Euphytica (2007) 154:83–90
DOI 10.1007/s10681-006-9272-7
Constitutive expression of Cry proteins in roots and border
cells of transgenic cotton
Oliver G. G. Knox Æ Vadakattu V. S. R. Gupta Æ
David B. Nehl Æ Warwick N. Stiller
Received: 3 May 2006 / Accepted: 12 September 2006 / Published online: 1 October 2006

Springer Science+Business Media B.V. 2006
Abstract Transgenic cotton plants expressing
Cry1Ac and Cry2Ab, from the soil bacterium
Bacillus thuringiensis (Bt), provide effective con-
trol of certain lepidopteran pests, however, little
is known about the proteins below ground
expression. We used ELISA to quantify in vitro
expression of the Cry1Ac and Cry2Ab proteins in
mucilage, root border cells and root tips in five
transgenic cultivars of cotton compared to con-
ventional cultivar Sicot 189. Expression of Cry
O. G. G. Knox (&)
CSIRO Entomology, Locked Bag 59, Narrabri, NSW
2390, Australia
e-mail: Oliver.Knox@csiro.au
V. V. S. R. Gupta
CSIRO Entomology, PMB2, Glen Osmond, SA 5064,
Australia
D. B. Nehl
NSW DPI, Locked Bag 1000, Narrabri, NSW 2390,
Australia
D. B. Nehl
Cotton Catchment Communities Cooperative
Research Centre, Locked Bag 1001, Narrabri, NSW
2390, Australia
W. N. Stiller
CSIRO Plant Industry, Locked Bag 59, Narrabri,
NSW 2390, Australia
proteins in roots and border cells of the transgenic
cotton cultivars was constitutive and at detectable
levels, with Cry1Ac and Cry2Ab protein expres-
sion ranging from <20 ppb to >100 pbb. To
determine if genetically modified cotton demon-
strated simple differences in properties of the
root, when compared to an elite parental line
(cv. Sicot 189), we enumerated border cells on
seedling radicles. Border cell counts of 14 culti-
vars ranged from 0.2 to 1.1 · 104 cells per root tip
with an average of 5 · 103 border cells. Border
cell production in the transgenic cultivars was
generally similar to that of both donor and elite
parents, the exception being the cultivar Sicot
189, which had substantially more border cells
than all of its transgenic derivatives. Comparison
of border cell number with varietal disease
resistance ranking found a limited relationship
(r2 = 0.65, n = 7) between border cell numbers
and the commercial resistance rank against Fusa-
rium wilt of cotton. The implications of differ-
ences in cotton cultivar border cell number
and root tip expression of Cry proteins for
plant–microbe interactions in the rhizosphere
and the soil ecosystem are yet to be resolved.
Keywords Border cells Æ Cry protein Æ Exudates Æ
Mucilage Æ Gossypium hirsutum Æ Fusarium
oxysporum f. sp. vasinfectum Æ Verticillium dahliae
123
84
Introduction
Genetically modified (GM) plants have the
potential to affect the chemistry and biology of
the rhizosphere through changes in root exudates,
expression of introduced genes in roots, and
changes in agrochemical use (Bruinsma et al.
2003). GM cotton (Gossypium hirsutum), engi-
neered to express insecticidal Cry proteins from
Bacillus thuringiensis (Bt) can decrease the use of
chemical insecticides, thereby developing more
sustainable farming systems with reduced non-
target environmental impacts (Azevedo and
Araujo 2003; Betz et al. 2000). The majority of
research on the expression of Bt proteins in GM
cotton has concentrated on Bt expression in
above ground plant tissues and little experimental
data is available on Bt production belowground
(Gupta et al. 2001).
In most plant species, living cells at the
periphery of the root cap, known as border cells,
become detached as individuals or small groups
of cells (Bowers et al. 2003). Since free-living root
border cells have a critical role in rhizosphere
community dynamics, possibly contributing the
majority of the carbon-rich exudates released
from roots (Hawes et al. 1998), the expression of
transgenic products in these cells merits investi-
gation. Cotton has been credited with producing a
high number of border cells, reportedly producing
around 104 per root tip (Hawes et al. 2000).
Originally border cells were thought to act as
protective barrier for the growing root tip,
reducing friction between the soil and the root
surface (Haberlandt 1914). However, it is now
known that their structure and function are far
more complex. As border cells differentiate they
undergo changes in structure, size and gene
expression, the latter of which results in produc-
tion of several border cell specific proteins and
signal molecules that are predominantly exported
to the surrounding soil (Baluska et al. 1996;
Brigham et al. 1995). Once in the soil, these
excreted compounds have the potential to influ-
ence the direction of root growth, soil chemistry
and plant–microbe interactions in the rhizosphere
(Brigham et al. 1998; Hawes et al. 2000). The
ability of root caps, and border cells, to both
receive and transmit signals that influence the
123
Euphytica (2007) 154:83–90
direction of root growth indicates their involve-
ment as primary determinants of root architecture
(Aiken and Smucker 1996; Hawes et al. 2000).
There is also evidence for the production of
specific extracellular signals by border cells that
directly influence microbial gene expression, and
growth and attraction of fungi and nematodes
(Sherwood 1987; Hawes et al. 2000; Rodger et al.
2003). Plants release root exudates of different
quantity and quality, and thus influence which
microbes colonise the rhizosphere (Nehl et al.
1997; Hawes et al. 2000, 2003). Notable growth of
fungi and bacteria has been reported to occur in
the presence of clusters of detached border cells
(Hawes et al. 2003). Active movement toward
border cells by zoospores of Pythium (Goldberg
et al. 1989) and fungal species (Gunawardena and
Hawes 2002) have been demonstrated to occur
with a high degree of tissue specificity. Sacrificing
border cells to pathogen invasion and the
subsequent detachment of the dead border cell
and fungal hyphae offers a mechanism for pro-
tection of the root tip from infection (Hawes et al.
1998). Similarly, evasion of plant parasitic nem-
atodes by root tips due to border cell production
of a nematode immobiliser has been proposed as
a disease resistance mechanism (Hawes et al.
2000; Zhao et al. 2000).
In addition to establishing if five commercially
grown cotton cultivars are capable of exuding Cry
proteins, we investigated border cell numbers as a
potential indicator of changes in root structure/
function as a result of either Bt gene insertions,
gene expression or breeding of transgenic varie-
ties. This work compared the production of
border cells by conventional elite parent lines
and their transgenic offspring. In order to main-
tain a field relevance to the study, the cotton
varieties used represented a range of commer-
cially available varieties. None of the assessed
varieties would be categorised as near isogenic,
and were developed using two to four backcross-
es. In an attempt to establish if differences in
border cell numbers relate to differential disease
resistance, cotton varieties were screened for
border cell production relative to susceptibility
for the economically important soil borne patho-
gens Fusarium (spp.) and Verticillium (spp.).
Finally, we assessed levels of Cry1Ac and Cry2Ab
Euphytica (2007) 154:83–90
proteins expressed from border cells, the sur-
rounding mucilage and the root tip to investigate
potential for ‘belowground’ production of these
proteins by GM cotton cultivars.
Materials and methods
Preparation of seedlings
Cotton seeds of all varieties were acid-delinted
prior to use. Batches of 20 seeds of different
cotton cultivars were surface sterilised with a
3 min wash in a solution of ethanol (50% v/v) and
sodium hypochlorite (0.4% m/v available chlo-
rine), followed by three washes in sterile distilled
water. The surface sterilised seeds were germi-
nated on a moist filter paper in the lid of Petri
dishes, sealed with Parafilm, and incubated in
the dark at 30C for 72 h.
Cry protein assay
Germinated seedlings were suspended, with the
radicle downward, above a sterile multichannel
pipette reservoir using masking tape, and 2 ml of
1 · ELISA Extraction buffer (Envirologix, USA)
used to wash border cells and mucilage from a
total of 20 germinated seedlings per replicate. The
washings were transferred to a sterile syringe,
passed through a 0.22 lm filter, and the filtrate
retained as the ‘mucilage’ fraction. An aliquot
(5 ml) of phosphate buffer (4 g NaCl, 0.085 g
KH2PO4 and 0.3 g K2HPO4 per l of water) was
then washed through the filter. The filter was
removed from its housing, transferred to a 2 ml
microfuge tube. A 1 ml aliquot of 1 · ELISA
extraction buffer was added and the border cells
were liberated from the filter by rapid agitation
on a Disruptor Genie (Scientific Industries, USA)
for 5 min. This 1 ml border cell suspension,
including the filter, were retained as the ‘border
cell’ fraction. After washing, the terminal 5 mm
of 10 root tips were abscised into 1 ml of ELISA
extraction buffer and homogenised by hand using
a micro-pestle. All recovered root-tip material
was allowed to imbibe for 1 h at room temper-
ature (~22C). The levels of the Cry1Ac and
Cry2Ab protein in the different root fractions
85
were assessed by quantitative analysis using the
Cry1Ac and Cry2Ab plates (Strategic Diagnostic
Inc. USA) according to the manufacturers’
instructions. Standards used for the ELISA were
prepared from GHD73 (Genesearch, Australia),
a 62 mg ml–1 spore/crystal suspension of Cry1Ac,
and ground corn-stem (Monsanto, Australia) that
contained 6.014 mg g–1 of Cry2Ab.
Enumeration of border cells
Border cells were liberated by immersing the
terminal 15–20 mm of the radicle of germinated
cotton seedlings in 1 ml of sterile deionised water
in a 1.5 ml microfuge tube, allowed to imbibe for
5 min and agitated with 10 repeated flushes of
a 200 ll pipette. The seedling’s radicle was
removed and the entire 1 ml suspension trans-
ferred to a Sedgewick-Rafter counting chamber
(Phyco-Tech) for enumeration under a compound
microscope (200· magnification). The number of
border cells present in 80 arbitrarily chosen, cells
on the grid were counted. Counts were carried
out on all germinated seedlings, and were
repeated for a minimum of three batches of seed
for each cultivar.
Assessment of recovery of border cells using
the ELISA recovery protocol
To assess the effectiveness of the filter technique
at recovering border cells, compared to the
‘enumeration’ technique, 100 ll of the washed
filter recovery was transferred to 900 ll of sterile
water, vortexed and transferred to the Sedgwick
Rafter counting chamber as described above.
Comparison of border cell number
to Fusarium and Verticillium disease ranking
and nematode resistance
Commercial disease resistance rankings, relative
to that of the cultivar Sicot 189, were obtained
from cotton seed distributors (CSD) for eight
cotton cultivars, for which border cells counts had
been made. Existence of a relationship between
border cell number and disease rankings was
tested using a correlation matrix analysis (Gen-
Stat version 7, VSN International Limited, UK).
123
86 Euphytica (2007) 154:83–90

Border cell numbers in American cotton varieties
that are resistant (cv. Auburn 623) and suscepti-
ble (cv. DP 16) to the root-knot nematode,
Meloidogyne incognita, were compared using
ANOVA (GenStat version 7).
Results and discussion
Analysis of the above-ground impact of Bt GM
technology has demonstrated it to be highly
efficient with no known deleterious environmen-
tal impacts occurring beyond the desired insecti-
cidal control (Fitt 2000). Below ground
implications for this technology are, however,
largely unknown due to the complexity of soil
ecosystems (Kowalchuk et al. 2003) and limited
knowledge on the mechanisms of release, fate and
accumulation of Cry proteins in soil (Saxena and
Stotzky 2001;Sims 1995; Sisterson et al. 2004).
The ELISA assay of Cry proteins in the
mucilage of cotton radicles provided sufficient
protein for detection. Detection of Cry1Ac and
Cry2Ab in the mucilage of the transgenic culti-
vars (Table 1) indicated that roots of all the GM
cotton cultivars assayed were exuding the trans-
genic proteins. Only one replicate of cv. Sicala
40BR failed to produce any detectable Cry
protein, but this replicate had poor germination
and a microbial contaminant was observed
growing upon the seeds. The release of Cry1Ac
protein by roots of two-week old seedlings of
transgenic cotton cultivars Sicot 289i and 289RRi
was previously observed in solution culture
experiments (Gupta and Watson 2004), although
it has also been reported that the roots of cotton
do not exude Cry proteins (Saxena et al. 2004).
There are several possible reasons for these
discrepancies, but most likely is that Cry exuda-
tion is variety-dependent. For example, this pres-
ent study and that of Gupta and Watson (2004)
used commercial cultivars, whilst Saxena et al.
(2004) used the non-commercial Coker 312. All
these studies used transgenic plant material
derived from the same insertion event
(MON531), meaning that the source of the
transgenic material and its initial point of inser-
tion were identical. These contrasts emphasise the
need to assess Cry protein release in root
exudates of cotton on an individual variety basis.
Since root border cells have a critical role in
rhizosphere microbial community dynamics, by
contributing to the exudates released from roots
(Hawes et al. 1998), expression of gene products
in border cells of transgenic plants may be crucial.
Detection of Cry proteins in the mucilage was
obviously associated with either passive leakage
or active excretion by either roots or border cells.

Table 1 Low, medium and high qualitative rating of of transgenic and commercial cultivars of cotton, based on
expression of Bacillus thuringiensis Cry1Ac and 2Ab quantitative ELISA in a series of three different recovery
proteins in root mucilage, root border cells and root tips tests
a
Protein Cultivar Protein expression
b c d
Mucilage Border cell Root tip

Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3

Cry1Ac 289B + + + + + ++ + ++ +++

289BR + + ++ + + ++ + ++ +++
DP50BGII + ++ + ++ ++ +++
40BR C ++ + C +++ +++
71BR ++ +++ +++ +++ +++ +++
Cry2Ab 289B + + ++ + +++
289BR + + + + +++
DP50BGII + + + + ++
40BR + + + ++ +++ +++
71BR ++ ++ ++ ++ +++ +++
a
Symbols are ‘+’ for >0–20 ppb, ‘++’ for >20–100 ppb and ‘+++’ for >100 ppb; C = contaminated by microbial growth.
b
Washed from 20 root tips into 2 ml of extraction buffer. c Recovered from 20 washed root tips into 1 ml of extraction
buffer. d From 10 homogenised root tips devoid of mucilage and border cells in 1 ml of extraction buffer

123
Euphytica (2007) 154:83–90
However, the possibility that the Cry protein
observed in border cells had been translocated by
them was discounted for two reasons. First, the
Cry proteins were considered to be large enough
to preclude passive uptake and we could discern
no logical reason for active uptake. Secondly,
based on estimates of 0.187 lg mass per border
cells and actual fresh weight measurements of
excised root tips, the level of Cry protein in
border cells was consistently greater (ranging
from 17% to 292% higher, data not shown) than
in their corresponding root tips. The presence of
Cry protein in border cells was clearly the result
of expression within the border cells and appears
to have been up-regulated in comparison to the
corresponding root tips. Our observations consti-
tute the first record of Cry proteins in the border
cells of transgenic plants. Cry protein expression
in border cells varied between varieties (Table 1)
and, although this was probably attributable to
phenotypic differences in expression rates, recov-
ery of border cells from the filter membranes was
considered to be another factor. Analysis of
border cells numbers recovered from filters, when
compared to our mean border cell counts for the
same cultivar, indicated only 80% recovery with
this technique (data not shown). Regardless of
the levels of expression, Cry protein was detected
in border cells of all tested GM cultivars desig-
nated as having the Bt insecticidal trait.
Cultivar Sicot 189 (189) was the elite parent in
the development of several transgenic cultivars
including Sicot 189 Roundup Ready (189RR),
Sicot 289 Bollgard II (289B) and Sicot 289
Bollgard II Roundup Ready (289BR). How-
ever, the donor parents were either Coker 312 or
DP50BGII, and methods used for the transfer of
the transgenic traits into the elite line, prior to
several cycles of backcrossing, differed (Table 2)
(Monsanto Australia Limited 2003). Comparisons
among 189, 189RR, 289B and 289BR in three
replicated counts showed that 189 had a signifi-
cantly higher (P < 0.001, df = 24) number of
border cells than the other lines. Sicot 189 was
the only cultivar producing >104 border cells per
root tip (Fig. 1), which was a similar number to
that previously reported for cotton (Hawes et al.
2003). DP50 and DP50BGII did not have signif-
icantly different border cell numbers from each
87
other or from Coker 312 (P = 0.05, df = 56).
Similarities in border cell counts between DP50
and DP50BGII was taken as an indication that
these gene insertion events played a minimal role
in determination of border cell numbers. Other
gene insertion events would have to be assessed
on a case by case scenario for their potential to
influence border cell number.
In order to evaluate further the effect of
genetic modification on border cell frequency,
border cell production in two more elite, conven-
tional lines, Sicot 71 and Sicala 40 was compared
to that in their transgenic derivative lines, Sicot 71
Bollgard II Roundup Ready (71BR) and Sicala
40 Bollgard II Roundup Ready (40BR). In
these cultivars the border cell counts were not
significantly different (P = 0.20, df = 24) (Fig. 1),
indicating that border cell number was not always
lower in the transgenic than the elite parent.
Again border cell production was well below that
previously reported for cotton. There is, to our
knowledge, no previous study reporting differ-
ences in border cell numbers between cotton
cultivars, especially transgenic varieties.
Border cells have potential to affect interac-
tions between plants and pathogens in the root
environment (Goldberg et al. 1989). Following
our observation of differences in border cell
number among commercially available cotton
cultivars, we attempted to determine whether or
not these differences might relate to environmen-
tal fitness, namely resistance against soilborne
pathogens (Fig. 2). Border cell number was not
correlated with commercial disease resistance
rankings for Fusarium wilt, caused by Fusarium
oxysporum f.sp. vasinfectum, (r = 0.07, P = 0.66)
or Verticillium wilt, caused by Verticillium dahliae
(r = –0.10, P = 0.66). A major outlier in this
analysis, due to its high number of border cells,
was Sicot 189, which is used as the standard
cultivar for Fusarium wilt resistance with a
ranking of 100 and against which all other
cultivars are compared (Allen 2003). The other
major anomaly was Sicala 40BR, which has a
Verticillium wilt resistance rank (V rank) of 165,
the highest disease ranking of any cultivar against
Verticillium wilt. Removal of these outliers and
re-evaluation with a limited range of border cells
from 2 to 6 · 103 produced a significant positive
123
88 Euphytica (2007) 154:83–90

Table 2 Details of cotton varieties, parentage and their transgenic events used in this study
Variety Elite parent Event/s Trait/s

Conventional Sicot 189

Sicot 71
Sicala 40
DP50
DP16
Coker 312
Auburn 623
Transgenic 189RR Sicot 189 MON1445 CP4 ESPS (RR)
289i Sicot 189 MON531 Cry1Ac
289RRi Sicot 189 MON531/1445 Cry1Ac/CP4 ESPS (RR)
289B Sicot 189 MON531/15985 Cry1Ac/Cry2Ab
289BR Sicot 189 MON531/15985/1445 Cry1Ac/Cry2Ab/CP4 ESPS (RR)
DP50B DP50 MON531 Cry1Ac
DP50BGII DP50B MON531/15985 Cry1Ac/Cry2Ab
Sicot 71BR Sicot 71 MON531/15985/1445 Cry1Ac/Cry2Ab/CP4 ESPS (RR)
Sicala 40BR Sicala 40 MON531/15985/1445 Cry1Ac/Cry2Ab/CP4 ESPS (RR)
The singular insertion events MON1445 and MON531 were transformed into Coker 312, with event MON15985
transformed into DP50B

Fig. 1 Mean border cell 12000

numbers per cotton root
tip for a number of 10000
Border cells/root tip

varieties, based on
Sedgwick Rafter counts of 8000
border cells from 72 h old
seedlings recovered into 6000
1 ml of water. Grey filled
bars represent GM lines. 4000
Error bars represent the
standard error of the 2000
mean
0

Auburn 623
40

71
189B

289B
189

Coker 312

DP50

DP16
189RR

289BR

40BR

71BR
DP50BGII

Variety

correlation between resistance to Fusarium wilt Mendgen 1995). Chlamydospores and other rest-
and border cell number (r2 = 0.65, n = 7). ing structures of soilborne pathogens are known
Although there were insufficient points to extrap- to be stimulated by root exudates and, in field-
olate further, this result suggests that within this grown cotton, F. oxysporum f.sp. vasinfectum has
range, border cell frequency might have some been demonstrated to attain maximum colonisa-
role in resistance against Fusarium wilt. Fusarium tion within 4 mm of the root tip (Beckman 1987).
oxysporum has been previously reported to grow These observations suggest that some properties
in the presence of border cells of a number of host of border cells, such as their frequency or
plant species including cotton, however, the expression of signal molecules, could potentially
importance of this early interaction in disease interact with F. oxysporum f.sp. vasinfectum.
epidemiology is still under investigation (Guna- In the USA, soilborne nematodes cause disease
wardena et al. 2005; Rodriguez-Galvez and and substantial losses in cotton production

123
Euphytica (2007) 154:83–90
180
160
Disease F and V Rank
140
120
100
80
60
40
20
0
0 2000 4000 6000 8000
Border cells/root tip
Fig. 2 Plot of Fusarium (¤) and Verticillium (h) resis-
tance rankings for Sicala 40, 40BR, and Sicot 71, 71BR,
189, 189RR, 289B and 289BR against the mean number of
border cells produced by each cultivar
(Gazaway and Mclean 2003; Koenning et al.
1999). Border cells have been implicated in
plant–nematode interactions, potentially offering
protection of the root tip and possibly the plant
(Hawes et al. 2000; Zhao et al. 2000). In light of
this, we assessed the level of border cells pro-
duced on cultivars Auburn 623 and DP16, which
respectively are classed as resistant and suscepti-
ble, to the root-knot nematode Meloidogyne
incognita (Robinson and Cook 2001). Counts
did not reveal any significant difference
(P = 0.23, df = 28) in the number of border cells
produced by the two lines supporting the hypoth-
esis that a border cell-specific signal is involved in
the immobilisation of plant parasitic nematodes
(Hawes et al. 2000; Zhao et al. 2000).
Our observations of border cell frequency in
different cotton cultivars indicated that, in some
instances, transgenic lines have the potential to
differ from the elite parent in simple aspects of
their root morphology and, by implication, phys-
iology. Our results produced a value of 5 · 103
border cells per root tip, which was substantially
lower than the 104 previously reported for cotton
and only reached in our study by Sicot 189 (Hawes
et al. 2000, 2003). Hence, border cell production
can differ considerably among cotton cultivars,
although the consequences of this variation for
plant fitness are not currently understood.
While the presence of Cry proteins in roots
of established plants (cotton, maize) has been
89
previously reported (Saxena et al. 2004; Gupta
and Watson, 2004), our observations of Cry
proteins in radicles of very young seedlings
indicates that introduced gene expression occurs
immediately following seedling germination. The
detection of expression of the Cry proteins in the
root tips and border cells, and their excretion in
mucilage, of cotton suggest that Cry proteins from
cotton cultivars transformed with the Bt trait will
enter the soil ecosystem, not only from the above-
10000 12000
ground plant residues but also as a result of
release from damaged roots, root turnover and
exudation. In this study we have not attempted to
address issues of potential effects on non-target
organisms, increased selection pressure for devel-
opment of Cry resistant pest species, if Cry
protein exudation was passive or active, and the
potential fate and persistence of the proteins in
soil.
Differences in the level of detection of the Cry
proteins between cultivars point to the need for
assessment of all transgenic protein expression
by GM crops on a cultivar by cultivar basis.
The potential environmental consequences of
exudation of Cry genes and differences in border
cell numbers of cotton requires further investi-
gation.
Acknowledgements This work was carried out under
funding provided by the Cotton Research and Development
Corporation, and with the support of CSIRO Entomology,
Land and Water, and Plant Industry, and the NSW
Department of Primary Industries. The help of Dr Stephen
Allen and Mr Brett Ross (Cotton Seed Distributors,
Wee Waa, Australia) is acknowledged for providing disease
resistance rankings for commercially unavailable cotton
cultivars and help with ELISA.
References
Aiken RM, Smucker AJM (1996) Root system regulation
of whole plant growth. Annu Rev Phytopathol
34:325–346
Allen S (2003) Fusarium restistance ranking protocol for
cotton cultivars in Australia. Proceedings of the
Beltwide Cotton Conference
Azevedo JL, Araujo WL (2003) Genetically modified
crops: environmental and human health concerns.
Mutat Res 544:223–233
Baluska F, Volkmann D, Hauskrecht M, Barlow PW
(1996) Root cap mucilage and extracellular calcium as
modulators of cellular growth in postmitotic growth
zones of the maize root apex. Bot Acta 109:25–34
123
90
Beckman CH (1987) The nature of wilt diseases of plants.
St. Paul, Minesota, USA: APS Press
Betz FS, Hammond BG, Fuchs RL (2000) Safety and
advantages of Bacillus thuringiensis—protected plants
to control insect pests. Regul Toxicol Pharmacol
32:156–173
Bowers JE, Chapman BA, Rong JK, Paterson AH (2003)
Unravelling angiosperm genome evolution by phylo-
genetic analysis of chromosomal duplication events.
Nature 422:433–438
Brigham LA, Woo HH, Nicoll SM, Hawes MC (1995)
Differential expression of proteins and messenger-
RNAs from border cells and root-tips of pea. Plant
Physiol 109: 457–463
Brigham LA, Woo HH, Wen F, Hawes MC (1998)
Meristem-specific suppression of mitosis and a global
switch in gene expression in the root cap of pea by
endogenous signals. Plant Physiol 118:1223–1231
Bruinsma M, Kowalchuk GA, Van Veen JA (2003) Effects
of genetically modified plants on microbial communi-
ties and processes in soil. Biol Fertil Soils 37:329–337
Fitt GP (2000) An Australian approach to IPM in cotton:
integrating new technologies to minimise insecticide
dependence. Crop Prot 19:793–800
Gazaway WS, Mclean KS (2003) A survey of plant-
parasitic nematodes associated with cotton in Ala-
bama. J Cotton Sci 7:1–7
Goldberg NP, Hawes MC, Stanghellini ME (1989) Specific
attraction to and infection of cotton root cap cells by
zoospores of Pythium dissotocum. Can J Bot-Rev Can
Bot 67:1760–1767
Gunawardena U, Hawes MC (2002) Tissue specific local-
ization of root infection by fungal pathogens: role of
root border cells. Mol Plant-Microbe Interact
15:1128–1136
Gunawardena U, Rodriguez M, Straney D, Romeo JT,
VanEtten HD, Hawes MC (2005) Tissue-specific
localization of pea root infection by Nectria haema-
tococca. Mechanisms and consequences. Plant Physiol
137(4):1363–1374
Gupta VVSR, Roberts GN, Neate SN, Crisp P, McClure S,
Watson SK (2001) Impact of Bt-cotton on biological
processes in Australian soils. In: Akhurst RJ, Beard
CE, Hughes P (eds) Proceedings of the 4th Pacific
Rim conference on the biotechnology of Bt-environ-
mental impacts. CSIRO, Australia pp. 191–194
Gupta VVSR, Watson SK (2004) Ecological impacts of
GM cotton on soil biodiversity. A final report for the
Australian Government Department of the Environ-
ment and Heritage by CSIRO Land and Water. Glen
Osmond, SA
Haberlandt G (1914) Physiological plant anatomy London:
McMillan and Co
Hawes MC, Bengough G, Cassab G, Ponce G (2003)
Root caps and rhizosphere. J Plant Growth Regul
21:352–367
123
Euphytica (2007) 154:83–90
Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y (1998)
Function of root border cells in plant health: pioneers
in the rhizosphere. Annu Rev Phytopathol 36:311–327
Hawes MC, Gunawardena U, Miyasaka S, Zhao X (2000)
The role of root border cells in plant defence. Trends
Plant Sci 5:128–133
Koenning SR, Overstreet C, Noling JW, Donald PA,
Becker JO, Fortnum BA (1999) Survey of crop losses
in response to phytoparasitic nematodes in the United
States for 1994. J Nematol 31:587–618
Kowalchuk GA, Bruinsma M, Van Veen JA (2003)
Assessing responses of soil microorganisms to GM
plants. Trends Ecol Evol 18:403–410
Monsanto Australia Limited (2003) Bollgard II Cotton
Technical Manual
Nehl DB, Allen SJ, Brown JF (1997) Deleterious rhizo-
sphere bacteria: an integrating perspective. Appl Soil
Ecol 5:1–20
Robinson AF, Cook CG (2001) Root-knot and reniform
nematode reproduction on kenaf and sunn hemp
compared with that on nematode resistant and
susceptible cotton. Ind Crop Prod 13:249–264
Rodger S, Bengough AG, Griffiths BS, Stubbs V, Young
IM (2003) Does the presence of detached root border
cells of Zea mays alter the activity of the pathogenic
nematode Meloidogyne incognita? Phytopathology
93:1111–1114
Rodriguez-Galvez E, Mendgen K (1995) The infection
process of Fusarium oxysporum in cotton root tips.
Protoplasma 189:61–72
Saxena D, Stewart CN, Altosaar I, Shu Q, Stotzky G
(2004) Larvicidal Cry proteins from Bacillus thuringi-
ensis are released in root exudates of transgenic
B. thuringiensis corn, potato, and rice but not of
B. thuringiensis canola, cotton, and tobacco. Plant
Physiol Biochem 42:383–387
Saxena D, Stotzky G (2001) Bacillus thuringiensis (Bt)
toxin released from root exudates and biomass of Bt
corn has no apparent effect on earthworms, nema-
todes, protozoa, bacteria, and fungi in soil. Soil Biol
Biochem 33: 1225–1230
Sherwood RT (1987) Papilla formation in corn root-cap
cells and leaves inoculated with Colletotrichum gra-
minicola. Phytopathology 77:930–934
Sims SR (1995) Bacillus thuringiensis var Kurstaki [Cry-
Ia(C)] protein expressed in transgenic cotton: effects
on beneficial and other non-target insects. Southwest
Entomol 20: 493–500
Sisterson M, Biggs R, Olson C, Carriere Y, Dennehy
T, Tabashnik B (2004) Arthropod abundance and
diversity in Bt and non-Bt cotton fields. Environ
Entomol 33:921–929
Zhao X, Schmitt M, Hawes MC (2000) Species dependent
effects of border cell and root tip exudates on
nematode behavior. Phytopathology 90:1239–1245