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Summer Research Fellowship Programme of India's Science Academies

Segregation of Root Architecture traits in a Recombinant Inbred Lines of Ragi (Eleusine coracana Gaertn.) and QTL discovery using GBS data

S. Suraaj

Agricultural College and Research Institute, TNAU, Coimbatore 641003

Dr. M. S. Sheshshayee

Department of Crop Physiology, University of Agricultural Sciences, Bengaluru 560065

Abstract

Ragi is an important millet grown and consumed predominantly in the arid and semiarid regions of the world. Since major area of ragi cultivation is under rainfed conditions its yield is considerably low, increasing its production by improving drought adaptive trait such as root is important. Comprehending the root growth pattern has always been a challenging task to perform due to the geotropic and photosensitive nature of the roots. Architecture of roots is crucial for water mining and uptake of minerals and nutrients which aids the crop to sustain growth in water limiting environments. Therefore, our current research is focused on development of high throughput phenotyping methods for measuring root traits. A simple strategy of growing plants in culture bottles filled with a transparent gel, Clerigel, has been standardized at the center. This system provides an excellent option to capture the progressive changes in root architecture such as length and number of primary and secondary branches through imaging. A convenient algorithm to compute these root raits and their growth rates has been developed at the host institute. A mapping population comprising of 226 Recombinant inbred lines developed by crossing GE208 (high root) and GE156 (low root) is being used for determining the segregation pattern of root traits. The parental lines are presently being examined for root growth rates. These RILs have already been characterized for molecular diversity through Genotyping by sequencing approach (GBS). Root growth pattern and the segregation of these patterns among RILs will be used for identifying QTL associated with root traits in ragi.

Keywords: RIL, root growth pattern, root phenotyping, GE208, GE156, root traits

INTRODUCTION

Finger Millet [ Eleusine coracana Gaertn.] is an annual cereal grown in arid and semiarid areas in Africa and Asia. It belongs to PACMAD clade (for Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae) and is a Chloridoideae which is the only millet belonging to this sub family and undergoes C4 metabolism as that of pearl millet, sorghum and maize. It is an allotetraploid (2n=4x=36, AABB) with Eleusine coracana subsp.africana as its wild progenitor and it has been confirmed that Eleusine coracana subsp.coracana has evolved from the cross between Eleusine indica (AA Genome donor) and Eleusine floccifolia (probable BB Genome donor). It is a free tillering, robust and grows up to 1.7m tall with slender, erect and compressed stem. Leaf blades are linear to lanceolate, Inflorescence are digitate and those branches are slender. The digitately arranged branches may spread out and may turn reflexed, erect and incurved forming a fist like structure. Finger millet is well known for its rich nutritional composition viz., calcium (0.34%), dietary fibre (18%), phytates (0.48%), protein (6%–13%) minerals (2.5%–3.5%), phenolics (0.3%–3%) and it is also a rich source of thiamine, riboflavin, iron, methionine, isoleucine, leucine, phenylalanine and other essential amino acids.

Finger millet is well known drought adaptable and temperature tolerant cereal species. Constitutive traits such as root length, root diameter, number of laterals, number of secondary roots, root biomass determines the root architecture of the plant which is correlated with the drought tolerance of the plant. It is mandatory to understand the root traits and their association with crop productivity under several conditions.

Root system architecture (RSA) refers to the shape and spatial arrangement of a root system in the soil. Root Architecture is based on the growth rate, root angle, root length and the number of laterals and secondary roots formed and it is mandatory for plant anchorage and efficient uptake of water and nutrients from the soil. Even though the shoot drives uptake of water throughout the plant, root system architecture determines the access to water and thereby limits the functioning of shoots. Root architecture helps to comprehend the root functional traits and relation of these traits to that of whole plant strategies which may increase the crop productivity under drought conditions. Some of the root traits associated with drought includes small fine root diameters, long specific root length, root density at different depths of the soil. In order to have a better acquisition of water from different layers of soil deep roots and large xylem diameters are essential.

Studying the root system architecture in the field condition is challenging and entails the intervention of modern imaging tools. Imaging processing tools are advantageous over traditional methods in non-invasive capturing of various plant root characteristics like root diameter, length of roots, root volume to root surface area. Predictive techniques provide particulars about root systems by analysing information from soil cores and root crowns of field-grown plants (Trachsel et al., 2011). At present, several in situ methods which include rhizotron, magnetic resonance, and computed tomography techniques facilitate non-destructive spatial and temporal investigations into root systems grown in soil, but the current scale, resolution, throughput, and cost efficiency of these techniques limit their utility. Apart from them, simulation and modelling studies aids the researchers to predict evaluate, and target beneficial root traits or genotypes for specific growth environments. WinRHIZO is an image analysis system designed for automatic root measurement in different forms which includes the total root length, projected area, surface area, average diameter, root tips, branching points, and root length based on the different width intervals chosen by the user.

Temperature Induction Response is an efficient method to increase the plants tolerance to high temperature (Bhavana et al). Exposure to moderate heat stress before challenging them to severe heat stress aids in developing acquired thermotolerance. Temperature induction may aid in screening the thermotolerant lines in a root trait based segregating population and facilitate the identification of high root type lines with thermotolerance.

Statement of the Problems

Phenotyping of the roots have always been a tedious process to screen under field conditions and root growth is limited significantly by soil environment-genotype interactions. Many traditional studies focus on destructive techniques, which ultimately measure the root length, volume and density. These High throughput techniques are expensive and time consuming. So it is necessary to understand the Root system architecture temperature tolerance of finger millet using high throughput screening techniques.

Objectives

1) Development of appropriate high throughput phenotyping Technique to measure the Root System Architecture traits.

2) Phenotyping of Recombinant Inbred Lines (RILs) to screen for variability in Root System Architecture traits using Image Analysis Software

3) Screening the Recombinant Inbred Lines for temperature tolerance

LITERATURE REVIEW

The initiation of root and its growth in the plant is different from that of shoot of the plant. During root growth and development, meristematic cells in the root tip divide, elongates and differentiates and then the lateral organs are formed which aids in spreading of roots in different profiles of soil. The basic characteristics of root development have been analysed by dividing the root tip into different parts such as root cap, the meristematic zone, elongation zone and maturation zone. Root growth occurs in the root tip which consists of highly dividing meristematic cells. The root apical meristem tissue contains several types of root initials and the quiescent center (QC) is the source of all the tissues of the root. It is stated that the lateral root development occurs some distance back from root apical meristem ( Malamy and Benfey, 1997). The lateral root growth and development is mainly influenced by endogenous factors like hormones. Among all the hormones, different concentrations of auxin play a major role in root development and external factors such as water and nutrients availability in soil. Poor soil fertility and environmental stress reduce crop yields in many parts of the world ( Rogers and Benfey. 2015).

In the monocots, seminal root axes grow out of the developing embryo, while nodal axes such as the secondary, adventitious, crown or brace roots arise from the shoot and they gradually replace the seminal axes. The number, length, and spatial distribution of roots may be different between even closely related species and may change with developmental age and environmental conditions. Root topology can also be related to shoot development and physiology. So a combination of topological analysis with measures of root length and allocation, in relation to shoot biomass, provides a robust way of describing the architecture, growth, and response to the environment of a root. Branching of roots is due to the response of both endogenous and exogenous factors. The ratio of auxin and cytokinin concentrations is thought to be important for the regulation of developmental steps for the complete formation of lateral roots. Water uptake is also enhanced by increased root surface area, even though its transport may be limited by the hydraulic conductivity of the xylem elements in some circumstances.

Root system architecture (RSA) refers to the shape and spatial arrangement of a root system within the soil and it is pivotal for plant anchorage and efficient uptake of water, macro and micro nutrients from the soil and can have a major impact on fertilizer usage and yield in crops worldwide. It is highly plastic in nature and may get altered based on the environment and results in different root types with specific functions. RSA is shaped by the interactions between genetic and environmental components that establish a framework with which the plant explores the soil and responds to the external environment that dictates future growth patterns. Root system architecture in specific environmental differs and directly influences the above ground parts of the plant that impact yield.

Several techniques have been used to measure the root architecture traits to understand its relation with that of the plant productivity. Some methods used for measuring root growth characteristics include:

Field Grown methods such as Photographs or drawings, Trench, Pinboards, Auger and these methods aids in measuring the length, weight, diameter distribution patterns and may be a destructive technique to measure these traits.

Rhizotrons were first designed by Rogers at East Malling Research Station, England. It employs non-destructive technique so it is possible to observe successive root growth which helps to understand the growth rate. But costs for construction of a rhizotron will be greater and not affordable. Similar to this, Minirhizotron proposed by Bates where a mirror and a battery operated lamp mounted on the end of a stick to see roots intersecting a glass tube in the ground. Later these were modified with colour video camera with a right-angle viewing attachment that can be lowered into the underground tube, and images can be recorded. Limitations of this technique may be more number of tubes are required and it is labour and time consuming process.

With further modifications In 1985, James et al. suggested a non-destructive root measurement technique similar to the rhizotron, which minimized the cost and requirement for specialized equipment. It is generally constructed of two 20 cm × 20 cm × 0.5 cm transparent plexiglass plates held one cm apart by plastic tubing. Later Neufeld et al. created a root box similar to James et al. which was slightly larger and grew plant roots between a plexiglass sheet and a nylon sheet with soil medium on the other side of the nylon so complete view of the roots are possible. As there was innovation over the years, Pan et al. developed a new portable rhizotron system called mesorhizotron, to observe root growth in different cropping systems, soil conditions and environments. The mesorhizotron has a transparent face on a box that is buried in the soil, and a portable hand scanner can be placed into the box to scan the transparent wall view. The large-volume rhizotrons were developed which mimics in-ground conditions, including enhanced drainage for evaluating effects of soil moisture deficits on root growth. Rhizotrons have been employed in Characterization of Pearl Millet Root Architecture by Sixtine et al.

Container Grown Methods include Root washing, Root Rating, Horhizotron, Rhizometer, Hydraulic Conductance Flow Meter, Transparent Containers and Substrates. Root washing is a destructive method and there are chances for losing of data such as root length, root diameter classes and root weights. In this method much of the fine roots and root hairs are lost and it is time consuming.

Root rating is a simple and easy way to qualitatively describe root balls of container-grown plants, washed roots and propagative rooted cuttings. It can evaluate root density, appearance, branching and distribution. Root ratings can also be measured with the rhizotrons, minirhizotrons and Horhizotron™ by estimating the root density observed through the transparent walls. This method was found by Walters and Wehner for determining root growth rate in cucumber.

Another method that required careful washing and soil removal from seedling root systems is a photoelectric device termed the rhizometer. Rhizometer can be used to estimate root surface areas of seedlings by using a light source on which the root system is placed and a photocell to measure the reduction in light due to the roots and a galvanometer which in turn measures the decrease in output from the photocell. It is a rapid method for measurement, but sources of error may occur if roots cross one another may lead to underestimates of the root area, as well as the possibility of actively growing roots being translucent and therefore not give the same light reduction compared to opaque roots. But the limitation is natural architecture of the roots are lost once removed from the natural growing environment and other complex and interesting root measurements may also be lost as well.

The Horhizotron™ is also an non-destructive technique used to measure lateral root growth from an original root ball of a container-grown plant which can be used for post-transplant assessment. The centre of the Horhizotron™ holds the root balls and the plants are placed in the centre with eight panes of glass that extend away from the root ball in a four-pointed star shape. The substrate in each quadrant can be modified in various ways in order to examine the effects of different rhizosphere conditions. Each quadrant can be filled with a different substrate, or the quadrants can be divided with one type of substrate on the lower half and a different substrate on the upper half. Limitations include: Glass panels can move and crack, the shade box may not restrict the light completely which falls on the root system and only large container plants can be used to observe root growth.

The Hydraulic Conductance Flow Meter (HCFM; Dynamax, Inc., Houston, TX, USA) can measure both root and shoot hydraulic conductance with minimal disturbance to the root system, although the measurement is destructive to the plant as a whole, because the shoots are excised from the rootstock. The shoot stem or rootstock is fitted with water filled tubing of the HCFM and once connected, the HCFM uses consistently increasing pressure to cause water to flow into the root or shoot system. The pressure versus the water flow measurement is used to estimate the hydraulic conductance. With the root hydraulic conductance, it is possible to correlate it with the root mass and may possibly reduce the need to destructively wash and extract roots from the substrate.

As there are advances in technology, photographs or scanned images can be used by computer programs to evaluate several root measurements. The resolution of digital imaging combined with the objectivity of automation may aid in broad and productive sets of data. Numerous computer programs have been developed so far both commercially and freely available aids in processing of these digital images. Several of these programs include RootLM, RootReader 2D, EZ-Rhizo, WinRHIZO and WinRHIZO Tron.

Root image processing has been performed by Thomas et al. to identify seedling root traits linked to variation in seed yield and nutrient capture in field-grown oilseed rape (Brassica napus L.). They have employed RootReader2D which automatically calculates primary root length, lateral root length of all laterals, and lateral root number (LRN). Clark et al. have also used RootReader2D to study root systems on Rice and Maize.

Some methods have been developed to measure the 3D root systems such as:

X-ray computed tomography (CT) and nuclear magnetic resonance imaging (NMRI), are non-invasive and non-destructive methods which aids in studying the root growth over time leaving the roots undisturbed. This method uses X-rays to measure the photo-electrical absorptions or scattering to scan the roots growing in soil/substrate contained in PVC tubes and produces a 3D image. The sample will be rotated between an X-ray source and a detector, and a series of 2D projections can be recorded, from which a 3D volume dataset can be reconstructed. Even lateral root development or root elongation rates can also be measured. However a major drawback is other structures surrounding the roots, such as water-filled pores, can lead to low contrast hindering straightforward segmentation of the roots from the background. CT image data can be used to quantify root length, volume, surface area, mean diameter, root tip diameter and vertical root depth. Long scanning times are necessary to acquire high quality images. Characterization of rhizosphere hydraulic properties or soil aggregate properties is also possible. X-Ray Microcomputed Tomography has been used by Sixtine et al to image the root structure and they have analysed the images using VGStudio Max Software.

The nuclear magnetic resonance imaging (NMRI) method uses proton signal intensities to measure spatial array and produces an image of the root system. But it is mandatory to distinguish protons in roots from protons in soil in order to measure a correct image of the root system. Applications of NMRI range from phytopathology, storage root internal structures and water mobility in roots. Roots can appear to be much thicker in the NMRI compared to CT, and this is caused by the much coarser spatial resolution of the NMRI. Metzner et al. reported that the thinnest roots detected with NMRI were about 250 μm in diameter. It is recommended to CT for measuring fine root diameters as it provides higher spatial resolution and NMRI large diameter due to strong root-to-soil contrast achievable by NMRI.

Mapping population can be phenotyped digitally in 3D by combining the use of 2D rotational image series to estimate root traits with the aid of GiA Roots, a standalone, graphical user interface-based, freely distributed software with enhanced semi-automated image processing and an implementation of an algorithm that generates 3D reconstructions from rotational image series taken in optical correction tanks as described in Zheng et al.

Several high throughput phenotyping platforms have been developed among them Plant root monitoring platform (PlaRoM) consists of an imaging platform and a root extension profiling software application which enables high throughput monitoring of growing seedlings with high spatial and temporal resolution and detection of root elongation profiles. Imaging platform consists of a custom designed phytochamber and the enclosed measuring head. Two software applications were developed for PlaRoM, imaging application controls the imaging platform (constant growth condition and image acquisition) and the root extension profiling application calculates the growth profiles from obtained images and plots the results. Root extension profiling, Sequential image processing, Dynamics of primary root extension, Comparison of root extension profiles among different genotypes, Root hair development and Kinetics of lateral root development can also be measured.(Yazdanbakhsh et al. 2009)

WinRHIZO system can measure the total length, projected area, surface area, root tips, branching points, and root length based on the different width intervals chosen by the user. According to Fang et al., WinRHIZO is relatively inexpensive and suitable for both large and small-scale experiments. Villordon et al. used WinRHIZO to classify lateral root growth of sweetpotato. Sweetpotato roots were washed, placed in water and scanned to be analysed by WinRHIZO. Root type classification was based on predetermined diameter intervals designed by the researchers, and used to show different root stages. While the scanner allows for clearer images, the roots must be washed and the substrate removed, and measurements of the root system over time cannot be observed with this program. Washing roots can cause a loss of fine roots and disturb the natural architecture of the root system. Unless the roots are floated in water, as done by Villordon et al., the natural architecture is gone. Another program WinRHIZO Tron is used to analyse images from rhizotrons, minirhizotrons or other transparent wall techniques. Root tracings can also be scanned into the WinRHIZO Tron program, and root length can be converted to root mass. However, the images to be used with WinRHIZO Tron are often unfocused and blurry, so the user has to manually select the roots on the image and trace the length for the program to know what to measure. Other programs are available that also used images from scanners; Benjamin and Nielson used a flat-bed scanner to digitize images of roots and used the commercially-available image analysis software Sigma-Scan™ to determine surface area of the roots; scanners can also be used to acquire images of seedling roots growing along an agar surface, aeroponically grown systems and rhizotrons.

Temperature Induction Response (TIR) is an efficient technique which increases the thermotolerance by induction. After exposure to heat stress plants acquire tolerance which includes synthesis of small heat shock proteins(Queitsch et al.2000).Small heat shock proteins are expressed in mitochondria, endoplasmic reticulum, cytoplasm, nucleus and chloroplasts after exposure to heat stress in vegetative, reproductive stages.in earlier studies it has been demonstrated that seedlings which were exposed to sub-lethal temperature had better growth during recovery than the seedlings which were directly exposed to severe temperature(Srikanthbabu et al.2002).

METHODOLOGY

Concepts

Mercuric Chloride acts as an antimicrobial agent and it is an effective disinfectant even at low (0.1%) concentrations. Bavistin is a broad spectrum systemic fungicide containing 50% WP Carbendazim and it is effective against plant pathogenic fungi and it may reduce the growth of fungi in the media.

ClerigelTMSuper is an substitute for agar which is a purified, anionic hetero polysaccharide produce from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. It produces a clear and colourless gel which aids in visual detection of the rooting pattern in the plants. Strength of the gel is not affected over a wide range of pH and it is free of phenolic contaminants. ClerigelTMSuper is chemically inert to most of the biological growth additives. Contaminants can be easily detected at early stages compared to agar.

For Media Preparation & Inoculation autoclaved distilled water must be used for preparing media inorder to avoid contaminants and inoculation must be done in a sterile environment.

Root zone must be devoid of light so the culture bottles must be covered with an opaque sheet. Documentation must be done in a dark background so that the roots appear pale in colour. This is done to ensure that the software recognizes the roots without any interference. Photograph of the roots must be taken from a fixed distance with a fixed scale.

Temperature Induction Response (TIR) is a simple technique used to screen plantlets for high temperature stress tolerance and in this technique the seedlings are first exposed to an optimal induction temperature before being subjected to a severe challenging temperature, and subsequently it is allowed to recover at room temperature. The thermotolerant genotypes are the ones which exhibited maximum survivability and higher growth at the end of the recovery period.

2,3,5-Triphenyltetrazolium chloride stains the live tissues and this occurs by enzymatic reduction of TTC to insoluble red formazan by metabolically active tissues.

Methods

Plant material

Finger Millet [Eleusine coracana Gaertn.] Recombinant Inbred Lines obtained by crossing GE208 and GE156 were used in this study. Seeds were surface sterilized with 0.1% Mercuric Chloride for one minute, rinsed with sterile water, followed by rinsing with Bavistin for two minutes and rinsed again with sterile water. Seeds were then put up in sterile petridishes containing wet filter paper for 48 hours at 30°C in the dark for germination.

Preparation of nutrient media

Hoagland’s Nutrient Solution was prepared with the below concentration mentioned in Table 1 using sterile water and 2.5 g of ClerigelTMSuper was added to one litre of Nutrient solution to obtain media with 0.25 % Clerigel. Media was autoclaved and cooled down before inoculation of the ragi seedlings.

TABLE 1: COMPOSITION OF MODIFIED HOAGLAND’S NUTRIENT SOLUTION

HOAGLAND'S STOCK SOLUTION
STOCK SOLUTIONg/L

MOLARITY

OF STOCK SOLUTION

ml stock/LWORKING CONCENTRATION
Macronutrients
Ca(NO3)2.7H2O236.81 M2.5

2.5 mM Ca

5.0 mM N

KNO3101.11 M2.5

2.5mM K

2.5 mM N

MgSO4.7H2O246.41 M1.0

1.0 mM Mg

1.0 mM S

KH2PO4136.01 M0.5

0.5 mM K

0.5 mM P

Micronutrients
H3BO32.8646.2 mM1.046.2 µM B
MnCl2.4H2O1.819.14 mM9.14 µM Mn
ZnSO4.7H2O0.220.76 mM0.76 µM Zn
CuSO4.5H2O0.0510.2 mM0.2 µM Cu
H2MoO4.H2O0.090.5 mM0.5 µM Mo
Na2MoO4∙2 H2O0.120.4 mM0.4 µM Mo
Fe-EDTA
FeSO4.7H2O5.6

0.02 M

1.530 µM Fe
Na2EDTA7.45

Note: The pH of the nutrient solution was adjusted to 5.8 with 0.1 M NaOH

Inoculation

Two days old ragi seedlings were inoculated into culture bottles with 0.25 % ClerigelTMSuper containing Hoagland Nutrient Solution and they were incubated at Growth Chamber with 16 hours Photoperiod at 24°C for 18 days. Exposure of roots to light were minimised by covering the transparent media.

Documentation

Culture bottles containing ragi seedlings were documented from a fixed distance with a specified scale for root growth everyday up to 18th day and they were analysed using WINRHIZO Pro software which measures the root parameters.

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    WINRHIZO Pro

    Image analysis

    Images acquired everyday was sequentially fed into WINRHIZO Pro (Image analysis software) and ensured that the roots are pale and background is dark to ensure proper detection of root systems. (Figure 1)

    Temperature induction response

    Parents (GE208 and GE156) of RILs along with nine high root type and nine low root type Recombinant Inbred Lines were surface sterilized and kept for germination at 30°C with 60% Relative Humidity in the incubator. Three days uniform seedlings were selected from each line and they were transferred to aluminium trays. Trays were then subjected to sub lethal temperatures (28°C to 53°C) for 5 hours in the BOD incubator and they were subsequently subjected to lethal temperature (53°C) for 2.5 hours. Another subset of 3 days old uniform seedlings were directly exposed to lethal temperature (53°C).Both the Induced and non-induced ragi seedlings were the allowed to recover at 30°C with 60% relative humidity for 72 hours. Control trays were also maintained at 30°C without being exposed to sub lethal and lethal temperatures. After 72 hours of recovery per cent reduction in root and shoot growth was measured.

    Triphenyl Tetrazolium Chloride (TTC) viability assay

    0.1% of 2,3,5-Triphenyltetrazolium chloride solution was prepared using autoclaved distilled water and then the seedlings subjected to lethal and sub-lethal temperatures were incubated in solution for 48 hours and observed for staining.

    RESULTS AND DISCUSSION

    RIL Population was raised in Culture bottles (Fig.2), Root Structure (Fig.3) and Test Tubes (Fig.4) and it was inferred that Test tubes allows the growth of tap roots in length but limits the spreading of the roots. Culture bottles favours better spreading of roots and it can be used effectively until the primary root reaches the bottom and it is difficult to analyse the root pattern if the root starts to swirl at the bottom. Root structures made of Polypropylene facilitate longer root growth and spreading but it is quite tedious to handle them. So measurement of root growth can be done effectively up to 18 days in Culture bottles.

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      CULTURE BOTTLE
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        ROOT STRUCTURE
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          TEST TUBE

          Root lengths of the RIL Population were documented in Culture bottles up to 18th day and then the acquired images were analysed sequentially in WINRHIZO Pro software. Parameters provided by WINRHIZO Pro include Total root length, Surface Area, Average diameter and Root Volume. From the analysis we obtained the following data.

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            COMPARISON OF GE208 AND GE156
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              COMPARISON OF RIL 90 AND RIL 120

              From Fig.5 & Fig.6 we found that the total root length of the GE208 is higher compared to GE156 with the difference of around 11 cm. Similarly the average diameter, surface area and Volume are found to be better in GE208 than GE156.

              Root length of parental lines GE208, GE156 and Recombinant Inbred Lines raised in culture bottles were measured after 18 days and we have observed the following variation in the RIL Population.

                ROOT LENGTH
                  SHOOTLENGTH

                  From the above frequency distribution (Fig.7 & Fig.8) regarding root length and shoot length we observe that both the parents have similar characters in their root and shoot length and the distribution of shorter roots are more compared to the distribution of the longer roots in the above population. But the frequency of population exhibiting high shoot length is distributed among different categories.

                  From the above data we can infer that even though both the parents has similar root length based on manual measurements but based on the data from WINRHIZO Pro GE208 has better total root length compared to GE156 and it is due to the presence of more lateral roots in GE208.

                  Screening of RIL through Thermo Induced Response (TIR) treatment
                  ORDERPer cent reduction in root growth (%)Per cent reduction in shoot growth (%)
                  GE20833.3134.57
                  GE15623.8817.42
                  RIL23953.0418.30
                  RIL 21020.20-7.14
                  RIL 1209.477.15
                  RIL 8335.17-6.91
                  RIL 27647.174.47
                  RIL 7831.67-37.02
                  RIL 6651.814.38
                  RIL 2953.5612.14
                  RIL 234.28-13.58
                  RIL 846.373.64
                  RIL 3361.3219.13
                  RIL 2259.4416.30
                  RIL 7666.41-4.59
                  RIL 27949.811.11
                  RIL 28246.942.05
                  RIL 28453.819.74
                  RIL 9057.581.98
                  RIL 10341.59-8.81

                  Temperature Induction Response

                  Nine High root type and Nine Low root type Recombinant Inbred Lines obtained from GE208 and GE156 were screened for intrinsic tolerance using the standardized Thermo Induction Response (TIR) protocol. The experimental data were recorded and presented in Table 2. The per cent reduction in root growth varied from 9.47%(RIL 120) to 66.41%(RIL 76) and the per cent reduction in shoot growth varied from -37.02%(RIL 78) to 18.30%(RIL 239).TIR Response of high root type and low root type revealed that RIL 120 has shown the lowest per cent reduction in root growth. It has been also evident from the following table that GE156 has lower per cent reduction in root growth compared to the high root type parent (GE208).

                    Thermally Induced Seedlings

                    TriphenylTetrazolium Chloride (TTC) Viability Assay

                    TTC Assay was performed on the Control, Induced and directly exposed Lethal seedlings. It was found that the tissues are alive in Control and Induced seedlings, but the tissues in seedlings which were directly exposed to lethal weren’t stained.(Fig 10-Fig 13)

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                      CONTROL SEEDLING
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                        SUB-LETHAL SEEDLING
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                          LETHAL SEEDLING
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                            LETHAL SEEDLING

                            CONCLUSION

                            Growing of seedlings in a transparent gel and further phenotyping of the root traits using a software aids in comprehending the daily growth rate of the plants. As the growth conditions are limiting for these seedlings they can be phenotyped only during the early stages of root development. But this method of phenotyping is less expensive with reliable results. Development of phenotyping techniques at each and every stage of the plant may reveal much more validation regarding the root system architecture traits and further Development of automated Plant phenotyping techniques and their integration with Biology may enable us to understand all the factors which modulates the root architecture. Further screening of the RIL by TIR may identify the high root type lines which are thermotolerant and TIR is very constructive in identifying the thermotolerance during early stages of development.

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                            ACKNOWLEDGEMENT

                            With great pleasure I express deep sense of gratitude and profound indebtedness to Dr.M.S.SHESHSHAYEE, Department Of Crop Physiology, University Of Agricultural Sciences, Bengaluru for providing me the opportunity to work in his laboratory.

                            I express my heartiest thanks to the lab members at the Molecular Marker Lab who gave their valuable suggestions, encouragement and timely help which assisted me to learn things.

                            I have gained more basic knowledge about the experiments, handling of instruments and time management in research. Lab members also help me to understand not only the experimental knowledge,but they also shared the theoretical knowledge about the experiments.

                            I take this opportunity to thank my host institute mentor, Dr.M.Raveendran, Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore and Dr. E. Kokila Devi, Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore who has taken the utmost care on this internship training program to end in a successful manner.

                            I wish to express my gratitude to all the faculty staffs, Professors and Assistant Professors of Tamil Nadu Agricultural University, Coimbatore for their support getting blessed with this golden chance.

                            I would like to thank Indian National Academy of Sciences for offering me with the Summer Research Fellowship Program.

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