assessment of bt protein level in soil and effect of ... · summary the effect of transgenic...

Assessment of Bt protein level in soil and effect of transgenic

Bt brinjal expressing cry1 Ac gene on soil microflora,

nematodes, collembola and earthworms

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Effect of transgenic brinjal expressing Bacillus thuringiensis cry1 Ac

gene on soil microflora, nematodes, collembola and earthworms.

Objective

The objectives of this study were:

1) to evaluate the effect of Bt brinjal expressing cry1Ac gene on soil microflora,

nematodes, collembola and earthworms, and

2) to determine the Cry1Ac protein levels in the Bt brinjal grown soil.

Study conducted by

Maharashtra Hybrid Seeds Co. Ltd., Jalna (Maharashtra).

Summary

The effect of transgenic brinjal expressing the cry1Ac gene from Bacillus

thuringiensis (Bt) on the soil microflora, nematodes, collembola, and earthworms has

been studied. Soil and insect samples were collected from the Bt brinjal experimental

trials conducted at Jalandhar, Mirzapur, Ahmad nagar, Bhopal, Dharmapuri,

Dharwad, and Karnool during 2004-05. Microflora populations were measured by

dilution plating method. There were no consistent significant differences between Bt

and non-Bt treatments in the numbers of total culturable bacteria and fungi.

Nematodes were extracted using Cobb’s decanting and sieving method. Collembola

populations were measured using pitfall traps There were no significant differences in

populations of nematodes and collembola between Bt and non-Bt treatments.

Earthworms were observed in all the seven locations studied. The Cry1Ac protein,

determined by insect bioassays with Helicoverpa armigera, was not detected in any

soil samples. These findings indicated that transgenic brinjal expressing the cry1Ac

gene does not have any adverse effect on the microflora, nematodes, collembola and

earthworms in soil.

Introduction

The incorporation into plants of genes from Bacillus thuringiensis (Bt) that code for

the production of insecticidal toxins reduces many problems associated with the use

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of chemical pesticides, as the toxins are produced continuously within these plants

and exhibit relatively high specificity. However, there is concern that genetically

engineered crops may pose ecological risks to natural and agricultural ecosystems

(Conway, 2000; Hails, 2000; Stotzky, 2000). While efforts have been made to

examine the environmental impacts of genetically modified plants “ above ground”,

comparatively little research has been directed at impacts of genetically modified

crops on soil organisms and processes, perhaps because of the greater difficulties

involved in the study of soil invertebrates and soil microorganisms. Concerns about

impacts of GM crops on soil biota have been raised, in part because of the chemical

and biological properties of soil (McGregor and Turner 2000). Soil materials have

large sorptive capacities for biological molecules, including insecticidal bacterial

proteins and DNA. Laboratory studies (Crecchio and Stotzky, 1998, 2001; Saxena and

Stotzky 2001; Saxena et al. 2002; Tapp et al. 1994; Tapp and Stotzky, 1995, 1998)

have shown that insecticidal Cry proteins from B. thuringiensis are readily adsorbed

at equilibrium and bound to clay minerals and humic acids, and persist in soil for up

to 350 days, the longest time studied. Zwahlen et al. (2003a) investigated the

degradation of Bt toxin in transgenic corn leaves under field conditions and reported

the persistence of the Cry1Ab protein up to 240 days. Consequently, the issue of the

impact of the Bt proteins released to soil from the roots and biomass of Bt crops on

soil organisms including soil microflora, collembola and earthworms is an important

one. In this study we evaluated the impact of this Bt brinjal on soil microflora,

nematodes, collembola, and earthworms. Also, we tried to quantify the Cry1Ac

protein levels in the Bt brinjal grown soils.

Materials and methods

Locations for sample collection

Soil and collembola samples were collected from the Bt brinjal experimental trials

conducted at Jalandhar, Mirzapur, Ahmad nagar, Bhopal, Dharmapuri, Dharwad and

Karnool during 2004-05. The field trial the following treatments:

a. Bt treatment : MHB 80 Bt/ MHB 4 Bt/ MHB 9 Bt or MHB 99 Bt

b. Near-isogenic non-Bt treatment: MHB 80 Bt/ MHB 4 Bt/ MHB 9 Bt or MHB 99,

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Plant

Zone for non-rhizosphere

soil sampling

Zone for rhizosphere

soil sampling

1

34

5

2

c. Non-Bt local check : Pusa purple round/ Gondegaon/ Arka Shirish/ Manjari kota

or Green +white local, and

d. Non-Bt commercial check: Navakiran/ Ajay/ Extra green long/ Manju or Harit.

Each treatment had four replications. Samplings were made in three replications

for each treatment.

Soil sampling

Pre-harvest soil samples were collected at 0, 30, 90, and 150 days after

transplantation. For sampling purpose, the area around the plants selected was divided

into root/rhizosphere and non-rhizosphere zones as shown in the following figure.

The root zone for rhizosphere sample collection extended to 20cm area around the

plant, and the non-rhizosphere zone represented the 20-40cm area around the plant.

To get one sample, five core samples (each from a 15-cm deep and 7.5-cm diameter

area) were taken around the plant and mixed thoroughly. From this mixture, 100 g of

soil was drawn as a representative sample. During the pre-harvest stage, a total of 3

rhizosphere and 3 non-rhizosphere samples per treatment were collected at every

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sampling time-point. Samples were transported to the Biotechnology R&D Center,

MAHYCO, Dawalvadi (Maharashtra) for laboratory analysis.

Determination of total bacterial and fungal population

To determine the numbers of total culturable bacteria and fungi, 10 g of rhizosphere

or non-rhizosphere soil was added to 90 ml of sterile distilled water and shaken for 20

min at 250 rpm on a Gyrorotary shaker. Soil suspensions were diluted further with

sterile distilled water and 100µl of 10-3

dilution was spread-plated onto selective

medium. Luria Bertani (LB) containing 100 mg/l cycloheximide (Sigma Chemical,

St.Louis, MO) and 1% soil extract, and Rose bengal streptomycin agar (Saxena and

Stotzky 2001) containing 50 mg/l streptomycin sulphate (Sigma Chemical, St.Louis,

MO) were used to enumerate bacteria and fungi, respectively. Total culturable

bacteria and fungi were counted following incubation at 28οC for 3 and 7 days,

respectively. Differences in numbers of culturable bacteria and fungi among

treatments were analyzed with Analysis of Variance (ANOVA) (SAS Institute, 1989).

Analysis of soil invertebrates

Populations of earthworms and collembola were enumerated in all the Bt and non-Bt

treatments. Earthworms were sampled at five time-points during the pre-harvest

season, i.e. 0, 30, 90, 120 and 150 days after transplantation. For measurement of

earthworm populations, holes (30-cm diameter and 90-cm deep) were dug in soil

along two rows of plants. The soil from each hole was spread on a plastic sheet, and

earthworm counts were made in the field.

Collembola populations inhabiting the soil surface were measured using pitfall traps.

The pitfall traps, placed along two rows of plants, consisted of a 300 ml plastic jar

with an inserted funnel made by cutting the bottom out of a 100 ml disposable plastic-

drinking cup. A solution containing 10% formalin and 1% copper sulphate was used

to trap the collembola. Each trap was inserted in the soil so that the lip of the cup was

level with the soil surface. Three traps were used per treatment. The traps were

removed 3 days after installation, capped, and brought to the laboratory where the

contents were poured into a Petri dish and collembola were counted.

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Nematodes were extracted using Cobb’s decanting and sieving method (Ayoub,

1980), from the soil samples collected at 0, 30, 60, 90, 120 and 150 days after

transplantation. Soil samples for nematode analysis was taken in the root zone. Each

sample was collected by initially taking five core samples (each from a 20 cm deep

and 10 cm diameter) around a plant and then mixing them thoroughly. All soil

samples were stored at 15ο

C to minimize the changes in nematode populations.

Nematodes were extracted within 7 days after sampling. Numbers of nematodes in

each treatment were counted in 250 g soil. An analysis of variance (ANOVA) was

carried out using the SAS statistical software (SAS Institute 1989).

Level of Bt protein in soil:

The level of Bt protein in soil samples was determined by insect bioassays with

Helicoverpa armigera.

Bioassays with soil samples: To assay for Cry1Ac protein, soil samples were

incorporated into the artificial diet and then presented to H. armigera neonates. Five

grams soil from each sample was thoroughly slurried with 20 ml of water in a 100 ml

centrifuge tube on a vortex mixer, of which 2.8 ml was mixed with artificial diet to

bring it up to a total volume of 14 ml. The soil-diet mixture of each sample was

added to 16 wells in the 128-well tray. After the soil-diet mixture was cooled and

solidified, one neonate of H. armigera was introduced into each well.

Standard mortality bioassays: To serve as a reference standard, standard mortality

bioassay was done, that involved exposure of neonate larvae to various concentrations

of diet incorporated Cry1Ac protein that caused 0-100% mortality and also to

calculate the percent surviving second instar larvae. The stock solution of Cry1Ac

was made in 0.2% agar solution and dilutions were made in deionized water. The

source of Cry1Ac protein used in the bioassays was the commercial formulation,

MVP II ® (Mycogen Corp., USA), which contained 19.7% (by weight) Cry1Ac.

Various dilutions of Cry1Ac were later mixed in the H. armigera diet for the

bioassays. There were 32 larvae per replication with a total of four replications for

each Cry1Ac concentration. The bioassays were done in 128-well trays with each

well consisting of 750 µL diet with Cry1Ac.

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Bioassay trays were kept in dark at a temperature of 26 ± 1°C and 55-65% RH.

Mortality and instar stage of surviving larvae were recorded on seventh day. Larvae

that did not move when disturbed were considered to be dead.

Results

Microflora analysis

Total numbers of culturable bacterial and fungal colonies were counted, and analysis

of variance was done to determine the variations between Bt and non-Bt soils. No

consistent differences were observed in the culturable bacterial populations (Figures

1-7) between Bt, and non-Bt soil samples. The types of colonies, which grew from Bt

and non-Bt treatments, were similar.

The total fungal populations for the soil samples collected at pre- harvest stages are

presented in Figures 8-14. The differences in the total culturable fungal population

observed among the Bt and non-Bt treatments were not consistent. Species of

Aspergillus, Penicillium, and Rhizopus were the dominant fungi that were found from

these soil samples. Besides these, Trichoderma, Fusarium, Gliocladium,

Cephalosporium, Myrothecium, Cladosporium, Alternaria, Verticillium and

Rhizoctonia were some of the other fungi also seen in all six treatments.

Earthworms

Earthworm populations were measured at 0, 30, 60, 90, 120, and 150 days after

transplantation (DAT). The results are presented in Tables 1-3. At Jalandhar location

earthworms were observed only at 120 DAT. No significant (p>0.05) difference in the

earthworm populations was observed at other locations during the pre-harvest

sampling period. (Tables 1-3).

Enumeration of whole nematode communities, extracted from soil samples collected

from the Bt and non-Bt experimental plots, are presented in Figures 15-21. The

nematode population (Mean ± SE) estimated during the pre-sowing (0 DAT)

sampling period was 121 ± 8.9, 94.7 ± 3.03, 140 ± 6.2, 218 ± 6.7, 94.8 ± 12.3, 371

±13.3 and 243± 9.1 for Jalandhar, Mirzapur, Ahmad nagar, Bhopal, Dharmapuri,

Dharwad and Karnool, respectively. No significant difference in the total nematode

population was observed between Bt and non-Bt treatments irrespective of the

locations and sampling-timepoint.

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Collembola

Collembola were observed in all the seven locations studied. The mean (± SE)

number of collembola observed during the pre-transplantation time point at Jalandhar,

Mirzapur, Ahmad nagar, Bhopal, Dharmapuri, Dharward, and Karnool was

7.75±0.75, 4.75±0.43, 15.6±0.93, 19.6±0.74, 93.5±3.5, 26.4±1.48, and 5.42±0.47,

respectively. No significant (p > 0.05) differences in the number of collembola were

observed among Bt and non-Bt treatments irrespective of the locations during

different sampling time-points (Figures 22- 28).

Level of Bt protein in soil:

The level of Cry1Ac protein present in the soil samples from Bt brinjal field plots was

determined by insect bioassays with H. armigera. Results of the standard mortality

bioassay showed that 100% mortality occurs at 1.0 µg /ml of protein in the diet and

the lowest concentration (0.001 µg /ml) produced approximately 19% mortality

(Figure 29). Also, 46.50% of larvae were third instars at the lowest protein

concentration in the diet (Figure 30). Seventy nine percent of the larvae in the control

were third instars after six days of incubation and no mortality was observed in the

control treatment.

The results of insect bioassays with soil sample-diet mixture were presented in figures

31 to 37 for soil samples collected from different locations. Soil samples from

different locations when mixed individually with artificial diet produced no larval

mortality after six days incubation over all the sampling time points (Figures 31 – 37).

Also, the percent third instars were either equal to or higher in Bt soil samples than in

non-Bt, Local check and/or commercial check soil samples in all locations. The

average percent surviving third instar larvae were between 78.69-86.63% and 76.25-

88.0% when insect bioassays were done with pre-sown soil samples and control diet,

respectively.

The average percent surviving third instar larvae over all sampling time points was

around 87.5 % in Bt rhizosphere samples and 88.0% in Bt non-rhizosphere samples of

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North Zone (Jalandhar and Mirzapur), respectively (Figures 31 A, B and 32 A, B).

These values were comparable to 89.0% and 88.0 % in respective control samples

with no soil. The average percent surviving third instar larvae over different sampling

time points was around 81.5% in Bt rhizosphere and Bt non-rhizosphere soil samples

collected from Ahmadnagar, which was equal to the larvae obtained from control

(81.25%) (Figures 33 A and B). Similar trend was observed in the insect bioassays

containing Bt rhizosphere and Bt non-rhizosphere soil samples from Bhopal and

control sample, where there were 88.5% surviving third instars (Figures 34 A and B).

In the insect bioassays with soil samples collected from Dharmapuri, the average

percent third instar larvae was higher when Bt rhizosphere (86.0%) and Bt non-

rhizosphere (85.4%) soil samples were taken than when non-Bt soil samples and diet

control (84.0%) were taken (Figures 35 A and B). The percent third instars were 85-

87% in Bt rhizosphere and Bt non-rhizosphere samples from Dharwad and Kurnool as

compared to 87.0% when control (diet) was taken (Figures 36 A and B and 37 A and

B).

Discussion

An important aspect of the risk assessment of pesticidal transgenic plants encoding Bt

insecticidal genes is their impact on the soil ecosystem. We evaluated this by

measuring the populations of total culturable bacteria and fungi, numbers of

earthworms and collembola, and the level of Cry1Ac protein in soil on which Bt, and

non-Bt brinjal had been grown. We observed minimal differences, and none that was

persistent between the Bt and non-Bt brinjal treatments in the total population levels

of culturable soil bacteria and fungi. This is in agreement with our previous year study

where, we did not observe any significant effect of Bt brinjal on soil microflora (

“Effect of transgenic brinjal expressing Bacillus thuringiensis cry1 Ac gene on soil

microflora, collembola, and earthworms” - Report submitted to RCGM during July

2004). Even though no previous published reports on the effect of Bt brinjal on soil

bacteria and fungi are available, the effects of transgenic cotton and corn plants

expressing the Bt proteins have been studied. Donegan et al. (1995) evaluated the

effect of Bt cotton on the soil microflora by placing leaves of transgenic cotton plants

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in soil and monitoring numbers and species of indigenous soil bacteria and fungi.

They found that transgenic cotton lines expressing the cry1Ac gene caused a transient

increase in total bacteria and fungi population levels that were significantly higher on

several sample days than in the non-transgenic treatment. These authors suggested

that the genetic manipulation or the tissue-culturing of the plants might have resulted

in a change in plant characteristics that, other than production of Cry1Ac toxin, might

have impacted the soil microflora. Saxena and Stotzky (2001) showed that Bt corn

expressing the cry1Ab gene did not have any effect on culturable bacteria (including

actinomycetes), fungi, protozoa, nematodes, and earthworms. These findings

substantiate the findings of this study: i.e., the culturable microbial populations were

not adversely affected by Bt brinjal plants.

Collembola and earthworms aid in the decomposition process by fragmenting and

conditioning plant debris before further breakdown by microorganisms. The data of

our study indicated that soil collembola were not adversely affected by Bt brinjal

expressing cry1Ac gene. Our data are in relatively good agreement with the findings

of other researchers. Yu et al. (1997) evaluated the effect of transgenic potato

expressing Cry3A protein and transgenic cotton expressing cry1Ab or cry1Ac genes

on collembola and found that the time to attain reproductive maturity, egg production,

and final body length did not differ between collembola fed with transgenic or non-

transgenic plant tissue of these crops. Sims and Martin (1997) evaluated the dietary

toxicity of four Bt insecticidal proteins (Cry1Ab, Cry1Ac, Cry2A, and Cry3A) against

collembola and reported that purified Bt insecticidal proteins, equivalent to proteins

expressed in transgenic plants, posed no identifiable toxicological risk to soil-

inhabiting collembola. But, United States Environmental Protection Agency (EPA

2000) reported that the Cry1Ab protein found in one type of transgenic corn (event

176), when added to an artificial soil diet mix, caused significant mortality to

collembola and significantly reduced reproduction of the survivors. The LD50 was 240

mg Bt maize leaf protein/ kg of soil (95% CL 210-280). The lowest observed effect

level (LOEL) was 250 mg/ kg and the no observed effect level (NOEL) was 125 mg/

kg of soil. Even though EPA reported this non-target effect, they concluded that the

higher dose of Cry1Ab protein in corn (event 176) could have caused the adverse

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effect, and that there is a 200-fold safety factor in the levels of toxin that would occur

in the field.

Except in Jalandhar location, earthworms were observed at other locations during all

the pre-harvest sampling time-points of this study (Table 1-3). Published reports

indicate that Bt proteins do not have any adverse effect on earthworms (Saxena and

Stotzky 2001; Zwahlen et al. 2003b). Saxena and Stotzky (2001) did not find any

significant differences in mortality and weight of earthworms exposed to root

exudates or plant biomass of transgenic Bt corn or the near-isogenic control hybrid

after 40 or 45 days, respectively. Similarly, Zwahlen et al. (2003b) did not find any

significant differences in mortality between earthworms fed with biomass of Bt and

non-Bt corn. The nematode data of this study (Figures 15-21) indicated that Bt brinjal

appears not to be toxic to nematodes. This is in agreement with the published reports

(Saxena and Stotzky, 2001; Al-Deeb et al., 2003) indicating that Bt proteins do not

have any toxic effect to nematodes.

The absence of mortality in larvae released on soil-diet mixture of all locations clearly

suggests that soil samples do not contain any accumulated levels of Bt protein under

actual field conditions. Also, in insect bioassays done with soil samples from

different locations, the growth of the larvae as indicated by surviving percent third

instar larvae is equal to the growth observed when control (artificial diet alone) was

taken. This again suggests that there are no Bt accumulations that would cause

growth inhibition or mortality among the larvae. Other studies, performed under

actual conditions, have shown that Bt proteins produced in Bt transgenic crops are

rapidly degraded in soil. Palm et al. (1996) reported that in most of the experiments

with transgenic Bt cotton (Cry1Ab or Cry1Ac) and purified Bt toxin (Cry1Ac), an

initial rapid decline in extractable toxin concentration during the first 14 days,

followed by a slow decline until the end of the experiment. At the end of the

experiment, Bt toxin from transgenic cotton plants was

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and the half life of the protein in transgenic Bt cotton plant tissue was 41 days. Studies

of the environmental fate and degradation of other Bt proteins, such as Cry3A and

Cry1Ab, found that the half-lives of these proteins in soil was generally less than 20

days (Ream et al., 1994; Sims and Holden, 1996; Palm et al., 1996). Head et al.

(2002) reported that the quantity of Cry1Ac protein that accumulated as a result of

continuous culture of transgenic Bt cotton and subsequent incorporation of plant

residues into the soil by post-harvest tillage was low and did not result in any

detectable biological activity.

Conclusion

Our findings demonstrate that transgenic brinjal expressing Bt cry1 Ac gene does not

have any adverse effect on soil microflora (both fungi and bacteria), and soil

invertebrates such as earthworms, and collembola. This is not surprising since B.

thuringiensis and Bt proteins have shown to act specifically on the target insect pests

and do not have any deleterious effect on non-target organisms. Also, no Bt protein

was detected in any of the soil samples from Bt brinjal field plots.

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Figure 1. Effect of Bt brinjal on total rhizosphere and non-rhizosphere soil bacteria.

Colony forming units (± Standard error) x106

gram-1

soil. Location: Jalandhar (north

zone). Treatment details: T1 = MHB80 Bt, T2 = MHB80 non-Bt, T3 = non-Bt local

Check, T4 = Non-Bt commercial Check. P values for rhizosphere populations: 30

days after transplantation (DAP) = 0.0036, 60 DAP = 0.0407, 90 DAP = 0.1619, 120

DAP = 0.0301, and 150 DAP =0.0001. P values for non-rhizosphere samples: 30

DAP = 0.2469, 60 DAP = 0.0177, 90 DAP =0.7223, 120 DAP = 0.0011, and 150

DAP = 0.0001.

Jalandhar : rhizosphere bacteria

1.4

1.6

1.8

2

2.2

30 60 90 120 150

Sampling time-point: days after transplantation

Colo

ny f

orm

ing u

nit

s x

10

6 g

-1

soil

T1

T2

T3

T4

Jalandhar : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

2.2

30 60 90 120 150

Sampling time-point : days after transplantation

Colo

ny f

orm

ing u

nit

s x 1

06 g

-1

soil

T1

T2

T3

T4

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Colony forming units (± Standard error) x106 gram

-1 soil. Location: Mirzapur (north


Check, T4 = Non-Bt commercial Check. P values for rhizosphere populations: 30

days after transplantation (DAP) = 0.0027, 60 DAP = 0.0001, and 90 DAP = 0.0008.

P values for non-rhizosphere samples: 30 DAP=0.0056, 60 DAP = 0.0001, and 90

DAP = 0.0022.

Mirzapur : rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

2.2

30 60 90


Co

lon

y f

orm

ing

un

its

x 1

06 g

-1

soil

T1

T2

T3

T4

Mirzapur : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

30 60 90


Co

lon

y f

orm

ing

un

its

x 1

06 g

-1

soil

T1

T2

T3

T4

of 53



-1 soil. Location: Ahmad nagar

(central zone). Treatment details: T1 = MHB 4 Bt, T2 = MHB 4 non-Bt, T3 = non-Bt

local check- Gondegaon, T4 = non-Bt commercial check - Ajay. P values for

rhizosphere populations: 30 days after transplantation (DAP) = 0.0252, 60 DAP =

0.0004, 90 DAP = 0.0658, 120 DAP = 0.0001, and 150 DAP = 0.0802. P values for

non-rhizosphere samples: 30 DAP = 0.7792, 60 DAP = 0.0111, 90 DAP = 0.3459,

120 DAP = 0.1133, and 150 DAP = 0.0148.

Ahmad nagar : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

30 60 90 120 150


Co

lon

y f

orm

inh

un

its

x 1

06 g

-1 s

oil

T1

T2

T3

T4

Ahamad nagar: rizosphere bacteria

1

1.2

1.4

1.6

1.8

2

2.2

2.4

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

06 g

-1

soil

T1

T2

T3

T4

of 53



-1 soil. Location: Bhopal (central


check – Pusa purple round, T4 = non-Bt commercial check Navakiran. P values for


0.0208, 90 DAP = 0.0174, 120 DAP = 0.1157, and 150 DAP =0.0126. P values for


120 DAP = 0.0078, and 150 DAP = 0.2863.

Bhopal : rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

60 90 120 150


Co

lon

y f

orm

ing

un

its

x 1

06 g

-1

soil

T1

T2

T3

T4

Bhopal : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

30 60 90 120 150

Sampling time-point : days afer transplantation

Co

lon

y f

orm

ing

un

its

x1

06 g

-1 s

oil

T1

T2

T3

T4

of 53



-1 soil. Location: Dharmapuri

(south zone). Treatment details: T1 = MHB 9 Bt, T2 = MHB 9 non-Bt, T3 = non-Bt

local check- Arka Shirish, T4 = non-Bt commercial check- Extra green long. P values

for rhizosphere populations: 30 days after transplantation (DAP) = 0.0135, 60 DAP =


non-rhizosphere samples: 30 DAP = 01421, 60 DAP = 0.0039, 90 DAP = 0.6974, 120

DAP = 0.0041, and 150 DAP = 0.0001.

Dharmapuri : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

06 g

-1

soil

T1

T2

T3

T4

Dharmapuri : rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

2.2

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

06 g

-1

soil

T1

T2

T3

T4

of 53



-1 soil. Location: Dharward (south

zone). Treatment details: T1 = MHB 10 Bt, T2 = MHB 10 non-Bt, T3 = non-Bt local

check- Manjari kota, T4 = non-Bt commercial check- Manju. P values for



non-rhizosphere samples: 30 DAP=0.0036, 60 DAP = 0.386, 90 DAP = 0.0534, 120

DAP = 0.8172, and 150 DAP = 0.2765.

Dharward : rhizosphere bacteria

1

1.5

2

2.5

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x10

6 g

-1 s

oil

T1

T2

T3

T4

Dharward : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

2.2

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x10

6 g

-1 s

oil

T1

T2

T3

T4

of 53



-1 soil. Location: Karnool (south

zone). Treatment details: T1 = MHB 99 Bt, T2 = MHB 99 non-Bt, T3 = non-Bt local

check - green + white local, T4 = non-Bt commercial check- Harit. P values for




120 DAP = 0.1486 and 150 DAP = 0.3102.

Karnool : non-rhizosphere bacteria

1

1.2

1.4

1.6

1.8

2

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

06 g

-1 s

oil

T1

T2

T3

T4

Karnoo l : rh izo sphere bact eria

1

1 .2

1 .4

1 .6

1 .8

2

2 .2

2 .4

30 60 90 12 0 1 50

Sam plin g t im e-p oin t : day s aft er t ransp lan t at io n

Colo

ny f

orm

ing u

nit

s 10

6 g

-1 s

oil

T 1

T 2

T 3

T 4

of 53

Figure 8. Effect of Bt brinjal on total rhizosphere and non-rhizosphere fungal

population. Colony forming units (±Standard error) x104

gram-1

soil. Location:

Jalandhar (north zone). Treatment details: T1 = MHB80 Bt, T2 = MHB80 non-Bt, T3

= non-Bt local check – Pusa purple round, T4 = non-Bt commercial check- Navakiran.

P values for rhizosphere populations: 30 days after transplantation (DAP) =0.0248,

60 DAP = 0.2375, 90 DAP = 0.3870, 120 DAP = 0.5408, and 150 DAP =0.0065. P

values for non-rhizosphere samples: 30 DAP = 0.7072, 60 DAP = 0.3283, 90 DAP =

0.4548, 120 DAP = 0.0417, and 150 DAP =0.0123.

Jalandhar : rhizosphere fungi

15

20

25

30

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x

10

4 g

-1

soil

T1

T2

T3

T4

Jalandhar : non-rhizosphere fungi

15

20

25

30

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

of 53


population. Colony forming units (±Standard error) x104 gram

-1 soil. Location:

Mirzapur (north zone). Treatment details: T1 = MHB 80 Bt, T2 = MHB 80 non-Bt,

T3 = non-Bt local check – Pusa purple round, T4 = non-Bt commercial check-

Navakiran. P values for rhizosphere populations: 30 days after transplantation (DAP)

= 0.0689, 60 DAP = 0.4574, and 90 DAP = 0.0055. P values for non-rhizosphere

samples: 30 DAP = 0.798, 60 DAP = 0.6673, and 90 DAP = 0.0417.

Mirzapur : rhizosphere fungi

15

20

25

30

30 60 90


Co

lon

y f

orm

ing

un

its

x 1

04 g

-1

soil

T1

T2

T3

T4

Mirzapur : non-rhizosphere fungi

12

16

20

24

28

30 60 90


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

of 53


population. Colony forming units (±Standard error) x104 gram

-1 soil. Location:

Ahmad nagar (central zone). Treatment details: T1 = MHB 4 Bt, T2 = MHB 4 non-Bt,

T3 = non-Bt local check- Gondegaon, T4 = non-Bt commercial check - Ajay. P values




120 DAP = 0.8311, and 150 DAP = 0.0006.

Ahmad nagar: rhizosphere fungi

10

14

18

22

26

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

Ahmad nagar : non-rhizosphere fungi

10

12

14

16

18

20

22

24

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

of 53


population. Colony forming units (±Standard error) x 104 gram

-1 soil. Location:

Bhopal (central zone). Treatment details: T1 = MHB 80 Bt, T2 = MHB 80 non-Bt, T3

= non-Bt local check- Pusa purple round, T4 = non-Bt commercial check - Navakiran.

P values for rhizosphere populations: 30 days after transplantation (DAP) = 0.2749,

60 DAP = 0.0015, 90 DAP = 0.4667, 120 DAP = 0.0405 and 150 DAP = 0.1848. P

values for non-rhizosphere samples: 30 DAP = 0.0611, 60 DAP =0.3230, 90 DAP =

0.0382, 120 DAP = 0.0002, and 150 DAP = 0.2670.

Bhopal : rhizosphere fungi

10

15

20

25

30

30 60 90 120 150


Co

lon

y f

orm

ing

un

its

x 1

04 g

-1

soil

T1

T2

T3

T4

Bhopal : non-rhizosphere fungi

10

15

20

25

30

30 60 90 120 150

Sampling time-point : days afer transplantation

Colo

ny f

orm

ing u

nit

s x10

4 g

-1 s

oil

T1

T2

T3

T4

of 53


population. Colony forming units (±Standard error) x104

gram-1

soil. Location:

Dharmapuri (south zone). Treatment details: T1 = MHB 9 Bt, T2 = MHB 9 non-Bt,

T3 = non-Bt local check- Arka Shirish, T4 = non-Bt commercial check – Extra green

long. P values for rhizosphere populations: 30 days after transplantation (DAP) =

0.0094, 60 DAP = 0.0180, 90 DAP = 0.9485, 120 DAP = 0.3392, and 150 DAP =

0.4556. P values for non-rhizosphere samples: 30 DAP=0.0896, 60 DAP = 0.0478, 90

DAP = 0.0001, 120 DAP = 0.5526, and 150 DAP = 0.7728.

Dharmapuri : rhizosphere fungi

10

15

20

25

30

35

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

Dharmapuri : non-rhizosphere fungi

10

12

14

16

18

20

22

24

26

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

of 53


population. Colony forming units (± Standard error) x104 gram

-1 soil. Location:

Dharward (south zone). Treatment details: T1 = MHB 10 Bt, T2 = MHB 10 non-Bt,

T3 = non-Bt local Manjari Kota, T4 = non-Bt commercial check - Manju. P values




120 DAP = 0.9811, and 150 DAP = 0.2882.

Dharward : rhizosphere fungi

15

20

25

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x10

4 g

-1 s

oil

T1

T2

T3

T4

Dharward : non-rhizosphere fungi

14

18

22

26

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s x10

4 g

-1 s

oil

T1

T2

T3

T4

of 53


population. Colony forming units (± Standard error) x 104 gram

-1 soil. Location:

Karnool (south zone). Treatment details: T1 = MHB 99 Bt, T2 = MHB 99 non-Bt, T3

= non-Bt local check - Green+white local, T4 = non-Bt commercial check - Harit. P

values for rhizosphere populations: 30 days after transplantation (DAP) = 0.1240, 60

DAP = 0.1255, 90 DAP = 0.0605, 120 DAP = 0.0021, and 150 DAP = 0.141. P

values for non-rhizosphere samples: 30 DAP = 0.4103, 60 DAP = 0.1124, 90 DAP =

0.5024, 120 DAP = 0.0001, and 150 DAP = 0.0327.

Karnool : rhizosphere fungi

10

15

20

25

30

30 60 90 120 150


Colo

ny f

orm

ing u

nit

s 10

4 g

-1 s

oil

T1

T2

T3

T4

Karnool : non-rhizosphere fungi

10

14

18

22

26

30 60 90 120


Colo

ny f

orm

ing u

nit

s x 1

04 g

-1

soil

T1

T2

T3

T4

of 53

Figure 15. Effect of Bt brinjal on soil nematode population. Total number of

nematodes (±Standard error) 250 gram-1

soil. Each value is a mean of three

replications. Location: Jalandhar (north zone). Treatment details: T1 = MHB 80 Bt,

T2 = MHB 80 non-Bt, T3 = non-Bt local check – Pusa purple round, T4 = non-Bt

commercial check - Navakiran. P values: 30 days after transplantation (DAP) =

0.4541, 60 DAP = 0.9879, 90 DAP = 0.8970, 120 DAP = 0.7529, and 150 DAP =

0.1656.

Jalandhar

0

50

100

150

200

250

300

350

30 60 90 120 150


Nu

mb

er o

f n

emat

od

es 2

50

g -1

so

ilT1

T2

T3

T4

of 53




replications. Location: Mirzapur (north zone). Treatment details: T1 = MHB 80 Bt,

T2 = MHB 80 non-Bt, T3 = non-Bt local check – Pusa purple round, T4 = non-Bt

commercial check – Navakiran. P values: 30 days after transplantation (DAP) =

0.9692, 60 DAP = 0.8043, and 90 DAP = 0.0002.

Mirzapur

0

50

100

150

200

30 60 90


Nu

mb

er o

f n

emat

od

es 2

50

g-1

soil

T1

T2

T3

T4

of 53




replications. Location: Ahmad nagar (central zone). Treatment details: T1 = MHB 4

Bt, T2 = MHB 4 non-Bt, T3 = non-Bt local check Gondegaon, T4 = non-Bt

commercial check- Ajay. P values: 30 days after transplantation (DAP) = 0.7676, 60

DAP = 0.7554, 90 DAP = 0.7928, 120 DAP = 0.9147, and 150 DAP = 0.8990.

Ahmad nagar

0

50

100

150

200

250

30 60 90 120 150


Nu

mb

er o

f n

emat

od

es 2

50

g-1

soil

T1

T2

T3

T4

of 53




replications. Location: Bhopal (central zone). Treatment details: T1 = MHB 80 Bt,

T2 = MHB 80 non-Bt, T3 = non-Bt local check – Pusa purple long, T4 = non-Bt

commercial check- Navakiran. P values: 30 days after transplantation (DAP) =

0.6761, 60 DAP = 0.7015, 90 DAP = 0.5386, 120 DAP = 0.0838 and 150 DAP =

0.0326.

Bhopal

0

100

200

300

400

500

600

30 60 90 120 150


Nu

mb

er o

f n

emat

od

es 2

50

g-1

soil

T1

T2

T3

T4

of 53




replications. Location: Dharmapuri (south zone). Treatment details: T1 = MHB 9 Bt,

T2 = MHB 9 non-Bt, T3 = non-Bt local check – Arka Shirish, T4 = non-Bt

commercial check – Extra green long. P values: 30 days after transplantation (DAP)

= 0.3694, 60 DAP = 0.5745, 90 DAP = 0.0583, 120 DAP = 0.8492, and 150 DAP =

0.2438.

Dharmapuri

0

50

100

150

200

250

300

350

400

450

30 60 90 120 150

Sampling time-point: days after transplanation

Nu

mb

er o

f n

emat

od

es 2

50

g-1

so

ilT1

T2

T3

T4

of 53




replications. Location: Dharward (south zone). Treatment details: T1 = MHB 10 Bt,

T2 = MHB 10 non-Bt, T3 = non-Bt local check- Manjari kota, T4 = non-Bt

commercial check- Manju. P values: 30 days after transplantation (DAP) = 0.3092,

60 DAP = 0.6835, 90 DAP = 0.6861, 120 DAP = 0.1006, and 150 DAP = 0.3296.

Dharward

0

100

200

300

400

500

600

30 60 90 120 150


Nu

mb

er o

f n

emat

od

es 2

50

g-1

soil

T1

T2

T3

T4

of 53




replications. Location: Karnool (south zone). Treatment details: T1 = MHB 99 Bt,

T2 = MHB 99 non-Bt, T3 = non-Bt local check –Green + white local, T4 = non-Bt

commercial check- Harit. P values for rhizosphere populations: 30 days after

transplantation (DAP) = 0.4283, 60 DAP = 0.5717, 90 DAP = 0.1112, 120 DAP =

0.8720, and 150 DAP = 0.032.

Karnool

0

100

200

300

400

500

600

30 60 90 120 150


Nu

mb

er o

f n

emat

od

es 2

50

g-1

soil

T1

T2

T3

T4

of 53

Figure 22. Effect of Bt brinjal on soil Collembola. Each value is a mean of three

replications. Location: Jalandhar (north zone). Treatment details: T1 = MHB80 Bt, T2

= MHB 80 non-Bt, T3 = non-Bt local check - Pusa purple round, T4 = non-Bt

commercial check- Navakiran. P values: 30 days after transplantation (DAP) = 0.359,


Jalandhar

0

5

10

15

20

25

30 60 90 120 150


Co

llem

bo

la p

op

ula

tio

n :

Mea

n ±

SE

T1

T2

T3

T4

of 53


replications. Location: Mirzapur (north zone). Treatment details: T1 = MHB80 Bt, T2



60 DAP = 0.981, and 90 DAP = 0.0383.

Mirzapur

0

4

8

12

16

20

30 60 90

Sampling time-point; days after transplantation

Co

llem

bo

la p

op

ula

tio

n :

Mea

n ±

SE

T1

T2

T3

T4

of 53


replications. Location: Ahmad Nagar (central zone). Treatment details: T1 = MHB 4

Bt, T2 = MHB 4 non-Bt, T3 = non-Bt local check- Gondegaon, T4 = non-Bt

commercial check - Ajay. P values: 30 days after transplantation (DAP) = 0.433, 60

DAP = 0.847, 90 DAP = 0.121, 120 DAP = 0.734, and 150DAP = 0.1296.

Ahmad nagar

0

10

20

30

40

50

60

30 60 90 120 150


Co

llem

bo

al p

op

ula

tio

n:

Mea

n ±

SE

T1

T2

T3

T4

of 53


replications. Location: Bhopal (central zone). Treatment details: T1 = MHB80 Bt, T2




Bhopal

0

1

2

3

4

5

30 60 90 120 150


Co

llem

bo

la p

op

ula

tio

n :

Mea

n ±

SE

T1

T2

T3

T4

of 53

Figure 26. Effect of Bt brinjal on soil Collembola population. Each value is a mean of

three replications. Location: Dharmapuri (south zone). Treatment details: T1 = MHB

9 Bt, T2 = MHB 9 non-Bt, T3 = non-Bt local check- Arka Shirish, T4 = non-Bt

commercial check- extra green long. P values: 30 days after transplantation (DAP) =

0.385, 60 DAP = 0.683, 90 DAP = 0.708, 120 DAP = 0.564, and 150 DAP = 0.2561.

Dharmapuri

0

5

10

15

20

25

30 60 90 120 150


Co

llem

bo

la p

op

ula

tio

n :

Mea

n ±

SE

T1

T2

T3

T4

of 53

Figure 27. Effect of Bt brinjal on soil Collembola population. Each value is a mean of

three replications. Location: Dharward (south zone). Treatment details: T1 = MHB

10 Bt, T2 = MHB 10 non-Bt, T3 = non-Bt local check- Manjari kota, T4 = non-Bt

commercial check- Manju. P values: 30 days after transplantation (DAP) = 0.71, 60


Dharward

0

1

2

3

4

30 60 90 120 150


Co

llem

bo

la p

op

ula

tio

n :

Mea

n±

SE

T1

T2

T3

T4

of 53


replications. Location: Karnool (south zone). Treatment details: T1 = MHB 99 Bt,

T2 = MHB 99 non-Bt, T3 = Non-Bt local check - Green + white local, T4 = non-Bt

commercial check- Harit. P values: 30 days after transplantation (DAP) = 0.86, 60


Karnool

0

1

2

3

4

5

6

7

30 60 90 120 150


Co

llem

bo

la p

op

ula

tio

n :

Mea

n ±

SE

T1

T2

T3

T4

of 53

Figure 29. Dose-response curve for mortality of H. armigera neonate larvae exposed

to various concentrations of diet-incorporated Cry1Ac protein (MVP II) (n = 896)

Figure 30. Percent second instar larvae of H. armigera surviving post-exposure to

various concentrations of diet-incorporated Cry1Ac protein (MVP II). This data was

obtained from the standard mortality bioassay of which the mortality results are

presented in Figure 29.

0

10

20

30

40

50

60

70

80

90

100

0.001 0.01 0.1 1

Cry1Ac (ug/mL diet)

Pe

rce

nt

Mo

rta

lity

0

10

20

30

40

50

60

70

80

90

100

0 0.001 0.004 0.012 0.037 0.111 0.333 1

Cry1Ac (ug/ml diet)

Perc

en

t T

hir

d I

nsta

r L

arv

ae

of 53

Figure 31. Insect bioassays with Helicovepa armigera to detect the levels of Cry1Ac protein

present in the A) rhizosphere and B) non-rhizosphere soil samples collected from transgenic

brinjal field plots at Jalandhar (Kharif 2004) at different crop growth stages. T1, T2, T3, T4

and C represent MHB-80 (Bt), MHB-80 (Non-Bt), Local Check (Pusa Purple Round),

Commercial Check (Navakiran - Sungro seeds) and control, respectively. Neonates of H.

armigera were fed with artificial diet +/- soil samples to detect Cry1Ac protein. Growth stages

of larvae were recorded on 7th day of release.

0

10

20

30

40

50

60

70

80

90

100

T1 T2 T3 T4 C T1 T2 T3 T4 C T1 T2 T3 T4 C T1 T2 T3 T4 C

Treatments (Rhizosphere)

Pe

rce

nt

thir

d i

ns

tar

larv

ae

30 DAT 60 DAT 90 DAT 120 DAT

Jalandhar (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100


Treatments (Non-Rhizosphere)

Pe

rce

nt

thir

d i

ns

tar

larv

ae


Jalandhar (Kharif '04) B

of 53



brinjal field plots at Mirzapur (Kharif 2004) at different crop growth stages. T1, T2, T3, T4

and C represent MHB-80 (Bt), MHB-80 (Non-Bt), Local Check (Pusa Purple Round),

Commercial Check (Navakiran - Sungro seeds) and control, respectively. Neonates of H.

armigera were fed with artificial diet +/- soil samples to detect Cry1Ac protein. Growth stages

of larvae were recorded on 7th day of release.

0

10

20

30

40

50

60

70

80

90

100

T1 T2 T3 T4 C T1 T2 T3 T4 C T1 T2 T3 T4 C


Pe

rce

nt

thir

d i

ns

tar

larv

ae

30 DAT 60 DAT 90 DAT

Mirzapur (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100

T1 T2 T3 T4 C T1 T2 T3 T4 C T1 T2 T3 T4 C


Pe

rce

nt

thir

d i

ns

tar

larv

ae

30 DAT 60 DAT 90 DAT

Mirzapur (Kharif '04) B

of 53

Figure 33. Insect bioassays with Helicovepa armigera to detect the levels of Cry1Ac protein present in

the A) rhizosphere and B) non-rhizosphere soil samples collected from transgenic brinjal field plots at

Ahmadnagar (Kharif 2004) at different crop growth stages. T1, T2, T3, T4 and C represent MHB-4

(Bt), MHB-4 (Non-Bt), Local Check (Gondegaon Local), Commercial Check (Ajay Hybrid - Ankur

seeds) and control, respectively. Neonates of H. armigera were fed with artificial diet +/- soil samples

to detect Cry1Ac protein. Growth stages of larvae were recorded on 7th

day of release.

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d in

sta

r la

rva

e


Ahmadnagar (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d in

sta

r la

rva

e


Ahmadnagar (Kharif '04) B

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Figure 34. Insect bioassays with Helicovepa armigera to detect the levels of Cry1Ac protein present in

the A) rhizosphere and B) non-rhizosphere soil samples collected from transgenic brinjal field plots at

Bhopal (Kharif 2004) at different crop growth stages. T1, T2, T3, T4 and C represent MHB-80 (Bt),

MHB-80 (Non-Bt), Local Check (Pusa Purple Round), Commercial Check (Navakiran - Sungro seeds)

and control, respectively. Neonates of H. armigera were fed with artificial diet +/- soil samples to detect

Cry1Ac protein. Growth stages of larvae were recorded on 7th

day of release.

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d in

sta

r la

rva

e


Bhopal (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d in

sta

r la

rva

e


Bhopal (Kharif '04) B

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brinjal field plots at Dharmapuri (Kharif 2004) at different crop growth stages. T1, T2, T3,

T4 and C represent MHB-9 (Bt), MHB-9 (Non-Bt), Local Check (Arka Shirish), Commercial

Check (Extra green long - Sungro seeds) and control, respectively. Neonates of H. armigera

were fed with artificial diet +/- soil samples to detect Cry1Ac protein. Growth stages of larvae

were recorded on 7th day of release.

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d i

ns

tar

larv

ae


Dharmapuri (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d i

ns

tar

larv

ae


Dharmapuri (Kharif '04) B

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brinjal field plots at Dharwad (Kharif 2004) at different crop growth stages. T1, T2, T3, T4

and C represent MHB-10 (Bt), MHB-10 (Non-Bt), Local Check (Manjari gotta), Commercial

Check (Manju - Syngenta seeds) and control, respectively. Neonates of H. armigera were fed

with artificial diet +/- soil samples to detect Cry1Ac protein. Growth stages of larvae were

recorded on 7th day of release.

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d i

ns

tar

larv

ae


Dharwad (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100



Pe

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nt

thir

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ae


Dharwad (Kharif '04) B

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Figure 37. Insect bioassays with Helicovepa armigera to detect the levels of Cry1Ac

protein present in the A) rhizosphere and B) non-rhizosphere soil samples collected

from transgenic brinjal field plots at Kurnool (Kharif 2004) at different crop growth

stages. T1, T2, T3, T4 and C represent MHB-99(Bt), MHB-99 (Non-Bt), Local Check

(Green +White Local), Commercial Check (Harit - Syngenta seeds) and control,

respectively. Neonates of H. armigera were fed with artificial diet +/- soil samples to

detect Cry1Ac protein. Growth stages of larvae were recorded on 7th

day of release.

0

10

20

30

40

50

60

70

80

90

100



Pe

rce

nt

thir

d i

ns

tar

larv

ae


Kurnool (Kharif '04) A

0

10

20

30

40

50

60

70

80

90

100



Pe

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nt

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tar

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Kurnool (Kharif '04) B

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Table 1. Earthworm populations measured in the Bt brinjal experimental plots at

North zone locations.

Mean number of earthworms a


Location Treatmentsc

0 b

30 60 90 120 150

T1 0.00 0.00 0.00 0.00 1.70 0.00

T2 0.00 0.00 0.00 0.00 0.00 0.00

T3 0.00 0.00 0.00 0.00 0.33 0.00

T4 0.00 0.00 0.00 0.00 0.00 0.00

Jala

ndhar

P value 0.00 0.00 0.00 0.00 0.22 0.00

T1 1.33 1.33 1.33 0.33 YDd

YD

T2 1.33 1.00 1.00 0.00 YD YD

T3 2.00 1.33 1.30 0.33 YD YD

T4 0.67 1.33 0.67 0.33 YD YDMir

zapur

P value 0.7583 0.9849 0.7091 0.8022 YD YD

a. Each value is a mean of three replicationsb. Earthworm populations measured in the respective plots before transplantation.c. Treatment details :

Jalandhar: T1 = MHB 80 Bt, T2 = MHB 80 non-Bt, T3 = non-Bt

local check – Pusa purple round , and T4 = non-Bt

commercial check – Navakiran.

Mirzapur: T1 = MHB 80 Bt, T2 = MHB 80 non-Bt, T3 = non-Bt

local check – Pusa purple round, and T4 = non-Bt


d. YD = yet to be done.

of 53


Central zone locations.




0 b

30 60 90 120 150

T1 0.33 3.33 2.67 3.00 5.00 1.00

T2 0.33 1.67 2.33 3.67 1.67 0.33

T3 0.67 1.33 1.00 3.67 1.33 0.67

T4 0.00 2.00 2.33 4.00 1.67 1.00

Ahm

ad

Nag

ar

P value 0.4547 0.7487 0.4547 0.8652 0.1050 0.7718

T1 0.33 6.33 2.33 2.00 1.33 0.33

T2 2.00 1.67 1.00 0.33 0.33 0.33

T3 0.67 1.67 0.33 0.33 0.33 0.00

T4 1.67 4.00 1.33 0.67 0.00 0.33Bhopal

P value 0.7537 0.4333 0.3533 0.1984 0.112 0.4757

a. Each value is a mean of three replicationsb. Earthworm populations measured in the respective plots before transplantation.c. Treatment details :

Ahmad nagar: T1 = MHB 4 Bt, T2 = MHB 4 non-Bt, T3 = non-Bt

local check – Gondegaon , and T4 = non-Bt

commercial

check – Ajay.

Bhopal: T1 = MHB 80 Bt, T2 = MHB 80 non-Bt, T3 = non-Bt

local check – Pusa purple round, and T4 = non-Bt


of 53


South zone locations.




0 b

30 60 90 120 150

T1 0.67 0.00 1.33 1.67 2.33 2.33

T2 0.67 1.33 3.33 0.00 4.33 1.33

T3 0.67 2.00 1.67 0.67 4.33 1.00

T4 0.67 3.67 2.33 0.00 2.67 2.33

Dhar

map

uri

P value 0.8927 0.6030 0.8363 0.8363 0.6414 0.3556

T1 7.67 2.33 2.67 0.00 1.67 2.33

T2 17.00 2.67 3.33 0.00 0.00 1.00

T3 12.67 6.33 5.00 0.33 0.33 0.00

T4 10.00 5.00 6.00 0.00 0.00 1.00

Dhar

war

d

P value 0.6536 0.4925 0.0295 0.45 0.2241 0.1889

T1 0.00 0.00 1.33 2.33 1.33 1.67

T2 0.00 0.33 4.00 3.00 3.00 2.67

T3 0.00 0.00 2.67 3.00 2.67 3.00

T4 0.00 0.00 3.33 1.67 2.33 2.67

Kar

nool

P value 0.00 0.4547 0.0590 0.2287 0.1692 0.5376

a. Each value is a mean of three replicationsb. Earthworm populations measured in the respective plots before transplantation.c. Treatment details:

Dharmpuri: T1 = MHB 9 Bt, T2 = MHB 9 non-Bt, T3 = non-Bt

local

check – Arka Shirish, and T4 = non-Bt commercial

check – Extra green long.

Dharward: T1 = MHB 10 Bt, T2 = MHB 10 non-Bt, T3 = non-Bt

local

check – Manjari kota, and T4 = non-Bt commercial

check – Manju.

Karnool: T1 = MHB 99 Bt, T2 = MHB 99 non-Bt, T3 = non-Bt

local

check – Green + white local, and T4 = non-Bt

commercial

check – Harit.

Bt brinjal- pollen flow study

Page 1 of 17

Assessment of Pollen Flow from Bt brinjal


Page 2 of 17

CONTENTS

S. No Particulars Page no

1 Introduction 3

2 Materials and Methods 5

2.1 Sampling procedures for pollen flow studies 5

3 Results and Discussion 7

3.1 Estimation of Pollen Dispersal 7

4 Conclusions 8

5 Summary 8

6 References 9

7 Tables 1 – 8 12


Page 3 of 17

1. Introduction

In India among the Solanaceous vegetables, brinjal Solanum melongena Linn. is the

most important vegetable crop. It occupies 0.39 m.ha.(Sardana, 2002) with an

average production of 76 tonnes per ha. In recent years, however, the production of

brinjal not only in the Indian sub-continent but also in East and South Africa, Congo,

Malaysia, Thailand, Germany, Sri Lanka and Burma has been seriously affected due

to steady increase in insect pest infestation, especially the shoot and fruit borer,

Leucinodes orbonalis (Guen.). The young larvae of the pest bore in to petioles and

midribs of large leaves and tender shoots causing shoot tips to wilt and later they

bore in to flower buds and fruits. The affected fruits loose their market value besides

considerable reduction in yield as well as vitamin 'C ' content. The pest poses a

serious problem because of its high reproductive potential, rapid turnover of

generations and intensive cultivation of brinjal both in wet and dry seasons of the

year. In India it has been estimated that shoot and fruit borer causes damage to fruits

ranging from 25.8 - 92.5 % and yield reduction from 20.7 – 60% (Mall et.al., 1992).

Brinjal crop is also known to be attacked by a range of sucking pests, which include

leafhoppers, aphids, white flies, thrips and mites. Losses due to these sucking pests

range from 25 – 40% (Natarajan et.al., 1986, Anonymous, 1999).

Farmers all over the world use large quantities of chemical insecticides singly or in

combination to get blemish free fruits, which fetch premium price in the market. This

practice of indiscriminate use of insecticides has resulted in the build up of pesticide

residues in the produce, destruction of natural enemies, pest resurgence and

environmental pollution. As such published reports are not available on the

quantification of the chemicals used in brinjal for shoot and fruit borer control.

However, based on a sample estimate that the brinjal crop receives 5-10 sprays in a

season against the pest and each spray costing Rs.875 per ha, for the area of 0.39

m ha the total cost of insecticide sprays will be in the range of Rs. 1706-3412 million.

Considering 50% of the total insecticidal sprays in brinjal crops are targeted against

Fruit & Shoot borer, for a minimum of 2 - 5 insecticide sprays the cost would come in

the range of Rs. 1750 to 4375 per ha. If the Bt brinjal is adopted over 40% of 0.39m

ha brinjal area in India, it can

be projected that, there can be a national level saving of Rs 273 - 683 million

annually.

Crop protection to control insect pests in several crops through transgenic approach

is effectively used around the world. The development of transgenic cotton that


Page 4 of 17

expresses insecticidal crystal protein genes from Bacillus thuringiensis (Bt) var.

kurstaki had resulted in lines with improved resistance to lepidopteran insect pests

(Perlak et al., 1990). Fischhoff et al., (1987), developed insect tolerant transgenic

tomato plants showing resistance to tobacco hornworm ( Manduca sexta L.) and

tobacco budworm (Heliothis virescens F.). Perlak et al., (1993) developed genetically

improved potatoes producing Cry IIIA proteins having protection from the damage by

Colorado potato beetles. Several successful attempts at introducing insect resistant

genes into other crops such as corn (Koziel et al., 1993), tobacco (Warren et al.,

1992) and others have been reported. Rao et al., (1999) studied the relative efficacy

of seven lepidopteran specific Bt ä-endotoxin genes on Leucinodes orbanalis and

concluded that cry1A(c) gene was effective.

Plant transformation protocols have been developed for many crops using both

particle bombardment method and Agrobacterium mediated transformation methods.

We have been able to optimize a protocol for Brinjal transformation using

Agrobacterium system with relatively high frequency. Using this system, many

transgenic plants have been obtained and characterized. To reduce pests-linked

damage in brinjal crop as well as to protect the environment from adverse affects of

pesticides, our scientists at Maharashtra Hybrid Seed Company have developed

transgenic brinjal lines expressing the lepidopteran specific cry1A(c) gene under the

control of enhanced CaMV 35 S promoter for high level expression in brinjal to

provide an effective built-in control for shoot and fruit borer. This would result in

bringing down the cultivation costs of brinjal, as contribution of chemical pesticides to

brinjal cultivation is sizable. In addition, as the protein acts only on the target pests

(lepidopteran pests), it is not reported to reduce the population of beneficial insects,

which also helps to keep the destructive pest population under control.

The study briefed here aims to assess Pollen flow from transgenic Brinjal and the

performance of Bt brinjal transgenic lines under two different agro-climatic regions


Page 5 of 17

2. Materials and Methods

A field trial consisting of four brinjal lines was laid out during the K-02 season of

2002-03 in a randomized block design with four replications at Mahyco farms at

Mandwa, Jalna District, Maharashtra. Two transgenic brinjal lines, TB2 and TB3

which had a single insert of cry1Ac gene along with their non-Bt counter part (brinjal

variety Mahyco line 60208) with check (Manjri gota) were evaluated under two agro-

climatic regions which were approved by RCGM on 9.6.03, Ref No: BT/BS/17/02/94-

PID(Vol.III). Brinjal seedlings were transplanted in a plot of 29.4 X 18 m and spaced

at 90 X 60 cm. Each treatment plot was laid out with 5 rows of 10 plants each on a

plot of 4.5 X 6 m. The transgenic block was surrounded by rows of non-transgenic

trap rows as shown in Fig 1. Recommended agronomic practices, which include

basal dose of NPK (80:40:40 kg/ha) and subsequent top dressings, were applied.

Sprayings were initiated on attaining ETL of the sucking pests (2 leafhoppers or 5

white flies per leaf). A total of two pesticide sprays were imposed, with one

application of each selected spray for providing protection against sucking pest

complex. (Confidor-200SL @ 0.5 ml/liter for white flies and Dicofol @ 2.7 ml/liter for

control of mites).

2.1 Sampling procedures for pollen flow studies

Thirteen squares were planted with a non-spiny brinjal variety Pusa Kranti

surrounding the Bt experimental plots at varying distances (Fig 1). Fruit samples

were drawn from each row from plants at a distance of five meters on all side of the

square thrice at an interval of one month. The seeds were extracted from the fruits.

The seeds from plants were planted on the nursery beds for the squares from each

of the pickings. Data was recorded on the progeny rows grown from these plants for

all the three pickings. The

numbers of spiny seedlings were counted and their percentage was worked out to

know the out crossing.


Page 6 of 17

Fig 1. Schematic field layout (not to scale) for the estimation of pollen dispersal

by honeybees from transgenic Bt brinjal.

Central 29.4m x 18.0m Replicated Trial Block Containing Bt Brinjal Having Spiny Character

Row 6 Rows 1 to 5

Row 7

Row 8

Row 9

Row 10

Row 11

Row 12

Row 13

BeeHives

Rows 1 to 13 are planted with non-Bt, non-spiny brinjal plants forming concentric pollen-trap rows. Nos. 1 to 5 is 2 meters apart, at 2 to 10m distances and Nos. 6 to 13 is 5 m apart, at 15 to 50m distances from the transgenic plot.

Note: indicates honey bee hives at the four corners of the transgenic trial block.

Bee hives


Page 7 of 17

3. Results and Discussion

3.1 Estimation of Pollen Dispersal

3.1.1 At Mandwa

First Harvest

Out crossing was observed to an extend of 6.25 % on the western side in the eighth

Square and no out crossing was observed on other three sides in this square.

No out crossing was observed in squares 9 to 13. (Table 1).

Second Harvest

No out crossing was recorded in any of the square in the fruits harvested from

second Harvest study. (Table 2).

Third Harvest

No out crossing was recorded in any of the squares in the fruits harvested from third

harvest study. (Table 3).

A total of 1351 plant progenies (450 progenies from I, 451 progenies from II and 450

progenies from III picking) were observed for outcrossing. One progeny in the eighth

square has shown 0.6% outcrossing. Out of the 1351 progenies studied over squares

8 to 13, only one progeny has shown out crossing thus the outcrossing comes to

0.07%. Only one outcrossing event has been recorded in the eighth square, which is

situated at a distance of 25 meters from the source (Spiny Plants). No outcrossing

were recorded in squares 9,10,11,12 and 13 situated at a distance of 30, 35, 40, 45

and 50 meters from the source. (Table 4).

3.1.2 At Ranebennur

First Harvest

Out crossing was observed to an extend of 9.1 % on the western side in the eighth

square.

No out crossing was observed on other three sides to this square. No out crossing

was observed in squares 9 to 13. (Table 5).


Page 8 of 17

Second Harvest

No out crossing was recorded in any of the squares in the fruits harvested from

second harvest study. (Table 6).

Third Harvest

No out crossing was recorded in any of the squares in the fruits harvested from third

harvest study. (Table 7).

A total of 1183 plant progenies (395 progenies from I, 394 progenies from II and 395

progenies from III picking) were observed for out crossing. One progeny in the eighth

square has shown 0.8% out crossing. Out of the 1183 progenies studied over

squares 8 to 13, only one progeny has shown out crossing thus the outcrossing %

comes to 0.08%. Only one out crossing event has been recorded in the 8 th square

which is situated at a distance of 25 meters from the source (Spiny Plants). No

outcrossing were recorded in squares 9,10,11,12 and 13 situated at a distance of 30,

35, 40, 45 and 50 meters from the source. (Table 8).

Conclusions

4.1 Studies on Pollen Dispersal

Pollen dispersal studies indicate that maximum pollen transfer takes place up to 2

meters only, followed by 4 meters and 6 meters. The least dispersal was observed

at 20 meters at Mandwa location and at 15 meters at Ranebennur location from the

transgenic Bt brinjal lines (spiny plants).

Summary

Crop protection to control insect pests through transgenic approach is very effective

and forms an integral component of the integrated pest management (IPM)

strategies. The maximum amount of pollen flow from transgenic brinjal was found to

occur upto six meters and the extent of dispersal was upto a distance of 20 meters

only. It is in the best interests of the farming community the benefits of such

technology be conserved and extended for the longest possible time.


Page 9 of 17

6. References

Clark, M.F., Lister, R.M., and Bar-Joseph, M. 1986. ELISA techniques, Methods in

Enzymology 118:742-766.

Dogan, E. B., Berry R. E., Reed, G. L. and Rossignol, P. A. 1996. Biological

parameters of convergent lady beetle (Coleoptera: Coccinellidae) feeding of

aphids (Homoptera: Aphididae) on transgenic potato. J. Econ. Entomol. 89: 1105-

1108.

Fischhoff, D.A., Bowdish, K.S., Perlak, F.J., Marrone, P.G., McCormick, S.M.,

Niedermyer, J.G., Dean, D.A., Kusana-Kretzmer, K., Mayer, E.J., Rochester, D.E.,

Rodgers, S.G., and Fraley, R.T. 1987.Insect tolerant transgenic tomato

plants. Biotechnology 5:807-813.

Johnson, M. T., and Gould, F. 1992 Interaction of genetically engineered host plant

resistance and natural enemies of Heliothis virescens (Lepidoptera: Noctuidae) in

tobacco. Environ. Entomol. 21:586-597.

Khan, R. and Rao, G. R., 1976. Some results of interspecific pollination with

Solanum melongena, a popular vegetable crop, in Indo-Soviet Symp. Embryol. Crop

Plants Abstracts, New Delhi.

Koziel, M.G., Beland, G.I., Bowman, C., Carozzi, N.B., Crenshaw, R., Crossland, L.,

Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D.,

McPherson, K., Meghji, M.R., Merlin, E., Rhodes, R., Warren, G.W., Wright, M., and

Evola, S.V. 1993. Field performance of elite transgenic maize plants expressing an

insecticidal protein derived from Bacillus thuringiensis toxins. Biotechnology 11:194-

200.

Mall, N. P., Pande, R. S., Singh, S. V. and Singh S. K., 1992. Seasonal incidence of

insect pests and estimation of losses caused by shoot and fruit borer on brinjal.

Indian Journal of Entomology. 54(3): 241 – 247.

Natarajan, K., Sundaramurty, V. T. and Basu, A. K., 1986. Meet the menace of white

fly on cotton. Indian Farming 36(4): 37-44.


Page 10 of 17

Nishio, T., Mochizuki, H., and Yamakawa, K., 1984. Interspecific crosses between

eggplant and related species. Bull. Veg. Ornam. Crops Res. Sta. A12, 57.

Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate,

J.T., and Fishholl, D.A. 1990. Insect resistant cotton p

assessment of bt protein level in soil and effect of ... · summary the effect of transgenic...

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