CALLUS INDUCTION AND REGENERATION IN WHEAT CULTIVARS UNDER SODIUM AND CALCIUM CHLORIDES SALT STRESS

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1 Sarhad J. Agric. Vol. 23, No. 3, 2007 CALLUS INDUCTION AND REGENERATION IN WHEAT CULTIVARS UNDER SODIUM AND CALCIUM CHLORIDES SALT STRESS Naheed Akhtar *, Sajjad Ur Rehman Chughtai ***, Zubeda Chudhry **, Mohammad Hanif Munawwar *** and Mozammil Hussain *** ABSTRACT The study was conducted at the Agricultural Biotechnology Programme, National Agricultural Research Centre, Islamabad in 1995 to ascertain differences between four Pakistani wheat varieties Arz, Pak-81, Lu-26 and Pavon, with respect to their tolerance to salinity of sodium chloride (NaCl) and calcium chloride (CaCl 2 ) stresses. In vitro callogenesis and organogenesis responses were studied. Murashige and Skoog (MS) modified medium supplemented with different concentrations of chloride salts was used. Results clearly indicate striking differences in callus inductions and regeneration under salinity stresses of sodium chloride and calcium chloride salts on four wheat varieties. The most tolerant cultivar was LU- 26 followed by Pavon, Pak- 81 and Arz respectively. INTRODUCTION Of the world s available land area (14 billion ha), only around 3.2 billion ha are arable and 2 this arable land is subjected to excess salinity, a problem both in arid regions and in some areas near the ocean (Nabors and Dykes, 1985). Salinity has created an alarming situation in Pakistan. The poor crop growth in saline soils is thought to be due to such causes as imbalance in mineral nutrition, reduction in water uptake and/or direct toxicity of salts to plants. Agricultural crops and their varieties differ considerably with regard to salt tolerance at various growth stages, germination and seedling being generally the most critical ones (Brown and Hayward, 1956). Soil salinity at the time of seedling and emergence of rice resulted in greater decrease in grain yield than a similar level of salinity induced when the plants were six weeks old. The reduction in yield was attributed to a decrease in germination percentage in selecting wheat varieties for saline soils, particular attention should be paid to salt tolerance during emergence because poor crops frequently result from failure to obtain a satisfactory stand. At higher concentrations, calli lost water content and represented degenerated tissue, and even died. (Kapp, 1947). Seedlings of Triticum aestivum cultivars Lyallpur-73, Pak-81 and Lu 26-S were grown in solution culture at varying concentrations of NaCl for 8 days (Hanif and Davies, 1998). Increasing concentrations of NaCl reduced the size of apical meristem and caused the root hairs to form closer to the root tip in all the cultivars. One month old calli of wheat cultivars Lu-26 and Potohar in MS liquid medium were subjected to different salt concentrations to assess the effects of salinity stress on the growth and ion accumulation in the calli (Furrukh, 2002). The growth rate of calli decreased with increasing salts concentrations. Callus induction in Potohar variety showed higher growth rate reduction than LU-26. Callus induction decreased with increased, + Na and Cl whereas + K increased with increase in salt concentrations, with LU-26 recording the highest + Na and - Cl increment and Potohar recording the highest + K reduction.. Embryogenic calli of three varieties of wheat, initiated and maintained on Murashige and Skoog (MS) basal medium supplemented with 3 mg/l 2, 4-D (2, 4- dichloro phenoxy acetic acid) when stressed with varying levels of sodium chloride showed resistance in the three varieties in the order of Hyderabad-81 greater than Pak-81 greater than Lyallpur-73 (Tahir et al., 2002). Tissue culture can increase the stress tolerance of plants because large cell suspension or callus (a solid disorganized mass of plant cells) contains perhaps 50,000 cells. Thus tissue culture miniaturizes and streamlines the selection process (Nabors and Dykes, 1985; Zair et al. 2003). Abbas et al. (1983) studied the effect of sodium chloride and calcium chloride on callus induction in two cultivars of rice. At lower concentrations sodium chloride reduced callus formation while at moderate concentration it increased the frequency of callus induction. At higher concentrations, the inhibitory effect of calcium chloride was gradual but more pronounced than that of sodium chloride. A number of studies have been conducted to see the effect of NaCl on the tissue culture of wheat cultivars but little information is available on the in vitro effects of calcium chloride. Data are lacking regarding the effect of salt on tissue culture under salt stress in recommended wheat varieties in Pakistan. The present study was * ** *** Integrated Pest Management Programme, National Agricultural Research Centre, Islamabad - Pakistan Agriculture Biotechnology Programme, National Agricultural Research Centre, Islamabad - Pakistan Maize, Sorghum & Millet Programme, National Agricultural Research Centre, Islamabad - Pakistan

2 Naheed Akhtar et al. Callus induction and regeneration in wheat 650 conducted to investigate the effects of two salts (Sodium and Calcium Chlorides) on the callogenesis and organogenesis in four Pakistani wheat varieties. The objective was to improve stress tolerance in wheat varieties by culture under different concentrations of salts through callus initiation (callogenesis) and regeneration (organogenesis) responses. The information obtained will be helpful in breeding of salt tolerant wheat in Pakistan. MATERIALS AND METHODS The study was conducted at Agricultural Biotechnology Research Labs., National Agricultural Research Centre, Islamabad to ascertain varietals differences between four wheat varieties Arz, Pak-81, Lu-26 and Pavon, with respect to their tolerance to salinity of sodium chloride (NaCl) and calcium chloride (CaCl 2 ) as indicated by callogenesis and organogenesis. The explants taken were from 200 seeds of these varieties, which were exposed to different concentrations of NaCl and CaCl 2 salts. The seeds provide an excellent material to start with, because a rigorous surface sterilization can be applied to seeds, which results in minimal contamination of the resultant callus culture. The explants were sterilized with 5 % calcium hypochlorite followed by 3 subsequent rinsing of 15 minutes each with autoclaved distilled water under aseptic conditions. The medium used was developed by Murashige Skoog (1962), and modified by Kao and Michayluk, (1974). After preparation the medium was poured in test tubes plugged with cotton plugs and autoclaved at 120 C and 15 lb pressure for 15 minutes. Both types of media liquid and solid were used. Salt concentrations used were-50 mm, 100 mm, 150 mm and 200 mm. The cultures were placed in environmental chamber where a photoperiod of 16 hours and C (day/night) temperature were maintained. The calli produced on callus inducing media were transferred to the regenerating media. The seed derived callus system was achieved with these steps, the callus was induced from surface sterilized wheat seeds on modified (MS) Medium. The four-week-old calli were subcultured using the same medium for callus proliferation. The proliferated calli were divided into small pieces (50 mg each) and the pieces were transferred separately to the media with stress of NaCl and CaCl 2. Using NaCl and CaCl 2 enriched medium during the subculturing developed salt tolerant callus lines. This step was repeated 3-6 times after 25 days interval. After four weeks time the calli were finally transferred to regenerating media for plant regeneration. The cultures were placed in environmental chamber where a photoperiod of 16 hours and C (day/night) temperature were maintained. The three kinds of culture media used in the seed derived callus culture system were modifications of MS medium. Medium I and II contains 2, 4-D (2, 4- Dichloro phenoxy acetic acid), with concentrations of 1 and 2 mg/l, to induce callus, while medium III contains Naphthalene acetic acid (NAA) with concentrations of 2 mg/l. The medium II and III were supplemented with NaCl and CaCl 2 stresses along with high concentration of sucrose. RESULTS AND DISCUSSION The factors that affected callus induction and regeneration during sub-culturing were studied. These factors were Variety, Incubation time, Effect of light and effect of Sodium chloride and calcium chloride salts. Variety Marked varietal differences for callogenesis were observed (Table I), the frequency of callus induction was 25% in Arz, 34% in Pak-81, 51 % in Pavon and 76% in Lu-26, while regeneration was 5, 12, 23 and 30 in these cultivars, respectively (Table I). Wheat variety Lu-26 showed comparatively good Callogenesis and organogenesis while Arz variety showed comparatively less Callogenesis and organogenesis both. Farrukh (2002) reported that the relative water content of both varieties Potohar and LU-26 decreased with increasing salt concentrations. Incubation Time Most varieties developed a healthy callus after four weeks of incubation. Extended incubation generally resulted in decreased efficiency of callus induction (Table II). The callus induction efficiency after 24 weeks incubation period was 76 % calli in Pavon followed by LU-26 (55 %), Pak 81, (28 %), and least in Arz (25%). There was decrease in embryogenesis ability at high salt concentrations in most of the calli of durum wheat viz soft wheat, which showed necrosis and disappeared at (10 and 15 g litre-1). (2002). However soft wheat tolerated more salt than durum wheat where the best regeneration results were obtained in media containing 0 or 2.5 g litre-1 of salt. Browning started to show on the seedlings at 5 g litre-1 and was complete at 15 g literl Oudija et al. Similar studies were conducted earlier

3 Sarhad J. Agric. Vol. 23, No. 3, by Ansari et al. (1977) they showed that, the varieties of local origin (H-68. C-591) were comparatively better than their Mexican counterparts. Light The effect of light was highest (78 %) in Pak 81, followed by LU-26 (70 %), Pavon (60 %), and Arz (22%), (Table-III). In darkness callus induction efficiency was comparatively less (72 %) in Pak 81, followed by LU-26 (68 %), Pavon (58 %), and in Arz (18 %). Effect of light was highest (78 %) in Pak 81, followed by LU-26 (70 %), Pavon (60 %), and in Arz (22%). Results showed that effect of light was highest in Pak-81 and least in Arz. Effect of Sodium Chloride and Calcium Chloride Salts on Efficiency of Callogenesis and Organogenisis Formation When Sodium Chloride and Calcium Chloride Salts were added to the medium in concentration of 50, 100, 150 and 200 mm, percentage of calli and regeneration were decreased with increase in the concentration of salts (Table 1V). In case of both salts, the calli grown on control medium without addition of two salts were transferred to medium II with a stress of 150 and 200 mm concentration. Few NaCI and CaCI 2 calli turned dark brown, an indication of necrosis. However, some portions of the callus managed to grow, when step was repeated more than 3 times vigorously growing callus emerged from the necrotic callus. These salt tolerant callus lines continued improved growth in subsequent subcultures. Efficiency of plant regeneration declined with callus age. When NaCI and CaCI 2 were not added to the medium, efficiency of plant regeneration from callus was much higher. However, this higher regeneration efficiency was counter balanced by the earlier decreased growth of callus. As a consequence over all efficiency of plant regeneration per unit of callus initially used for subculture was lower when both salts were added to the medium. It is obvious that for every variety of wheat with increase in salt concentration, there was a decrease in percentage of Callogenesis and Regeneration. Lu-26 was most tolerant amongst the four varieties studied for both salts, as compared to Arz which was least tolerant (Table 1V). There were more percentage of Callogenesis and Regeneration in LU- 26 wheat variety followed by, Pavon, Pak-81 and Arz. Effect of CaCI 2 was less deleterious than NaCl stress but was harmful with its increasing concentrations for Callus formation and Regeneration both. We can say that with increase in NaCl and CaCI 2 salts concentrations there was decrease in callus induction and plant regeneration from callus. There is considerable evidence that increase in salinity reduced seed germination and growth in many crop plants (Wahhab, 1961; Younis and Hatata, 1971). Salinity conditions have been reported reduced crop yield and delayed flowering (Hassan et al. 1970). It is well established that NaCl at certain limit is more toxic than CaCl 2 (Storgonov, 1970). Na+ is toxic for many physiological functions of plants (Abdullah and Ahmed, 1981), as well as Cl _ is responsible for decrease in chlorophyll mainly due to higher accumulation of chloride under salt stress (Basslavskaya and Syroesshkina, 1936). In case of CaCl 2, Ca ++ is a beneficial cation responsible for many physiological functions like cellulose integrity and for binding of phenolic compounds under stress situation (Deckock et al., 1975). Khan and Patel (1973), Naqvi et al. (1975) and Ansari et al. (1977) stated that wheat cultivars screened to determine their inherent capacity of salt tolerance, showed that varieties of local origin (H-68. C-591) were comparatively better than their Mexican counterparts. Further studies indicated that H-68 and C-591 exhibited more amylase activity than Mexi-pak and that salinity reduced this activity in both the cases. Callus induction frequency increased at 50mM to 100mM NaCl; however Proliferation of the Calli Similar Callogenesis response in wheat cultivars under NaCl stress was observed by Mahmood and Quraishi (1985) and in rice by Abbas et al. (1983). Hanif and Davies (1998) reported that 8-day exposure to NaCl reduced the length of root apical meristem in Secale cereal to a greater extent than Triticum aestivum. NaCl induced also root hair formation much closer to the root cap boundary than in the control roots. Zair et al. (2003) concluded that plant regeneration from callus initiated on high NaCl levels might be a valid method of selection for salt tolerance. Cho-Un-Haing, and Kasha, (1995) studied the addition of the ionophore A23187 (0.5 micro M) with CaCl 2 (1 mm) further improved the embryoid formation in both wheat and barley. Our results like above-mentioned researchers indicated that degree of Callogenesis and organogenesis vary not only from variety to variety but also from salt to salt. According to our results CaCl 2 was less deleterious than NaCl at

4 Naheed Akhtar et al. Callus induction and regeneration in wheat 652 low concentrations but it was more harmful at high concentrations in Pak-8 and Arz while NaCl was more harmful at high concentrations in Pavan and LU-26. There was decrease in both Callogenesis and organogenesis with increase in salinity levels in both Sodium Chloride and Calcium Chloride salts stresses. LU-26 was most tolerant to both salts while Arz was least tolerant to both salts. These results will add information regarding response of wheat varieties to salt tolerance through tissue culture. Programme, NARC), Miss Shagufta Aziz (SO, National IPM Programme, NARC) Mr. Muhammad Irshad (Sr.Entomologist, National Insect Museum Programme, NARC) and late Dr. Azra Quraishi, (CSO, Agriculture Biotechnology Programme, NARC), and Dr. Hamid Rashid (SSO, Agriculture Biotechnology Programme, NARC) for their constant encouragement for accomplishing this work. Acknowledgements Acknowledgements are due to Dr. Zaib Abdullah, (PSO, Plant Stress Phisiology Labs., NARC), Dr. Tahira Yasmeen, (SO, Cereal Diseases Research Table I. Varietal Differences in Callus Formation and Regeneration from of Wheat Without Salt Stress. Callus formation (%) Arz 25 5 Pak Pavon Lu Regeneration (%) Mature Seeds Table II. Effect of Incubation Time on the Efficiency of Callus Formation in Wheat Cultivars Callus formation (%) 4 weeks 6 weeks Arz Pak Pavon Lu Table III. Effect of Light On Callus Induction In Wheat Cultivars Callus formation (%) Darkness Arz Pak Pavon Lu Light (16 hrs.)

5 Sarhad J. Agric. Vol. 23, No. 3, Table IV. Effect of Sodium Chloride Salt and Calcium Chloride Salts on Efficiency of Callus Induction and of Regeneration from Mature Seeds of Wheat. Effect of Sodium Chloride Salt Effect of Calcium Chloride Salt Arz Pak-81 Pavon Lu-26 Salt concentration (mm) callus Induction Regener ation callus Formatio n Regenera tion REFERENCES Abbas, S.T., N. Mahmood, and A. Quraishi, Effect of NaCl and CaCl 2 on callus induction in Oryza sativa L. In: Quraishi, A. and John I. (eds), Plant Tissue Culture. NARC. pp Abdullah, Z.N. and R. Ahmed Effect of saline soils, amended with Calcium Nitrate on the growth and chemical constituents of potato plants. Pak. J. Bot. 13(1): Ansari, R., S.M. Naqvi and A. R. Azmi Effect of salinity on germination, seedling growth and amylase activity in wheat. Pak. J. Bot. 9(2): Basslavskaya, S. and M. Syroesshkina, Influence of the chloride ion on the content of chlorophyll in the leaves of potatoes. Plant Physiol. 11: Brown, J.W. and H. E. Hayward Salt tolerance of alfalfa varieties. J. Agro.48: Chaudhuri, I.I. and H.E. Wiebe Influence of calcium per treatment on wheat germination on saline media. Plant and Soil 28: Cho-Un-Haing and K.J. Kasha The effect of calcium on ethylene production and microspore-derived embryogenesis in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) anther cultures. J. Plant Physiol. 146(5/6): Deckock, P.C., P.W. Dyson and A. Hall and F. Grabowaska Metabolic changes assoiated with Calcium deficiency in potato sprouts. Potato Res. 18: El-Shafey, Y.E. and S.M. Salem Production of salt tolerant line through wheat calli retreatment with fast neutrons. 45(1): Farrukh, J In vitro salt tolerance in Wheat. I. Growth and ions accumulation. Int l. J. Agric. Biol. 4(4):

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