Progress report for GS23-295
Project Information
Southwest Texas has been experiencing years of drought that is expected to continue. Cotton is the major crop of West Texas that requires less water comparatively, but extreme conditions affect its growth, yield, and development. Water saving technologies are needed to improve water efficiency in crops. A major bottleneck in water-saving technologies that cotton faces is the lack of mechanistic understanding of diversity in root systems for further genetic improvement. It is well-studied that genetic differences in root system architecture (RSA) and associated traits such as root biomass, depth, and overall morphology, can positively overcome the water deficit conditions in crops. Studies have shown that genetic differences in root system architecture can positively overcome water-deficit conditions in different crops. However, the adaptation of root systems to a semi-arid environment and underlying genetic mechanisms is not well studied in many crops, especially cotton. Cotton possesses a deep taproot but lacks active shallow roots which can be a sustainable strategy in the West Texas region. When limited rainwater is available for a short period of time, it cannot reach the deep root zones. Active shallow roots could improve water and nutrient uptake near the surface and sub-surface regions. Considering the advantages of root systems and underlying genetics, our project will investigate the root phenotypic plasticity of diverse landraces, commercial cultivars and lines grown in West Texas. A comprehensive evaluation will lay a foundation to optimize the root system architecture and offer sustainable solutions to improve water efficiencies under water-deficit conditions.
1) Identify a subset of Upland cotton accessions (175-200) and elite commercial cultivars (15) by selecting from the 600 accessions grown in the greenhouse.
2) a) Evaluate the root system architecture (RSA) of the diverse germplasm using image-based root phenotyping platform under controlled greenhouse conditions. The plants will be grown in the greenhouse for about 2 weeks before scanning their RSA with the scanner and software program. b) Select a subset of the 5 most promising and the 5 least promising of the exotic accessions in addition to the control and elite cultivars for contrast. To evaluate the RSA traits, under well-watered and water-deficit treatments in field conditions, three replications will be under irrigated conditions and the other will be rainfed only.
3) Based on the 1st year’s findings, lines with contrasting RSA will be further explored to identify QTLs or genomic loci using advanced genomic tools (GWAS) and additional field experiments to identify novel genes for optimizing the cotton RSA for enhanced water capture and efficiency.
4) Working in collaboration with soil physicist (Dr. Deb) to understand the mechanisms by which the RSA and root water uptake functions at deep soil layers and shallow zones. Investigate the water movement from soil to roots in relation to cotton’s variation of traits.
Research
In the first objective to determine the varieties of cotton to use for this project, 660 cotton accessions were ordered from USDA-GRIN. Those were planted in the greenhouse to grow for seed accession. Those were screened using phenotypes and their origins to reduce to 200 accessions to move to the next objective. The elite commercial cultivars grown in West Texas was collected from various seed industry partners including BASF, Bayer, Armor, Phytogen, and NexGen. These companies selected varieties that they were interested in testing in this project for drought tolerance. Some varieties already have some drought tolerance characteristics. The main goal of this objective is to get a diverse set of genetic variation to then compare root systems for the best and the worst RSA to compare water efficiency for sustainable water use. Traits related to yield, disease resistance and fiber quality will also be considered.
For the second objective, all the selected lines were screened for root morphology at the seedling stage, 2 weeks after planting. They were screened using a visual scoring and high-throughput phenotyping platform WinRhizo, Inc. WinRHIZO is an image analysis system specifically designed for measuring root morphological traits. We focused on total root length, diameter, and surface area among other characteristics. This process is according to Kadam et al., 2015. Dr. Jagadish’s lab has extensive experience working with root morphology and anatomy of various crops. These experiments will help us to narrow down the lines for in-depth analysis under well-watered and water-deficient conditions. 
The next phase of objective two is to identify lines with differential response to water deficit conditions in relation to an altered RSA. To achieve this goal, we plan on growing a subset of contrasting lines in controlled-environmental greenhouse conditions under well-watered 100% field capacity and water-deficit conditions around 40%-60% field capacity (Kadam, et al., 2015). Water deficit stress will be imposed after seedling emergence. Plants under the control treatment will be maintained at 100% field capacity throughout the experiments. Following the 30 days of stress, plants will be harvested around 50 days after sowing and traits associated with the RSA, shoot biomass, and physiology will be collected as detailed in Kadam, et al., 2015. With the anatomical studies the root diameter, stele diameter, and number of meta xylems will be considered.
Following the evaluation of RSA and anatomical traits from objective 2, cotton cultivars showing differential or contrasting phenotypes was further evaluated under field conditions. Uprooting cotton plants in the field would have been challenging, so to overcome this issue, large woven bags filled with field soil was used to proxy the field conditions. Watering was done manually by adding the recommended amount determined by the University of Georgia.
During the cotton growth, the morphological (shoot) traits were measured. These accessions were grown and were intended to be harvested at the boll formation stage (i.e., the peak root biomass accumulation phase). However, a hailstorm destroyed some of the bags and some were damaged as a result. The time that it took to manually wash these took longer than expected. Therefore, this experiment was necessary to be repeated again over the winter in the greenhouse with the same field soil and the same bags.
The repeated experiment was done in the greenhouse because of the winter conditions. With the limited space in the greenhouse and the amount of soil needed to transfer to the greenhouse from the field, the number of genotypes was reduced to 12 instead of the original 24. This was intended so we could have 3 replications of 12 rather than fewer replications. Watering was done similarly to the field manually measured with the graduated cylinder. For the soil density, the volume and weight were measured, and the soil was set to fit within the same mass per volume to get the ideal density and compaction.
A comprehensive analysis of diverse germplasm and elite commercial cultivars grown in West Texas will allow us to identify the functional role of root tissue plasticity for adapting to water-deficit stress. It will be interesting to evaluate if current commercial cultivars in addition to deep root systems possess active shallow roots and does it help conserve soil moisture during vegetative and reproductive stage.
A method of measuring and understanding of the mechanisms of root water uptake in relation to the soil layers will be in collaboration with soil physicist. This investigation will include discovering a method of measure water movement from the soil to the roots in relation to the variety of cotton's traits. Overall, this information will be helpful to investigate, understand and optimize root plasticity in upland cotton and translate this knowledge to develop next generation cotton cultivars with better RSA under water-deficit conditions to improve sustainable agriculture. Commercial producers can apply this research to their ongoing projects to improve the future elite varieties which will provide farmers to reduce their water consumption and save money while increasing their yields.
Since September 2023 the following results have been evaluated with additional experiments being planned for the following year. The field experiment included a field of 550 Upland Cotton varieties that leaf samples were collected to genotype the germplasm for eventual association mapping. The genotyping is planned to take place later in the 2024 year. There were 24 genotypes selected for the above ground bag experiment. Only 2 replications could be completed with the lateness of the season getting them planted and the time and labor it took to fill the bags. The images of the roots were uploaded into the WinRHIZO software for the analysis of the traits with the ruler sticks set for image calibration. The results from the end of October with extracting the bags do show some differences in the genotypes and amount of root length and surface area. However, none can be statistically significant with the limited replications and missing data make it impossible to statistically validate these results and a repeated experiment will need to be done. Only two replications were able to be done with the time it took to fill and the heat of the summer. The two replications also have a lot of variation between the two because of the time difference of extracting the roots. Also, 12 samples were damaged in the hailstorm which could also account for some of the variation. The plants were also past their boll development in many of the plants because of the time it took to extract them. This could also have had an impact on the roots that were present. A lot was learned from this experiment and hopefully the repeated experiment did not repeat the same mistakes.
As one could tell by the raw data visually represented, not much can be concluded from this.
Beginning in January a repeated experiment was done with 12 genotypes and 3 replications because of the limitation of space in the greenhouse and wanting to have more than 2 genotypes less of the genotypes were used. The soil was also from the same research farm and the density was kept the same by weighing the amount of soil for a given space for each bag to be equalized. Each replication was planted and extracted within a week of each other, to reduce the time difference that happened in the past experiment. Many things were better in this experiment, but in our attempt to extract the roots after 6 weeks to have time for the next experiments, the roots were not as mature and were thinner and broke off easy in the extraction process. The roots were still collected to get the total root length and surface area, but the original architecture of how they were laid out in the soil was lost.
Some of the plants suffered from insects and possible infections in the greenhouse where other genotypes seemed to be unaffected. This could have contributed to differing in their development of each other in some of the plants.
The raw data is visualized by the graph series 1 being one replication. The plants were randomized throughout the greenhouse area and to see if just one replication did better than another, or if was random this graph shows it wasn't always one replication doing better than the rest. By the end of collecting the roots, I may have gotten better at collecting them and less roots may have been lost to cause some variation as well. More experimentation will need to be done before a conclusion can be met.
This compares to other conventional systems as it is cheaper than getting advance rhizotron devices and expensive PVC pipes or other methods of growing root architecture. Washing of the roots was able to get some of the main roots intact to get depth of the taproots. Additional experiments will be able to know the best way of extracting and when to extract the roots and how to best simulate the field conditions because other research that does RSA usually does not use field condition or field soil because of the difficulty and expensive cost of doing studies like this. To be able to have this information for the future and if it follows the same pattern as other studies than it can show that their results are similar to field conditions corroborating other RSA studies results.
The next step for this project is to have a little over 200 accessions in a drought field and the same in an irrigated field. About 5 replications of each of the 200 will be planted. In addition, 50 individual plants of the selected lines from previous experiments will be in these two field conditions to phenotype the above ground. The phenotypes and shoot growth will be documented as they develop in the drought conditioned field and the irrigated field.
Another experiment will be to have 5 replications of the 2 Gallon sized bags of those same 200+ accessions to get their RSA scanned and their traits analyzed for the association mapping.
Educational & Outreach Activities
Participation Summary:
Presented my research at the 10th Annual Water College in January 2024. Farmers, sponsors, and educators were present at the event of over 200 hundred people.
Lamb, M. L. (2024, January 24). Optimizing cotton's root system to improve response to water-deficit stress [PowerPoint slides]. Institute of Genomics for Crop Abiotic Stress Tolerance, Texas Tech University. https://texastechuniversity-my.sharepoint.com/:p:/g/personal/miclamb_ttu_edu/Ede1MWpL2SxNu8MjQb-bWEYBcdg5L-_jzAUcg3wI-n_O8Q?e=dWC8qk&nav=eyJzSWQiOjI1NiwiY0lkIjoxMDk4NTcyMjJ9
Project Outcomes
If we can figure out more about the roots and how they are related to shoot traits and the genetic relationships with the roots. It will save future generations so much time and labor and money to develop their crops with a better understanding of the Root System Architecture. Because it is very difficult to view the roots to know what traits are being bred for and what root traits will be beneficial or not in different abiotic stresses. This project's goal is to get additional understanding of methods of how to assess the RSA and also do it in an economically inexpensive way to better our understanding of the genetic mechanisms of the RSA.
We are still learning as we continue these few months since September 2023. I have been learning how much work is required to learn something unknown and also how much knowledge that comes from so many other people before is needed to build upon each other. I am excited for the next year of learning and hope to come up with new information to better serve society and agricultural partners.
I would like to wait on giving any recommendations until my further experiments and additional learning for the next year and see what I learned to share.