EFFECT OF HOMOBRASSINOLIDE ON THE GROWTH OF MUSTARD CROPS GROWN IN SEMI-ARID TROPICS OF NIZAMABAD

The effect of homobrassinolide (HBL) on the shoot growth (length, fresh weight, diameter, and dry weight) root growth (length, fresh weight, diameter, and dry weight) and foliar growth (plant fresh weight, leaves per plant and leaf area) of mustard plants grown in the semi-arid tropics of Nizamabad was studied. The soil in Nizamabad district is saline land black soil wherein the plants usually experience drought and saline stresses. Application of HBL was very effective in stimulating the shoot growth, root growth as well as the foliar growth of mustard plants. The promotion of shoot, root and foliar growth is an indicator that HBL mitigated the negative effect of the semi-arid conditions of the soil.


INTRODUCTION
Mustard (Brassica junceae) is an oil yielding plant that belongs to the family Brassicaceae which is grown in various parts of the globe. Mustard plant has very tiny seeds which are usually used as a condiment. Mustard oil is extracted from the seed which is widely used for cooking, massaging etc. A few varieties of mustard plants have edible leaves that are known as mustard greens. It is a well-established fact from time immemorial that plants are the critical components of dietary food chains in which they provide almost all the essential mineral and organic nutrients to humans Grusak and Dellapenna (1999). Brassinosteroids (BRs) are a new type of polyhydroxy steroidal Plant growth regulators (PGRs) with significant growth-promoting influence (Bajguz and Piotrowska-Niczyporuk, 2014;Vardhini, 2017). Mitchell and his co-workers (1970) discovered BRs which were later extracted from the pollen of Brassica napus L. by Grove et al. (1979). BRs can be classified as C 27 , C 28 or C 29 BRs according to the number of carbons in their structure (Vardhini, 2013). Haubrick and Assmann (2006) reported that 60 related compounds have been identified. However, Vardhini et al. (2006) reported that brassinolide (BL), 28-homobrassinolide (28-HomoBL) and 24-epibrassinolide (24-EpiBL) are the three bioactive BRs being widely used in most physiological and experimental studies. BRs are considered ubiquitous in plant kingdom as they are found in almost all the phyla of the plant kingdom like alga, pteridophyte, gymnosperms, dicots and monocots (Bajguz, 2009). Rao et al. (2002) stated that BRs are a new group of plant growth hormones that perform a variety of physiological roles like growth, seed germination, rhizogenesis, senescence, etc. and also confer resistance to plants against various abiotic stresses. Though, BRs were initially identified based on their growth promoting activities, subsequent physiological and genetic studies revealed additional functions of BRs in regulating a wide range of processes, including source/sink relationships, seed germination, photosynthesis, senescence, photomorphogenesis, flowering and responses to different abiotic and biotic stresses (Vardhini, 2013). Nizamabad district experiences a tropical dry and wet season with most of the rainfall in June to October. It usually experiences erratic rain fall. The soil is saline land black soil which is deep loamy to clay loam, moderately drained, neutral to alkaline in nature. The semi-arid condition directly poses a threat to the overall yield of the plants as they usually experience drought and saline stresses. The present study is undertaken to understand the effect of application of homobrassinolide (HBL) on the growth of mustard plants in terms of shoot, root and foliage grown in the semi -arid soils of Nizamabad. juncea L. var. Indian mustard "Tulasi" were procured from, National Seeds Corporation Limited, Hyderabad, Telangana State, India. Homobrassinolide (HBL) is a commercially available brassinosteroid and was procured from Bahar Agrochem & Feeds Pvt. Ltd, Ratnagiri, Maharastra, India, Ltd. It is marketed by Godrej Agrovet Ltd., Hyderabad, Andhra Pradesh, India.
Pot Culture: The Plants were grown under natural day length in the semi-arid tropics of Nizamabad town. The mustard seeds were sown in clay pots (40×50 cm in diameter 40×70cm in height) containing fresh sieved black soil which consisted of uniformly mixed soil and vermin-compost in the ratio of 10:1 ratio per pot to make the soil fertile. The experimental studies were done in triplicates'. Around 8 to 10 seeds were sown in each pot at a depth of 1.5 cm and watered daily. On the 10 th day, priming was done and only five plants were retained in each pot to facilitate the plant plants growth to its full potential.

Foliar
Application of Homobrassinolide (HBL): Homobrassinolide (HBL) was exogenously applied in 3 different concentrations viz., 0.5 µM, 1.0 µM and 2.0 µM. as foliar spray. Three replicates were maintained for each concentration of HBL. Control plants were also maintained in triplicates which were sprayed with distilled water. The pots were placed in open environment and the mustard plants were grown under natural day length. The three different concentrations of HBL were exogenously applied to the plants as foliar spray and the spraying was done in the early hours of the day by using sprayers. The HBL was treated three times to the mustard plants viz., on the 35 th , 40 th and 45 th day after sowing.

Length of Shoot and Root:
The pots were adequately watered and the plants were gently removed. All necessary caution was taken to see that the root system was intact. The shoot and root parts were washed with tap water (twice).The water adhering the shoot and the root parts were removed with the help of blotting paper. The shoot and root length was measured on 35 th , 45 th , 55 th and 65 th days with using a meter scale and their values were expressed in centimeters.

Fresh Weight of Shoot and Root:
After measuring the length, the fresh weights of the shoots and roots were recorded on 55 th and 65 th days. A meter balance was used for this purpose. The shoot and root fresh weights were expressed in grams.

Dry Weight of Shoot and Root:
The shoot and root were dried in the oven at 110 o C for 24 hours and their dry weights were recorded on 55 th and 65 th days. The dry weights were expressed in terms of grams.

Diameter of Fresh Stem and Root:
The diameter of fresh stem and root were recorded on 65 th day. A screw gage was used for this purpose. The fresh stem and root diameters were expressed in centimeters.

Diameter of Dry Stem and Root:
The stem was dried in the oven at 110 o C for 24 hours and the diameter of dry stem was recorded on 65 th day. A screw gage was used for this purpose. The diameter of the dry stem and root were expressed in centimeters.

Foliage of Mustard Plant:
The foliage of the mustard plants grown in semi-arid tropics of Nizamabad was recorded in terms of number of leaves per plant on 35 th , 45 th , 55 th and 65 th day.
Leaf Area of Plant: The leaf area per plant was determined by estimating in on the 55 th day after sowing following the formula of Carleton Foote (1965) which is given below: Leaf area (cm 2 ) = maximum leaf length × maximum leaf width × 0.75 (Where, 0.75 is the Correction factor) The leaf area was multiplied by total number of leaves to calculate total leaf area per plant. The leaf area was recorded on 35 th , 45 th , 55 th and 65 th day.

Shoot Length of mustard plant:
The effect of HBL on the shoot length of mustard plants are shown in Figure -1. All the treatments of HBL substantially increased the shoot length of mustard plants grown in semi-arid tropics of Nizamabad over the untreated control plants. 2.0µM HBL conc. caused maximum increase of 39.21 % followed by 1.0µM conc. of HBL which caused an increase of 32.35 % and 0.5µM conc. of HBL which caused an increase of 10.78 % over controls on 35 th day. 0.5 µM HBL conc. caused maximum increase of 56.43 % followed by 1.0µM conc. of HBL which caused an increase of 48.78 % and 2.0 µM conc. of HBL which caused an increase of 45.45 % over controls on 45 th day. 0.5 µM HBL conc. caused maximum increase of 90.12 % followed by 1.0µM conc. of HBL which caused an increase of 75.92 % and 2.0 µM conc. of HBL which caused an increase of 46.91 % over controls on 55 th day. 2.0µM HBL conc. caused maximum increase of 75.5 % followed by 1.0µM conc. of HBL which caused an increase of 68.5 % and 0.5µM conc. of HBL which caused an increase of 60.16 % over controls on 65 th day.      Table -3. All the treatments of HBL substantially increased the fresh root weight of mustard plant grown in semi-arid tropics of Nizamabad over the untreated control plants that were recorded on 55 th day. 0.5 µM HBL conc. caused maximum increase of 175.6 % followed by 1.0µM conc. of HBL which caused an increase of 100 % and 2.0 µM conc. of HBL which caused an increase of 87.8 % over controls on 55 th day.
Root dry weight of mustard plant on 55 th day: The effect of HBL on the root fresh weights of mustard plants recorded on the 55 th day is shown in Table -3. All the treatments of HBL substantially increased the dry root weight of mustard plant grown in semi-arid tropics of Nizamabad over the untreated control plants that were recorded on 55 th day. 0.5 µM HBL conc. caused maximum increase of 185 % followed by 1.0µM conc. of HBL which caused an increase of 120 % and 2.0 µM conc. of HBL which caused an increase of 115 % over controls on 55 th day.

Root fresh weight of mustard plants on 65 th day:
The effect of HBL on the root fresh weights of mustard plants recorded on the 65 th day is shown in Figure -3. All the treatments of HBL substantially increased the fresh root weight of mustard plant grown in semi-arid tropics of Nizamabad over the untreated control plants that were recorded on 65 th day. 0.5 µM HBL conc. caused maximum increase of 331 % followed by 1.0µM conc. of HBL which caused an increase of 300 % and 2.0 µM conc. of HBL which caused an increase of 173.77 % over controls on 65 th day.
Root dry weight of mustard plants on 65 th day: The effect of HBL on the Root dry weights of mustard plants recorded on the 65 th day are shown in Figure -3. All the treatments of HBL substantially increased the dry root weight of mustard plant grown in semi-arid tropics of Nizamabad over the untreated control plants that were recorded on 65 th day. 0.5 µM HBL conc. caused maximum increase of 343 % followed by 2.0µM conc. of HBL which caused an increase of 313 % and 1.0 µM conc. of HBL which caused an increase of 310 % over controls on 65 th day.

Diameter of the stem (fresh) of mustard plant:
The effect of HBL on the stem fresh diameter of mustard plants recorded on the 65 th day is shown in

Diameter of the root (fresh) of mustard plant
The effect of HBL on the root fresh diameter of mustard plants recorded on the 65 th day is shown in Figure -4. All the treatments of HBL substantially increased the diameter of fresh root top of mustard plant grown in semi-arid tropics of Nizamabad over the untreated control plants that were recorded on 65 th day. 2.0 µM HBL conc. caused maximum increase of 102.56 % followed by 1.0µM conc. of HBL which caused an increase of 85.38 % and 0.5 µM conc. of HBL which caused an increase of 59.74 % over controls on 65 th day.

Diameter of the root (dry) of mustard plant
The effect of HBL on the root dry diameter of mustard plants recorded on the 65 th day is shown in Figure -4

DISCUSSION
It is a well-established fact that BRs are a new group of PGRs which play a positive role in the growth and development of plants. BRs increased the height of two field-grown inbred lines of maize (Zea mays L.) during the vegetative and early reproductive phases of plant development during the early weeks after their application (Hola et al., 2010).
Shoot growth: Seed treatment and foliar application of BL at 0.05, 0.1 & 0.2 ppm resulted in significant increase in growth of tomato (Lycopersicon esculentum L.) in terms of length, fresh and dry weight of shoots (Nafie and El-Khallal, 2002). Foliar application of BRs as foliar spray enhanced the growth of ground nut plants (Vardhini and Rao, 1998) and tomato plants (Vardhini and Rao, 2003). Supplementation of BL improved the dry-weight and growth in mulberry shoots (Kuno, 1997). Supplementataion of 24-epiBL to the media resulted in stem elongation of sweet pepper (Franck-Duchenne et al., 1998). Kim et al. (2008) studied that supplementation of BL to the culture media influences the regeneration of adventitious shoots from cultured leaf discs of tobacco. 28-HomoBL and 24-epiBL improved the growth parameters in Lycopersicon esculentum (cv. K-21) sampled at 45 (24 h after spray) and 60 days after sowing (Hayat et al., 2011). Application of BRs enhanced the growth in terms of plant height and dry weight of shoots of Plectranthus forskohlii/ coleus (Swamy and Rao, 2011) and Pelargonium graveolens (L.) Herit. (Swamy and Rao, 2009). Exogenous application of BL resulted in improved growth and anatomical characteristics for stems of dwarf pear in vitro conditions (Chen et al., 2014). Even in the present study, application of HBL significantly increased the shoot growth in mustard plants grown in the semi -arid soils of Nizamabad.
Root Growth: 24-EpiBL played a distinctive role on the growth of onion (Allium cepa) root tips (Howell et al., 2007). 24-EpiBL played a pivotal role in the development of excised roots of tomato grown in vitro (Roddick et al., 1993). BRs triggered growth promotion of apical meristems in potato tubers (Meudt et al., 1983). Supplementation of BRs resulted in increased root nodulation in French bean (Upreti and Murti, 2004) and also improvement in root epidermal cells (Cheng et al., 2014). BRs increased the root nodulation and nitrogrenase activity in ground nut plants (Vardhini & Rao, 1999) and soya bean plants (Miao et al., 2013). Vardhini et al. (2012) reported that application of BRs increased the qualitative growth of radish in terms of root fresh weight, root dry weight, oxalic acid, ascorbic acid, folic acid and niacin. Application of 24-EpiBL (Platonova & Korableva, 1994) and BL (Korableva et al., 2002) promoted the growth of apical meristem of potato tubers. (22S, 23S)-HomoBL enhanced the rooting capacity and survival of adult Norway spruce cuttings (Rönsch et al., 1993). BL increased the growth and ginsenoside content of Panax quinquefolium hairy root (Zhou et al., 2003). Treatment with two synthetic BRs improved the in vitro growth of Yucca filamentosa, Dioscorea rotundata and Ananas comosus (Bieberach et al., 2000). Hayat et al. (2011) reported that 28-homoBL and 24-epiBL increased the growth of Lycopersicon esculentum. Imbibitions of 24-epiBL to pea (Pisum sativum L.) cv. Climax seeds subjected to sodium chloride stress exhibited increased germination, embryo axis length and most of the aspects of shoot and root growth at seedling stage (Shahid et al., 2011). Earlier studies clearly emphasized that external supplementation/application of BRs improved plant growth in the case of wheat (Sairam, 1994) and groundnut (Vardhini and Rao, 1998) which is in tune with the present study where foliar application of HBL substantially increased the root growth of mustard plants grown in semi-arid tropics of Nizamabad.

Foliar growth:
The enhanced shoot and root growth of HBL treated mustard plants was associated with increased foliar growth. Application of BRs resulted in proliferation of leaf cells (Nakaya et al., 2002) of Arabidopsis thaliana, leaf expansion in Solanum lycoperscion (Zhiponova et al., 2013) and improvement of leaf nutrition in Camellia oleifera (Zhou et al., 2013) was observed. Further, regeneration of adventitious shoots from cultured leaf discs of tobacco (Kim et al., 2008) was also reported. Arteca and Arteca (2001) also reported that BRs not only induce exaggerated growth in hydroponically grown Arabidopsis thaliana, but also control the proliferation of its leaf cells (Nakaya et al., 2002). Schlagnhaufer & Arteca (1985) studied that BRs induced epinasty in tomato (Lycopersicon esculentum cultivar Heinz 1350) plants. Oh and Clouse (1998) and Ho (2003) reported that BL accelerated the rate of cell division in isolated leaf protoplasts of Petunia hybrida. Application of 24-epiBL and 28-homoBL enhanced the growth in terms of leaf number, leaf area, and the dry weight of leaves of coleus (Swamy and Rao, 2011). BL improved the physiological characteristics of Brassica oleracea var. italica leaves (Wang and Yang, 2009). Exogenous application of BL resulted in improved growth and anatomical characteristics for leaves of dwarf pear in vitro conditions (Chen et al., 2014). The present study reveals that application of HBL on mustard plants as foliar spray promoted the shoot, root and foliar growth of mustard plants grown in semi-arid soils of Nizamabad. The soils of Nizamabad are saline and dry in nature inhibiting the growth of plants. BRs have the ability to promote growth of plant under stressful conditions. The present study reveals a new insight that application of HBL overcame the negative effect of the semiarid conditions of the soil (reflected in the control plants) and promoted the shoot, root and foliar growth (reflected in the HBL-treated plants) of mustard.