MATERIALS AND METHODS

Sites and Plant Material: The experiment was conducted first at Colorado State University (USA) in 1994 and later repeated at National Agricultural Research Centre (Pakistan) in 1998. Initially, seven genetically stable lentil lines and varieties, including winter and spring lentils, were selected from our world collection (Table 1-4). Later, this number was reduced to four as a manageable experiment. Of these four lines, Balochistan Local (B. Local) and ILL 5865 (currently released as SHIRAZ-96) are known for their winter and cold hardiness while the other two, Brewer and Laird (spring cultivars), for their non-cold hardiness (Ali et al., 1991; 1999). All seven genotypes were used for analysis of random shoot (whole) samples of acclimated plants (Table 5). Initially, shoots were sampled randomly. Later, it was observed that young and old shoots had different cold tolerance limits. Therefore, shoot tips (leaf primordia) and basal leaves from old and young shoots were sampled to examine the differences in carbohydrate content and freezing tolerance among these tissues (Table 1).

Growth conditions, acclimation and freeze testing procedures: A well established protocol for screening of lentil for cold hardiness under controlled conditions was used for acclimation and freeze testing of the genotypes (Ali et al., 1999).

Table 1:	Coacentrations (10–5 mol g–1) of sorbitol (Sor), glucose (Glu) and sucrose (Suc) in four different tissues of 6-week acclimated plants of two hardy (H) and non-hardy (NH) lentil genotypes and cold tolerance of the genotypes at -5°C freeze temperature where cold tolerance assessed on the basis of cold injury score (1 = no foliar injury to the plant and 5 = dead)

Table 2:	Correlation coefficients (± Standard Error) between cold injury, determined from a rating scale of 1-5 based on foliar injury and sugar content (10–5 mol g–1) in different acclimated plant samples of hardy and non-hardy lentil genotypes. Probability is in parenthesis

Table 3:	Concentrations (10–6 mol g–1) of sorbitol (Sor), glucose (Glu) and sucrose (Suc) in whole-shoot samples of non-acclimated-two hardy (H) and two non-hardy (NH) lentill genotypes

Table 4:	Concentrations (10–5 mol g–1) of sorbitol (Sor), glucose (Glu) and sucrose (Suc) in wholoolant (above-ground) samples of non-acclimated two hardy MI and two non-hardy (NH) lentil genotypes

Table 5:	Concentrations (10–5 mol g–1) of sorbitol (Sor), glucose (Glu) and sucrose (Suc) in whole-shoot samples of acclimated four hardy (H) and three non-hardy (NH) lentil genotypes
S.E. for genotypes = 1.65, sugars = 1.08 and their interaction = 2.86. d.f. = 147. C.V. = 45%. NS = Non-significant (p>0.05)

Trays containing plants not to be exposed to acclitnation -treatments were placed in the greenhouse at 25±3°C day and 10±3°C night temperatures with a 12 h photoperiod (Table 3-4). The 6-week acclimated and control plants were first sampled and then tested for freezing tolerance by reducing growth chamber temperature at the rate of 1°C h^–1 starting from 0°C to reach the minimum temperature of -5°C and then for 4 h holding at this temperature. The total time in freezer was 9 h for the plants tested. After freeze testing, plants were held at room temperature overnight and then transferred to the greenhouse at approximately 25°C with a 12 h photoperiod. Within 48 to 72 h of the freezing test, data for injury were recorded on the scale of 1 to 5 where 1 = no, foliardamage and 5 = dead (Ali et al., 1999; Table 1).

Sample preparation and ^,derivetizetien: Plant samples were frozen in a freezer at -^-70°C or in liquid nitrogen (-196°C) and freeze-dried. All samples were ground and passed through a 400 mesh (US sievadesignation) stainless steel sieve prior to derivatization. A modified trirnethylsilation procedure (Sweeley et al., 1963) was used. -A-known quantity of 0.5 to 1.5 mg of ground sample was placed in a screw-capped vial, in duplicate, containing 25 μg of internal standard. Then 400 μl of pyridine, 80 μl of hexamethyldisilazane (98%) and 40 μ1 of trirnethylchlorosilane were added in^- this order. The tightly capped tubes were heated at 80°C for 20 minutes. After heating, tubesAmere uncapped, blown dry at room temperature, and 0.5 ml-of^-_n-hexane was added to the dried-residue in each. The solvent was pipetted into a clean, dry tube. The n-hexane solution was again blow dried and 200 μl of fresh n-hexane were added. One μl of this solution was injected with a dropping needle injector into gas chromatograph. Total four (Table 1, 3, 4) and eight (Table 5) samples were taken and each sample was run twice. The amount of each analyte was determined using the internal standard method. Sorbitol, glucose, sucrose, raffinose and stachyose were chromatographed on an PIP-1 methyl siloxane capillary column (12 m, 0.20 mm, 0.33 μm). Since raffinose and stachyose were completely absent or below the detectable concentration in all plant samples, they were not taken into account for any consideration or, presentation.

Statistical analysis: A completely randomized design (CRD, two or three-factor with four or eight replications) was used to analyze the data. Least Significant Difference (LSD) range test was used for mean separation. To determine association between carbohydrate concentrations and freezing ^,tolerance, a simple correlation was run between concentration of sorbitol, glucose and sucrose and visible freezing injury in randomly sampled whole shoots and ^-the data from tips and basal leaves of young and old^-shoots (Table 1, 2, 5).

RESULTS

The genotypes were highly significantly (p<0.01) different for cold tolerance at -5°C. The two winter hardy genotypes had no cold injury at this minimum temperature but 75% foliar damage was observed in the two winter non-hardy genotypes when freeze tested after complete acclimation under controlled conditions (Table 1). The control (non-acclimated) plants of all genotypes were completely killed at this temperature (data not presented). It shows the variability for freeze tolerance among the genotypes when acclimated.

Sorbitol concentrations in the tips and basal leaves of young and old shoots of fully acclimated plants were consistently and highly significantly (p<0.01) higher in the non-hardy cultivars than in the cold-hardy lines. There was no other trend found for other endogenous sugars in the genotypes (Table 1). A highly significant (p<0.001) positive correlation (0.74 ± 0.09) was found between cold injury and sorbitol in tips and basal leaves of young and old shoots of acclimated plants. There were no other significant (p>0.05) correlations between cold injury and other sugars in this study (Table 2). There were nonsignificant (p>0.05) differences for sorbitol among the genotypes when they were not hardened (Table 3, 4).

When whole shoots of acclimated plants were sampled at random, there were no significant (p>0.05) differences for sugar content among four hardy and three non-hardy genotypes. A high coefficient of variation was found for sugar content among genotypes (Table 5). In control (non-acclimated) plants, two non-hardy cultivars (Brewer and Laird) were significantly (p<0.01) higher in glucose and sucrose than one of the cold hardy lines (B. Local) but not higher than the other cold-hardy line (ILL 5865) (Table 3). Overall, there were no trends that suggested an association of endogenous sugars to cold hardiness when whole-shoots or whole-plants were sampled (Table 3-5).

DISCUSSION

After complete acclimation, the lentil plants show similar cold tolerance under controlled conditions as they show under natural environments (Ali et al., 1991; 1999). The cultivars or lines used in the study were pure, homozygous and genetically stable. The seeds were also obtained from the genotypes grown in the same environment. Furthermore, the plants were grown under controlled conditions where a minimum, micro-enviroment effect may be expected and a few tissues of the plant were sampled whereas the other similar tissues were left on the same plant for cold injury observations. Therefore, there are almost no chances of having any differences among the genotypes due to any environmental variation or handling the genetic material.

A positive correlation between cold injury and sorbitol was found indicating that higher sorbitol content was associated with lack of cold hardiness (Table 2). This is probably the reason that non-significant (p>0.05) differences for sorbitol were found among the genotypes when they were not hardened (Table 3, 4). But when hardened, these concentrations were highly significantly (p<0.01) higher in the non-hardy cultivars than the hardy lines and showed significant (p<0.05) differential response to freeze tolerance (Table 1). These data show that high concentrations of sorbitol are associated with lack of cold hardiness. It may be that during cold acclimation carbon resources are directed to some unknown protective metabolite in hardy tissues, but not in non-hardy tissues. It is also possible that sorbitol, in some unknown way, is associated with undesirable freezing events. The role of sorbitol in drought tolerance of faba bean (Vida faba L.), common bean (Phaseolus vulgaris L.) and chickpea (Cicer arietinum L.) has been reported by Amede and Schubert (1997). The concentration of sorbitol was substantially increased in faba bean during drought stress and the response of all species was accompanied by an increase in the solute pool which created a substantial decrease in osmotic potential and rate of cell division and cell expansion. The low temperature stress also affects the plant growth and in response to this environmental change, the winter hardy plants go under dormant conditions but winter non-hardy plants keep their growth continued and do not prepare themselves for the forthcoming undesirable events and hence die in the low temperatures (Levitt, 1972; 1980; Sakai and Larcher, 1987). One or both alternatives may be worthy of further consideration, but this study was not designed to explore these mechanisms. It does, however, suggest a starting point for further metabolic studies concerning a possible role of carbohydrate metabolism in cold survival of lentil.

A high coefficient of variation in whole-shoot samples suggested a high endogenous variability in sugar content among samples (Table 5). It is clearly evident that the metabolic changes in the plant body in response to environmental change are extremely important for its survival (Levitt, 1972; 1980; Sakai and Larcher, 1987). It is concluded that lack of cold hardiness was associated with increased concentration of sorbitol.

ACKNOWLEDGMENTS

The financial support of USAID and PARC under MART/AZR project for Ph.D. program and the research, work reported in this paper, and the seed provided by Dr. W. Erskine (ICARDA) and Dr. F. J. Muehlbauer (USDA, WSU) are gratefully acknowledged.

A part taken from thesis submitted to the Academic Faculty of Colorado State University in partial fulfillment of the requirements for the degree of Ph.D.