Evaluation of transgenic cotton lines expressing an insecticidal fern protein against whitefly, Bemisia tabaci (Gennadius)

Abstract

Background

Transgenic research in crops involves using genetic engineering techniques to introduce specific genes of interest from other organisms, or even entirely new genes into plant genomes to create crops with desirable traits that wouldn't be possible through conventional breeding methods. Transgenic crops have been developed for various traits globally. Whitefly, Bemisia tabaci (Gennadius) is one of the major sucking pests of cotton that cause significant damage to the cotton production. To combat whitefly infestations, researchers have developed four transgenic cotton lines expressing the fern protein. And those transgenic lines need to be evaluated for their performance against the target pest—whitefly. The evaluation was designed as controlled trials in polyhouse or muslin cloth cages under open-choice and no-choice conditions by comparing four transgenic cotton lines (A, B, C, and D) with three control groups, including untransformed cotton plants with a same genetic background of the transgenic line, conventionally bred whitefly-resistant cotton, and whitefly-susceptible cotton. In order to study the generational effect, the evaluation also involved studies on whitefly development in laboratory, muslin cloth cage, and polyhouse conditions.

Results

Both open-choice and no-choice experiments had shown that all the four transgenic cotton lines (A, B, C, and D) expressing the fern protein reduced adult whitefly numbers significantly compared with the control lines, except for the no-choice conditions in 2021, where the transgenic line C was non-significant different from the resistant control line. Notably, the nymphal population on the resistant control line was relatively low and non-significant different from the transgenic line C in 2021; and the transgenic lines A and C in 2022 under open-choice conditions. Under no-choice condition, the nymphal counts in the resistant control line was non-significant different from transgenic lines C and D in 2021; and transgenic line D in 2022. All transgenic lines showed significant decrease in egg hatching in 2021 and nymphal development in 2022, except for the transgenic line C which had no significant different in the nymphal development comparing with non-transgenic control lines in 2022. Adult emergence rates in both years of evaluation showed significant decrease in transgenic lines A and B comparing with the control lines. Additionally, the results showed a significant reduction in cotton leaf curl disease and sooty mold development in all the four transgenic lines compared with susceptible control under open-choice conditions, indicating potential benefits of transgenic lines beyond direct effect on whitefly control. Furthermore, the research explored the generational effects of the fern protein on whitefly which revealed the lowest fecundity in the transgenic line C across F₀, F₁ and F₃ generations, lower egg hatching in F₁ and F₂ generations in transgenic lines A and B, shorter nymphal duration in F₁ and F₂ generations in transgenic line B, and the least total adult emergence in the transgenic line C in F₀ and F₃ generations.

Conclusions

These findings suggest that the transgenic cotton lines expressing fern protein disrupts whitefly populations and the life cycle to a certain extent. However, results are not consistent over generations and years of study, indicating these transgenic lines were not superior over control lines and need to be improved in future breeding.

Background

The advancement of biotechnological approaches for developing transgenic plants, along with effective targeted gene delivery methods coupled with improvement in plant regeneration procedures, has led to the development of transgenic plants for various traits of interest (Munaweera et al. 2022). In cotton, various specific traits of interest, such as pest and disease resistance, herbicide tolerance, improved nutrition, and abiotic stress tolerance, have been exploited and commercialized under field conditions. Among these traits, genetically modified crops resistant against insect-pests have been most widely used and provide a unique opportunity for effective management of key pests pertinent to different agro-climatic conditions. The development of transgenic cotton expressing genes encoding cry toxins (proteins) from Bacillus thuringiensis (Bt) causing direct larval mortality, offered a remarkable success for management of bollworms (Wu et al. 2008). The introduction of transgenic cotton with cry toxins reduced the usage of broad spectrum insecticides targeted towards management of bollworms, but leads to the increased incidences of sucking pests (Lawo et al. 2009). Whitefly, Bemisia tabaci (Gennadius), is the polyphagous sucking pest that infests several hundred-plant species, including the economically important crop, cotton (Li et al. 2021). Whitefly cause damage by sucking phloem sap that results in early wilting, premature defoliation, stunted growth, and ultimately lead to yield loss. Beyond the direct sap feeding, whiteflies inflict significant indirect damage to cotton. Their sugary excrement, honeydew, acts as a breeding ground for sooty mold fungus, which coats leaves and opened cotton bolls  and the lint. The resulting discoloration and stickiness severely deteriorate the quality and marketability of the cotton. Out of a total of 114 reported plant viruses transmitted by whiteflies, 111 are carried by the B. tabaci alone (Jones 2003), including cotton leaf curl virus which can infect plants and cause cotton leaf curl disease (CLCuD) and resulting in complete loss of yield. The increased incidence of whitefly was observed since 2012 and severe outbreaks were noticed during 2015 (Kumar et al. 2020) and 2022 cotton seasons in North India (Prabhulinga et al. 2023).

The use of chemical insecticides still remains an indispensable option in pest management. Morpho-physiological traits also offer durable resistance to insect pests, but in the scenario of numerous pest species attacking single crop, it is difficult to breed for insect resistance in cotton. Genetic enhancement of cotton through conventional breeding in incorporation of insect resistance traits, such as antixenosis, antibiosis or tolerance, has attained little success due to the genotypes lack of the required level of resistance, or to an unfavorable trait to an insect species may be supportive to other insect species. Despite the measures for controlling insects by the application of chemical pesticides, about 30% of cotton production is lost to various pests (Oerke 2006). The growing awareness of the problems associated with pesticide applications and lack of desired traits in the cotton germplasm heightened the interest to adopt transgenic technology for pest management (Chakravarthy et al. 2014).

Plants are natural sources of lectins and protease inhibitors, which are known to be part of their defense mechanisms against insects. These proteins may play a role in lectin toxicity, potentially harming insects by various mechanisms, such as interfering with digestion or binding to insect gut cells. Several characteristics of ferns and mosses make them generally less favorable as food sources for insect herbivore compared with flowering plants. The protein identified from edible fern Tectaria macrodonta, named as Tma12 was used to develop transgenic insect resistant cotton lines (Shukla et al. 2016). Lectins ravage insects by binding to specific receptors on the brush border membrane of gut epithelial cells, which disrupts the normal function of these cells, and ultimately leading to insect mortality (Vasconcelos et al. 2004). Studies on lectin introgressed transgenic plants under no-choice conditions have been evaluated against several insect pests (Rao et al. 1998; Yao et al. 2003; Wu et al. 2006). Similarly, mannose-specific, Allium sativum leaf agglutinin genes, ASA and ASAL, conveyed marked resistance against homopteran pests when expressed in rice (Yarasi. 2008; Saha et al. 2006). Further studies through in-planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on brown plant hopper, green leaf hopper, and white backed plant hopper, as evident from significant reduction in the fecundity, development and survival of these insects. Rice engineered with combined ASAL and Galanthus nivalis (GNA) lectin genes exhibited superior resistance against major sap-sucking pests (Bharathi et al. 2011). The engineered rice lines were evaluated by in-planta bioassays, using brown plant hoppers, green leaf hopper and white-backed plant hopper insects on F₂ and F₃ pyramided rice lines, parental transgenic lines and untransformed control plants. In the present investigation, the evaluation of transgenic cotton lines expressing fern protein against whitefly, B. tabaci, was undertaken under open choice, no-choice conditions, in-planta bioassay including study on generational effect on whitefly biology were carried out over four generations.

Methods

Maintenance of nucleus culture

Whiteflies (B. tabaci) were collected from cotton (Gossypium hirsutum L.) grown on farms at Indian Council of Agricultural Research—Central Institute for Cotton Research (ICAR-CICR), Regional Station, Sirsa, Haryana, India during two years' evaluation (2021 and 2022). The population (nymphs/adults) was established in a contained environmental conditions in polyhouse on susceptible cotton plants to maintain a nucleus colony. Freshly emerged adults emerging from this colony were then used for various experiments (Fig. 1).

Fig. 1

Maintenance of whitefly in polyhouse. a Whitefly infestation on single leaf of cotton, b Single plant arranged in muslin cloth cage, c Maintenance of whitefly on several plants in polyhouse

Evaluation of transgenic cotton lines

The transgenic cotton lines were evaluated against whitefly under open-choice, no-choice conditions in muslin cloth cages. The generational effect was evaluated through biology studies under laboratory conditions, using potted plants raised in muslin cloth cages. The transgenic lines of Tma12 expressing fern protein were designated as A, B, C, and D. And the control lines were untransformed cotton plants with a same genetic background (non-transgenic, Coker-312), conventionally bred whitefly-resistant cotton (LPS-141), and whitefly-susceptible cotton (HS-6). Both open-choice and no-choice experiments were conducted under confinement in muslin cloth cages, taking consideration of the environmental issues related to the pollen out flow. Observations on population dynamics were recorded in the confined cage conditions from 30^th standard meteorological week (SMW) onwards during cropping season of 2021 and 2022. In order to study the in-planta effect on growth and developmental parameters, eggs and 1^st instar nymphs were marked under no-choice experiment. The biology of whitefly was studied under laboratory conditions by marking the eggs on transgenic lines and control lines to understand the effect of fern protein on the growth and developmental parameters over different generations.

Open-choice assay

The settling preference of whitefly on different cotton genotypes including transgenic cotton lines and control lines was evaluated using open-choice assay. The effectiveness of Tma12 transgenic lines over non-transgenic lines was assessed by planting them alongside of control lines simultaneously with uniform spacing (67.5 cm × 60 cm) covered with 40 × 40 mesh count muslin cloth cage in a single chamber (W×L×H: 9 m × 16 m × 3 m) at same time. Once the plants geminated and reached 40–45 days stage (at least five true leaf stage), five pairs of whitefly adults were released per plant within cloth cage. The whiteflies were free to move and choose between different genotypes for settling and ovipositional preference. After 72 h, whitefly preference was assessed by counting whitefly number settled on the upper, middle and lower canopy of 10 randomly selected plants from each of transgenic lines and control plants. After 20 days of adult release, whitefly adults and nymphs were recorded from both fixed and random sets of plants (3 each) at weekly intervals from fully formed upper, middle, and lower leaves. Simultaneously, nymphs (all stages) were recorded from fully formed leaf at 5^th main stem node from the top. The observation were recorded up to 150 days-old cotton. Additionally, at 100 days after sowing (DAS), data were collected on the incidence of cotton leaf curl disease (CLCuD) and the presence and severity of sooty mold development associated with whitefly infestation (Fig. 2).

Fig. 2

Field layout of open-choice and no-choice experiments. a Field view depicting open -choice (front) and no-choice (back) cage layout in the field, b Closer view of no-choice cage in the field, c Closer view of open-choice cage in the field, d. Inside view of no-choice cage in the field, e Inside view of open-choice cage in the field, f Marking of 1^st instar nymphs on leaf, g Marking of 1^st instar nymphs on leaf (Closer view), h Development of sooty mold, i Appearance of CLCuD

No-choice assay

The efficacy of Tma12 transgenic cotton lines was confirmed through no-choice assay wherein plants introgressed with protein were challenged with whitefly adults, to feed and complete its lifecycle under separate muslin cloth cage compartments. Twenty plants of each transgenic line (A, B, C & D), along with control lines (non-transgenic, susceptible and resistant cotton), were sown concurrently (spacing 67.5 cm × 60 cm) into separate compartments (W×L×H:3 m ×6 m× 3 m) made of 40 × 40 mesh count muslin cloth to ensure no whitefly was escaped. Five pairs of whitefly adults per plant were released in each compartment at 40–45 days after sowing (at least five true leaf stages). In order to investigate whitefly population dynamics on various transgenic lines and control lines, whitefly adults and nymphs populations were monitored weekly up to 150 DAS. This involved counting adults and nymphs on the fully formed leaves from upper, middle, and lower stems of 3 fixed and 3 randomly selected plants from each line. Additionally, adults and nymphs were counted from fully formed leaf at 5^th main stem node from the top. Due to lack of prior confirmation regarding whitefly virulence, data on CLCuD incidence under no-choice conditions is excluded from this report (Fig. 2).

In-planta bioassays

In-planta bioassays were conducted to study the effect of introgressed transgenic lines on growth and developmental parameters of whitefly. After 7–10 days of adult release under no-choice conditions, 50 eggs of each transgenic line/control line were marked to study the effect of Tma12 transgenic lines on egg hatching. Similarly, after 20 days of whitefly adult release, fifty 1^st-2^nd instar nymphs were marked. And the marked nymphs were observed till emergence on alternate days for mortality, deformity, and parasitization. The next lot was marked 20 d after the first marking. Eggs were marked four times whereas nymphal marking were repeated five times on each transgenic line and control lines.

Analysis of control effect among different generations of whitefly

This study aimed to assess the potential impact of protein (toxin) expressed in transgenic lines on successive generations (F₀ to F₃) of whitefly. While immediate mortality was not observed in the initial whitefly population exposed to the transgenic plants, the protein might be interfering with the biological process. To investigate this possibility, whitefly biology was studied across four generations using potted plant in no-choice chambers. For each transgenic/control line, twenty plants were grown in plastic containers (1 L capacity). When plants attain 30–40 days stage, they were used for biology studies. A single, previously unexposed adult whitefly pair was confined to a single leaf using the clip cage or cup cage on each plant. Ten such clip cages were prepared and each clip cage was tagged on a separate plant. This resulted in a total of 10 whitefly pairs per transgenic/control line (5 pairs were used for fecundity studies and 5 for developmental studies). After whiteflies removal, the plants were transferred to a whitefly-free screen house. Following a five-day exposure period, the number of eggs laid and hatched were recorded. Fecundity was calculated by counting the total number of egg laid per female. The hatching percentage was calculated by using total egg hatched divided by total eggs marked, and then multiplying hundred. First-instar nymphs were marked to track their development. The time taken for emergence and the duration of each developmental stage (egg, nymph, and pupa) were recorded. To assess potential transgenerational effect, adult whiteflies (male/female) from the F₀ generation were then introduced to fresh plants of their original respective transgenic and control lines. This process was repeated for three generations (F₁-F₃). In each generation, new, un-infested plants of the same age were used for both transgenic and control lines (Figs. 3 & 4).

Fig. 3

Preparation of clip cages, release of whitefly and marking of stages for biology study. a Whitefly male (right) and female (left), b Release of whiteflies in clip cages for tagging on leaf, c Cotton leaf tagged with whitefly for biology study, d Closer view of whitefly eggs laid on leaf, e Marking of newly emerged nymphs for biology study, f Full grown pupae ready to emerge as adult

Fig. 4

Whitefly developmental stages. a Egg mass, b 1^st instar nymph, c 2^nd instar nymph, d 3^rd instar nymph, e 4^th instar nymph (Pre-pupae), f Whitefly adults with egg mass

Statistical analysis

To evaluate the statistical significance of difference between treatments, the one-way analysis of variance (ANOVA) was performed in completely randomized design, with treatments (transgenic and control lines) as fixed effects whereas replications as random effect. The analysis was performed using WASP 2.0 statistical software (Jangam et al. 2004). The data were appropriately transformed wherever required using square root/ arc sin transformation (Gomez et al. 1984). Tukey's honestly significant difference test at P ≤ 0.01 or P ≤ 0.05 was used for post-hoc comparison of treatment means. Standard error of mean (SEM) was calculated to quantify the variation within each treatment group for biological parameters.

Results

Open-choice assay

The open-choice assay revealed a significantly higher number of whitefly nymphs establishing on leaves of the susceptible control line compared with both the non-transgenic and resistant control line. The number of established nymphs in the resistant control line (53.25 ± 0.86 nymphs per 3 leaves in 2021, 48.31 ± 1.32 nymphs per 3 leaves in 2022) was not significantly different from the transgenic line C (52.27 ± 0.75 nymphs per 3 leaves) during 2021 at P < 0.01, the transgenic line A (45.57 ± 0.23 nymphs per 3 leaves) and the transgenic line C (47.91 ± 0.12 nymphs per 3 leaves) during 2022 at P < 0.01. However, the adult population establishment in all the control lines including the resistant line differed significantly from all four transgenic lines in both years of evaluation (2021 at P < 0.01; 2022 at P < 0.01). The average population reduction in two years of evaluation in transgenic lines compared with non-transgenic cotton ranged from 16.86%–33.92% in the nymphal population and 8.26%–22.81% in the adult population (Table 1).

Table 1 Whitefly nymphal and adult counts (Mean ± SEM) in transgenic and control cotton lines under open-choice conditions during 2021 and 2022

No-choice assay

The results of no-choice assay clearly depict higher whitefly nymphal and adult establishment on leaves in the susceptible control line followed by non-transgenic cotton and resistant control line. The nymphal establishment in rthe esistant control line (53.57 ± 0.74 nymphs per 3 leaves in 2021, 49.56 ± 0.25 nymphs per 3 leaves in 2022) was significantly different to transgenic line A (49.71 ± 0.94 nymphs per 3 leaves in 2021, 43.18 ± 1.26 nymphs per 3 leaves in 2022) and transgenic line B (45.89 ± 0.40 nymphs per 3 leaves in 2021, 42.55 ± 0.72 nymphs per 3 leaves in 2022). In the adult population establishment during 2021, the resistant control line (42.05 ± 1.42 adults per 3 leaves) was non-significant to the transgenic line C (39.05 ± 0.67 adults per 3 leaves) but significantly different from the transgenic line A (36.54 ± 1.33 adults per 3 leaves), B (35.34 ± 1.43 adults per 3 leaves) and D (35.17 ± 1.15 adults per 3 leaves). However, in the adult population establishment during 2022, all the control lines including resistant line differed significantly from all four transgenic cotton lines at P = 0.01. The average population reduction for both the years of evaluation compared with non-transgenic cotton ranged from 26.69%–40.63% in the nymphal population and 11.19%–26.47% in the adult population (Table 2).

Table 2 Whitefly nymphal and adult counts (Mean ± SEM) in transgenic and control cotton lines under no-choice conditions during 2021 and 2022

In-planta bioassay

In-planta bioassays were conducted to study the impact of transgenic lines on whitefly growth and developmental parameters. Egg hatching success rate was evaluated by marking a total of 200 eggs per transgenic/control line across both evaluation years. The egg hatching success rate differed significantly among the control and transgenic lines in the first year (2021) at P = 0.01. However, in the second year (2022), egg hatching rate in the resistant control line (75.00% ± 1.29%) was non-significant to all the four transgenic lines at P = 0.01 (Table 3). Red-eyed nymph mortality data revealed significant differences among the transgenic and control lines. In the first year (2021), the transgenic line A (48.00% ± 0.89%) caused significantly higher mortality than the control lines at P = 0.01. And the transgenic line D recorded significantly lower mortality than the resistant control line and non-transgenic control line, but was not significant from the susceptible control line. Red-eyed nymph mortality of the transgenic line C was not significant from non-transgenic line and resistant control lines in 2021. This trend continued during the second year (2022) with the transgenic lines A (56.40 ± 2.48%), B (57.60 ± 2.32%), and D (51.60 ± 3.87%) showing significant higher mortality than all control lines at P = 0.01, while the transgenic line C has non-significant different mortality from the non-transgenic control line (Table 4). Significantly higher adult mortality and lower adult emergence rates were recorded in the transgenic lines A and B compared with control lines across both evaluation years (2021 and 2022). This suggests potential effect of transgenic lines on whitefly adult survival and reproduction (Table 4).

Table 3 Effect on egg hatching percentage in different transgenic and control cotton lines under no-choice conditions during 2021 and 2022

Table 4 Effect on nymphal development, mortality and adult emergence in transgenic and control cotton lines under no-choice conditions during 2021 and 2022

Reaction of cotton leaf curl disease and sooty mold incidence

Open-choice assay revealed the highest incidence of CLCuD (91.67% ± 0.00%) and sooty mold (100.00% ± 0.00%) on the susceptible control line which differed significantly from the rest of transgenic lines and control lines at P = 0.01. The sooty mold appearance exhibited a similar trend, with highest incidence on the susceptible control line (96.97% ± 3.03%) even under no-choice condition. No-choice data showed no significant difference in whitefly nymphs on the 5^th main stem leaf among all transgenic and control lines (Table 5).

Table 5 Cotton leaf curl virus disease, sooty mold incidence, and average whitefly nymphs on 5^th main stem leaf in transgenic and control cotton lines during 2022

Studies on generational effect on whitefly biology

The life cycle parameters of B. tabaci were investigated across four successive generations. Separate muslin cloth cages contained potted plants of various genotypes and transgenic lines allowing for the study of generational effect of the fern protein on whitefly within the same genetic background (the transgenic line or control line). The generation wise fecundity across F₀, F_1, and F₃ clearly depicts that the transgenic line C recorded significantly lower fecundity values at P < 0.01 compared with all the three control lines. The susceptible control line displayed the highest fecundity (Table 6). Similar to fecundity, egg hatching was highest in susceptible control line across generations. Transgenic lines A (F₁: 72.61% ± 3.55%; F₂: 70.21% ± 0.71%) and B (F₁: 74.64% ± 2.91%; F₂: 69.88% ± 3.84%) showed significant difference from all control lines in terms of egg hatching percentage during F₁ and F₂ generations at P = 0.01 (Table 7). This suggests that transgenic lines A and B negatively affect hatching.

Table 6 Generation wise fecundity in transgenic and control cotton lines

Table 7 Generation wise hatching percent in transgenic and control cotton lines

In F₁ generation, the transgenic lines B (13.67 d ± 0.88 d) and C (14.00 d ± 0.58 d) were recorded significantly shorter in the nymphal duration than the rest of control lines at P = 0.01 (Table 8). Similarly, in the F₂ generation, the transgenic line B (15.00 d ± 0.58 d) recorded the shortest nymphal duration which was significantly different from control lines at P = 0.01 (Table 8). The overall effect on total life cycle of whitefly was non-significant among transgenic lines and non-transgenic control lines, except in F₁ generation where the transgenic line B and the resistant line were significantly different from the rest of transgenic and control lines. In F₃ generation, the resistant control line was significantly different from all transgenic lines in terms of duration required to complete the life cycle (Table 9). However, in terms of total adult emergence, the least adult emergence was reported in the transgenic line C (12.25 ± 1.75) in F₀ generation which was non-significant different from transgenic lines A, B, and D at P = 0.01 and significantly different from all control lines. Transgenic lines A (8.00 ± 0.58) and C (9.00 ± 0.58) were recorded significantly lower adult emergence in F₁ generation than the rest of the transgenic lines and control lines at P = 0.01, while the susceptible control line (17.00 ± 1.58) recorded the highest adult emergence which was significant different from the rest of control and transgenic lines. All the transgenic lines differed significantly from control lines in F₂ generation at P = 0.01 in terms of total adult emergence (Table 10). Overall, the generational effect of transgenic lines was not consistent in terms of its effect on biological parameters of whitefly, such as fecundity, egg hatching, nymphal duration, overall developmental period, and adult emergence. Transgenic line C recorded the lowest fecundity in F₀, F₁ and F₃. Transgenic lines A and B recorded lower egg hatching than the rest lines in F₁ and F₂ generations showing its effect on hatching. Transgenic lines B and C recorded shorter nymphal duration in F₁ generation. Transgenic line B recorded shorter nymphal duration in F₂ generation. Transgenic line C in F₀ and transgenic lines A and C in F₁ had lower total adult emergence than all the rest.

Table 8 Generation wise nymphal durations (in days including red eyed nymphs) in transgenic and control cotton lines

Table 9 Generation wise life cycle (d) in respective generations of transgenic and control cotton lines

Table 10 Generation wise effect on total adult emergence number in transgenic and control cotton lines

Discussion

New tools has introduced to study the development of transgenic crops and potentially manipulate the interactions between plants and herbivores. By incorporating genes from other organisms, researchers can engineer plants to produce proteins that interfere with the biology of insect pests. The study of transgenic lines' performance on traits of interest will facilitate the selection of superior lines for subsequent breeding programs. These studied parameters in insects include factors of population establishment, fecundity, egg hatching, nymphal development time, adult mortality, etc. By monitoring these biological parameters across generations of the insect population, one can gain valuable insights into the long-term effects of transgenic crops on pest populations and develop strategies for sustainable pest management.

The evaluation of transgenic lines is a crucial step in the development of genetically engineered crops for agricultural applications. This process involves a series of tests designed to assess the effectiveness of the transgenic lines in producing desired traits. Evaluation typically involves a combination of laboratory experiments, greenhouse trials, and field studies. Data analysis using statistical methods helps researchers determine whether the transgenic line shows potential for field application. A successful evaluation paves the way for further development and regulatory approval for commercial use.

In the present investigations, four transgenic lines were evaluated for their performance compared with counterpart line, including control resistant line and control susceptible line. These lines showed varied response to the whitefly. Previous studies conducted by Shukla et al. (2016) identified and evaluated the Tma12 protein derived from the edible fern T. macrodonta as an insecticidal agent against whitefly. The study indicated that the protein showed strong insecticidal activity in in-vitro feeding assays with a median lethal concentration as low as 1.49 μg/ml. Furthermore, even sub-lethal doses of this protein also disrupts the whitefly life cycle. Contained field trials conducted with transgenic cotton lines expressing Tma12 at a concentration of approximately 0.01% of total soluble leaf protein demonstrated resistance to whitefly infestation without any observable yield penalty. Moreover, these transgenic cotton lines were also appeared to be protected from whitefly-borne cotton leaf curl viral disease (Shukla et al. 2016). The results of the study conducted by Shukla et al. (2016) revealed that fern protein expressed in transgenic lines evaluated in the present study does not cause direct mortality of whitefly, but in-vitro assay with pure protein showed whitefly mortality at concentration as mentioned above, Hence in the present study, detailed experiments were conducted to evaluate the effect of Tma12 transgenic lines on whitefly establishment, growth, development and biological parameters.

The virulence of Chit1 from Metarhizium anisopliae (Tn-25) was compared between the fourth instar nymphal and adult stages of B. tabaci revealed that the nymphal stages of B. tabaci were more susceptible compared with the adult stages (Anwar et al. 2019). Luan et al. (2013) developed a new approach for gene silencing in whiteflies, involving the delivery of dsRNA through plant leaf feeding. Five mated female adult whiteflies were introduced into a silencing system where detached tomato leaflets were soaked in a solution containing dsRNA targeting the genes. The survival rate of adult whiteflies was not significantly affected by dsRNA feeding. Additionally, the fecundity of adult whiteflies fed with Cyp315a1, Cyp18a1, and E75 dsRNA remained unchanged compared with the control group. However, whiteflies fed with dsRNA targeting the EcR gene laid significantly fewer eggs compared with the control whiteflies. Tomato plants that have been genetically modified to express two genes, shZIS (7-epizingiberene synthase) and zFPP (z-z-farnesyldiphosphate), in their glandular trichomes, showed enhanced repellence against whiteflies. These transgenic tomato lines also experience a significant decrease in the ability of whiteflies to reproduce and survive in bioassays, while maintaining the normal flavor and yield of the fruit (Bleeker et al. 2012).

In the present study, we as well employed five whiteflies per plant in both open and no-choice environments to evaluate the effectiveness of various transgenic cotton lines. In both open-choice and no-choice assays, four Tma12 transgenic cotton lines exhibited better performance compared with control lines in terms of whitefly population establishment, survival, and reproduction. Specifically, these transgenic lines demonstrated lower whitefly adult and nymph populations, reduced egg hatching rates, and increased red-eyed nymph mortality. Additionally, transgenic lines A and B exhibited higher adult mortality and lower adult emergence rates. Notably, the susceptible control line consistently experienced the highest incidence of CLCuD and sooty mold. Overall, the generational effects of the transgenic lines were not congruous. The lowest fecundity across F₀, F₁, and F₃ was recorded in the transgenic line C, showing the effect of this transgenic line on key biological parameters of whitefly. Post egg laying, the lowest hatching in F₁ and F₂ generations was reported in transgenic lines A and B. In terms of nymphal duration, transgenic lines B and C in F₁ generation and the transgenic line B in F₂ generation were recorded shorter than the rest. However, the transgenic line C in F₀ generation and transgenic lines A and C in F₁ generation had lower total adult emergence than the rest.

Similar to previous studies conducted by various researchers on different crops, our research underwent a rigorous evaluation process for selecting most promising transgenic cotton lines for further breeding program. We employed standard procedures, including open-choice, no-choice along with in-planta evaluation and biology studies, to assess the impact of transgenic cotton lines expressing fern protein. This evaluation encompassed the study on the effect of transgenic cotton lines in terms of direct insect reduction (mortality) and potential indirect effects (biological parameters and generational effect). This comprehensive dataset obtained from the present study enables us to evaluate the suitability of these transgenic lines for future applications. Given the inconsistent performance of evaluated transgenic lines across generations and years of study on growth and developmental parameters of whitefly, it's uncertain whether they would offer significant advantage. Furthermore, as whiteflies are vectors of CLCuD, even small surviving populations on transgenic cotton lines could transmit the disease, rendering them unsuitable for future breeding programs.

Conclusion

This study investigated performance of transgenic cotton lines containing an insecticidal protein from a fern (T. macrodonta) to combat whitefly (B. tabaci) infestations. In open-choice experiments, a significant difference between all transgenic lines compared with all control lines in terms of whitefly adult population establishment was reported during both the years of evaluation. A similar trend of whitefly adult establishment was observed even under no-choice condition where all transgenic lines differed significantly from all control lines except transgenic line C which was non-significant with resistant control line during 2021. The highest whitefly establishment was reported in the susceptible control line under both open and no-choice experiments in both years of evaluation except in 2021, in which the resistant control showed the highest adult whitefly count but was not significantly different from the susceptible control line under open choice conditions. In-planta tests showed that the Tma12 transgenic lines A and B had the lowest egg hatch rate and the highest nymphal mortality compared with the controls. Additionally, evaluations of disease susceptibility revealed that the susceptible control line had significantly higher level of cotton leaf curl disease and sooty mold development compared with all four transgenic lines, the resistant control, and non-transgenic control lines. The generational effect of transgenic lines expressing fern protein was demonstrated as disrupting growth and developmental parameters of whitefly such as fecundity, egg hatching, nymphal duration, overall developmental period, and adult emergence. However, the lack of consistent results across generations and years of study ed the use of these transgenic cotton lines for further breeding applications, as they did not exhibit consistent superior performance compared with control lines.