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Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.
Keywords: Brca1, Brca2, mrna, quantitative, splicing, next-generation sequencing, targeted next-generation sequencing, mrna isoforms Targeted RNA-seq Assessment of BRCA1/2 Splicing Frontiers in Oncology | www.frontiersin.org May 2018 | Volume 8 | Article 140 inTrODUcTiOn At least 20% of hereditary breast and ovarian cancer cases contain germline pathogenic variants in breast cancer susceptibility genes BRCA1 (MIM #113705) or BRCA2 (MIM #600185) (1). Functioning as tumor suppressor genes, BRCA1 and BRCA2 repair single and double-stranded breaks in DNA, a process which can be compromised when variants that disrupt pre-mRNA splicing to create aberrant splice isoforms are present (2)(3)(4). These variants may directly disrupt splice sites or splicing regulatory regions, such as exonic splicing enhancers or exonic splicing silencers (5). Resulting splicing aberrations, such as major deletion/retention events and frame shifts, can lead to loss of function through the introduction of premature termination codons, leading to non-functional isoforms that are generally destroyed by nonsense-mediated decay (NMD), or via the production of truncated proteins (6). In addition, variants located at splicing regulatory regions, such as exonic splicing enhancers, have been shown to significantly alter the abundance of natural BRCA1/2 isoforms (7,8). We and others have recently employed nextgeneration sequencing technologies to explore the expression of mRNA isoforms in BRCA1 and BRCA2 variant carriers (9)(10)(11). A better understanding of expression level changes that reflect normal variation in BRCA1/2 splicing patterns between individuals would improve our understanding of isoform regulation for identifying variability that is likely to be of clinical relevance. In-depth qualitative data published for BRCA1 and BRCA2 allows for normally expressed mRNA isoforms to be distinguished more easily from aberrant isoforms (12,13). The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium developed a 5-tier classification system, which uses mRNA splicing information to help interpret the pathogenicity of possible spliceogenic variants (14). Splicing assays for BRCA1 and BRCA2 mRNA isoforms have historically employed a PCR-based qualitative (or semi-quantitative) approach, with only very recent work expanding into a quantitative analysis. To assess key elements for splicing assay design and the integrity of published splicing data, a multicentre quality control investigation was conducted by the ENIGMA Splicing Working Group (15). This study highlighted the need to standardize splicing protocols between laboratories after raising a number of methodological issues associated with current PCR-based protocols, including (1) primer design that encompasses only a subset of the BRCA1 and BRCA2 exons, (2) non-standardized use of NMD inhibitors, (3) isoforms infrequently confirmed by sequencing, and (4) a qualitative or semi-quantitative approach to assess mRNA expression patterns, as opposed to quantitative assessment (14,15). Although this study demonstrated variation in analytical sensitivity between samples, and the same sample between participating laboratories, the impact of underlying experimental factors remained unclear.
Targeted RNA-seq technologies potentially address many of the difficulties currently associated with PCR-based assays. For example, RNA-seq platforms enable detection of mRNA isoforms both qualitatively and quantitatively (16,17), thus producing comprehensive transcript profiles across the entire gene(s). Assessment of the analytical sensitivity of targeted RNA-seq to both qualitatively and quantitatively measure BRCA1/2 isoform expression in relation to experimental factors would provide a deeper understanding of the sources of mRNA isoform variation in these genes, but has yet to be evaluated.
In this study, we carried out targeted RNA-seq to assess BRCA1/2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously utilized by the ENIGMA-led multicentre study (15). We describe a comprehensive assessment of naturally and aberrantly occurring BRCA1 and BRCA2 mRNA isoforms in relation to experimental factors. Our results show that quantitation of relative levels of naturally occurring transcripts is not significantly impacted by key elements of cell-storage and culture protocols, with the possible exception of NMD-inhibition. We provide recommendations for future use of targeted RNA-seq for the analysis of variants that may disrupt RNA splicing.

MaTerials anD MeThODs
This study was approved by the Southern Health and Disability Ethics Committee (12/STH/44). samples 27 LCLs derived from 17 BRCA1 or BRCA2 rare variant carriers, and 10 healthy controls ( Figure S1 and Table S1 in Supplementary Material) were obtained from Kathleen Cuningham Consortium for Research into Familial Breast Cancer. Eighteen of the cell lines (sample IDs LCL1-8 and LCL18-27) were previously used in a multi-center methods splicing analysis study coordinated through the ENIGMA consortium (15).
Variants included in this study are referred to by the recommended Human Genome Variation Society 1 nomenclature, including use of A in the ATG translation initiation codon to start the nucleotide numbering for BRCA1 (GenBank accession-NM_007294.3) and BRCA2 (GenBank accession-NM_000059.3) (18).
Cell lines were cultured in RPMI 1640 media, with fetal calf serum (10%) and penicillin-streptomycin (1%), while incubated at 37°C in a 5% CO2 atmosphere. RNA was isolated from cycloheximide treated (4 h 100 µg/mL) and untreated cells using the Qiagen RNeasy Mini Kit. RNA was reverse transcribed into cDNA using Superscript III First Strand Synthesis System (Invitrogen), according to the manufacturer's instructions.

rna-seq analysis
Targeted RNA-seq was undertaken on all cycloheximide treated and untreated LCLs using the Illumina Truseq Targeted RNA Expression kit. The targeted sequencing assay was custom designed in Illumina's design studio using 34 BRCA1 and 28 BRCA2 oligonucleotides chosen from a database of validated predesigned probes (Tables S2 and S3 and Figures S2 and S3 in Supplementary Material). Capture of the targeted cDNA sequences was performed according to the manufacturer's specifications ( Figure S4 in Supplementary Material). Sequence analysis was carried out using the Illumina MiSeq platform.  Whole transcriptome sequencing analysis was also carried out for one control (LCL24). Using these data, BRCA1 and BRCA2 mRNA isoform splicing expression patterns were also obtained. RNA from LCL24 was sequenced with and without cycloheximide treatment on a HiSeq2000 using the Truseq ® Stranded mRNA kit (Illumina).

Read Mapping and Processing
Targeted RNA-seq and whole transcriptome read mapping was undertaken as previously described (19). Briefly, the Homo_ sapiens.GRCh37.72 reference genome was downloaded from Ensembl 2 and the chromosomes arranged into lexicographic order prior to mapping. Sequence reads were mapped using the two pass approach of the Spliced Transcripts Alignment to a Reference (STAR) aligner using the default settings unless specified otherwise (20). Maximum intron length was set to 100,000 nucleotides to accommodate for splice junctions that span the length of each gene. Detected splice junctions for each sample were extracted from STAR's SJ.out file for further analysis. Sample-specific read counts are listed in Table S4 in Supplementary Material.

Targeted RNA-seq Data Analysis: Normalization
Raw read counts of all alternative splicing events were normalized to measure the relative number of individual spliced exon/exon junctions in each sample. This was achieved by calculating the read depth of the full-length transcript (Figure 1). To normalize read depth, the total read count between two adjacent exons (exons 2 and 3 in BRCA1 and BRCA2, respectively) was used as the reference junction (RJ) (hereon referred to as "reference junction") to calculate the relative expression of the full-length and alternative mRNA transcripts for both BRCA1 and BRCA2, respectively. To achieve this, the sum of all non-overlapping exon 2-3 alternative splicing events was deducted from the RJ for each gene independently. This leaves a proportion of reads that solely represent the full-length transcript (Figure 1Bi). To determine the relative proportion of each splice junction, the total number of reads for each sample was first calculated. This consisted of the sum of the reads encompassing the alternative splicing events together and the previously calculated full-length transcript (using RJ exons 2-3, see above) (Figure 1Bii). The relative proportion of the individual isoforms in each sample is determined by dividing its respective read count by the total number of reads for that sample (Figure 1Biii). These expression values were incorporated into a comparative expression analysis, using the mean and SE (95%) of each isoform across the controls. This approach does not account for the possibility that some of the detected alternative splicing events may occur concurrently. Relative expression ranges were calculated based on the criteria that at least two control samples expressed the transcript with more than 10 reads, each sample was represented by more than 10,000 reads per gene, and each sample expressed at least two minor transcripts for the studied gene.
Alternative events were excluded from these calculations if they had questionable probe efficiency (such as was observed for BRCA1 Δ9-10) or were common NAG events (21). These are Targeted  shown to commonly co-occur with the other events detected, and so are not deducted as separate alternative splicing events when calculating the full-length transcript. The resulting proportions were compared using complementary log-log confidence intervals (22).

Quantitative Pcr (qPcr) Validation of splicing events
The expression levels observed for the BRCA1 exon 10-11, Δ10, and Δ9-10 junctions were assessed using Taqman qPCR assays and Roche LightCycler ® 480 platform. Primers were designed to encompass each targeted junction type, with the corresponding probe spanning the junction ( Of the previously reported naturally occurring isoforms not detected in BRCA1 and BRCA2 using targeted RNA-seq (12,13), the majority (28/32 in BRCA1 and 13/17 in BRCA2) were due to target probe placement (restricted to the options predesigned by Illumina), while the remainder (11/32 BRCA1 and 4/17 BRCA2) were presumably not expressed at levels that were detectable in our cell lines (Tables S6 and S7 in Supplementary Material). From our observations, and those reported in previous publications (12), all BRCA1 mRNA exons have been shown to be spliced out in at least one naturally occurring alternative mRNA transcript. By contrast, six BRCA2 exons (8, 14, 21, and 24-27) were not found to be involved in an exon skipping event in this study or by Fackenthal et al. (13). Nineteen alternative transcripts (11 BRCA1 and eight BRCA2) were detected solely in the samples treated with a NMD inhibitor (Tables S6-S9 in Supplementary Material). Of the 19, 14 are out of frame.
To assess the targeted RNA-seq method for evaluating transcript profiles in rare variant carriers (LCL1-8), we compared our data with those previously reported from the PCR-based ENIGMA multicentre study (15) (Table S10 in Supplementary Material). In this study, a total of 37 BRCA1 and 11 BRCA2 alternative splicing events were identified by targeted RNA-seq in addition to those detected by reverse transcriptase-PCR (RT-PCR) from the multicentre study (Table S10 in Supplementary Material). We found that 33/37 BRCA1 and all 11 BRCA2 of these events fell outside the region targeted by the RT-PCR assays. By comparison, 26 BRCA1 and 12 BRCA2 splicing events were exclusively detected in these samples in the multicentre study. However, 21 of these events were not detected by the Truseq Targeted RNA Expression platform due to the absence of probes targeting those regions, while 11 were due to the events involving multiple separate regions, which are not detectable together using this platform. Of the remaining six events not detected by Targeted RNA-seq (BRCA1, Δ5-6, Δ9, Δ9-11, Δ9-12, Δ11-12, Δ22), three (Δ9, Δ22, and Δ9-11) were respectively identified by three laboratories, which always included the two laboratories utilizing capillary electrophoresis for detection. Further to this, BRCA1 Δ5-6 was identified solely by laboratories utilizing capillary electrophoresis, which was the most sensitive detection method used in the multi-center study (15).
Targeted RNA-seq identified another five splicing events described by Whiley et al. (15) that were solely present in four out of the eight LCLs each carrying a known spliceogenic rare variant (Table S10 in  In contrast to the multicentre study, targeted RNA-seq was unable to detect the Δ5 event associated with the pathogenic variant BRCA1 c.135−1G>T, likely as a result of a low read count (Table S10 in Supplementary Material). These data further highlight the complexity associated with detection and interpretation of BRCA1 and BRCA2 splicing patterns when assays are designed across the whole transcript.

Quantitative assessment of Brca1 and Brca2 Transcripts
To derive quantitative information from the targeted RNA-seq data, we separately calculated the relative expression range for BRCA1 (n = 25) and BRCA2 (n = 14) transcripts from the study LCLs that did not contain known splice disrupting variants in the respective gene assayed. The number of alternative splicing events detectable across multiple LCLs was double in cycloheximide treated cell lines compared to that found in non-treated cells (Figures 2A,B), thus the following quantitative data corresponds to treated cells only. A correlation was observed between the number of detected alternative events and the total read count per sample for BRCA1 (R 2 = 0.68) and BRCA2 (R 2 = 0.69) ( Figure  S5 in Supplementary Material). The questionable probe efficiency observed for alternative event BRCA1 Δ9-10 using targeted RNA-seq was confirmed with qPCR to be overinflated (Table S11 in Supplementary Material) and so was excluded from the normalization calculations.
The full-length transcripts were found to be the most highly expressed mRNA isoforms for both BRCA1 and BRCA2 when comparing the relative levels of all mRNA isoforms expressed for each gene, while they also had the greatest expression variability (Figure 2). No BRCA1 splicing events were expressed above 20% of the total number of detected transcripts, whereas the expression ranges of BRCA2 Δ9-10, Δ12, and ▾20 exceeded this level.  Despite a high mRNA expression variability detected for natural isoforms, results still highlighted notable isoform expression differences between variant carrier and control LCLs not measured by PCR-based methods in the ENIGMA multicenter study (15). The most significant difference was for Δ10 in LCL5 (BRCA1 c.[594−2A>C; 641A>G]), which expressed an 8.8-fold increase compared to controls. The isoforms Δ15 and ▾21 were also upregulated in this sample (2.8-and 5.0-fold increase, respectively). Expression differences were also found for ▾25 (4.5-fold increase) for LCL8 (BRCA2 c.8632+1G>A); ▾20 (2.3-fold increase) and ▾25 (2.4-fold increase) for LCL1 (BRCA2 c.426-12_426-_8delGTTTT); and ▾21 and Δ11 (1.3fold increase) for LCL4 (BRCA1 c.671−2A>G) (Figures 2B,D).
The effect of lcl storage and culture conditions on Brca1 and Brca2 mrna isoform expression A key observation from the ENIGMA multicentre study was the variability in BRCA1 and BRCA2 isoform detection between laboratories using different cell processing and assay protocols (15). However, it was unclear whether these inter-laboratory differences are due to untested aspects of the laboratory protocol.
To explore this possibility, we assessed the effect of cell culture and storage conditions on BRCA1 and BRCA2 isoform expression in RNA extracted at six time points from LCL7 with fortnightly freeze/thaw cycles. RNA was sequenced using targeted RNA-seq with technical triplicates (Figure 3). Detection of the more prominently expressed alternative splicing events (for example, BRCA1 Δ9-10 and Δ1Aq) was more consistent across time points and between technical replicates than it was for the minor events (Figures 3 and 4). Testing for significant isoform expression differences by time point using the linear model found no consistent effect for BRCA1 and BRCA2 in either NMD inhibitor treated or untreated samples ( Figures S6-S9 in Supplementary Material). Together these results suggest that expression variability does exist at the intra-laboratory level and this variability is greatest for mRNA  Table S1 in Supplementary Material) treated with an nonsense-mediated decay inhibitor. A freeze-thaw process was undertaken after time points two and four. Three technical replicates are listed under each time point. isoforms detected at low levels, such as the samples not treated with NMD inhibitors (Figures 3 and 4; Figures S10 and S11 in Supplementary Material). However, there was no evidence for systematic effects relating to the number of freeze/thaw storage cycles or whether the cells have been analyzed after 1 or 2 weeks growth.

DiscUssiOn
BRCA1 and BRCA2 mRNA splicing assays are often carried out in a diagnostic and research setting to assess the effects of variants of uncertain clinical significance. To date, PCR-based mRNA splicing assays have been the method of choice to assess mRNA transcripts qualitatively. While genetic variation has been suggested to induce abnormal isoform expression changes (23), such aberrations are not easy to detect using non-quantitative or semi-quantitative techniques. Here, we have utilized a targeted RNA-seq approach to qualitatively and quantitatively assess the expression profile of BRCA1 and BRCA2 mRNA isoforms in LCLs previously assayed in a multicentre study (15). This whole-gene approach is much more comprehensive than traditional PCRbased splicing assays, as evidenced by the detection of 55 mRNA isoforms (30 known and 25 novel) using only a small fraction of the samples used for the reported catalog of 80 BRCA1/2 isoforms (12,13). Several transcripts were not detectible due to limitations with the capture design and/or reduced sensitivity, however, lowly expressed BRCA1 and BRCA2 isoforms that have not been identified by previous studies were able to be detected using this platform (12,13). Additional splicing events may be present, but at very low levels, requiring a much higher sequencing depth for detection. Further work is required to determine if such transcripts are clinically important, and so establish whether their detection is required for an understanding of breast cancer risk. The targeted RNA-seq platform utilized in this work was able to overcome previously reported limitations of PCR-based assays (14,15) by providing multiple exon coverage across BRCA1 or BRCA2 for each assay, sequence confirmation of splicing event, and quantitative assessment of isoform expression patterns. Moreover, our study demonstrated that the use of NMD inhibitors is important for obtaining detectable levels of full length and alternative splicing events using the Illumina Truseq Targeted RNA Expression platform.
We show the utility of targeted RNA-seq to quantitatively identify previously reported upregulated splicing events, such as Δ10 (BRCA1 c.[594−2A>C; 641A>G]) and Δ11 (BRCA1 c.671−2A>G) (Figure 2), while the expression levels of many of the other isoforms in the variant carriers were within the range seen in controls. Our study also identified higher levels of Δ15 and ▾21 for LCL4 (BRCA1c.[594−2A>C; 641A>G]) than expected in controls, which have not previously been reported, likely because these small differences are not easily observable with semi-quantitative technologies. However, these changes are not associated with pathogenicity in BRCA1 c.[594−2A>C; 641A>G] carriers (9), and any association between the variant and each splicing event remains unclear. It is possible that future research with additional control samples may show that these small expression changes are within the natural expression range. Interestingly, the relative expression ranges observed for all alternative events appeared to be more tightly regulated in BRCA1 than BRCA2. While they do not appear to overlap any important domains, three BRCA2 mRNA isoforms were expressed at greater levels than those associated with BRCA1 (Figure 2). These differences suggest that greater variability in expression for some BRCA2 isoforms is tolerated in LCLs, however, further research is required to established BRCA1 and BRCA2 isoform expression patterns in cancer specific tissue, such as normal breast and ovarian epithelia.
Splicing data from this study, in addition to those from recent reports (10)(11)(12)(13), show that every exon in BRCA1 and 20/27 exons for BRCA2 are skipped in at least one natural isoform. This highlights how quantitative assessment of aberrant splicing would be very beneficial in these highly variable genes as it would provide a more comprehensive detection method of the splicing changes present. It also suggests that seven BRCA2 exons are likely to be highly conserved, so any changes involving these exons are likely to be detrimental in the cell.
To identify technical factors that also contribute BRCA1 and BRCA2 mRNA expression differences between samples, we assessed the effect of cell culture and storage conditions on BRCA1 and BRCA2 isoform expression across the six experimental time points. Our results showed variability in the isoforms expressed at any given time, irrespective of the number of storage events and culture time. Moreover, variability is greater when RNA-seq assays generate relatively low number of sequence reads (Figures 3  and 4; Table S12 in Supplementary Material).
Targeted RNA-seq platform utilize short fragmented library reads to detect mRNA splicing events. Such platforms are, therefore, limited in their ability to determine whether multiple events occur on the same transcript. Recently, we carried out a study using the MinION™ (Oxford Nanopore Technologies, Oxford, UK) long read sequencer and obtained whole transcript information for BRCA1 in a normal sample, which showed evidence of co-occurring splice events in BRCA1 (24). Further work involving long read sequencing of BRCA1 and BRCA2 would help to further distinguish transcript exon structure regarding all deletion and retention events for variant carriers. Results from such research will also accurately define out-of-frame transcripts which are prone to NMD.
Here, we utilize targeted RNA-seq technology to provide a comprehensive review and quantitative assessment of naturally occurring mRNA isoforms in BRCA1 and BRCA2. While qualitative analysis alone has been assumed to be sufficient for identifying aberrant events, the more quantitative RNA-seq offers improvements to the accuracy and capabilities of PCRbased assays, overcoming many of the detection limitations presented by the earlier technologies. These results lead us to make the following recommendations: (1) technical replicates (n > 2) of the variant carrier are necessary to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) NMD inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. While advances in probe design, and possibly the uptake of long read sequencers, are essential to allow detection of all expressed mRNA isoforms using this platform, the decreasing costs of RNA-seq technology, alongside an increasing understanding of bioinformatics capabilities, will likely increase the progression away from PCR-based assessment of gene expression, as evidenced by recent work by Davy et al. (10). In addition, the advanced capabilities of RNA-seq promise to aid in evaluating the clinical significance of variants in BRCA1 and BRCA2, but further exploration is required to determine whether these variants are influencing expression. Quantitative assessment of BRCA1/2 isoforms in a greater number of control samples using other RNA-seq platforms will further improve our understanding of "normal" expression and provide an invaluable reference for establishing the occurrence of aberrant splicing when assessing genetic variants. Furthermore, careful assay design will be crucial for obtaining data across the gene, thus enabling an interpretation of splicing changes for the entire transcript as opposed to selected regions.

aUThOr cOnTriBUTiOns
Conception or design of the work-VL, LW, JP, BR, MC, and AS. Resources-kI and LW. Data collection and drafting the article-VL and LW. Data analysis and interpretation-VL, JP, and LW. Critical revision of the article and final approval of the version to be published-all authors.

acKnOWleDgMenTs
We thank all members of the ENIGMA consortium Splicing Working Group for the useful suggestions relating to the study execution and interpretation. We would also like to thank


Assessment the Influence and Efficacy of Some Biocides on Rice Plants

Rice is an important crop in southern part of Odisha. About 70 % people depend on cultivation of rice for their livelihood. To maximize rice yields, sustainable management of the crop is needed to control the main pests that limit rice growth and development. It is necessary to use biocides and pesticides. Neem oil act as biocides due to its active ingredient azadirachtin substances with pesticidal properties are found in all parts of the neem tree. Azadirachtin consists of more than 25 different but closely related compounds act as pesticides which are currently available. Azadirachtin can act as a feeding deterrent against a number of insect pests including beetles. It reduces the level of the insect hormone Ecdysome by disrupting the insect's molting process so that the immature larvae cannot develop into adults.

Introduction
Rice is an important crop in southern part of Odisha. About 70 % people depend on cultivation of rice for their livelihood. To maximize rice yields, sustainable management of the crop is needed to control the main pests that limit rice growth and development. It is necessary to use biocides and pesticides. Neem oil act as biocides due to its active ingredient azadirachtin substances with pesticidal properties are found in all parts of the neem tree. Azadirachtin consists of more than 25 different but closely related compounds act as pesticides which are currently available. Azadirachtin can act as a feeding deterrent against a number of insect pests including beetles. It reduces the level of the insect hormone Ecdysome by disrupting the insect's molting process so that the immature larvae cannot develop into adults.
After treatment with neem-based pesticides insects are either die or crippled with distorted wings or the immature larvae and nymphs remain in an immature stage and then die. In tropical countries rice has been identified as one of the crops that are particularly susceptible to the negative impacts of pesticide use. In addition to the physicochemical nature of the pesticide, tropical climatic conditions and agricultural practices play important roles in determining the fate and distribution of pesticides in the tropical paddy field ecosystem. The assessment of toxicological effects of pesticides in the tropical paddy has been evaluated through seed germination percentage, vegetative parameters and chlorophyll content. The seedlings are the crucial stage of the plant, so, the experimental study evaluated 14 days old seedling's vegetative growth parameters and chlorophyll content and percentage of seed germination influenced by different biocides (neem oil, carbosulfan and Oxadiargyl). The experimental results found that all concentrations of (0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 ml/l) neem oil, carbosulfan and oxadiargyl decreased the percentage of seed germination, vegetative parameters and chlorophyll content. Out of these three biocides, neem oil was found less toxic towards the test cultivar. Effective training of the paddy farmer has been identified as the key to successful implementation of integrated pest management strategies for more productivity. sexual communication may be disrupted which results in reduced fecundity (Schmutter, 1990). Neem oil works in a number of different ways. The oil forms a coating on the insect's body, blocking the breathing openings and suffocating the insect. It also has a repellent effect on certain insects and mites. Neem oil prevents the germination and penetration of some fungal spores. One percent neem oil treatment was effective in managing powdery mildew on hydrangeas, lilacs and phlox.
Carbosulfan shows a good agronomic efficiency, this insecticide has environment restrictions due to the risks of contamination. One problem frequently associated to the carbofuran is the death of forest animais if they contaminated with it. Dietrich et al., (1995) found similar problems for the carbofuran granules applied in field crops in Swiss. According to Crepeau and Kuivila (2000), the continuous presence of carbofuran in water channels and rivers might cause severe, sub-lethal or chronic damages to aquatic organisms. Gupta (1994) observed that carbofuran is toxic to micro crustaceous and vertebrates of a same food chain. Norberg-King et al., (1991) affirm that carbofuran is a toxic insecticide to the aquatic wildlife. The use of carbofuran in agriculture is prohibited in some countries (Anonymous, 1996a;Anonymous, 1996b;James, 1995) due to its eco-toxicity. The carbofuran persistence in the paddy water and in soil solution has been studied in tablelands and channels, because carbofuran is degraded mainly by hydrolysis (Seiber et al., 1978;Tejada andMagallona, 1985 andTrevizan et al., 2002). The degradation process of carbofuran (2, 3dihydro-2, 2-dimethyl-7-benzofuranol methylcarbamate) molecule (represented as RX) occurs when it reacts with the water molecule originating a new C-N bond and breaking a C-X bond in the original molecule. The net reaction is essentially a direct displacement of X by OH (Seiber et al., 1978 andMabury andCrosby, 1996). The volatilization, photolysis and oxidation are important factors in the process of carbofuran dissipation (Seiber et al., 1978;Deuel et al., 1979). Carbosulfan (2, 3-dihydro-2, 2dimethyl-7-benzofuranyl (di-nbutylaminosulfenyl) (methylcarbamate) as substituted carbofuran as insecticide, because of environmental reasons and of its lower persistence. According to Marsden et al., (1982) and Umetsu and Fukuto (1982), carbosulfan presents lower toxicity for the aquatic wildlife than carbofuran. Tejada and Magallona (1985) demonstrated that carbosulfan was not detected in samples of paddy water and soil solution collected few hours after its application. Hydrolysis is the main path of carbosulfan to carbofuran degradation, its first metabolite, but in some cases, th is latter might present longer persistence in a low pH environment (Ramanand et al., 1991). Other environmental factors, such as temperature variation, might directly influence the carbosulfan degradation process (Sahoo et al., 1990).
Oxadiargyl acts as protoporphyrinogen IX oxidase inhibitor. It is pre-emergence and early post-emergence herbicide active on broad-leaved weeds (Amaranthus, Bidens, Chenopodium, Malva, Monochoria, Polygonum, Portulaca, Potamogeton, Raphanus, Solanum, Sonchus, Rotala), grasses (Echinochloa, Leptochloa, Brachiaria, Cenchrus, Digitaria, Eleusine, Panicum and wild rice) and annual sedges, in rice, upland crops (sunflower, potato, vegetables and sugar cane) and perennial crops (fruit trees and citrus). Plants Studies on lemons, sunflowers and rice demonstrated very low levels of residues at harvest, mainly parent compound (Leake et al., 1998). Soil/Environment DT50 (lab., aerobic) 18-72 d (20-30 °C), forming two major metabolites (one of which is herbicidal) which are, in turn, steadily degraded, resulting in mineralisation to CO 2 and a soil-bound residue. Oxadiargyl dissipates rapidly from water into the sediment phase and is readily degraded under anaerobic conditions. Strongly adsorbed to soil (Koc 1000-3000); oxadiargyl and its two major soil metabolites show low mobility in 4 soil types and are unlikely to leach. Field results were consistent: DT50 9-25 d, mean DT90 90 d; for oxadiargyl and its two major metabolites, DT50 was 9-31 d, DT90 65-234 d; >95% of oxadiargyl residues remained in the top 10 cm of soil, and no residues were found below 30 cm. Oxadiargyl is a potential herbicide in crops such as lettuce and capsicums (Dickmann et al., 1997).
Investigation of these pesticides is of the utmost importance for a better understanding of their dynamics in the environment. The literature available the effect of neem oil, carbosulfan and Oxadiargyl on rice crop is very scanty hence the aim of this study was undertaken to evaluate the impacts of biocides on seed germination, seedling vigor and chlorophyll contents of 14 day's old seedlings.

Materials and Methods
Pure line seeds of above mentioned cultivars of rice were procured from Regional Agricultural Research Station, Odisha University of Agriculture and Technology (O.U.A.T), Brahmapur. Bio-pesticides i.e. neem oil, Carbosulfar 25%E.C. (Marshal) and Topstar (Oxadiargy1 80% w.p.) were used in this present piece of investigation.

Preparation and mode of application of test chemicals
The common recommended doses, of different concentrations of the test chemicals were prepared by using distilled water as solvent. In this piece of investigation, the preliminary concentrations 0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 ml/l or gm/kg of all the bio-pesticides i.e. Neem oil, carbosulfan 25% E.C. (Marshal) and Topstar (oxadiargy1 80% W.P.) were prepared.

Experimental schedule
Seed germination and seedling growth experiments of test plant were conducted in the laboratory condition and study the vegetative growth parameters and chlorophyll contents.

Surface Sterilization
All the glass wares including seeds have been properly sterilized. Prior to germination, all the seeds were surface sterilized by treating with 5% concentration of sodium hypochloride solution for 12 minutes and then thoroughly washing it with distilled water for 3 to 4 times to remove completely the sodium hypo-chloride solution. Glass materials, absorbent cotton, filter papers and vermiculites were properly sterilized and used for germination of test plant rice seeds.
Control set of petridishes were prepared by taking same amount of water. Watering was allowed in all petridishes at an interval of 12 hr with 10 ml of water.

Germination chamber
Specially designed germination chamber was meant to study the vigoursity of germination of test seeds under different pesticides i.e. treatment with scheduled concentration of test pesticides. Light is an inhibitory factor for the process of the germination, hence precaution were taken to make the germination chamber light proof throughout the course of investigation.
The duration of germination experiment limited to 72 hr. for first observation of germination was recorded after 12 hr. treatment and subsequently at an interval 12 hr. duration the germination strategy of scheduled i.e. 72 hr. Emergence of radical through the coleoptiles extending the length of 0.1 cm was considered as the standard of germination.

Seedling Chamber
A special type of chamber has been designed for the growth of germination seed designated as seedling chamber. The chamber is partitioned by wooden plates to keep the petridishes. Besides maintaining of thermostability, Photo-stability has been allowed with 2000 ± 200 lux of fluorescent light throughout the course of seedling growth. Temperature constancy maintained at 25 0 C during the course of seedling growth investigations.
Petridishes with germination seeds were transferred to the seedling chamber. Growth of radical and plumule of the germinating seeds were allowed for seven days.
Throughout the period of seedling growth aeration under the laboratory conditions was kept intact. Random collection of days old seedlings was made from the seedling chamber for the different vegetative parameters and chlorophyll content.
The collected seedlings were washed properly with water and separated root and shoot portion. The separated shoot and root portions were soaked with blotting paper and measured length and fresh weight. The shoot and root were dried in a woven with 60 o centigrade and measured dry weight.

Extraction and estimation of chlorophyll
In order to measure the chlorophyll content in leaves, ten seedlings of 14 days old were taken randomly from each petridish of both control and treated a sets, leaves were separated from seedlings, washed thoroughly, dried the surface water by soaking on a blotting paper, cut in to small pieces and weighted for 50 mg. then this weighted samples were homogenized with 1-2 ml. of 80% ethanol(v/v) with mortar and pestle and the homogenate were centrifuged at 3000 xg for 20 minutes at 28± 10c.
The pellets were again homogenized with 80% ethanol and centrifugation was repeated till colorless pellets obtained which were further used for other chemical analysis. All the supernatants were pooled together and a total volume of 10ml. was prepared with 80% ethanol. The optical density (O.D) of the extract was measured with help of Elico digital spectrophotometer (model CL-27) at 663 and 645 nm, taking 80% ethanol as blank. Estimations were calculated following the formulae suggested by Arnon (1949).
= v mg/g (fresh weight) Where, V= Total volume of extract in C.C W= Weight of samples in gram And O.D. = Optical density of the extract

Effect of neem oil
Treatment of Paddy seeds was done in the germination bed containing 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0ml./lit and controlled set. The time of exposure for germination of seeds was allowed for 24h, 36h, 48h, 60h and 72h. Rate of germination of rice seeds with respect to time of treatment in different concentration of neem oil and controlled condition was studied. After an interval of 24h the first observation were made. The highest rate of germination percentage was found in the controlled condition comparing with test chemicals. The rate of germination percentage in rice seeds progressively declined with the increasing concentration of neem oil. Thus the percentage of germination is considered to be inversely proportional to the concentration of neem oil treatment. Under controlled condition percentage of germination was found to be 27.2% subsequently 25.27, 23.2%, 20.3%, 17.9%, 16%, 14.2%, 12.3%, 10.3%, 7.6% and 3.4% of seed germination were observed with the treatment of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0ml./lit concentration of neem oil respectively after 24 h incubation. Percentage of seeds germination after 72h were 97.4%in control and 87.8%, 78.2%, 63.2%, 58.5%, 44.8%, 36.4%, 32%, 22.9%, 16.4% and 8.2% in 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0ml./lit concentration of neem oil respectively. Other concentration exhibited intermediate value ( Table 1).
The correlation coefficient values were calculating between the concentration of neem oil and percentage of germination of the different duration of observation. Significance values (p) were also calculated taking degree of freedom (df) for the different hours of treatment. The r values were after 24h. r = -0.974 and the value of p≤0.001, after 36h the value of r= -0.967 and p≤0.001, after 48h. r = -0.962 and p≤0.001, after 60h r = -0.985 and p≤0.001 and after 72h. r = -0.954 and p≤0.001 (Table 1).
(Marshal). Thus the percentage of germination is considered to be inversely proportional to the concentration of Carbosulfan 25% E.C.
The correlation coefficient values were calculating between the concentration of Carbosulfan 25% E.C. (Marshal) and percentage of germination of the different duration of observation. Significance values (p) were also calculated taking degree of freedom (df) for the different hours of treatment. Thus after 24h. r= -0.985 and the value of p≤0.001, after 36h the value of r=-0.978 and p≤0.001, after 48h. r=-0.972 and p≤0.001, after 60h r= -0.969 and p≤0.001 and after 72h. r=-0.959 and p≤ 0.001.
The correlation coefficient values were calculating between the concentration of Topstar (Oxadiargyl 80% W.P.) and percentage of germination of the different duration of observation. Significance values (p) were also calculated taking degree of freedom (df) for the different hours of treatment. Thus after 24h. r= -0.988 and the value of p≤0.001, after 36h the value of r=-0.982 and p≤0.001, after 48h. r=-0.975 and p≤0.001, after 60h r= -0.970 and p≤0.001 and after 72h. r=-0.962 and p ≤0.001.

Effect of neem oil
All the concentration of neem oil considerably checked the root length of rice seedling. The seedling of control set exhibited maximum length of root 4.1 ± 0.02 cm at 14 days. The maximum root length of rice seedling exhibited the length of 3.8 ± 0.01cm with treatment of 0.5 ml./lit. concentration at 14 days however minimum root growths 1.2 ± 0.01 cm in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of neem oil. Statistical methods for mean length of root growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.975 at 14 days old rice seedling and at p≤ 0.001 obtained with d.f. 20. Shoot length exhibited more or less same trean as it found in root length and their values The seedling of control set exhibited maximum length of root 8.4 ± 0.04 cm at 14 days however the shoot length of rice seedling exhibited the length of 7.9 ± 0.03cm and 2.7 ± 0.01 cm in 14 days old rice seedlings treated with 0.5 and 5.0 ml./lit. concentration of neem oil respectively. Statistical methods for mean length of shoot growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.981 at 14 days old rice seedling. However, p≤ 0.001 obtained with d.f. 20 (Table 4).

Effect of carbosulfan 25% E.C. (Marshal)
Carbosulfan 25% E.C. (Marshal) checked the root length of rice seedling at all the concentration ranging from 0.5 to 5.0 ml/l. The seedling of control set exhibited maximum length of root 4.1 ± 0.02 cm whereas minimum root growths 0.6 ± 0.01 cm in 14 days old rice seedlings treated with 5 ml./lit. concentration of carbosulfan 25% E.C. (Marshal).Statistical methods for mean length of root growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.958 at 14 days old rice seedling and at p≤ 0.001 obtained with d.f. 20. The shoot length of seedling of control set exhibited maximum length of shoot 8.4 ± 0.04 cm at 14 days whereas the shoot length of rice seedling exhibited the length of 7.1 ± 0.03cm and 8.5 ± 0.01 cm in 14 days old rice seedlings with treatment of 0.5 Ml./lit. and 5 ml./lit. concentration of carbosulfan 25% E.C. (Marshal) respectively. Statistical methods for mean length of shoot growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.961 at 14 days old rice seedling at p≤ 0.001 obtained with d.f. 20 (Table 5).

Effect of Topstar (oxadiargyl 80% W.P.)
All the concentration of Topstar (oxadiargyl 80% W.P.) considerably checked the root length of rice seedling. The seedling of control set exhibited maximum length of root 4.1 ± 0.02 cm at 14 days however at same period of incubation the root length of rice seedling exhibited the length of 2.9 ± 0.01cm with treatment of 0.5 ml./lit. and 0.6 ± 0.01 cm in treatment with 5 ml./lit. concentration of Topstar (oxadiargyl 80% W.P.). Statistical methods for mean length of root growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.957 at 14 days old rice seedling at p≤ 0.001 obtained with d.f. 20. The effect of Topstar (oxadiargyl 80% W.P.) on shoot length exhibited similar trend as it found in Carbosulfan 25% E.C. The seedling of control set exhibited maximum length of shoot 8.4 ± 0.04 cm at 14 days however the shoot length of rice seedling exhibited the length of 6.9 ± 0.03cm and 1.3 ± 0.01 cm in 14 days old rice seedlings treated with 0.5 Ml./lit. and 5 ml./lit. concentration of Topstar (oxadiargyl 80% W.P.) respectively. Statistical methods for mean length of shoot growth ± standard deviation adopted taking 100 random samples in each case, value of r found -0.966 at 14 days old rice seedling at p≤ 0.001 obtained with d.f. 20 (Table 6).

Effect of neem oil
All the concentration of neem oil considerably checked the shoot fresh weight of rice seedling. The seedling of control set exhibited maximum fresh weight 13.8 ± 0.12 mg at 14 days however the shoot fresh weight of rice seedling exhibited the length of 12.0 ± 0.11mg and 4.7 ± 0.02 mg in 14 days old rice seedlings treated with 0.5 ml./lit. and 5 ml/lit. concentration of neem oil respectively. The seedling of control set exhibited maximum shoot dry weight 4.1 ± 0.04 mg at 14 days whereas the shoot dry weight of rice seedling exhibited the length of 3.3 ± 0.06mg and 1.4 ± 0.02 mg in 14 days old rice seedlings treated with 0.5 ml./lit. and 5 ml./lit concentrations of neem oil respectively.
All the concentration of neem oil considerably checked the root fresh weight of rice seedling. The seedling of control set exhibited maximum root fresh weight 7.2 ± 0.06 mg at 14 days. However the root fresh weight of rice seedling exhibited 6.7 ± 0.05 mg and 3.1 ± 0.02 mg in 14 days old rice seedlings treated with 0.5 ml./lit. and 5 ml/lit. concentration of neem oil respectively. The seedling of control set exhibited maximum root dry weight 2.7 ± 0.05 mg at 14 days. However the root dry weight of rice seedling exhibited the length of 2.1 ± 0.04 mg with treatment of 0.5 Ml./lit. concentration of neem oil at 14 days. Similarly root dry weight 0.8 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 Ml./lit. concentration of neem oil (Table 4).

Effect of Carbosulfan 25% E.C. (Marshal)
All the concentration of carbosulfan 25% E.C. (Marshal) considerably checked the shoot fresh weight of rice seedling. The seedling of control set exhibited maximum fresh weight 13.8 ± 0.12 mg at 14 days. However the shoot fresh weight of rice seedling exhibited the length of 9.5± 0.08 mg with treatment of 0. Carbosulfan 25% E.C. (Marshal) considerably checked the root fresh weight of rice seedling at all treated concentration. The seedling of control set exhibited maximum root fresh weight 7.2 ± 0.06 mg at 14 days. However the root fresh weight of rice seedling exhibited the length of 5.8 ± 0.04 mg with treatment of 0.5 ml./lit. concentration of carbosulfan 25% E.C.(Marshal) at 14 days. The root fresh weight 2.3 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of carbosulfan 25% E.C.(Marshal). The seedling of control set exhibited maximum root dry weight 2.7 ± 0.05 mg at 14 days. However the root dry weight of rice seedling exhibited 1.6 ± 0.03 mg with treatment of 0.5 ml./lit. concentration of carbosulfan 25% E.C.(Marshal) at 14 days. The root dry weight 0.2 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of carbosulfan 25% E.C.(Marshal) ( Table 5).

Effect of Topstar (Oxdiargyl 80% W.P.)
All the concentration of Topstar (Oxadiargyl 80% W.P.) considerably checked the shoot fresh weight of rice seedling. The seedling of control set exhibited maximum fresh weight 13.8 ± 0.12 mg at 14 days. However the shoot fresh weight of rice seedling exhibited the length of 9.3± 0.08 mg with treatment of 0.5 Ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) at 14 days. The shoot fresh weight 3.0 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 Ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.).
The seedling of control set exhibited maximum dry weight 4.1 ± 0.04 mg at 14 days. However the shoot dry weight of rice seedling exhibited the length of 2.6± 0.05 mg with treatment of 0.5 ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) at 14 days. The shoot dry weight 0.6 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 Ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) Different concentrations of Topstar (Oxadiargyl 80% W.P.) considerably checked the root fresh weight of rice seedling. The seedling of control set exhibited maximum root fresh weight 7.2 ± 0.06 mg at 14 days. However the root fresh weight of rice seedling exhibited the length of 5.6 ± 0.04 mg with treatment of 0.5 ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) at 14 days. The root fresh weight 2.2 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) The seedling of control set exhibited maximum root dry weight 2.7 ± 0.05 mg at 14 days. However the root dry weight of rice seedling exhibited the length of 1.4 ± 0.02 mg with treatment of 0.5 ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) at 14 days. The root dry weight 0.25 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of Topstar (Oxadiargyl 80% W.P.) ( Table 6).

Effect of neem oil
The different concentrations of neem oil considerably checked the chlorophyll contents of rice seedling. The seedling of control set exhibited maximum chlorophyll -a contents 3.04 ± 0.06 mg at 14 days. However the chlorophyll -a contents of rice seedling exhibited 2.68 ± 0.06mg with treatment of 0.5 ml./lit. concentration of neem oil at 14 days. The chlorophyll -a contents 0.89 ± 0.02 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of neem oil.
The seedling of control set exhibited maximum chlorophyll -b contents 0.89 ± 0.02 mg at 14 days. However the chlorophyll -b contents of rice seedling exhibited the length of 0.83 ± 0.02mg with treatment of 0.5 ml./lit. concentration of neem oil at 14 days. The chlorophyll-b contents 0.37 ± 0.01 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of neem oil.
The seedling of control set exhibited maximum total chlorophyll contents 3.93 ± 0.09 mg at 14 days. However the total chlorophyll contents of rice seedling exhibited the length of 3.51 ± 0.08mg with treatment of 0.5 ml./lit. concentration of neem oil at 14 days. Total chlorophyll contents 1.26 ± 0.03 mg in 14 days old rice seedlings were found in treatment with 5 ml./lit. concentration of neem oil (Table 4).

Effect of Carbosulfan 25% E.C. (Marshal)
All the concentration of Carbosulfan 25% E.C. (Marshal) considerably checked the chlorophyll contents of rice seedlings. The seedling of control set exhibited maximum chlorophyll -a contents 3.04 ± 0.06 mg at 14 days. However the chlorophyll -a contents of rice seedling exhibited the length of 2.46 ± 0.05mg with treatment of 0.5 Ml./lit. concentration of Carbosulfan 25% E.C. The seedling of control set exhibited maximum total chlorophyll contents 3.93 ± 0.09 mg at 14 days. However the total chlorophyll contents of rice seedling exhibited 3.14 ± 0.07mg with treatment of 0.5 Ml./lit. concentration of Carbosulfan 25% E.C. (Marshal) at 14 days.

Seed germination
Generally all seed plants come across three important stages during their lifetime such (a) germination of embryo from seed (b) seedling stage and vegetative growth and (c) reproductive stage. During above three developmental stages various internal and external environmental factors affect the number of physiological biochemical and other vital functions that control the growth and development of concerned plants. During the process of germination activation and morphogenesis of dormant embryo take place, which are controlled by several internal and external conditions. According to Evenari (1961), germination is "sum of all the physiological processes occur inside the seed that start with the inhibition of water and ends with the protrusion of embryonic root in dicot and coleorhiza in monocots". Saxena (1979) has reviewed on germination growth and yield of many crop seeds in relation to seed soaking (presoaking and presowing treatments). Muralikrisna and Saxena (1991) reported that time taken from seed soaking or presoaking to germination or seedling establishment vary with different crops and nature of internal and external conditions. Delayed emergence of redicle of coleorhizae may be due to deterioration of seedbed conditions such as water logging or scarcity, physico-chemical nature of soil i.e. temperature, pH, and chemical composition of soil, attacked by mircorganisms, soil borne pests and other damages (Singh and Saxena, (1991). Bell (1993) and Vanhezewijk et al., (1993) reported that the process of germination hold better under appropriate and favourable environmental condition which include water, air, temperature, free from high salt concentrations an inhibitors and sometimes the spectral quality of light. The most important events of germination are enhanced by which the seeds imbibe till optimum level of water content is attained, hence any alternation or adulteration in water content affects the germination process. It is also reported that osmotic potential helps in making all the preparation in germination (Bhumbia et al., 1968;Singh, 1987 andSaxena, 1988) reported that high osmotie pressure caused due to high salt concentration the soil water cause the suppression of seed germination.
Reported are available on the correlation coefficient between germination and emergence of embryonic root or colcohiza (Chen et al., 1968;Heydeker, 1972 andLatito andQuagliotti, 1993) reported that following inhibition germination is an enzymatically activated process, mediated through on RNA.
The control of enzyme production should be at the time of DNA transcription into m-RNA. The hormones perform a vital role in regulating the enzymatic activation during germination as determined by nucleic acid.
The role endogenous growth regulators during the seed germination can't be ruled out. The reserved starch present in endosperm is simplified by the action of various amylase enzymes. Hess (1975), suggested that αamylase in the endosperm is secreted by aleurone layer. Paleg (1960a and1964) reported that application of GA 3 stimulates the development of α-amylase activity resulting in acceleration of seed germination in Barley. Paleg (1960a and1964), Chrispeels and Jones (1980) reported that GA 3 may act by affecting the substances causing mobilization of stored material localized in the storage tissue.
GA 3 also increases the percent of polyribosomes resulting more protein synthesis that are utilized for morphogenesis (Frankland et al., 1971). Like GA 3, IAA also influenced the germination process in various crop seeds. (Pandey and Sinha, 1988;Robertson et al., 1976, Acharya, 1994.The present experimental findings marked that Neem-oil, marshal (Carbosulfan 25% E.C). Top star (oxadiargyl 80% w.p.) considerably altered the process of germination in experimental rice cultivar.
Delay or inhibit the process of germination caused by different concentration of Topstar in test cultivar might have checked the amylase, proteolase and lipase activities or activation of hydrolytic enzymes in seeds such as luithirase, hyaleuronidase those are assist the breakdown of embryonic tissue.
Multi neem oil being a neem based pesticide, contains vast array of biological active chemicals such as protolimonoids, limonoids and non-isoprenoids, which act on crop pests by its larvicidial activities. They regulate different growth and development in insects and act effectively against many fungal diseases like smut, rust, powdery mild few and wilt (Singh, 1994). At molecular level both plant and animals are more or less similar and carryout same type of chemical and physiological activities during their growth and development and neem oil is hazardous to insects and pests, there is no guarantee that it might have not affecting on plant systems. Based on results obtained by the application of different concentrations of neem oil the possible mechanism of inhibitory affect on seed germination of test crop plant is discussed below.
Considerably checked of seed germination by all concentrations of neem oil/ranging from 0.5-5 ml/l suggested limonoids might have checked or inhibited various physiological and biochemical processes associated with seed germination. Joshi and Prakash (1992) reported that leaf litter leachate of Melia azardichate exhibited inhibitory effects on germination of wheat, maize and pea. Similar reports suggested by Melkania (1984) on seed germination of turnip, barnyuard millet, finger millet, dock wheat and soyabean. Hence, the active ingredients present in neem-oil especially limonoids might have significantly inhibited the seed germination of three test crops. Since there are no much reports are available on the mode of actions on physiological and biochemical processes of neem-oil further in depth studies in this line are needed.

Seedling growth and change in chlorophyll content
Seedling establishment is considered as one of the crucial stage, after germination, where various morphogenesis activities are established along with changes in physiological and biological processes. During this period the seedlings try to become self-established by the development of root and shoot systems. The growth and development of shoot and root are controlled by various metabolic activities carried out in leaves, hence changes in shoot and root length of seedlings and their fresh and dry weights and changes in total chlorophyll contents are considered important parameters of seedling establishment.
All the concentration of neem-oil, marshal (carboslfan 25% E.C) and Top star (Oxadiargyl 80% W.P.) considerably reduced the seedling growth due to interaction of phytotoxins with photophosphorylation pathway and inhibition of light activated mg +2 ATPase activities (Moreland, 1980). Though the carbohydrate synthesis mainly depends on the efficacy of photosynthetic processes, the accumulation of carbohydrate in plants is directly proportional to the amount of chlorophyll present in the leaves.
The decrease in various parameters of seedling growth in the test crop is might be due to controlling of enzyme activities responsible for synthesis of plant growth regulators like GA 3 and IAA that control shoot and root growth. Butler (1973) and Mishra and Adhikary (1986) reported that plant growth regulators, at lower concentrations induce synthesis of macromolecules resulting better seedling growth whereas higher concentration act as inhibitors.
Abdl Baki and Anderson (1972) suggested that many physiological and bio-chemical processes such as respiration fatty acid synthesis, protein synthesis, oxido-reduction reaction and several enzyme activities are associated with seedling vigor.
Vegetative growth of the plant body also depends on different environmental factors such as change in temperature, humidity, photoperiod and application of different agrochemicals (Bremner, 1969 (a &b) andCannel, 1969). It is quite common the most visible tillers die without producing grains and there may be many buds which never reach the visible stage (Davidson, 1964), Vergara et al., (1966) opinion that plant height is prime importance while working on the crop plants. Cleland (1964) suggested that gibberellins play important role on the growth of plants. Jones and Phillips (1966) suggested that in many plants highest level of gibberellins occur in apices of stem and root, young leaves, embryos and endosperm of developing seeds.
The application of different synthetic pesticides, herbicide and other biocides is a common and modern agricultural practice by the farmers for better yields, which more or less selectively interfere with growth of plant species. Further these chemical have a high toxicity on wide range of plants while others are very resistance to degradation in the environment and accumulate in the plants creating problems faster than they can solved. Moore (1965) suggested that all the biocides are not hazardous but most of them are toxic. A vast reports are available on effect of biocides several crop plants such as soya bean (Hsia and Cao, 1978), ragi Khan, 1982 andAcharya, 1994). Further due to advancement of biotechnologies, pesticides of plant origin (biological pesticide or biopesticides active) are developed and recorded for use. Neem oil is one of such biological derived pesticide having pesticidal active indegredients "Limonoids" which include azadiractuin, nimbocinal, epinimbocinal, nimbin, solamin and meliontriol. There are limonoid compound contain complex of active ingredients which act on crop pests by its larvicidal activities, disrupts, the growth process of insects, repellant or antifeedant to insect and act afectively against many fungal diseases like smut, rust, powdery mildew etc because of the above features neem oil is approved by the integrated pest management (IPM). As insects and pathogens die by the application of biocides, there is no guarantee that crop plants affected. These days may be significant in first generation but certainly these are physiologically are genetically hazardous as the plants and animals are more or less similar at molecular or sub-molecular level. Based on the results obtained by the application of different concentration of neem oil seedling growth of test cultivar the discussion are described below.
The seedling length, fresh and dry weight and chlorophyll content of the seedling of rice were significantly reduced by all concentrations of neem oil, carbosulfan and oxadiargyl which might be due to inhibiting and/or checking of protein, nucleic acids and carbohydrate synthesis. Such type of results have been reported by Kohli et al., (1988)on green gram, Padhy et al., (1992) on sesame, Jayakumar et al., (1990), Panda(1994) on ground nut by the eucalyptus globulus leaf leachate which contain allelochemics like phenolics, terpenoid and flavonoid which are more or less similar in structure and function of neem oil and other biocides. Further, it was reported that influence of biopesticide in relation to cell multiplication to auxin production in cells, influence of auxins on translocation of carbohydrates from source to sink are needed for better understanding on physiological and biochemical changes that occurs during seedling growth. Such type of related allelochemics present in neem oil might have suppressed various metabolic activities resulting decrease in seedling growth.
The decreased in height of plant suggests that the metabolites might have synthesized less and could not properly trans-located from source to sink. Murty et al., (1973) suggested that leaf thickness or specific leaf weight has significance correlation with photosynthetic rate per unit area in rice. Yoshida and Ahn (1968) observed that carbohydrate content of grain is largely depends on the amount of photosynthates formed after flowering.
The lower concentration of biocides caused higher rate of seed germination, but higher concentration exhibited negative correlation with germination process. The parameters of seedling growth (length, fresh an dry weight of both shoot and root and chlorophyll content of leaves, were significantly inhibited. Among different pesticides neem oil was found less toxic followed by carbosulfan and Oxadiargyl. Hence, effective training should be imparted to the paddy farmer on successful implementation of integrated pest management strategies for more productivity of crop and sustainable agriculture.