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EFFECT OF NaCl STRESS ON CALLUS MORPHOLOGY AND GROWTH OF SUGARCANE CALLUS CULTURES (cv. SPF 234 AND cv. HSF 240).

Byline: N. Munir and F. Aftab

ABSTRACT: Salt stress is one of the major abiotic stresses limiting crop productivity. Growth of sugarcane which is an important cash crop is affected by various biotic and abiotic stresses. As sugarcane is a glycophyte it do not thrive well in saline soils. Response of two sugarcane cultivars (cv. SPF 234 and cv. HSF 240) was observed in response to various salt levels during the present study. Callus cultures were transferred to the standardized media for callus formation supplemented with various NaCl concentrations (MS + 13.5 uM 2, 4-D + 0-160 mM NaCl; 9 treatments). A variation in callus morphology was observed in both the tested sugarcane cultivars upon transfer to NaCl- containing media. It was found that the callus cultures become brown and necrotic when subjected to salt stress. A general decrease was observed in fresh weights of callus cultures when subjected to salt stress. However less fresh weight reduction was recorded in SPF 234 callus cultures as compared to callus cultures of cv. HSF 240.

Hence SPF 234 appeared to be more salt tolerant as compared to cv. HSF 240 under in vitro conditions. This study therefore, is an important step forward in improving salt tolerance of sugarcane by in vitro techniques.

INTRODUCTION

Soil salinity is a major problem that threatens the productivity of crops in many countries of the world including Pakistan (Ashraf, 1994). It has been reported that 397 million hectares of land throughout the world are salt affected (FAO, 2005). Sugarcane is an important industrial cash crop in Pakistan but its annual yield is far below the existing potential. Soil salinity is one of the reasons that result in low productivity of sugarcane (Akhtar et al., 2003). Furthermore, it also decreases the quality of sugarcane juice by decreasing its sucrose content (Lingle and Weigand, 1996). Salt stress is expressed by many morphological and metabolic changes Plant tissue culture techniques provide a useful tool to produce stress tolerant plants including the plants resistant to soil salinity. These techniques provide a promising and feasible approach to develop salt tolerant plants (Jain, 2001).

Not much work has been reported on in vitro selection and response of callus cultures towards salt stress in Sugarcane cultivars growing in Pakistan. The present investigation was conducted to observe the in vitro response of two sugarcane cultivars (cv. SPF 234 and cv. HSF 240) towards NaCl stress.

MATERIALS AND METHODS

Establishment of Callus cultures: Callus cultures were established from explants ranging from 5-8 mm in diameter (2-3 mm thick) derived from young inner 2-3 whorls of leaves of two sugarcane cultivars (SPF 234 and HSF 240) on MS (Murashige and Skoog, 1962) medium

supplemented with 13.5 M 2,4-D. The young inner 2-3 whorls of sugarcane leaves, wrapped deeply within the mature ones were used as explants. The inner leaves were surrounded by many layers of leaves and therefore, did not require any particular disinfection. However, to minimize the chances of contamination, during explant preparation, 20-30 cm long field-grown plant material was initially surface sterilized with 95 % ethyl alcohol

The calluses were induced under dark conditions and later transferred to 16-h photoperiod (35 mmol m-2 s-

1) provided by cool fluorescent tube lights 27 +- 2 oC

Salt stress to callus cultures: Different concentrations of NaCl were used in the present study and added directly into already prepared MS medium. Medium was supplemented with 9 different salt concentrations (0-160 mM) to observe the effect of salt stress on sugarcane callus culturesCallus morphology and fresh weights (g) were recorded at day 90, 120 and 150.

Experimental Design: The experiment was perfomed in complete randamized design with 20 replicates per treatment. Each experiment was repeated thrice.

Statistical Analysis: The data was analyzed by univariate analysis of variance by using SPSS version 11. The values were also compared statistically by Duncan's multiple range test.

RESULTS

During the present study, it was observed that sugarcane callus cultures underwent several morphological changes with the addition of salt in the medium over successive subcultures. Morphology of sugarcane callus cultures (cvs. SPF 234 and HSF 240) at various NaCl levels is shown in Table 1 and 2, respectively. It is evident from the data that 60-days-old callus cultures of cv. SPF 234 maintained on MS basal medium supplemented with 13.5 mM 2,4-D were greenish-yellow, friable and granular. When 60-days-old calluses were shifted to the same medium containing various NaCl levels (0-160 mM), variation in callus morphology of both the cultivars was observed. It was observed that calluses during different subcultures (at day 90, 120 or 150) had no signs of necrosis in the absence of salt in the medium. However on NaCl containing medium (20-160 mM NaCl) callus cultures turned brown and complete necrosis of callus culture was obtained at 160 mM NaCl at day 90 and further maintenance was not possible due to necrosis.

For cv. HSF 240, the callus color and morphology was also found to be affected with increasing salt concentration. The callus cultures before NaCl treatment at day 60 were greenish-yellow and friable. Like the other cultivar, most of the callus cultures of cv. HSF 240 maintained at 0 mM NaCl level did not show any sign of necrosis during different subcultures at day 90, 120 or 150. Upon shifting of these callus cultures to MS medium with various salt concentrations, changes in morphological characteristics were recorded. It was observed that with an increase in salt concentration as well as with the passage of time (during successive subcultures), greenish-yellow callus started turning brownish in color. For this cultivar (unlike SPF 234), complete browning of the callus cultures, however, was observed when MS medium was supplemented with 140 mM NaCl level or above. Representative pictures of callus cultures at 0mM NaCl level and at various salt levels are given in Fig 1.

A significant effect of the tested media was recorded on fresh weights of callus cultures at day 90,120 and 150 after NaCl treatment (Table 3). It was observed that callus cultures of both the cultivars showed a gradual decrease in fresh weights with an increase in salt concentration at day 90, 120 or 150 as compared to the control. During different subcultures, fresh weights of the calluses of both sugarcane cultivars maintained at 0 mM NaCl level (control), increased slightly up to day 150, i.e., the final day of data collection. The callus cultures of cv. SPF 234 at day 120 up to 60 mM NaCl level had slightly greater fresh weights as compared to callus cultures maintained on the same NaCl level at day 90. At day 150, once again reduction was observed in fresh weights of callus cultures subjected to various NaCl concentrations. Almost similar growth trend was observed in callus cultures of cv. HSF 240.

However, at 40 or 60 mM NaCl levels, fresh weight of callus cultures at day 120 had similar values (1.06 gs cultivar, once again reduction was observed in fresh weights of NaCl-treated callus cultures as compared to the control at day 150. The overall significance thus depicts reduced callus growth with an increasing salt level.

Table 1: Morphology of sugarcane callus cultures (cv. SPF 234) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150 a

###NaCl

###NaCl treatment###after NaCl treatment

###(mM)

###At day 60###At day 90###At day 120###At day 150

###Greenish-yellow, friable,###Greenish-yellow,###Greenish-yellow,###Whitish-yellow,

###0

###granular###friable, granular###friable, granular###friable, granular

###Greenish-yellow, friable,###Greenish-yellow,###Greenish-yellow with some whitish###Brownish-yellow,

###20

###granular###friable, granular###portion, granular###friable, granular

###Greenish-yellow,###Brownish-yellow, friable,###Greenish-brown,

###40###Greenish-yellow, friable, granular

###friable, granular###granular###smooth surfaced

###Greenish-yellow,###Greenish-yellow###Yellowish-brown,

###60###Greenish brown, friable, granular

###friable, granular###granular###friable,granular

###Greenish-yellow,###Off-white with some brown Off-white with some reddish- brown###Blackish-brown,

###80

###friable, granular###portion, granular###portion, granular###friable, granular

###Greenish-yellow,###Brownish-off white, friable,###Yellowish-brown,

###100###Yellowish-brown, friable, granular

###friable, granular###granular###translucent

###Greenish-yellow,###Brownish-off white, friable,###Blackish-brown,

###120###Blackish-brown, necrotic

###friable, granular###granular###necrotic

###Greenish-yellow,###Brownish-off white, friable,###Brownish-black,

###140###Blackish-brown

###friable, granular###granular###necrotic

###Greenish-yellow,

###160###Brownish-black###ND b###ND b

###friable, granular

a Callus morphology is based on 60 culture vessels per NaCl treatment.

b ND: Not determined because callus cultures could not be maintained at these salt levels over successive subcultures

Table 2: Morphology of sugarcane callus cultures (cv. HSF 240) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150 a

###Callus###morphology###Morphology of sugarcane callus cultures

###NaCl

###before NaCl treatment###after NaCl treatment

###(mM)

###At day 90###At day 90###At day 120###At day 150

###Greenish-yellow,###Greenish-yellow,###Greenish-yellow,###Translucent,

###0

###friable, granular###friable, granular###friable, granular###yellowish-green

###Off white with some

###Greenish-yellow,###Greenish-yellow,###Yellowish-brown,

###20###brown portion,

###friable, granular###friable, granular###friable, granular

###friable, granular

###Greenish-yellow,###Brownish-yellow,###Greenish-brown, friable,###Greenish-brown,

###40

###friable, granular###friable, granular###granular###smooth

###Greenish-yellow,###Brownish-yellow,###Brownish off white,###Yellowish-brown,

###60

###friable, granular###friable, granular###friable, granular###smooth

###Brownish with some

###Greenish-yellow,###Brownish-yellow, friable, Brownish off-white,

###80###off-white portion,

###friable, granular###granular###friable, granular

###friable, granular

###Greenish-yellow,###Brownish-off white,###Blackish-brown, friable, Brownish-black, smooth

###100

###friable, granular###translucent###granular###surfaced

###Greenish-yellow,###Brownish off-white,###Brownish-black,

###120###Blackish-brown, necrotic

###friable, granular###friable, granular###necrotic

###Greenish-yellow,

###140###Blackish-brown, necrotic###ND b###ND b

###friable, granular

###Greenish-yellow,

###160###Brownish-black, necrotic###ND b###ND b

###friable, granular

a Callus morphology is based on 60 culture vessels per NaCl treatment.b ND: Not determined because callus cultures could not be maintained at these salt levels over successive subcultures.

Table 3: Effect of different NaCl levels (0-160 mM) supplemented to MS medium on fresh weights of sugarcane

(cvs. SPF 234 and HSF 240) callus cultures under dark conditions at day 90, 120 and 150A Data presented here are the means of 60 culture vessels per NaCl treatment (20 replicates per treatment and the experiment was repeated thrice).

###Fresh weight of callus cultures A

###(g)NaCl Level###Cultivars

(mM)###SPF 234###SPF 234

###Days###Days

###90###90###90###90###90###90

0###1.24 a###1.29 a###1.29 a###1.22 a###1.26 a###1.30 a

20###1.14 b###1.20 b###1.17 b###1.12 b###1.18 b###1.02 c

40###1.13 b###1.17 bc###1.04 c###1.13 b###1.06 c###1.0 d

60###1.13 b###1.19 c###1.05 c###1.12 b###1.06 c###1.0 d

80###1.10 c###1.07 d###1.02 d###1.04 c###1.07 c###1.05 b

100###1.04 d###1.05 d###1.01 d###1.01 cde###0.99 d###0.98 d

120###1.04 d###1.02 e###1.01 d###1.02 cd###1.02 cd###0.98 d

140###1.03 d###1.02 e###0.98 e###0.90 de###ND B###ND B

df###8 and 261###7 and 232###7 and 232###8 and 261###6 and 203###6 and 203

Different letters within a specific column represent significant difference at P= 0.05 according to

Duncan's Multiple

Range Test.

Data were transformed using 3[?]y (where y is the fresh weight) to normalize the data. Non-transformed mean values are presented.

B ND represents that the value could not be determined due to complete callus necrosis. , NS Significant at 1% level ( ) or non-significant (NS) according to F test with df mentioned against each.

DISCUSSION

It is evident from the results of this study that60-days-old callus cultures on MS basal medium were greenish yellow, friable and granular. When 60-days-old calluses were shifted to various NaCl levels (0-140 mM), variation in callus morphology was quite distinct and data to that effect were recorded at day 90, 120 and 150. Thus our results conform to the earlier findings of Gandonou et al., (2005 a, b) who have also reported that salt stress affect callus morphology. It is not surprising to note that these results are also true for many other members of the family Poaceae, e.g., wheat (Karadimova and Dambova,1993). Arzani and Mirodjagh (1999) have also observed similar results and reported brown coloration, necrosis and inhibited growth of callus cultures of wheat at higher NaCl concentrations.

Jaiswal and Singh (2001) observed in chick pea that the control treatment (without stress) could establish green and friable callus while NaCltreatments resulted in varying degrees of browning andnecrosis depending on the NaCl concentration used.

During the present work, a decrease in fresh weight of callus cultures was also observed with increasing salt concentration in the medium. Decline of callus growth due to NaCl stress as observed in the present study is a usual phenomenon in many plant tissues subjected to stress (Greenway and Munns, 1980; Reddy and Vaidyanath, 1986; Rains, 1989; Cushman et al., 1990). This retardation of growth may be due to the fact that certain amount of the total energy available for tissue metabolism is channeled to resist the stress (Cushman et al., 1990).

In sugarcane, Gandonou et al., (2005 a, b; 2006) and Errabii et al., (2006) studied the effect of salt on sugarcane callus cultures and found that fresh weight of calluses decreased with the corresponding increase in the concentration of NaCl in the culture medium. A gradual decrease in fresh as well as dry weights under salt stress has also been reported by Rahnama et al., (2003) and Agarwal and Pandey (2004) in potato and cassia plants, respectively. Similarly, Nasir et al., (2000) also observed a same decrease in fresh weight of sugarcane seedlings under salt stress.).

It was observed that the two sugarcane cultivars used during the present study (cv. SPF 234 and cv. HSF240) were different in their salt tolerance level. This was quite evident by the data on fresh weight of calluscultures after salt stress treatments. Similar findings were reported for rice (Lutts et al., 1996; Basu et al., 2002), wheat (Barakat and Abdel-Latif, 1996) and sunflower (Alvarez et al., 2003) where NaCl stress was shown toreduce callus growth and the fact that different genotypesresponded differently.

In conclusion, results of the study indicate that the different response of cultivars towards salt stress that can be an important step forward in using the cultivars for in vitro selection and improving their salt tolerance.

Acknowledgement: Authors are thankful to Higher Education Commission for awarding Merit scholarship for PhD studies to Ms. Neelma Munir that facilitated the accomplishment of this research work.

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Department of Biotechnology, Lahore College for Women University, Lahore

Department of Botany, University of the Punjab, Q. A. Campus Lahore-54590, Pakistan.

Corresponding Author E-mail: neelma.munir@yahoo.com
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