The experimental material was GK 4602 hybrid restorer line, i.e. GK 4602 R, one of the Ganga Kaveri Seed Company’s top hybrid; this hybrid is susceptible to Bacterial Leaf Blast (BLB), Blast (BL) and Brown Plant Hopper (BPH). Since the hybrid have a good agronomical attributes and popular amongst the farmer, therefore it was a candidate variety for introgression of resistance to biotic stresses viz., BL, BLB and BPH. Biotechnology team along with rice breeding team has introgressed BLB genes viz., xa13, Xa21, the line thus generated named GK4602 BLB and this was the experimental material.

The improved GK4602 BLB (which introgressed the BLB genes viz., xa13 and Xa21 from donor IRBB60) used in this investigation as a recipient parent and two donors viz., NLR 145 (donor for Pi54 blast gene) and IR 71033 (donor for Bph20 & Bph21 genes) for Blast and BPH.

The parental material recipient variety Improved GK 4602 BLB and donor parents NLR 145 and IR 71033 were planted in crossing block for generating F₁s that later used for back crossing. For making the crosses the experimental material was planted in three staggered sowing dates for optimized the synchronization of flowering of the lines to get sufficient amount of F₁ seeds.

The breeding scheme followed using two sets of crosses; in the first set the cross was made between the GK 4602BB improved and IR 71033, and in other set this recipient was crossed with NLR 145.

Two sets of the crosses were made between donor and recipient parents as per the crossing scheme mentioned in Figure 1.

The crosses thus generated were sown in crossing block. The F₁ from both the sets were backcrossed with recipient parent and from each cross 5000 seed were generated. The foreground selection was done from BC₁F₁ in batches; the heterozygous plants were selected for further back crosses, the resultant BC₁F₁ of positive plants were sown along with the recipient parent and this processed were continue till BC₃F₁, along with the foreground selection, the background selection was also made. The plants that show the phenotypic similarity with the recipient parent are positive were advanced from BC₂F₁. In BC₃F₁, these positive plants (having all the introgressed genes) and similar to the recipient parent were selfed to produced BC₃F₂ from both the sets, these two sets of seed was planted in breeding block and they were intercrossed, the F₁ thus generated was advance to F₂, the five gene positive plants through markers were selected that showed the recipient parent plant characters. The recovery of genome of recipient parent was 99% for all the morphological traits. Primers used for foreground and background selection were mentioned in Table 1 and Table 2.

Table (1):
Foreground primers used and their sequences

Table (2):
Background polymorphic primers used for BLB, BLAST and BPH

Obtained 5 gene positive plants were evaluated in multilocation trial for agronomic and yield traits. Seed of each positive plant which has in the background of recipient parent were harvested and were divided in four sets, for multilocation trial as well as glass house trials. The hot spot of BLB, BL and BPH were selected where sufficient pressure of the pest and disease was prevalent. The multilocation trial was conducted at Ambikapur (for blast), Dhamtari for BLB and at Rajahmundry for BPH. The trial was conducted in RBD, the row to row distance was 20 cm and plant to plant distance was 15 cm each selected plant grown in 6 m row length in 12 row plots.

The seeds of five gene positive was planted in Glass house as well as hot spots of Bacterial Leaf Blight, Blast and BPH for phenotypic evaluation.

The following observations were made-

1. Days to 50% flowering (days)
2. No. of panicles/plant
3. Panicle length (average of 10 panicles in cm)
4. Yield/ha
5. Disease pest reaction in control condition
6. Disease and pest reaction in hot spot

Screening Against the Bacterial Blight Pathogen
Pyramided lines of both genotypes were screened using the bacterial blight (BLB) isolates available at Ganga Kaveri Seeds Pvt Ltd, Hyderabad, India, under natural conditions in the field. At maximum tillering stage, the plants’ top leaves were clip-inoculated with bacterial suspension at a density of 109 cells/ml²³ to test the reaction of pathogen on plant. The isolate produced average lesion lengths of between 0.1 and 4.8 cm in resistant differentials and between 6.0 and 18 cm in susceptible differentials for evaluating the resistance of introgressed lines developed in this work. Introgressed lines were planted with a distance of 15 to 20 cm (between plants and rows) spacing. Each of the examined lines were undergone clip-inoculations of nine leaves from three distinct plants (three leaves per plant), and 20 days later, both visual score and the measurement of lesion length (LL) were studied to record the phenotypic reactions of the lines. A measurement of 5 cm of LL was used as a differentiating factor between resistant lines and susceptible lines. Plants with LL of 5 cm or less were rated as resistant, and those with LL of 5 cm or more were rated as susceptible, with minor changes.^{24, 25} For the screening process, three sets of plants from each line containing 18 plants were used. Screening using clip inoculation method in field is shown in Figure 2.

Screening Procedure for Blast Pathogen
A Uniform Blast Nursery (UBN) method was used to screen blast disease for every entry including both pyramided line and parents in a single row of 50 cm and with a 10 cm of spacing between row to row. Local susceptible check and resistant checks were sown in between the nursery (after every 10 entries). HR 12, most commonly used susceptible variety was spread as border in two rows for the entire plot, which in result helps in spreading of disease effectively. Disease scoring was done using 0-9 scale. Susceptible control HR12 and test entries were seen in Figure 3.

Artificial Inoculation
The nursery was planted during weather conditions conducive to blast disease development, ensuring favourable conditions for infections and the polycyclic growth of the disease. To intensify the incidence of blast, we introduced additional inoculum by harvesting diseased leaves, cutting them into small fragments, and evenly distributing them across the nursery. This process could be carried out, particularly during prolonged wet weather periods. The assessment of blast severity was conducted using the SES scale, with a minimum of two evaluations performed at 10-day intervals spanning from day 25 to day 30.^{26, 27} The screening involved three separate sets of plants for each line, with each set comprising 18 plants.

BPH Bioassay for the selected NILs and their Hybrids
BPH introgressed lines were evaluated for resistance in a net house. For this experiment IR71033 and TN1 were used as resistant and susceptible controls, respectively. The BPH samples employed in the test were initially gathered from the field and subsequently maintained through continuous rearing on TN1 in the laboratory. A modified seedling bulk test, in accordance with the SSST protocol, was conducted for the bioassay.^28,29 Thirty pre-germinated seeds of each entry, comprising both the improved lines and controls, were uniformly planted in plastic plates measuring 7 × 7 × 8 cm. This process was replicated three times. Once the seedlings had advanced to the second leaf stage, a thinning process was carried out, reducing the number to 20 plants per plate. Upon reaching the third-leaf stage, the seedlings underwent infestation with 2^nd  to 3^rd-instar BPH nymphs, with an application rate of 8 to 10 insects per seedling. Throughout the evaluation period, the water level was consistently maintained at approximately 0.5 cm above the root. Twenty-four hours after infestation, all seedlings received a gentle pat to ensure the even distribution of nymphs. Release of BPH nymphs on seedlings were shown in Figure 4. Subsequently, once all TN1 plants had died, the remaining plants underwent examination, and assessments were assigned according to the following modified scoring criteria, which are based on the Standard Evaluation System for Rice (IRRI 2002):

1. No damage observed.
2. Very slight damage detected.
3. Most plants displayed yellowing in their first and second leaves.
4. Yellowing was widespread, with nearly half of the plants wilting or succumbing.
5. More than half of the plants had perished, and the remaining ones exhibited severe stunting.
6. All plants had died.

In accordance with the criteria established by IRRI (2002), an average resistance score falling within the range of 0.1 to 1.9 was categorized as highly resistant (HR), while scores between 2.0 and 3.9 were classified as resistant (R). Scores ranging from 4.0 to 5.9 were denoted as moderately resistant (MR), whereas scores between 6.0 and 7.9 were considered susceptible (S). Finally, scores in the range of 8.0 to 9.0 were characterized as highly susceptible (HS).

The fifteen introgressed lines having all five genes multiplex were planted at Dhamtari (CG), Ambikapur (CG) and Rajahmundry (AP). The Dhamtari is a hot spot of BLB, Ambikapur is a hot spot for Blast and Rajahmundry is a hot spot for BPH.

Fifteen introgressed lines along with recipient, donor parent and susceptibly check was the experimental material. The experiment was conducted in all the three locations in RBD design, each entry were planted in 20 rows of 6 m in 20 cm row to row and 15 cm plant to plant distance in four replications. The susceptible variety fence 2 meter in all sites of the experimental plots. As per the IRRI standard scale was used for observing the incidence of BLB, Blast and BPH

Evaluation for Agronomic performance and Grain yield
In all the three locations, viz., Dhamtari, Ambikapur and Rajahmundry in middle rows ten individual plants randomly selected for taking observations for agronomic characters, the days to 50% flowering was recorded in plot basis.