Acta Physiologiae Plantarum

, Volume 33, Issue 3, pp 905–910

Effect of ethephon and calcium chloride on growth and biochemical attributes of sugarcane bud chips

Authors

    • Plant Physiology and Biochemistry DivisionIndian Institute of Sugarcane Research
  • S. Solomon
    • Plant Physiology and Biochemistry DivisionIndian Institute of Sugarcane Research
  • A. K. Shrivastava
    • Plant Physiology and Biochemistry DivisionIndian Institute of Sugarcane Research
  • A. Chandra
    • Plant Physiology and Biochemistry DivisionIndian Institute of Sugarcane Research
Original Paper

DOI: 10.1007/s11738-010-0617-4

Cite this article as:
Jain, R., Solomon, S., Shrivastava, A.K. et al. Acta Physiol Plant (2011) 33: 905. doi:10.1007/s11738-010-0617-4
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Abstract

The present study was aimed at improving sprouting and establishment of bud chip seed stocks of sugarcane cultivar CoSe 92423 by pre-planting soaking in growth-promoting chemicals viz ethephon (0.1 g dm−3) and calcium chloride (1 g dm−3) along with water-soaked control for 24 h. Treated bud chips recorded higher bud sprouting, shoot height, root number, fresh weight of leaves, shoot and roots, and plant vigor index. In both the treatments, reducing sugars contents, acid invertase, and ATPase activity increased in developing sprouts; increase was about 86.5 and 40.7% in reducing sugars, 28 and 70% in acid invertase, and 15 and 23% in ATPase activities over control by ethephon and calcium chloride treatment, respectively. Reducing sugars contents and activity of acid invertase and ATPase enzymes of sprouted buds exhibited significant positive correlation with bud sprouting and plant vigor index. These findings indicate that soaking of bud chips in growth-promoting chemicals viz ethephon (0.1 g dm−3) and calcium chloride (1 g dm−3) solutions helps in enhancing bud sprouting, root growth, and plant vigor by altering some of the key biochemical attributes essential for the early growth and better establishment of bud chips under field conditions which is otherwise poor in untreated chips.

Keywords

Bud chipReducing sugarsAcid invertaseATPaseCalcium chlorideEthephonSugarcane

Abbreviations

ATPase

Adenosine triphosphatase

EDTA

Ethylenediaminetetraacetic acid

HCl

Hydrochloric acid

TCA

Trichloroacetic acid

KCl

Potassium chloride

FW

Fresh weight

Introduction

Sugarcane (Saccharum spp., Poaceae) is vegetatively propagated crop widely grown in a zone around the world within 30° of the equator. The first “plant” crop is generally harvested from 12 to 24 months after planting; thereafter, “ratoon” crops may be harvested at shorter to equal time periods. Ratoons may be grown from one to several cycles. The fully mature stalks contain juice of 18–20% sucrose. More than 80 countries grow sugarcane, producing 111.8 million metric tons of white sugar. About 75% of the world’s sugar (sucrose) supply is from sugarcane, and the other 25% from sugar beet. Sugarcane is used also for the production of ethanol and biomass which are excellent sources of alternative energy.

The commercial cultivation of sugarcane (Saccharum spp. hybrid) is carried out through vegetative propagation using stalk cuttings or setts, and nearly 8–10% of total produce is used as planting material. Crop establishment is therefore proving to be uneconomical as cost of “Seed Cane” used for replanting accounts for over 20% of the total cost of production. In conventional system prevailing in India, about 6–8 tons seed cane/ha is used as planting material, which comprises about 25–30 cm stalk pieces having 2–3 buds. This large mass of planting material poses a great problem in transport, handling, and storage of seed cane that can undergo rapid deterioration and reducing the viability of buds. A viable alternative to reduce the mass and improve the quality of seed cane would be to plant-excised axillary buds of cane stalk, called bud chips, which are less bulky, more economical, and more easily transportable seed material. A famous sugarcane physiologist van Dillewijn (1952) stated that a small volume of tissue and a single root primordium adhering to the bud are enough to ensure germination in sugarcane. He has also stated that where growing conditions are favorable, stalk cutting with only one viable bud performed well as seed material. Studies carried by Narasimha Rao and Satyanarayana (1974) and Ramaiah et al. (1977) showed the feasibility of eliminating the internode part of the seed piece and using only buds for commercial planting. However, their efforts were limited to small plot experiments.

Extensive work has been done using different types of seed cane materials such as single bud settlings, bud chip raised seedlings, 1–3 bud setts for crop establishment, and then determining the effect of the planting material on growth and yield of sugarcane in India (Reddy et al. 1986). It was observed that, due to saving in seed material, the maximum net returns were obtained with bud chip raised seedlings. Earlier studies established that about 80% by weight of the sett-planting seed material can be saved by planting bud chips (Narasimha Rao 1977; Gokhale 1977; Narendranath 1992; Iqbal et al. 2002; Prasad and Sreenivasan 1996; Tamilselvan 2006).

Earlier studies carried out at various locations in India have established the potential of bud chip as planting material, but this technology could not be scaled up to commercial levels due to poor sprouting of bud chips and subsequent establishment under field conditions. Keeping in view, a research project has been started at the Indian Institute of Sugarcane Research, Lucknow to work out the physio-biochemical basis of bud chip viability for long-duration storage, its treatment, storability, raising of seedlings, and their establishment in field. Under this project, experiments were conducted to maintain the viability of bud chips for long-duration storage. Results obtained indicated that bud chips stored in polyethylene bags after fungicide treatment and stored at low-temperature conditions (10 ± 1°C) exhibited about 80% bud germination even after 10 days of storage than one stored at room temperature (about 40%). In bud chips, moisture content was within the range of 70–77% during storage similar to 0 day moisture content. Present study was aimed to assess the stimulatory effect of growth-regulating chemicals viz calcium chloride (1 g dm−3) and ethephon (0.1 g dm−3) on bud sprouting and early growth characteristics of bud chip raised sugarcane plants and related biochemical attributes.

Ethephon, an ethylene-generating compound is a versatile growth regulator. Ethylene has been implicated as a factor that controls the timing of seed germination, the rate and dimensions of etiolated seedling growth and leaf expansion, the initiation and progressing of abscission and fruit ripening, and the expression of a number of stress-related responses in plants (Anthony and Schaller 1996; Cassab et al. 1988). In sugarcane, the growth-stimulating effects of ethephon and calcium chloride have been demonstrated on sprouting of aerial and underground stubble buds of winter-initiated ratoons (Solomon et al. 1998; Jain et al. 2009).

Calcium is an essential plant nutrient which stimulated the growth and development of plants. It is required for structural roles in the cell wall and membranes as a counter-cation for inorganic and organic anions in the vacuole and as an intracellular messenger in the cytosol (Burstrom 1968; Marschner 1995; White and Broadley 2003). It plays an important role in various physiological processes by acting as a second messenger in the transduction of endogenous and exogenous signals (Hepler and Wayne 1985). Calcium is necessary for cell elongation and known to activate a number of enzymes such as phospholipase-D, lecithinase, ATPase, and amylase (Davidson and Long 1958; Kalckar 1944; Chrispeels and Varner 1967; Hepler 2005).

Materials and methods

A soil tray culture experiment was conducted in a completely randomized design with three replications to evaluate the response of growth-promoting chemicals viz ethephon and calcium chloride separately on bud sprouting and early growth of sugarcane bud chips at Indian Institute of Sugarcane Research, Lucknow, India. The bud chips from a commercially cultivated sugarcane (Saccharum sp. hybrid) variety CoSe 92423 were scooped out from freshly harvested sugarcane stalk with the help of a hand-operated bud scooping device. These bud chips were immediately submerged in freshly prepared aqueous solutions containing ethephon (0.1 g dm−3) and calcium chloride (1 g dm−3) for 24 h at room temperature (25 ± 1°C). The treated bud chips along with water-soaked control were planted in large plastic trays containing farm soil and organic manure commonly used in conventional sugarcane planting. These trays were kept under net house conditions (day temperature 31.1–37.6°C, night temperature 15.6–19.7°C), and periodic observations were taken with respect to agronomic traits.

Sprouting percent and biochemical parameters viz reducing sugars, invertase, and ATPase activity were measured in growing sprouts after 1 week of planting.

Reducing sugars assay

For the estimation of sugars, finely chopped buds (0.5 g FW) were fixed in 10 cm3 boiling 80% ethanol (Nelson 1944). The material was ground and centrifuged at 6,000×g at room temperature. To determine reducing sugars, 0.5 cm3 aliquot was drawn from aqueous phase and mixed with 0.5 cm3 copper reagent and kept in boiling water bath for 10 min. After cooling, 0.5 cm3 arsenomolybdate reagent was added, and the volume was made up to 25 cm3 with distilled water, and absorbance was measured at 540 nm. The results were expressed as mg g−1 FW.

Acid invertase activity assay

The activity of acid invertase was determined in fresh buds after extracting the material (0.5 g FW) in 10 cm3 of 0.1 M citrate buffer (pH 5.4) by the method of Hatch and Glasziou (1963) with some modifications. The reaction was initiated by adding 0.5 cm3 of 0.2 M sucrose to a reaction mixture containing 1.0 cm3 of 0.1 M citrate buffer (pH 5.4) and 1.0 cm3 enzyme extract. The reaction was set at 30°C for 30 min and stopped by adding 1.5 cm3 saturated solution of sodium sulfate followed by 1.0 cm3 boiling ethanol. The reaction mixture was transferred to a boiling water bath and heated for 5 min. After removing ethanol by evaporation, the volume of the reaction mixture was made up to 10 cm3 with distilled water and centrifuged at 6,000×g for 15 min. The amount of invert sugars was estimated in 0.5 cm3 aliquot of the supernatant, according to Nelson (1944). The acid invertase activity was expressed as μg invert sugars mg−1 protein.

ATPase activity assay

The activity of ATPase was determined in fresh buds after extracting the material (0.2 g FW) in 2 cm3 grinding medium containing 0.05 M Tris–HCl buffer pH 7.5, 0.003 M EDTA, and 0.25 M sucrose by the method of Fisher and Hodges (1969). The reaction mixture containing 1 cm3 0.1 M Tris–HCl buffer pH 7.5, 0.05 cm3 1 M KCl, and 1.0 cm3 suitably diluted enzyme extract was stabilized at 38°C. The reaction was initiated by the addition of 0.2 cm3 0.05 M adenosine triphosphate (ATP) to the reaction mixture, and after 15 min the reaction was stopped by the addition of 0.5 cm3 20% (w/v)TCA. The reaction mixture was centrifuged at 2,000×g at 4°C for 10 min. The amount of inorganic phosphorus (Pi) was estimated in 0.5 cm3 aliquot of the supernatant, according to Fiske and Subbarow (1925). The activity of ATPase was expressed in μg Pi liberated mg−1 protein.

Soluble protein assay

Soluble protein was determined in enzyme extracts according to Lowry et al. (1951). The intensity of blue color developed was measured on a Systronics-117 spectrophotometer at 640 nm using bovine serum albumin as the calibration standard.

Growth parameters

Growth parameters, namely shoot height, root number, and fresh weight of different plant parts, were determined 35 days after planting. Plant vigor index was determined as per formula given by Bewly and Black (1982). Plant vigor index = sprouting percent × shoot height.

Statistical analysis

The experiment was conducted in a completely randomized design (CRD) with three replications. The data were recorded in three replications. Standard deviation (±SD) was calculated using means of three replicates (Panse and Sukhatme 1985).

Results and discussion

Effect of chemical treatment on growth of bud chip raised sugarcane plants is given in Fig. 1. Treatment with growth-promoting chemicals improved the sprouting of bud chip seed stocks which ranged from 32 to 36% over control due to ethephon and calcium chloride treatment, respectively (Fig. 2). Increase in sprouting with chemical treatment was closely associated with a corresponding increase in reducing sugars and acid invertase activity in sprouting buds. Earlier studies carried out in subtropical India using ethephon and calcium chloride as growth promoter has shown positive results on the sprouting of sugarcane sett and underground buds (Solomon et al. 1998; Jain et al. 2009). Solomon et al. (1998) have shown an enhancement of about 13–17% in sett sprouting in ethephon-treated seed cane pieces. A pre-harvest foliar application of ethephon improved stubble bud sprouting in winter-initiated ratoons (Solomon et al. 2001). Improvement in sprouting by calcium application may be due to enhanced activity of ATPase activity in presence of calcium ions (Kalckar 1944; Chrispeels and Varner 1967; Jain et al. 2009). Besides, calcium was seen to enhance the ability of cytokinin and to promote cotyledon expansion (Leopold et al. 1974; White and Broadley 2003).
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-010-0617-4/MediaObjects/11738_2010_617_Fig1_HTML.jpg
Fig. 1

Effect of ethephon and calcium chloride on growth of bud chip raised sugarcane settlings [control, ethephon (0.1 g dm−3), calcium chloride (1 g dm−3)]: a Initial plant material, b bud chips and leftover cane, c bud chip raised sugarcane settlings

https://static-content.springer.com/image/art%3A10.1007%2Fs11738-010-0617-4/MediaObjects/11738_2010_617_Fig2_HTML.gif
Fig. 2

Effect of ethephon and calcium chloride on bud sprouting, bud weight, and biochemical attributes of sprouted buds of bud chip seed stocks in sugarcane. T1 control, T2 ethephon (0.1 g dm−3), T3 calcium chloride (1 g dm−3)

Chemically treated bud chips showed higher contents of reducing sugars in sprouted buds as compared to water-soaked control (Fig. 2). Reducing sugars contents increased from 4.45 mg g−1 FW at control to 8.3 mg g−1 FW at ethephon and 6.26 mg g−1 FW at calcium treatment. Similar changes were also observed in the sprouted buds of winter-initiated ratoon of sugarcane subjected to a pre-harvest foliar treatment of ethephon (Shrivastava et al. 2006). This has also been corroborated by Solomon and Kumar (1987) who reported a progressive increment in acid invertase activity during sprouting of cane buds in different sugarcane varieties grown in north India. Increase in reducing sugars contents in the sprouted buds indicates higher transportation of hexoses towards the growing apical meristem due to ethephon and calcium chloride application.

The bud chips soaked in ethephon and calcium chloride solutions showed higher activity of acid invertase and ATPase enzymes in sprouted buds (Fig. 2). Increase in enzyme activity of acid invertase and ATPase activity indicates enzyme activation during bud sprouting by growth-promoting chemicals. Acid invertase hydrolyzes sucrose into hexoses (glucose and fructose) and ATPase liberated inorganic phosphorus to provide cells with carbon and energy for the synthesis of different compounds essential for sprouting and subsequent growth of the underground buds (Jain et al. 2007). Further, these hexoses may increase the osmotic pressure of cells, suggesting a possible function of acid invertase on cell elongation and plant growth. Hatch and Glasziou (1963) observed a linear correlation between the rate of shoot or internode expansion and acid invertase activity in sugarcane. Gayler and Glasziou (1972) further delineated a strong relationship between total invertase activity and elongation of immature internodal tissue in sugarcane, indicating a similar mechanism in chip buds that subsequently differentiate into tillers. Soluble protein content determined in enzyme extracts of sprouted buds decreased with chemical treatments (Fig. 2); decrease in its concentration indicated higher rate of protein hydrolysis during bud sprouting. Biochemical attributes viz reducing sugars, acid invertase, and ATPase activity of sprouted buds exhibited significant positive correlation with bud sprouting and plant vigor index (Table 1).
Table 1

Correlation between bud sprouting and plant vigor index with biochemical parameters of sprouted buds of sugarcane bud chips

Biochemical parameters

Bud sprouting (%)

Plant vigor index

Reducing sugars

0.790***

0.928***

Invertase activity

0.986***

0.903***

ATPase

0.969***

0.862***

*** P < 0.001

Treated bud chips showed significant increase in root number and weight; increase was about 38 and 8% in root number and about 160 and 137% over control in root weight by ethephon and calcium treatment, respectively (Figs. 1, 3). Similar to sugarcane, calcium and ethephon are known to stimulate root growth in other plants (Hepler 2005; Raven et al. 1992). Higher root emergence due to chemical treatment helps in early and better establishment of bud chip seed stocks under field conditions.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-010-0617-4/MediaObjects/11738_2010_617_Fig3_HTML.gif
Fig. 3

Effect of ethephon and calcium chloride on growth parameters of bud chip raised sugarcane settlings. T1 control, T2 ethephon (0.1 g dm−3), T3 calcium chloride (1 g dm−3)

The shoot height showed marked improvement in both the treatments; it was about 50 and 32% over control by ethephon and calcium treatment, respectively. Improvement in stalk length by ethephon spraying at early stage of tillering was also reported by Li and Solomon (2003). Similarly, leaf and shoot weight increased significantly due to both the treatments (Fig. 3).

The plant vigor of bud chip raised settlings was enhanced due to chemical treatment; this response was better with ethephon treatment (Fig. 3). Earlier reports have indicated that ethephon stimulated tillering and stalk formation with corresponding yield increase in cereal crops (Van Andel 1973).

The initial experiments carried out by our group under subtropical climate have indicated that soaking bud chip seed material in ethephon and calcium chloride solutions improves bud sprouting, root activity, and plant vigor. Higher contents of reducing sugars and acid invertase activity in sprouted buds indicates that both calcium chloride and ethephon promote cell division, growth, differentiation of cells, and synthesis of sucrose in growing meristem of sugarcane. Due to lower food reserves (1.2–1.8 g sugars/bud) in bud chip seed stocks compared to conventional 3-bud seed material (6.0–8.0 g sugars/bud), the food reserves and moisture in the bud chip depletes at a faster compared to 2 or 3 bud sett which is reflected in their poor sprouting and early growth. Treatment of bud chips with growth-promoting chemicals probably circumvent these conditions of stress by faster initiation of shoot, roots, early leaf development, higher photosynthetic rate, and activating essential biochemical reactions prerequisite for the establishment and survival of bud chip plantlet in the soil.

In conclusion, pre-planting soaking of sugarcane bud chip seed stocks in growth-promoting chemicals, calcium chloride (1 g dm−3) and ethephon (0.1 g dm−3), improves their survival and establishment under field conditions. Further research work in relation to impact on bud chip storage following treatment with growth-promoting chemicals is underway. The bud chip technique would help sugarcane farmers and personnel to transport their valuable cane seed any where with less risk, assured survival, and good establishment. Besides, sending the treated bud chips instead of canes from one location to other would largely reduce the chances of sett-transmitted diseases. This system will also help in rapid multiplication of seed of improved sugarcane varieties.

Acknowledgments

We would like to thank Director, Indian Institute of Sugarcane Research, Lucknow, India for his encouragement and critical reading of this manuscript.

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2010