Variation in Root Development Response of Napier Grass to Drought Stress

Global climate change and increasing agricultural activity are the main causes of biotic and abiotic stresses, which negatively affect the plant growth and crop yields. The plant root system is the ﬁrst organ for sensing the soil mois ture limitation; therefore root growth under elevated water deﬁcit is an important indicator for plant’s drought tol -erance. Although the previous studies focused on the morphological traits of Napier grasses under water stresses, the root growth changes due to drought levels remain largely unclear. In order to evaluate variation in root performance to respond to drought stress, four cultivars named “Cỏ voi thuần” (CVT), King grass, Packchong, and VA06 were grown for 10 days under drought conditions under polyethylene glycol 6000 (PEG6000): 0% PEG6000 as control, 5% PEG6000, 10% PEG6000, 15% PEG6000 and 20% PEG6000. As compared to control, the root growth of all cultivars was reduced under drought treatments; however, signiﬁcant variation in the root development response to drought levels was found. Among Napier cultivars, “Cỏ voi thuần” expressed drought-tolerant genotypes. The information on the root length, diameter, surface area and volume of the cultivars reveals interest-ing guidelines for further studies to explore the mechanisms behind root adaptation of Napier grasses to drought.


INTRODUCTION
Global climate change and increasing agricultural activity are the main causes of biotic and abiotic stresses, which negatively affect the plant growth and crop yields [Raza et al., 2019]. Abiotic stresses such as salinity, cold, waterlogging and drought have adverse impacts on crop yield and about 50-70% of crop yield reduction is attributed to such abiotic stresses [Francini & Sebastiani, 2019]. Drought stress is one of the most severe abiotic stresses that directly affect the growth and development of crop thus affecting its productivity [Yavad et al., 2019]. For instance, a recently published report indicates that between 1983-to 2005, 75% of global cultivated land (454 million hectares) experience drought-induced yield losses, which account to about 166 billion United States dollars [Kim et al., 2019].
The impact of drought on major crops has been reported in several studies, including meta-analyses and summary studies. Severe drought-induced crop yield losses of 14.0% and 21.8% were reported for maize and soybean, respectively [Wang et al., 2020]. Under drought, the wheat and rice yields decreased by 27.5% and 25.4%, respectively . Water stress reduced the yield attributes and grain yield in sorghum [Jabereldar et al., 2017]. Farooq et al. [2009] documented the economic yield reduction and critical stages of growth affected by drought stress in barley, maize, rice chickpea, and pigeon pea common beans, sunflower, canola, soybean and potato. Besides field crops, drought stress also affect grasses and fodder crop growth, yield and productivity. Severe drought reduces the nutritive quality of forage legumes [Kuchenmeister et al., 2013, Liu et al., 2018. Drought decreases the shoot and root biomass, plant height, tiller number and leaf growth of rhizomatous grasses (Pascopyrum smithii and Elymus lanceolatus) [Zhang et al., 2017]. Guinea (Panicum maximum) and Napier (Pennisetum purpureum Schumach.) grasses exhibit decreased plant height and herbage mass under drought stress [Purbajanti et al., 2012].
Napier grass (Pennisetum purpureum Schumach.), also known as elephant grass, is a major C 4 perennial forage crop grown in many tropical and subtropical regions of the world [Negawo et al., 2017]. Despite being native to East and Central Africa, Napier grass is currently distributed in Central and South America, Asia, Australia, Middle East and Pacific islands [Singh et al., 2013]. The factors behind the wide adoption of Napier grass as a fodder crop include; high forage productivity, rapid regeneration and fast growing characteristics, drought tolerance and high water use efficiency [Purbajanti et al., 2012, Kabirizi et al., 2015. Napier grass, like many other C 4 plants, has numerous drought-coping adaptation mechanisms [Lopes et al., 2011]. During limited water availability, Napier grass exhibits a larger root system in combination with a less restrictive stomata regulation to maximize carbon assimilation [Cardoso et al., 2015].
Plant root systems play a crucial role in detecting the changes in soil moisture; thus, they develop appropriate drought survival mechanisms [Zhang et al., 2017]. It has been indicated that altering the root structure of crops grown under water stress can increase their yield [Lynch et al., 2014]. In different crops, root traits such as small fine root diameters, specific root length, root length density, specific root area and root angle have been suggested as desirable traits for improving plant productivity under drought stress [

Materials
Four Napier cultivars named "Cỏ voi thuần" (CVT), King grass, Packchong and VA06 were used in this study. For each Napier grass, uniform stem cuttings were used, having 2 nodes that were 25 cm in length, 35 g in weight and 1.5 cm in diameter.

Experimental design
In order to induce root and bud development, the cuttings of each cultivar were inserted in humid sandy soil for 3 days. Then they were transplanted and fixed into position in the polystyrene board with soft silicone rubber. The board with cuttings was then placed and floated on a modified Kimura B nutrient solution (composed of 0. 36  O)) in a plastic container (61 cm × 42 cm × 20 cm). The root system in container was continuously aerated by two air pumps 1.0 L min −1 at opposite ends of the container. Seven days after transplanting (DAT), the plants were treated for 10 days under drought levels. In this study, polyethylene glycol 6000 was used to simulate drought tress. Five drought stress levels were 0% PEG6000 (control), 5% PEG6000, 10% PEG6000, 15% PEG6000 and 20% PEG6000 in the Kimura B solution (Fig. 1). The solutions were changed every 3 days to avoid nutrient depletion. The experiments were conducted in a greenhouse and designed with the randomized complete block design with 6 replications (6 boxes) per PEG6000 level treatment. A plant for each genotype in a box was used as a replication for data analysis.

Statistical analysis
The data were analyzed using R software. The effects of cultivar, drought, cultivar by drought interaction on the measured traits were analyzed by two-way ANOVA; separating mean values by Tukeys's honest significant difference test at P<0.05.

Effects of cultivar, drought level and their interaction on root dry weight (RDW, g plant -1 ) and shoot dry weight (SDW, g plant -1 )
RDW and SDW of almost cultivars were not significant different at the lower drought levels 5% PEG6000 compared with plants at 0% PEG6000, except for RDW in King grass (Fig. 3 and Fig. 4). Significant reductions were observed in RDW Table 1 shows the data on the effects of cultivar (C), drought treatments (T), and their interaction (T×C) on the measured traits. It was found that C, T, and T×C had significant effects on all measured traits except RD.   and SDW of all cultivars at 10% PEG6000. In the comparison among cultivars, "Cỏ voi thuần" showed higher drought tolerance; while Packchong and King grass were drought-sensitive ( Fig. 3 and Fig. 4).

Effects of cultivar, drought level and their interaction on root traits
Significant variations in root trait responses (RL, RSA, and RV) to drought level were found among cultivars (Fig. 5-7, Fig. 9, Table 2

-4).
Significance difference was not found in RD of cultivars under drought (Fig. 8, Table 5). Similar to RDW and SDW, "Cỏ voi thuần" showed higher drought tolerance; while Packchong and King grass were drought-sensitive (

DISCUSSION
Napier grass distribution and accessions in various gene banks around the world has been reported [Negawo et al., 2017]. Genetic variation and Napier grass germplasm characterization using various kinds of DNA markers has been reported in several previous studies [Lowe et al., 2003. Similarly, morphological characterization of Napier grass has also been studied. Budiman et al.
[2012] evaluated three Napier grass cultivars in regards to their above ground morphological characteristics, such as plant height, stem diameter and leaf stem ratio. However, there are fewer studies on variation on root performance under drought stress in Napier grass. In this study, four cultivars of Napier grass were evaluated for their root response to drought stress using polyethylene glycol 6000 (PEG6000) at 0% (control), 5%, 10%, 15% and 20% drought levels.
The obtained results indicated significant difference in root and shoot growth response to drought among the Napier grass cultivars ( Table  1). The four Napier grass cultivars exhibited reduced root length growth as compared to control (0% PEG6000). Among the four cultivars; CVT exhibited the highest root length (RL), RSA and RV development at all drought levels, thus indicating drought-tolerance traits, while King grass and Packchong were drought sensitive (Tables 2, 3 and 4, Figure 3-5). The ability of Napier grass    This study, therefore, indicates a Napier grass "Cỏ voi thuần (CVT)" could be an important genetic plant material resource for breeding of better Napier grass cultivars that are tolerant to drought and high yielding. Higher development (as root branching and elongating) in root responses to water stresses is more advantageous to plant acquiring water but also nutrient uptake [Palta et al., 2011]. Packchong and King grass presented drought-sensitive cultivars with rapid decrease of RL and RSA. In addition, it was found that "Cỏ voi thuần (CVT)" was better adapted to drought as compared to the other cultivars. These results suggest that by a combined analysis of root plasticity and its association with water uptake and water use efficiency a more mechanistic understanding of factors involved in Napier root responses to drought will be reached and this should be the next research step.