same species, but the number of codons edited varies in transcripts of the same gene in different species 4,29–32 (Table 1).
In plant, there is no published evidence that transcripts present in comparable abundance in different
tissues are differentially edited. Transcripts of psbL are
present in similar amounts in chloroplasts and chromoplasts of ripening bell pepper (Capsicum annum), and
editing occurs in both types of plastids 80 . When the degree of editing of spinach cp-psbF and cp-psbL transcripts was examined by direct sequencing of cDNAs,
Table 1. Altered codons in plant mitochondrial protein-coding
genes*
Mitochondrial gene (cms related)
atp6
atp9
CoxII
nad1
nad3
Pcf
rpl16
rps12
Rps19
mt-ATP 6 core region
Rice
Sorghum
Radish
Petunia
Ocnothera
Number of codons altered (%)
6
13
5
7
13
3
3
4
5
Number of codons changed (%)
4
6
0.4
6
8
*Adapted from Hanson et al. 64 with modification.
Figure 3. Construction of orf239 chimeric gene in transgenic tobacco (Nicotiana tabaccum). Strategy for cloning each gene construct into the transferred DNA binary vector pB1101 is shown.
Aocs, activator of the Octopine synthase gene (trimer); Amas 2′
gene, activator of mannopine synthase 2′gene; Pmas 2′
, promoter of
the mannopine synthase 2′gene; PLat 52, promoter of the tomato
pollen specific gene Lat 52. The boxed ATG is the first codon of orf
239. ? , Site specific cleavage; *, the amino acid altered as a consequence of cloning (Adapted from He et al. 19 ).
182
Figure 4. Schematic diagram of atp6 region of cytoplasmic male
sterile rice (Oryza sativa). The N-atp6 and B-atp6 genes isolated
from N(r), B (R), and C. The coding region is shown on a dotted box.
The probes used for nucleic acid hybridization are indicated by bars
(1–5). The sizes of probes 1–5 are 0.55, 0.4, 1.3, 0.45 and 1.7 kb,
respectively. The primers used for sequencing, PCR amplification
and primer extension experiment are shown as arrow heads (a–j). The
atp6 RNAs are represented by arrows. The transcript of B-atp6 is
altered, whereas that of N-atp6 is not changed by the presence or
absence of the Rf 1 gene. The restriction sites are shown on E, EcoR1;
H, HindIII; P, PstI; T, EcoT221, S, SalI and V, EcoRV. (Adapted
from Iwabuchi et al. 33 ).
Bock et al. 81 found that the extent of editing was lower
in seeds and roots than in leaf etioplasts and chloroplasts. The reverse transcription-polymerase chain reaction (RT-PCR) assay was applied to search for
differences in the extent of editing of petunia nad3 transcripts in different tissues, no developmental regulation
of the extent of editing was detected 21 . In the absence of
a transformation system in which sequences can be deliberately altered, editing can be assessed in abnormal
genes created by the rearrangement characteristic of
plant mitochondrial genomes 82 . In genes created by
such aberrant recombination events, pieces of the coding regions of mitochondrial genes are put into different
contexts, but editing events in the translocated regions
are usually still observed. Despite numerous recombination events placing portion of atp9, parts of the first and
second exon of Cox II, and an unidentified reading
frame (urf) together in the petunia pcf gene, almost all
sites are edited 83 . A 193-nucleotide segment of wheat
Cox II is edited when present as part of the nad3/rps12
transcript 27 . In the atp6 gene, found in maize T-cms, a
segment containing 30 nucleotides of the coding region,
plus some upstream sequence derived from the atp9
coding region was still edited even when located next to
an unidentified urf 84,85 (see Figure 2).
Nuclear-encoded genes required for normal mitochondrial gene expression have been described for several mitochondrial genes in yeast 86 . They regulate the
expression of mitochondrial genes at both the transcriptional and post-transcriptional levels. In higher plants
the expression of the nuclear and mitochondrial genomes must be coordinated to make a functional cell.
Cms is a loss of functional pollen in plants that can be
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
REVIEW ARTICLES
attributed to unique mitochondrial genomes. Cms is the
resultant of an incompatibility between nucleus and
mitochondrial genomes such that pollen is not properly
formed. However, there are nuclear genes designated Rf
or Fr (restorer of fertility) that can correct this incompatibility and allow the development of functional pollen. Thus, cms offers a system with which to study the
mechanism(s) by which nuclear genes directly regulate
mitochondrial gene expression. Among the several Rf
genes known to reduce or completely eliminate the cms
phenotype, some specifically affect the transcription
patterns of the cms gene. One of the restorers introduces
new processing sites into the mutant T-urf13 transcripts
and leads to almost complete reduction of the corresponding polypeptide synthesis 17,86 (see Figure 2). The
open reading frame (orf) of the mitochondrial complex
IV gene CoxI is extended by a recombination event in
sorghum cytoplasms and leads to a cms phenotype upon
high expression 4. As yet not known nuclear genes revert
the cms phenotype and compensate for the expression
of this aberrant protein 87 . In sunflower mitochondria, a
recombined novel orf correlated to a cms phenotype is
likewise influenced by specific nuclear gene expression 4.
Comparison of Northern blot analysis and run-on experiments has exhibited that the nuclear restorer genes
act at the post-transcriptional level and alter transcript
stability. Recent results suggest that RNA processing by
Rf 1 in rice-cms system does influence the sequential
post-transcriptional editing of the B-atp6 RNA 33 (see
Figure 4). The B-atp6 gene was identified as a cms gene
by Southern analysis of the mitochondrial genome. The
coding sequence of B-atp6 was identical to the normal
N-atp6 gene but its 3′
-flanking sequence was different
starting at 49 bases downstream from the stop codon 33 .
Northern analysis showed that B-atp6, transcribed into a
2.0 kb RNA, in the absence of the Rf 1 gene whereas two
discontinuous RNAs, of 1.5 and 0.45 kb were detected
in the presence of the Rf 1 gene. Iwabuchi et al. 33 interpreted these results by suggesting that the unprocessed
RNAs of B-atp6 are possibly translated into altered
polypeptides and that interaction of RNA processing
and editing plays a role in controlling cms expression
and the restoration of fertility in rice. The recent identification of a nuclear restorer gene in maize (rf 2) coding
from a mitochondrial aldehyde dehydrogenase
suggests that metabolic enzymes influence directly
through gain of function or indirectly by their metabolic
activity 16 .
Cms phenotype caused by mitochondrial
mutation
The cms phenotype is now thought to be caused by
mitochondrial mutations. Mitochondrial genomes of
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
higher plants are too large (over 200–2400 kb) to permit
identification of mutation by complete genomic sequencing. In plants, several strategies have been used to
locate mtDNA regions of interest for sequencing. In
maize, type cms-T, a genomic library was screened with
cDNAs from mitochondria of fertile vs male sterile
(cms) maize lines, to identify the gene region expressed
differentially under the influence of the nucleus 4. So far
twenty-nine mitochondrial cms related region(s) or
gene(s) (mostly chimeric in nature) in over twelve plant
species have been completely or partially characterized
(Table 2; also see Figures 1–4)). Mitochondrial genes
have been reported to be associated with cms cytoplasm
in maize, sunflower, petunia, sorghum and radish 88–96
(Table 2). In maize, petunia and sorghum a chimeric
mitochondrial gene which produces a novel protein has
been found in cms plant mitochondria 87,92,97 . In the mitochondria of cms sunflower a new open reading frame
is co-transcribed with the atp1 gene 98 and the protein
supposed to be encoded by this orf is uniquely present 99 . Among these examples only the T-urf13 (Figure
2) associated with maize cms-T cytoplasm has been
strongly correlated with the cms phenotype based on the
analysis of a number of mutants which are toxin resistant and fertile 100 . Although novel proteins uniquely
present in the cms mitochondria, an altered gene expression caused by mitochondrial genome rearrangments is thought to be responsible for cms, the precise
Table 2. Mitochondrial region/gene(s) involved in cytoplasmic
male sterile phenotype in several higher plant species
Plant species
Mitochondrial gene(s)/
chimeric mitochondrial gene
Rice (Oryza sativa)
Maize (Zea mays)
Sorghum (Sorghum
vulgare)
Sunflower (Helianthus
annuus)
Oilseed Rape (Brassica
napus)
Bean (Phaseolus vulgaris)
Teosinte (Wild relative of
maize)
Arabidopsis thaliana
Nicotiana tabacum
Brasssica tournefortii
Radish (Raphanus sativum)
Triticum timopheevi
Petunia (Petunia hybrida)
Brassica cybrids (Ogura
cytoplasm)
Atp 6 genes (N-atp 6
and B-atp 6), orf 25,
cox III
T-urf 13, orf 221, atp 6,
atp 9
orf 107, Cyto c oxidase
atp A, atp 9, Cob,
rrrn 26, orf 552
atp 6, orf 224, nad 4
References
33, 46
16, 21, 30,
34, 100, 116,
119–121
61, 87, 89
98, 99, 122
63
pvs sterility sequence,
orf 98 and orf 239
Cox II
13
11, 47
CHM
orf 25, orf 239
orf 3
atp A
atp 6, orf 25
pcf-a
orf 138, orf B
123
19, 107
108
96
110
124
125, 126
183
REVIEW ARTICLES
molecular mechanism underlying cms expression still
remains to be elucidated. Observation of altered electron transport in petunia and toxin-mediated membrane
disruption in maize plants, bacteria and yeast expressing
the maize urf13 gene product provide clues to possible
mechanism of disruption of pollen development or dysfunctional mitochondria in aborted pollen. Whether disruption in a particular mitochondrial function is at the
root of cms in all higher plant species, or whether defects in numerous mitochondrial activities can produce
pollen male sterility will only be unravelled by further
probing of physiological and biochemical defects present in cms genotypes.
Nuclear gene influence
The nucleus somehow affects mitochondrial genome
organization and function in higher plant systems. The
plant mitochondrial genome, now fully sequenced in
Arabidopois 101 and Marcantia 102 , is organized in a much
more complex and variable structure than is observed in
other higher plants or eukaryotes 103 . In most higher
plants, this organization is defined by the presence of
recombinationally active repeated DNA sequences that
allow for high- and low-frequency inter- and intramolecular recombination events to occur 104 . The
physical organization of the mitochondrial genome in
plants has proved to be rather difficult to define. Although most genomes map on circular molecules defined by overlapping clones, direct physical observation
by pulsed field gel electrophoresis, electron microscopy
and other procedures has revealed that the genome may
consist of both linear and circular forms, with molecules much larger than the multiple circles constructed
by clone analysis 105 . A multipartite genome organization exists in the mitochondria of most plant species,
with each molecule containing only a portion of the
genetic information. The relative copy number of the
various mtDNA forms and their recombinational activity appears to be under nuclear control. One of the most
pronounced examples is the observed loss of a mitochondrial genomic molecule in response to a single nuclear gene in common bean (Phaseolus vulgaris)13,106 .
The cms-associated mitochondrial mutation in common
bean, pvs-orf 239, appears to be maintained on a single
210 kb molecule within a tripartite mitochondrial genome organization 13 . Introduction of a single dominant
nuclear factor, Fr, (fertility restorer) results in a genomic shift of the pvs-orf 239-containing molecule to
substoichiometric levels within the genome, thus restoring pollen fertility/male fertility. The development of
alloplasmic lines, derived by recurrent backcrossing
strategies or protoplast fusion to combine different mitochondrial and nuclear genotypes, routinely gives rise
to changes in relative stoichiometries and mitochondrial
184
genomic rearrangements in Nicotiana 107 , Brassica 108,109
and Triticum 110–112 . In some cases, it has been possible
to identify specific nuclear loci essential to establish
compatibility in individual nuclear–cytoplasmic (intergenomic) combinations 113 . It may thus be possible to
predict particular nuclear–cytoplasmic intergenomic
interacting genetic combinations which would give rise
to high frequency of specific mitochondrial mutations.
Several nonchromosomal stripe mutations (ncs) affect
distinct loci and have arisen by what appears to be different molecular events 11,47,114 . A characteristic of cms
mutation in plants is the presence of chimeric genes or
chimeric locus and different open reading frames are
joined together or placed in proximal locations and cotranscribed with standard mitochondrial genes. Despite
much progress, and the identification of several mitochondrial loci that specify cms in a number of crop
plants (Table 2; also see Figures 1–4), the molecular
basis of this nuclear–mitochondrial interactive generelated genic defect is not well understood in any plant
species. Observations of altered electron transport in
wheat 115,116 and petunia and toxin-mediated membrane
disruption in maize plants 15,16 , bacteria, and yeast expressing the maize urf13 gene product, provide clues to
possible mechanisms for disruption of pollen development 117 . Whether disruption in a particular mitochondrial function 115,118,119 is at the root of cms in all
species, or whether many nuclear–mitochondrial intergenomic interaction-related genetic defects in numerous
key mitochondrial activities related to electron transport
and oxidative phosphorylation (ATP energies) can produce male pollen sterility, will only be revealed by further probing of physiological and biochemical defects
naturally or evolutionarily present in cms genotypes of
higher plants. Cms mutations have proven particularly
useful in defining regulation of mitochondrial respiratory functions. Data suggest that variation in promoter
strength is likely a primary influence on relative transcription rates 4. Nucleus genes regulating transcription
rate or promoter selection are difficult to detect. Perhaps the most compelling demonstration of nucleus control on mitochondrial gene transcription is described in
a Zea mays/Zea perennis alloplasmic line. A single nuclear gene, described Mct, influences promoter selection
in CoxII gene in maize 11,47,114 . Transcriptional initiation
at position 907 produces the predominant CoxII transcript of ~1900 nucleotides. The dominant nuclear Mct
allele apparently directs transcriptional initiation at a
second site (– 347) upstream to the CoxII locus. Interestingly, this alternate initiation site, detected as a
shorter CoxII transcript, does not conform to the consensus promoter sequence described for maize 4,15 . This
provided some circumstantial evidence that specific
nuclear gene produced cofactors may influence promoter selection in cms-related genomic region of plant
mitochondria. While nuclear genome is undoubtedly the
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
REVIEW ARTICLES
principal source of heredity and Mendelian gene transmission, the role played by cytoplasmic and maternally
and non-Mendelian inherited mitochondrial genome
through intergenomic interaction linked cotranscriptions
in giving rise to evolutionarily and speciation wise important cms trait and resultant hybrid seeds with heterosis having high proprietary, economic, and commercial
relevance in higher plant world needs further focusing
and mechanistic unravelling 115–118 .
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Received 2 February 2000; revised accepted 24 May 2000
Genetic transformation of rice: Current status
and future prospects
S. Ignacimuthu†,**, S. Arockiasamy † and R. Terada*
†
Entomology Research Institute, Loyola College, Chennai 600 034, India
*National Institute for Basic Biology, Okazaki, Japan
Genetic transformation of rice has been an important area of research in the past few years. PEG,
electroporation, microprojectile bombardment, and
Agrobacterium have been used to mediate gene transfer. Genes for insect resistance, fungal resistance,
virus resistance, herbicide resistance, bacterial resistance and nematode resistance have been utilized in
rice transformation. The methods involved, the genes
utilized and the promoters used in rice transformation along with integration and expression of transgenes are discussed in this review.
THE ever-increasing human population especially in the
developing countries and various abiotic and biotic
stresses have posed a challenge to boost the rice
production in limited cultivable land 1,2 . Genetically engineered plants with genes of direct interest can be produced in a relatively short time and can be of direct
value in the agri-food industry. Recently it has been
**For correspondence. (e-mail: [email protected])
186
recognized that marker-free transgenics may be more
acceptable commercially 3,4 . Some good reviews are already available 5–8. Initially rice was transformed with
direct transformation methods such as particle bombardment. Recently it has been shown that Agrobacterium tumefaciens can efficiently transform rice also. In
this review, we shall discuss the latest developments in
the transformation of rice. We shall focus on advances
in gene transfer techniques and we shall also mention
specific genes that have recently been introduced into
rice plants as well as various issues related to the integration and expression of foreign genes.
Methods of gene delivery
PEG-mediated rice transformation
In 1986, Uchimiya et al. 9 first obtained transgenic calli
after polyethylene glycol (PEG)-induced DNA uptake
of the nptII gene into root-derived protoplasts, followed
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
REVIEW ARTICLES
by selection on kanamycin. The first transgenic rice
plants were recovered by Zhang et al. 10 . Datta et al. 11
published the first report of the recovery of transgenic
indica rice plants from cultivar Chinsurah Boro II. In a
systematic study of factors that influence the efficiency
of PEG-mediated transformation and regeneration, Hayashimoto et al. 12 reported that increasing the PEG
concentration from 0 to 30% (w/v) reduced the plating
efficiency of Nipponbare protoplasts from 11 to 3% and
Taipei-309 protoplasts from 14 to 5%. However, the
number of hygromycin-resistant calli obtained increased
with increasing concentrations of PEG in both cultivars.
The transformation frequency of Taipei-309 protoplasts
was also found to increase with increasing concentrations of plasmid DNA up to the levels tested (20 µg
DNA per 2–5 × 104, based on the number of protoplasts
used).
Most reports of successful PEG-mediated transformation and subsequent regeneration of transgenic rice
plants have used 20–25% PEG, 10–30 min incubation
times, and 10–100 µg of DNA per ml protoplast. Detailed protocols have been described by Hodges et al. 13
and Li et al. 14 .
Electroporation-mediated gene transfer
Electroporation has also been widely used to introduce
naked DNA into protoplasts. Toriyama et al. 15 first used
this method for the production of Yamahoushi cultivar
transgenic rice via anther culture-derived protoplasts
with the aminoglycoside phosphotransferase II
(aph(3')II) gene, conferring resistance to the antibiotics
kanamycin and G418. Southern analysis showed the
correct size fragment upon hybridization to the aph(3')II
gene. In another early study, Zhang et al. 10 electroporated protoplasts from cell suspension of Taipei-309
leaf base calli with the 35S promoter fused to the nptII
gene. Out of six regenerated plants, two were positive
for NPTII activity. Biswas et al. 16 also successfully reported the recovery of transgenics from indica rice IRRI
breeding line IR58 protoplasts. Zu and Li 17 obtained
fertile transgenic indica rice plants using seed embryo
cells. Chaudhury et al. 18 observed transient expression
of gus gene in intact seed embryos of indica rice.
Microprojectile bombardment-mediated gene transfer
Microprojectile bombardment, also called the biolistic
method or the particle gun method has been used in
many laboratories. The concept has been described in
detail by Sanford 19 . The ability to deliver foreign DNA
into regenerable cells, tissues or organs appears to provide the best method for achieving genotypeindependent transformation bypassing Agrobacterium
host specificity.
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
In earlier experiments, Christou et al. 20 reported that
immature embryos were isolated from greenhouse
grown cultivars of japonica, javonica and indica rice.
The scutellar region of the embryo was bombarded and
transient activity for the introduced gene(s) was observed after 24 h of bombardment. Bombarded tissues
were plated on regeneration media supplemented with
appropriate selective agents; transformed embryos and
other transgenic organized tissues, such as shoots and
roots in addition to transformed plants were obtained.
Sivamani et al. 21 reported a gene transfer procedure
for the model indica rice variety TN1, using bombardment of embryogenic callus. They developed a procedure which allowed generation of highly homogenous
populations of embryogenic subcultured calli by selectively propagating a small number of regenerationproficient calli derived from seed. An efficiency of 3%
could be obtained on an average over a number of experiments. Zhang et al. 22 regenerated transgenic plants
from bombarded embryogenic suspensions of elite indica rice varieties, using procedures which are now
commonly used by many groups. They commented that
this genotype and environment-independent transformation system for indica rice that uses regenerable embryogenic suspensions as targets for bombardment may
now be used routinely for the introduction of useful
genes into elite rice varieties. Jain et al. 23 optimized the
biolistic method for transient gene expression and production of agronomically useful transgenic basmati rice
plants. Many biolistic devices (e.g. particle gun) have
been developed, including both commercial and labbuilt models. Moreover there is a possibility to get a
relatively inexpensive simple particle gun specially for
rice-producing developing countries 24 .
Agrobacterium-mediated gene transfer
The transformation of dicotyledons by Agrobacterium is
well established. But in the case of monocots it is not
the general process. In the past monocots, particularly
graminacious crop plants including important cereals
like rice and wheat were considered to be recalcitrant to
this technology and they were outside the Agrobacterium host range. However, transformation methods
based on the use of Agrobacterium are still preferred in
many instances because of the following properties: (i)
easy to handle, (ii) higher efficiency, (iii) more predictable pattern of foreign DNA integration, and (iv) low
copy number of integration.
Raineri et al. 25 described the production of transformed japonica cultivar by cocultivation of mature
embryos with Agrobacterium. Results of Southern blotting indicated the integration of the T-DNA into the
plant genome, but no transgenic plants were regenerated. In 1992, Chan et al. 26 obtained a few transgenic
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rice plants by inoculating immature embryos with a
strain of Agrobacterium. They proved the inheritance of
the transformed DNA to progeny plants by Southern
blot hybridization, although they analysed the progeny
of only one transformed plant. Hiei et al. 7 subsequently
reported a method for efficient production of transgenic
rice plants from calli of Japonica cultivars that had been
cocultivated with A. tumefaciens. Their evidence was
based on molecular analysis and genetic studies of a
large number of transgenic plants and the analysis of
sequence of T-DNA junctions in rice. Rashid et al. 27
reported the successful application of such a method to
basmati cultivars of indica rice after only minor modifications. In the same year, Dong et al. 28 also described
the successful applications of the method to Javanica
rice. Aldemita and Hodges 29 showed that immature embryos were also good starting materials for Agrobacterium-mediated transformation of indica and japonica
varieties. Park et al. 30 reported that the isolated shoot
apices are suitable explants for successful application of
this method. Uze et al. 31 and Toki 32 reported such types
of transformation with minor modifications. Abedinia et
al. 33 transformed an Australian cultivar by the Agrobacterium-mediated method. Mohanty et al. 34 reported the
successful application of this method in an elite indica
rice variety pusa basmati 1 and transmission of the
transgene to the R 2 progeny was also demonstrated.
Khanna and Raina 35 transformed indica rice cultivars by
the Agrobacterium-mediated method using binary and
super-binary vectors. Chen et al. 36 developed a protocol
for consistent and large-scale production of fertile transgenic rice plants. Yokoi et al. 37 produced chilling tolerance of photosynthesis and unsaturation of fatty acids
in rice by introducing the GPAT gene. Goto et al. 38
obtained rice seeds with iron fortification by using
soybean ferritin gene. Ye et al. 39 succeeded in engineering the provitamin A (b-carotene) biosynthetic pathway
into carotenoid-free rice endosperm.
Desired genes introduced into rice
Insect resistance
In the last few years various genes have been introduced
into rice, since the methods of transformation have been
well established in this species. Majority of the genes
that have been introduced into rice are related to disease
and insect resistance (Table 1). To our knowledge,
there are about eleven reports, in which Bacillus thuringiensis (Bt)-derived genes have been introduced in
rice against insect resistance by electroporation 40 , biolistic method 41–49 and by Agrobacterium method 50 .
Other genes such as oryzacystatin 51,52 , corn cystatin 53 ,
PINII 54 , trypsin inhibitor 55,56 , GNA 57,58 have also been
added.
188
Fungal resistance
Lin et al. 59 reported the production of rice transgenic
plants against a rice sheath blight pathogen (fungus)
Rhizoctonia solani. Stark-Lorenzen et al. 60 reported the
production of transgenic rice plants containing Stilbene
synthase gene to enhance resistance to the fungus
Pyricularia oryzae using PEG-mediated direct gene
transfer.
Virus resistance
Hayakawa et al. 61 have introduced the coat protein gene
of rice stripe virus into two japonica varieties of rice by
electroporation of protoplasts. Recently 62 transgenic
plants were produced showing the expression of coat
protein of rice dwarf virus. Sivamani et al. 63 introduced
the coat protein (CP) genes CP1, CP2 and CP3 of rice
tungro spherical virus (RTSV) individually or together
into indica and/or japonica rice cells by particle bombardment.
Herbicide resistance
Christou et al. 20 have recovered herbicide-resistant
transgenic rice plants from a number of commercially
important cultivars, including recalcitrant indica varieties using electric discharge particle acceleration.
Further, Cao et al. 64 have reported the production of
herbicide-resistant transgenic rice plants, in which the
plants were transformed by microprojectile bombardment with plasmids carrying the coding region of the
Streptomyces hygroscopicus phosphinothricin acetyle
transferase (PAT). In the same year Datta et al. 65 reported the introduction of the bar gene into protoplasts
of rice (IR72) by PEG-mediated transformation. Rathore et al. 66 produced herbicide-resistant rice plants by
introducing the bar gene into protoplasts derived from
immature embryos by PEG-mediated DNA uptake.
Recently a successful first rice field trial using a bar
gene was reported by Oard et al. 67 . They bombarded the
bar gene into immature embryos of commercial cultivars ‘Gulfomont’, ‘IR72’ and ‘Koshihikari’. Sankula et
al. 68,69 evaluated the application of Glufosinate on rice
transformed with the bar gene in a field trial study.
In one instance, introduction of the herbicide-resistant
bar gene in rice also provided protection from the sheath
blight pathogen, Rhizoctonia solani70. In several studies
the bar gene was used as a selectable marker gene30,71 .
Bacterial resistance
Zhang et al. 72 reported the introduction of the Xa21
gene into embryonic suspensions through the biolistic
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CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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190
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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method, which confers resistance to Xanthomonas
oryzae pv oryzae, a bacterial blight pathogen.
Nematode resistance
Vain et al. 73 recovered nematode (Meloidogyne incognita)-resistant transgenic plants, containing genes coding for an engineered cysteine proteinase inhibitor
(Oryza Cystatin-ID860; OC-I 8D86), using the particle
gun method.
Other genes
Xu et al. 74 introduced the barely LEA3 gene into a japonica rice variety, Nipponbare, using the biolistic
method and the transgenic plants showed increased tolerance to both water stress and salinity. Some other
genes have been recently introduced in attempts to improve the nutritional and other qualities. Phytoene synthase from daffodil (Narcissus pseudonarcissus) was
transferred into immature embryos of a rice model variety (Taipei-309) by microprojectile bombardment. Phytoene was unequivocally identified in several transgenic
rice endosperms 75 . Rice mature embryos were subjected
to the introduction of ribulose bisphosphate carboxylase
gene. The rice plants used nitrogen with enhanced efficiency during photosynthesis at saturating levels of CO 2
and high irrradiance 76 . Ku et al. 77 reported the high
level expression (12% of total soluble protein) of maize
phosphoenolpyruvate carboxylase (PEPC) gene in rice
plants. The PEPC gene, which catalyses the initial fixation of atmospheric CO 2 in C 4 plants was introduced
into the C 3 crop rice.
Promoters used in rice transformation
The success of plant transformation depends very much
on promoter sequences 78 . To express the transgenes in
plant cells, appropriate promoter sequences have to be
introduced alongside the gene to ensure efficient transcription of mRNA 79 . A large number of promoters have
been used in rice transformation (Table 2) and several
promoter sequences have been isolated from monocots
for specific use in cereal species for the
efficient expression of the transgenes. The promoter
CaMV 35S or its derivatives have been used
greatly 7,20,27,28,64,80–83 ; the level of gene expression of
this promoter has been reported to vary between different species of plants and different parts of the plant 84 .
Further, it has been proved to be useful particularly in
dicot species 78 . Unfortunately the levels of gene expression produced by this promoter in cereals are up to
hundred-fold less than in dicots 85,86 . A number
of monocot promoters (EMU 87,88 , Actin1 30,55,64,89,90 ,
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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Table 2. Constitutive and non-constitutive promoter genes used in rice transformation
Promoter
35S
Source
Gene
Pattern of gene expre ssion
Cauliflower mosaic virus, 35S RNA transcript
All plant tissues
EMU
Actin 1
Cauliflower mosaic
virus
Maize
Rice
Modified maize alcohol dehydrogenase 1 promoter
Rice actin 1 gene
All tissues
All plant tissues
Ubi-1
Adh
Maize
Maize
Maize ubiquitin 1 gene
Alcohol dehydrogenase II gene
His 3
LHCP
Wheat
Rice
rbCS
Rice, tomato
Pin II
Rol C
OSg6B
OsgrP1
Potato
Agrobacterium
rihzogenes
Rice tungro bacilli
form virus
Maize and rice
Rice
Histone 3 gene
Light harvesting chlorophyll binding gene
of photosystem II
Ribulose 1,5-bisphosphate carboxylase/oxygenase
small subunit gene
Wound inducible II gene
Open reading frame 12 (RF12) of the R 1 plasmid
T 2 DNA region
Rice tungro bacilli form virus, major transcript gene
All plant organs
Constitute root cap, anther,
filament, pollen, scutellum,
endosperm, embryo shoot
and root, Anerobically
induced in roots
Dividing root cells
Light inducible in leaves,
stems and floral organs
Light inducible in mesophyll
RSs1
PEPC
Rice
Maize
Rice sucrose synthase gene
Phosphoenol pyruvate carboxylase gene
Bp10 gene
GluA-2
GluB-1
GluA-3
RAG-1
GLb-1
NRP33
Ltp2
Brassica
Rice
Rice
Rice
Rice
Rice
Rice
Barley
Pollen gene
Glutelin gene
Glutelin 1-gene
Glutelin 3-gene
Albumin 1-gene
Globulin gene (Glb-1)
Prolamin (NRB 33)
Lipid transfer protein
RTBV
Glycine-rich gene
Glycine-rich cell well protein gene
ubiquitin-171,91–93 and Adh1 94,95 ) have also been evaluated to enhance gene expression in cereals. Among
these promoters, hpt and EMU hph constructs gave a
more dramatic expression than ubiquitin gene and any
of the bar containing constructs. The gene for Actin1
and ubiquitin-1 (gene of maize) has been shown to produce highest levels of expression in rice. Other promoters such as His 96 , LHcP 97 , reCS 98 , Pin 54,55 , Rolc 88,99 ,
RTBV 100 , O8gCB 101 , OsgrP1 102 , RSs1 57,58,103 , PEPc 43,77 ,
BP1050 , Glu 104 , Ltp 105 have also been used successfully.
Integration and expression of transgenes
The stable integration, expression and inheritance of
transgenes are of great importance in the application of
genetically transformed rice to agriculture, but they
have not been extensively studied 106 . Only the first generation of transgenic plants (R 0 progeny) were analysed
192
Reference
20, 27, 28, 64,
80–83
87, 88
30, 55, 64, 89,
98
71, 88, 91–93
94, 95
96
97
98
Wound inducible
Vascular tissue
and embryonic tissue
Leaf phloem tissue
54, 55
88, 89
Tapetum specific expression
Expressed in young stem,
leaves, vascular bundles,
epidermis, wound inducible,
and in water stress conditions
Phloem-specific expression
Green tissues, leaves and
leaf blades
Pollen-specific expression
Endosperm specific
Endosperm specific
Endosperm specific
Endosperm specific
Endosperm specific
Endosperm specific
Aleurone-specific gene
101
102
100
57, 58, 103
43, 77
50
104
75, 104
104
104
104
104
105
in many reports. Hiei and Komari 107 analysed the inheritance of transgenes as far as the R 4 generations successfully for eighteen transgenic plants that have been
produced by the Agrobacterium-mediated method and
stable integration of transgenes in the genome of all
progenies was demonstrated. In this study only a small
number (fewer than three) of copies of the transgene
integrated in a majority of the transformants. Due to this
advantage Agrobacterium-mediated transformation is
the method of choice. However in another study molecular analysis of genome DNA from engineered plants
indicated the presence of vector sequences outside the
T-DNA border 108 . Agrobacterium was also found to
persist on the surface and within tissues of transformed
plants in soil-grown plants up to twelve months following transformation 109 . These are the two potential problems in Agrobacterium-mediated transformation 110 . By
contrast, direct transformation technology will result in
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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transgenic plants containing multiple copies of transgenes, complicated pattern of integration 111 and high
occurrence of chimeric events 112 .
Takano et al. 113 analysed the transgenic plants produced from protoplasts through the calcium phosphate
method. One factor that contributed to the complex patterns of integration of foreign genes in transformation is
the probable activity of topoisomerase I or II. Because
of these illegitimate recombinations, accomplishing
rearrangements, large-scale deletions and translocations
were found at the sites of integration. Further, recognition sites for topoisomerase were identified in the rearranged sequences involved in the process of gene
silencing (expression of transgene and even native gene
is suppressed) 114 . The inactivation of gene expression is
known as co-suppression, when homologous coding
sequences are involved. Silencing of the waxy gene was
observed in rice that had been transformed with a
cloned waxy gene 115 . This is the first study of silencing
of a native gene and a transgene. A similar type of study
on silencing of transgenes and 5-azacytidine-mediated
reactivation of the transgene have been reported in
rice 116 . Silencing of the genes encoding b-glucuronidase
in transgenic rice was demonstrated by methylation 117 .
Such type of studies probably help to understand the
intrinsic mechanisms for the control of gene expression
in rice and also the molecular mechanisms of plants.
Conclusion
It is obvious that rice transformation system through
both Agrobacterium and particle bombardment has become a routine procedure in many laboratories for the
production of transgenic plants. Transgenic plants containing genes of agronomic interest including insect,
herbicide, virus and nematode resistance through direct
transformation techniques have been obtained. The next
challenge for rice improvement via genetic engineering
is to produce viral tungro disease resistance which can
help in preventing the annual loss in the range of 1.5
billion US dollars in south and south-east Asia as well
as to produce iron-rich rice, including introduction of
the apomictic trait which will allow to fix hybrid vigour
in the progeny without any further segregation.
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Received 23 February 2000; revised accepted 10 May 2000
RESEARCH ARTICLES
Resolution of the nonlocality puzzle in the EPR
paradox
C. S. Unnikrishnan
Gravitation Group, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India and NAPP Group, Indian Institute of
Astrophysics, Koramangala, Bangalore 560 034, India
Correlations of quantum-entangled multiparticle
systems have been widely discussed in the context of
the Einstein–Podolsky–Rosen (EPR) paradox. Bell’s
theorem prohibits a local realistic description of
these correlations. The standard quantum mechanical derivation as well as the interpretation of the experiments suggest that a mysterious nonlocality is a
basic feature of these correlations. We show that the
correlations of space-like separated entangled particles can be reproduced starting from local probability amplitudes. The use of complex number
amplitudes circumvents the widely discussed Bell’s
theorem. The result implies no-collapse-at-a-distance
and resolves the EPR puzzle.
THE Einstein–Podolsky–Rosen (EPR) nonlocality puzzle is one of the most discussed fundamental problems
in physics 1–4. EPR considered a two-particle quantum
e-mail: [email protected]
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
system entangled and correlated in position and momentum and argued that under the assumption of locality
and a reasonable definition of physical reality, the quantum mechanical description is incomplete. The conclusion of incompleteness was based on their reasoning
that suggested the need for simultaneous existence of
definite values for noncommuting observables for the
individual particles before a measurement was performed. The EPR analysis was motivated by the desire
to assign an objective reality to measurable properties in
the microscopic world independent of the observer or
apparatus. The Copenhagen interpretation of quantum
mechanics rejected any such objective reality. EPR proposed that if the value of an observable could be predicted with certainty without the disturbance of a
measurement, then there was an element of physical
reality associated with that observable for the system.
There is no way to probe experimentally such a physical
reality for the single quantum, for example the reality of
the spin component in a specific direction for a single
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RESEARCH ARTICLES
photon or an electron. But if multi-particle correlated
systems are considered, like two spin- 12 particles
propagating out from an initially spin-zero state, then it
is possible to explore the consequences of assuming
objective reality to observables.
Bell’s theorem
David Bohm transcribed the EPR argument to the spin
variables by considering the singlet entangled state of
two spin- 12 particles 5. This state is described by the
wave function
ψ
S
=
1
2
{| 1,− 1〉− | − 1,1〉},
(1)
where the state |1, –1〉 is a short form for |1〉1 |–1〉2, and
represents an eigenvalue of +1 for the first particle and
–1 for the second particle if measured in any particular
direction. y S is inherently nonlocal, describing both
particles together, even when they are far apart in
space-like separated regions. It is a superposition of
two-particle product states and there is no specific state
assigned to any of the particles individually. A measurement on one particle changes the whole wave function, collapsing the states of both particles to definite
values.
If the spin component is measured in any direction on
one of the particles, the same component can be predicted with certainty for the other companion particle
using the conservation law that the total spin is zero.
The EPR definition of reality then assigns objective
reality before measurement to this spin component.
Since the decision as to which component is to be
measured is arbitrary and could be delayed till the particles are far apart, the assumption of locality – that one
measurement should not influence the result of the
other – implies that there should be physical reality for
all the three components of the spin for the two particles
individually. This is not allowed by quantum mechanics
since the three spin components are mutually noncommuting. According to the EPR argument, this conclusion suggested the plausibility of a better theory,
possibly with additional hidden variables that would
assign specific values to the observables in each run of
an experiment such that the statistical predictions of
quantum mechanics are reproduced when averaged over
the distribution of the hidden variables. Such a theory is
a local (realistic) hidden variable theory.
John Bell analysed the EPR problem in the early sixties and established the Bell’s inequalities obeyed by
any local hidden variable theory of correlations of entangled particles 3,4,6 . He considered the measurement of
the spin components on the two particles in two differ196
ent directions, in contrast to the EPR analysis that involved measurements in the same direction (see Figure
1). The result of such a measurement is two-valued in
any direction. If A and B denote the outcomes +1 or –1
(written as + or –) and a and b denote the settings of the
analyser or the measurement apparatus for the first particle and the second particle, respectively, then the
statement of locality is that
A(a) = ± 1,
B(b) = ± 1.
(2)
The outcome for the first particle is decided by the local
setting a for the analyser with which the first particle
interacts, and for the second particle the outcome is decided by the local setting b. The statement of existence
of reality is that some hidden variable decides the outcomes even before the measurement, or equivalently the
outcome has an objective existence even if the measurement is not actually carried out. This is encoded as
A(a,h1) = ± 1,
B(b, h2) = ±1,
(3)
where h1 and h2 are hidden variables associated with the
outcomes. In an experiment involving the kind of measurement described, the experimenter can calculate a
correlation function of the outcomes defined by
P (a, b) =
1
N
∑ ( A B ).
i i
(4)
This is a classical correlation function obtained by averaging the products of the form (++ = +), (– – = +), (+–
= –) and (– + = –). Since the joint events (++) and (– –)
are coincidences and the events (+ –) and (– +) are anticoincidences (‘coincidence’ denotes both particles
showing the same value for the measurement and ‘anticoincidence’ denotes those with opposite values),
P(a, b) denotes the average of the quantity (number of
detections in coincidence – number of detections in
Figure 1. Schematic diagram of spin correlation measurements on
entangled particles. The outputs of the two analysers are +1 or –1
and these are correlated in a coincidence unit. a and b are the two
analyser directions. Dotted arrow on the right analyser shows the
direction a for reference.
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
RESEARCH ARTICLES
anticoincidence). The task of the theory is to calculate
this function starting from suitable basic ingredients.
Bell chose to calculate this correlation by multiplying
A(a, h1) and B(b, h2) and integrating over the distribution of the hidden variable h, since the assumption of
realism demanded that the outcomes were ‘there’ even
before the measurement was made. The Bell correlation
is then ∫dhr(h)A(a, h1)B(b, h2), where ∫dhr(h) = 1. The
essence of Bell’s theorem is that the function P(a, b)
has distinctly different dependences on the relative angle between the polarizers for a local hidden variable
description and for quantum mechanics.
The correlation predicted by a local realistic theory is
bounded by the Bell’s inequalities. The magnitude of a
particular sum of correlations P(a, b) for different combinations of a and b is bounded by the value 2, and the
same combination calculated in quantum mechanics
using the entangled wave function and spin operators
exceeds this bound, violating the inequality 3,6 . Also,
experimentally measured correlations agree with the
quantum mechanical predictions and violate the
inequality 4,7 . The concept of local realism is not tenable
in the Bell framework. These results have been interpreted as evidence for nonlocal influences across spacelike separated events. The measurement of an observable on one of the particles causes the other particle to
acquire a definite state consistent with the relevant conservation law. The standard quantum mechanical derivation of these correlations employs the nonlocal
multiparticle wave function, and measurement on one
particle is said to collapse the state of the companion
particle to a definite eigenvalue. Though this nonlocal
feature cannot be used for superluminal signal communication 8, there is a conflict with the spirit of relativity 9.
Also, such superluminal influences in the microscopic
physical world are bizarre and at present beyond any
understanding in terms of any physical mechanism.
The nonlocality puzzle can be resolved however, if
the correct physical input is used for the calculation of
the quantum correlations. The correct correlations can
be reproduced if we start with the description of the
relevant physical phenomenon, like the passage of the
quantum particle through a polarizer, using local probability amplitudes 10 . In the local hidden variable theories of the form Bell considered, the correlations are
calculated from eigenvalues and this procedure does not
preserve the phase information and wave-like characteristics of the quantum system. The situation has some
analogy to the description of interference in quantum
mechanics. Any attempt to reproduce the interference
pattern using locality and the information on ‘whichpath’ will fail since the phase information is lost or
modified in such an attempt. We calculate the correlation from the local amplitudes instead of from the eigenvalues. The wave nature of quantum systems is then
explicitly used in the calculation of the correlations and
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
the final probabilities are calculated by squaring a suitable inner product of the local amplitudes. (It is the use
of complex functions with a phase that is interpreted as
the ‘wave nature’ in this paper.) The realism in this
theory is at a deeper level, concealed as a phase and not
as actual outcomes before a measurement is made. This
possibility was not considered in earlier local realistic
theories, and turns out to be the crucial concept required
to resolve the EPR puzzle.
A new calculus for correlations
Consider the breaking up of a maximally entangled
state – photons entangled in orthogonal polarization
states or spin- 12 particles entangled in (up, down)
state – as in the standard Bohm version of the EPR
problem 4,5 . The two particles go off in opposite directions and are in space-like separated regions. Two observers make measurements on these particles
individually with time stamps such that these results can
be correlated later (Figure 1). We assume that strict locality is valid. Measurement on one particle does not
change either the magnitude or phase of the complex
amplitude associated with the companion particle. In
particular, measurement on one particle does not cause
the companion particle to acquire a definite state.
At each location the result of a measurement is twovalued, denoted by (+) and (–) for each particle (two
mutually exclusive outcomes), for any angle of orientation of the analyser. We prescribe the local rules or amplitudes for the transmission through an analyser for
these particles (we use the terms polarizer and analyser
in a generic way. They could be Stern–Gerlach-like
analysers for spin- 12 particles or polarizers for photons). The local amplitudes for events + and – for the
two particles individually are denoted by the complex
functions C1+ , C1–, C2+ and C2–. Amplitudes C1+ and C1–
are mutually orthogonal and similarly C2+ and C2– are
mutually orthogonal. The statement of locality is at the
level of these amplitudes, and can be written as
C1± = C1± (a, f1), C2± = C2± (b, f2),
(5)
where f1 and f2 are the ‘hidden variables’. These are
internal variables associated with the individual particles and appear in the amplitudes as a phase. These can
be thought of as the initial undetermined phase, associated with the spin of the individual quantum particles. A
definite value for these variables does not imply a definite state for the particles before the measurement.
The locality assumption also implies that
A(a, f1) = ±1,
B(b, f2) = ±1.
(6)
But, this has a meaning different from the one in the
standard local realistic theories. Here, it means that the
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RESEARCH ARTICLES
outcomes, when measured, depend only on the local
setting and the local internal variable.
In this framework the correlation function is not
P(a,b) = (1/N)∑ (Ai Bi ) or ∫dhr(h)A(a, h1)B(b, h2). We
calculate the correlations from the local amplitudes.
These correlations are of the form U(a, b) =
Real(NC i Cj*), where N is a normalization factor. It is
the square of this correlation function that would give a
joint probability. This new calculus of local amplitudes
ensures that all the probabilities are positive definite.
(There are local theories which reproduce the correct
correlations using probability distribution of the hidden
variables that are not positive definite. These theories
are neither rooted in quantum concepts nor in classical
concepts, and are really ‘out of this world’.) The correlation function is analogous to the two-point amplitude
correlations of two independent classical electromagnetic fields. The expression can be generalized to situations where there are more than two particles.
Now that we have outlined the general scheme and
assumptions as well as the point of departure in calculating the correlation, let us consider the maximally
entangled singlet system described by eq. (1), the
most widely discussed example in the context of nonlocality. We prescribe the local amplitudes as
C1+ = 1 exp{is(q1 – f1)} for the first particle at the
2
first polarizer and C2+ =
1
2
exp{is(q2 – f2)} for the
second particle at the second polarizer. There are corresponding amplitudes, C1– and C2– for the events denoted
by –, and they differ only in the phase for the maximally entangled state.
In these amplitudes, q1 and q2 are the directions of the
two polarizers, and s is the spin of the particle (1 for
photons and 12 for the spin- 12 particles). The explicit
dependence of the amplitude on the spin of the particle
is motivated by the fact that we are dealing with systems with phases and the phase associated with the spin
rotations (a geometric phase) is a necessary input in this
description 11 . The correlation at source is encoded in
the relative value or the difference f0 of the internal
variables. f0 is a constant for all the entangled pairs.
The locality assumption is strictly enforced since the
two amplitudes depend only on local variables and on
an internal variable generated at the source and then
individually carried by the particles without any subsequent interaction of any sort. The individual measurements at each end will now separately give the correct
result for transmission for any angle of orientation.
These probabilities are
C 1 C 1 * = C 2C 2 * =
1
.
2
(7)
Events of both types (++) and (– –) contribute to a ‘coincidence’. The correlation function for an outcome of
198
either (++) or (– –) of two maximally entangled particles is
U(q1, q2, f0) = 2Re(C1C2*) = cos{s(q1 – q2) + sf0}. (8)
It is normalized such that its square will give the conditional joint probabilities of the type ‘outcome + for the
second particle, given that the outcome for the first particle is +’, etc. All references to the individual values of
the internal variable f have dropped out.
We now derive the relation between this correlation
function and the experimenter’s correlation function
P(a, b) = (1/N)å(Ai Bi ). Since U2++ = U2–– for the maximally entangled state, U2(q1, q2, f0) is the probability
for a coincidence detection (++ or ––), and (1 – U2(q1,
q2, f0)) is the probability for an anticoincidence (events
of the type +– and –+). Since the average of the quantity
(number of coincidences – number of anticoincidences)
is
U2(q1, q2, f0) – (1 – U2(q1, q2, f0))
= 2U2(q1, q2, f0) – 1,
(9)
the correspondence between P(a, b) and U(q1, q2, f0) is
given by the expression,
P(a, b) = 2U2(q1, q2, f0) – 1
= 2cos 2{s(q1 – q2) + sf0} – 1.
(10)
Examples and applications
Singlet spin- 12 particles and photons
Consider the singlet state breaking up into two spin- 12
particles propagating in opposite directions to spatially
separated regions. Since the total spin is zero in any
basis we set q0 = p. Then the correlation function and
P(a, b) calculated from this function are
U(q1, q2, f0) = cos{s(q1 – q2) + sf0}
1

= cos  (θ1 − θ2 ) + π / 2
2

1
2
= − sin (θ1 − θ2 ),
(11)
1

P (a, b) = 2 sin 2  (θ1 − θ2 ) − 1
2

= – cos(q1 – q2) = –a×b.
(12)
This is identical to the quantum mechanical prediction
obtained from the singlet entangled state and the Pauli
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
RESEARCH ARTICLES
spin operators 4. We have reproduced the correct correlation function using local amplitudes.
For the case of photons entangled in orthogonal polarization states we get, by setting s = 1 and f0 = p/2 to
represent orthogonal polarization,
U(q1, q2, f0) = cos{(q1 – q2) + p/2}
= – sin(q1 – q2),
(13)
P(a, b) = 2sin2(q1 – q2) – 1 = – cos(2(q1 – q2)),
(14)
which is the correct quantum mechanical correlation.
Two-particle interferometry
The cases of particles entangled in other sets of variables like momentum and position, and energy and time
can be mapped onto the spin problem with two-valued
outcomes and the local amplitudes reproduce the correct
correlations.
Consider a pair of EPR-entangled particles which are
propagating in opposite directions. Each particle encounters a double slit arrangement (Figure 2) on their
way12 . It is easy to see that there will not be any single
particle interference pattern in this case on either
screen. Near-perfect momentum correlation implies an
extended source since a small source leads to uncontrollable uncertainties in momentum. Then the spatial coherence is not sufficient to produce the single particle
interference pattern. But there is a two-particle interference pattern observable in the coincidence in two detectors, which could be in space-like separated regions.
Usually the results in multiparticle interferometry are
interpreted as evidence of bizarre nonlocality 12 since the
pattern depends on the difference of the coordinates of
both the detectors, just as the spin correlations depend
on the difference in settings of the two analysers.
In the experiment, particles pass the slit planes and
are detected with two counting detectors, one on each
side. As in the case of spins, we assume the existence of
an internal variable, denoted by x0, and we assume that
the two correlated particles have the same value for the
internal variable. For the spin- 12 case, the relative rota-
Figure 2. Two-particle dual double slit experiment. S(A) and S(B)
are the double slits and D(A) and D(B) are the movable detectors.
The outputs of the detectors are correlated as in Figure 1.
CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
tion required to go from a maximum of transmission
through the analyser to a minimum is p. In the case of
double-slit interference, the situation is similar. The
relative rotation in phase from the center of the bright
fringe to the centre of the dark fringe is p, for the angular variable defined by q = akx, where k = 2p/l, and x is
the coordinate of the detector along the fringe pattern. a
is a scale factor representing the distance of the slits
from the source and the detectors. So, the double-slit
interference problem can be mapped to the spin- 12
singlet problem.
The corresponding local amplitudes are
C1 =
1
2
exp(iαk ( x1 − x0 ) / 2),
and
C2 =
1
2
exp(iαk ( x2 − x0 ) / 2),
(15)
where x1 and x2 are the detector positions. The single
particle detections on either side separately do not show
any interference. The correlation function is
U(x1, x2) = cos(ak(x1 – x2)/2).
(16)
Probability for coincidence detection is
P(x1, x2) = cos 2(ak(x1, x2)/2)
1
= (1 + cos kα ( x1 − x2 ) ).
2
(17)
This is the two-photon interference pattern (it is more
appropriate to call it a two-photon correlation pattern)
with 100% visibility. The photon which is being detected at one detector has no nonlocal influence on the
photon detected at the other detector.
Three-particle GHZ state correlations
So far we have been discussing examples in which the
statistical predictions of quantum mechanics were compared with the prediction from a local theory. There
have been examples where a single measurement of a
perfect correlation, assuming perfect detectors, etc. is
enough to demonstrate the conflict between quantum
mechanics and a local realistic theory 13,14 . Experiments
are yet to be done for these cases. We now show that
the theory with local amplitudes and the spin internal
variable deals with the correlations in such cases in a
remarkably simple and transparent way.
Consider the three-particle GHZ state 13 defined as
|ψ
GHZ 〉 =
1
2
(| 1,1,1〉− | 1,− 1,− 1〉),
(18)
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where the eigenvalues in the kets are with respect to the
z-axis basis. The conflict between a local realistic theory and quantum mechanics is the following statement:
The prediction from quantum mechanics for the measurement of the x-component of spin on all the three particles represented by the operator σ 1x ⊗ σ x2 ⊗ σ x3 is
given by
σ 1x ⊗ σ x2 ⊗ σ x3 | ψ
GHZ 〉 =
− |ψ
GHZ 〉.
(19)
Local realistic theories predict 13 that the product of the
outcomes in the x direction for the three particles should
be +1. This contradicts eq. (19).
We now show that the correct correlations can be reproduced using local amplitudes. The general idea is
that the three-particle correlation, analogous to our
scheme for two-particle states, is the real part of a complex number Z obtained as a suitable product of three
complex amplitudes. We choose the different phases
such that the correlation represented by N(Z–,–,–) is ±1,
to satisfy the condition that the joint probability for the
outcome (–, –, –) is unity according to eq. (19). N is a
normalization factor. For this we choose (Z–, –, –) to be
pure real. The rest of the correlations follow without
any additional input since flipping the sign once (for
example (Z+, –, –)) amounts to rotating Z through the
phase p/2, and the corresponding probability P(+,–,–) =
Real(Z+,–,–)) 2 = 0. This is because the amplitudes for +
and – are orthogonal. Clearly the joint probabilities are
unity for the outcomes containing an odd number of (–),
and zero for all other outcomes. This result is independent of the definition (once a definition is chosen the
phases can be chosen to get the desired outcomes) of
the complex correlation Z, though for convenience the
correlation can be defined as NReal(C1C2*C3*).
which an EPR effect has been discussed 16 . The analysis
using local amplitudes shows that the probability for
both the particles in the correlated beam decaying into
the same mode, say p0p0, at equal proper time is zero
without the nonlocal ‘EPR effect’.
Entanglement through measurement
The general framework presented here is suited also for
analysing entanglement correlations between particles
which have not interacted in the past, but get apparently
entangled due to joint measurements on their companion particles 17 . If one considers two ensembles of entangled particle pairs (1, 2) and (3, 4) such that (1, 2)
are entangled and (3, 4) are entangled, but (2, 3) or
(1, 4) are not entangled, it is possible to make
entanglement correlations between (1, 4) for example,
by making Bell-type joint measurements on the pair
(2, 3). In a Bell-state measurement, particles 2 and 3
are made to interfere on a beam-splitter and then it
is possible to pick out joint states of the type
| ψ 2,3 〉± = 1 (| 1, − 1〉 ±| − 1, 1〉). Though there is no prior
2
fixed relation between the internal variables of (1, 4),
the Bell measurement on (2, 3) chooses a sub-ensemble
in which there is an observed correlation between particles 2 and 3 and hence between particles 1 and 4, due to
fixed prior relationship in internal variables of particle
pairs (1, 2) and (3, 4). There is no nonlocality. The pair
(1, 4) picked out using the observation of |y 2,3 ñ– state,
for example, as a filter will also show the same behaviour as the pair (2, 3) in the state |y 2,3 ñ–. Only correlations at source and subsequent filtering into subensembles through Bell-type measurements or other
similar operations are needed. The various schemes are
yet to be studied in detail.
Other applications
Discussion
Hardy’s puzzle
Obtaining the correct correlations assuming locality
implies that the measurement on one particle does not
collapse the other particle to a definite state. There is a
distinction between a real measurement and ‘predictability with certainty’ of an outcome. Quantum correlation at source and a measurement on one of the particles
is enough to make this prediction using the conservation
laws. But till an actual measurement is made the companion particle does not acquire a definite state. There
is direct proof for this from the fact that while an actual
measurement of position on one of the particles disperses its momentum according to the uncertainty principle, this measurement and the resulting 100%
predictability of the companion particle’s position do
not cause corresponding dispersion in the momentum of
the companion particle. This is the lesson from Popper’s
experiment 18–20 .
Another important case is the one discussed by Hardy 15
involving two non-maximally entangled particles and
four observables, in which four separate correlations
predicted by quantum mechanics cannot be reproduced
in the local realistic hidden variable theory. The nonlocality demonstration uses three zero joint probabilities
and one nonzero joint probability constructed from
three of the measurement possibilities. The local amplitudes which give the correct joint probabilities for the
Hardy problem have also been constructed.
K meson beams
The analysis based on local physics presented in the
earlier sections can be applied to the K0 – K 0 beam for
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RESEARCH ARTICLES
Apart from resolving the nonlocality problem, we
have also found an answer to the original EPR paradox
of simultaneous reality of noncommuting observables.
The paradox arises only from the necessity to assume
reality for the outcomes before a measurement is made,
to reproduce the correct perfect correlation. Since we
found a way of getting these correlations without such
an assumption, and without nonlocality, there is no EPR
paradox. There are physical systems that are beyond the
scope of the EPR definition of reality.
The approach we have taken has locality as a basic
feature and the objective reality (reality of a physical
quantity independent of observer or apparatus) is at the
level of the internal variables or initial phases. There is
no objective reality to the outcomes before the measurement is made. One may explore the relation between
the reality of the phase variable and the actual outcomes, and that would be a major step towards understanding
quantum
measurements.
Such
an
understanding amounts to conceptual determinism in
quantum mechanics, even though the initial phase is
unmeasurable. It may turn out that observers in the classical world will not be able to grasp the mapping between such phases and actual outcomes. These issues
are being contemplated on.
In summary, we have reproduced the correct correlations of entangled particles under the assumption of
strict locality of amplitudes. The correlations agreeing
with experiments and quantum mechanical predictions
emerge without one measurement causing a collapse of
the state of the companion particle. This resolves the
EPR nonlocality puzzle. The results have significant
implications to the interpretation of all experiments involving entangled particles, including quantum teleportation and entanglement swapping. This approach
introduces a new point of view in the physical and philosophical understanding of the nature of reality in the
microscopic world.
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CURRENT SCIENCE, VOL. 79, NO. 2, 25 JULY 2000
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Received 1 April 2000; revised accepted 22 June 2000
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