SrEp AND Nursery TECHNOLOGY or Forest TREES [Proceedings of IUFRO Symposium 1997) Editors D.G.W. Edwards Forest Tree Boi 4018 Cavalin Court, Victoria S.C.Naithani Seed Biology Lab, Schoo! of Lite Sciences mt NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS. New Delhi, IndiaCHILLING-INDUCED DIEBACK IN SAL (SHOREA ROBUSTA Gaertn. f.) SEEDLINGS S. KESHAVKANTand S.C. NAITHANI Seed Biology Lab, School of Lite Sciences, Pt. Ravishankor Shukla University, Raipur 492 010, INDIA (Email: naithoni@gwri.dot.net.in) ABSTRACT ‘Between June and October, rapid increases in shoot lengths of sal seedings grown in 3 fd ‘wore accompanied by 12 {017 fold increases in weight. Numbers of leaves more tan doubled ‘and leat welghts increased 11 0 13old. Between November and March, when ai temperatures {el below 15°C, neay 50% of soot and leat welght was los. Constant exposure to femperaures that hovered between &° and 15°C caused desiccaton and necroses of leaves and shoots in 80-86% of the seedings. Lest chlorophyll content and protein content of shoots, leaves and roots decreased by two this during the ald period. Root growth was not greaty influenced by the tompersise fuctuatons, and rotshoot ratios Increased to 2.7% in January. and to 61 in March. However, only 12-17% of damaged seedlings prosuced new coppice shoo!s when air temperatures tobe above 15°% in Apri. In contas, when other seedlings were moved to a reenhouse in October, troughout he winter period shoot growth emained rapid and continuous root shoot rates did not nse above 3:1, and chlorophyll and protein contents continued to increase. The data are discussed to provide an explanation of the ciingsnsuced dieback Which was reduced by 20% in seedings placed in the greenhouse. INTRODUCTION Sal (Shorea robusta Gaertn, £) forests cover 14.2% of the total forest area of India oshi, 1980). The species is widely distributed from Sri Lanka, India and Nepal to the west through Burma and other countries of South East Asia, and the Philippines. ‘Nursery production and planting of seedlings on forest sites has not been successful because of slow growth and massive morality due to frequent dieback annually for the first few years (Joshi, 1980). This dieback phenomenon in sal seedlings is described as inhibition of shoot growth followed by the development of new multiple shoots (similar in size to that ofthe initial shoot) in the next season (Champion and Seth, 1968)262 Factors that have been associated with mortality and dieback of sal seedlings include increased soil pH (Krishnaswamy, 1951), poor soil aeration (Namdeo eal, 1989), low soil moisture (Gupta, 1954; Sahay, 1956), prolonged drought (Seth and Shrivastava, 1971), canopy density (Hole, 1921), excessive fire (Nath and Kamath, 1967), browsing (Lal, 1960), and weed growth (Bhatnagar, 1958). Except for sol moisture, however, no efor has been made to establish the relationship of these factors with mortality or dleback. Soil moisture over 107% or below 39% has been shown to cause a steep reduction in growth, causing dieback and mortality in sal seedlings (Hole, 1921). Preliminary studies on nursery-raised sal seedlings showed dieback and morality even when seedlings were well irigated (63-72% soil moisture), so soil moisture may not be the main cause (Keshavkant and Naithani, 1996). Infact, it was observed that shoot Browth of young sal seedlings ceased completely during winter season when femperatures fell below 15°C. Seedling damage was apparent as surface pitting and necroses in leaves and shoot tps, injuries that ae similar to chilling injury symptoms frequently reported in many tropical and subtropical plants (Markhart, 1986). No detailed growth analyses of sal seediings, either growing in the nursery or the forest have been published relating seasonal variations associated with changes in Browth patter, or to demonstrate and quantify the effect of low temperature with Erowth. The objectives of this study were to investigate changes in growth pattern ‘caused by low temperature of nursery-grown sal seedlings. MATERIALS AND METHODS Mature sal seeds (Chaitanya and Naithani, 1994) were collected from the Gariyabandh forest reserve, 91 kms, north east of Raipur (20°38' latitude, 82°04" longitude), Seeds were germinated immediately after collection in a basket between two wet jute bags After radical emergence, germinants were planted in polybags (6x9 inches, opaque) containing a mixture of black soil, sand and farm yatd manure (2:1:1), and grown in the “Sal Nursery” at the Botanical Garden, School of Life Sciences, Pt. Ravishankar ‘Shukla University, Raipur. Three seedlings were planted per polybag initially, then thinned to one seedling after 15 days, and approximately 14,000 seedlings were raised. Seedlings were irigated to field capacity twice a day, but fertilizer and insecticides were applied in the rainy season only (July to September) to boost seedling growth. Nearly 4000 sal seedlings were transferred to a greenhouse (temperature 35-37C, RH 70-76%) in the last week of October. The greenhouse was built of thin transparent- Plastic sheet thus allowing almost equal light radiation in the greenhouse (47,800 lux)263 as in the field (48,000 lux), Greenhouse-raised seedlings were transferred in April back to the field condition. Seedlings were sampled monthly in field and greenhouse. Growth and biochemical analyses were conducted on 10 replications, and repeated in 1996/97 and 1997-98, Air temperature and relative humidity records for the field condition was ‘obtained from the Department of Physics and Agrometeorology, Indira Gandhi Agriculture University, Raipur, while greenhouse conditions during November to March were recorded by a hygrothermograph. Seedling injury was recorded monthly in field and greenhouse by visual observations of surface pitting, browning and necrosis of leaves, and shoot-tip necrosis. Damaged seedlings were expressed as a percentage of total seedlings by the following formula Number of injured seedlings Total number of seedlings planted °% Injured seedlings = x 100 Rejuvenated seedlings, those that show formation of laterals or sprouts from the root-shoot collar following chilling injury, were similarly expressed as follows Number of seedlings showing regrowth % Rejuvenated seedlings = — x 100 Total number of injured seedlings ‘Thirty days after planting germinated seeds in the polybags, 10 seedlings were randomly sampled for biomass determination. Fresh weight of leaves, shoots and roots, ‘aswell as their dry weights after drying at 85°C for 72 hours, were recorded, together with the average length of roots, shoots, and leaf numbers. Root-shoot ratio (dry ‘weight basis) was calculated to determine the relative amounts of dry matter in root and shoot for each month, Known weights of leaves were homogenized in chilled 80% acetone. The homogenate \vas centrifuged at S000rpm for Smin. and the supernatant used for total chlorophyll estimation calculated by the formula given by Amon (1949). For total protein, the method of Plummer (1988) was used. 500mg tissue was homogenized in 3ml of 10% trichloroacetic acid (10% w/v) and centrifuged at 5000 rpm for 5 min. The sediment was dissolved in 3 ml of 0.1N NaOH and kept overnight with intermittent shaking, then centrifuged. The supernatant was used for total protein estimation by the method of Lowry eval, (1951).284 [All results were analyzed using SPSS Software. The growth and biochemical results of field and greenhouse grown seedlings were tested (test) for significant differences (P=0.05) as per Panse and Sukhatme (1967). RESULTS AND DISCUSSION “After planting at one month old, the seedlings grew vigorously from June to October (Table 2), From November, when night temperatures fell below 15°C, more than 86 of the seedlings not only ceased to grow, but were severely damaged. They remained so through March when, with summer approaching, approximately 17% that had desiccated shoots, with or without desiccated brown leaves, resumed vigorous growth by means of coppice shoots from April onwards (Table 1). Such periodic (or episodic) rather than continuous growth is almost universal among species belonging to Hevea, Theobroma, Quercus and Pinus (Zimmerman and Brown, 1971; Longman and Jenik, 1988). Table 1. Percentage of Injured and Rejuvenated (dicback) sl seedlings with the meteorological data prow under field and greenhouse conditions. Perentage injury and rejuvenation ‘vas recorded in sample populations of 100 seedings in each test conditions. Values re ‘means of + SE of 10 replications. Green house values indicated by * are significantly diferent from the field condition at P = 0.05. Month % Injured Seedlings "% Rejwenated Seedlings Min Temperature (C) Field Greenhouse ‘Field Greenhouse Field Greenhouse une 00 00 oo 09 28.020.02 July 00 0 00 00 2.40.01 Aug 00 oo 00 oo 24,980.05 Sep. 0840.12.00 00 00 23,640.18 Oa, 0920.16 00 oo 00 22.220.16 Nov, 1140.09 080.12" 00 00 14.1003 30,080.05 Dee 2440.21 0630.07" 00. ov 1084005 32,020.09 Jan 220.12 042021" 00 0 09,020.02 30.80.10 Fe, 1140.05 20.11" 00 0 10.740.06 31,520.11 Mar, 0420.08 00 oo 00 1464003 31,220.08, Ap. 00 00 0980.11 oss0.21% 22.90.05 May 00 00 0510.09 6e0.11* — 26.320.07 June 00 00 0340.17 280,16" 27.820.09285 ‘Table 2. Changes in the number of leaves, shoot and root lengths of sal seedlings grown for one year under field and greenhouse conditions. Velues are means of 10 replications + SE. ‘Greenhouse values indicated by * are significantly different from field at P = 0.05 eee cece gee eee eee Month Leaf Number ‘Shoot Length (em) Root Length (em) Field Greenhouse Field Greenhouse Field Greenhouse June 240.09 04.10.11 06.020.03, July 420.08 10.10.08 08.40.04, ‘Avg, $20.11 16.20.03 1054001 Sep. 640.07 1750.03 11,740.06 Oct, 740.11 19,640.06 12.420.11 Nov, 620.11 0740.08" 19,840.11 20.080.117 ——12.480.10 12.10.10 Dec 480.08 0820.03 20,080.11 20.940.11* 12,680.05. 12,780.08 Jan, 34003 0940.01 20.0008 21,020.08 12.80.05 13.10.09 Feb, 340.02 1080.01" 20,180.04 24,020.08% 13,080.03. 14,280.06 Mar, 240,03 1240.04 20.030,02 25.820.05* 14,580.07 15.50.08 ‘Apt, 34002 1280.03 04,340.09 26.50.08 15.1008 18.280. May 440.01 1340.03 11,040.11 27.1008% —15.280.09 18,950.11" June $40.01 1340.04 16,040.05 27.520.08* 15,340.08 19,080.05" eee Initially, the one-month-old seedlings, averaging 4.12 cm shoots and 0.099 fresh weight and 0.027g dry weight, grew rapidly for another four months and attained heights of 19.66 em, accompanied by a 12-fold gain in fresh weight and a 17-fold increase in dry weight (Tables 2-4). Leaf numbers more than tripled, leaf fresh weight increased 13-fold and dry weight [1 fold, which were the main reasons forthe overall rapid growth of seedlings from June to October. From November, a gradual loss in fresh and dry weight ofthe leaves led to an almost $0% decline by March. Simultaneously, fresh and dry weights of the shoots also decreased (Tables 3 and 4). These massive losses to the aerial parts was caused principally by their exposure to low temperatures that ranged from 9°-15°C. As they became chilled, leaves of sal seedlings developed surface pitting initially which was followed by necrosis of leaf margins; the necroses later spread 10 the remaining leaf area, Constant exposure of seedlings to low temperature for five months (November to March) led to desiccated, brown and necrotic leaves and shoots. CChilling- induced reduction in growth is an established indicator in sensitive tropical and subtropical plants (Wright, 1974; Wright and Simon, 1973). Chilling-injury symptoms such as brown spotting, discoloration and leaf necroses appear after brief exposure to low temperature in chilling-sensitive tropical plants such Zea mays, Dracaena sanderana and Cucumber (Lyons, 1973), terminating in necrotic lesions (Hallgren and COquist, 1990). Such manifestations are probably the end result of a complex series of events (Hallgren and Oquist, 1990),286 ‘Table 3 Changes inthe ftesh weight of leaves, shoots and roots of sl seedlings grown for one year under field and Green-house conditions, Values are means of 10 replications # SE. Green house values indicated by * are significantly different from field at Po0 05 level Month Lat @ Shoct (2) Root (@) Field Greenhouse Field Greenhouse “Field Greenhouse June 0.13840.02 0.09940.01 0.21540.10 July 0.57740.01 D.1ss0.08 0.52020 05, ‘Aug 1:24240.0 .6s4s0.02 1.109:0.05 Sep 157980.14 o.Rsts0.06 1 82610.11 Oct 1,79540.16 12tte0.12 21083009 Nov 14398003 1.88020.11¢ —1.1:026.08 1 3020.07 26308008 2.88920.09" Dec 1.341005 1.92520,06 0.98540.06 1434008" 2.83020.05 3.85080.03¢ Jan 1 15240,02 2.08680,05* 0.7480.09 1492001" 2.90020.09 4.20040.0K* Feb 6.$8040,06 234740.11 0.53020.07 2.1240.08" 3.24020.10 5.40340.08* Mar 0.22040.04 26848008" 0370=0.11 2.5220.11* 3.57040.05 5.68840.06" Apr US7780.0S 3.03149.05" 0.68720.10 2.6040.13* 39104008 6.19920.09% Muy 144240.11 3.31740.11 1.09180.12 2.8340.17% 41202005. 6.41520.10° June 1 52840.09 348040.09* 1.35040.09 2.9840.10" 4.25080.09 6.s9Re008" ‘Table 4. Changes inthe dry weigh of leaves, shoots and roots of sal seedlings grown for one year under field and Greenhouse conditions. Values are racans of 10 replications =SE ‘Groen house values indicated by * are signiicanly different frem field at P=0.05, Month Lat (o) ‘Shoot (=) oot (2) Feld Greenhouse Field Greenhouse—Field——_Greenhouse June 010482003, 0.02730.01 0.10730.19, dy 024820.02 0.18930,02 0.24840.02 Aug 0.43120.01, 0.27720.03 0.58020 08, Sep 0.54620.06 0.39510 02 o99se0.tt Oct 057440.08 0.49120.02 sh tsieo 09 Nov 0,5090.03 0.58820.09" 0.47920,04 0.49540.03" 1.297400 —1.34840.09 Dec 042020.05 0.6R040,05" 03613008 05842000" 1.34080.071.74620.05¢ Jan 0.36840.02 073440,05" 0.31740.06 0.60740.03% 1 $0080.06 2.055e0.08" Feb 0.27510.03 0.96940.02% 0.24640.11 095340.08" 1.70020.07 2.7600.07" Mar 0.17340.02 1,108+0.04 0.197803 1108#0.06" 1.88020.05 | 2.770e0.10" Apr 03884004 123080.11% 0.3612008 1.23080.07" 1 98080.07 2.97020.11" May 0.52720.06 1.54620.96" 0.43680,09148I20.06" 2.24080.04 3.1202006" Jone 0.75820.08 1.69240.08 0.59440,11 1.$4640.09% 2.360208 3.32020.08°267 When temperatures rose above 15°C in Aprit, between 12-1726 of the chilling-injured seedlings exhibited re-growth in the form of whippy, lateral shoots that gained rapidly in fresh and dry weight (Tables 3 and 4) with the formation of new leaves (Fable 2). Many woody species are capable of regenerating new shoots, known as coppicing, following stem destruction (Kruger and Reich, 1993) although its cause is uncertain. Shoots originate from lateral buds subtending the aborted shoot tip, oft resulted in bushy, or whippy branching, patterns (Halle et a!., 1978) In contrast, root growth was almost constant from November to March. Roots grew rapidly between June and October, doubiing in length (Table 2) accompanied by a 26-fold increase in fresh weight and a 48-fold increase in dry weight (Tables 3 and 4), From November through March. roots increased * less than Ig fresh weight and about 0.62 dry weight (Tables 3 and 4). This reauction appeared to have been ‘caused not by 0 the direct effect of chilling temperature, but more likely by the lack of photosynthate from damaged shoots. When four-month-old seedlings were moved to the greenhouse in November, dry weights of leaves, shoot and roots increased (able 4) together with substantial increases in length of shoots, roots and number of leaves over the nexrt four raonths (Table 2). The inerease in the number of leaves from 7 to 12 assured increased availability of photosynthats to support rapid shoot and root growth (fables 3 and 4). Root-shoot ratios were 1:1 intially in one-mionth-old seedlings (June), but increased to 2.1 in August and to 3:1 in October, whea the minimum air temperature was between 22-28°C. When field temperatures fell below 15°C, root-shoot ratios increased to 3.7:1 in January and to 6:1 in March because of the cessation of shoot growth during these months (Table 5). Higher roct-shoot ratios have been observed in several plant species when exposed to chilling temperature (Davis and Lingle, 1961 McMichael and Queesenberry, 199%), Chilling injury has been reported primarily as by chill closure, combined with reduced hydraulic conductivity of root membranes (Wilson, the result of excessive water loss cau: induced prevention of stomatal 1976), The ability of seedlings to change rootshoot ratios may be only one part of the overall mechanism to tolerate chitling-imposed stress (Klepper et al, 1973). Such high root-shoot ratios result in large food reserves in the roots which ereates the potential for coppicing (Reieh er al, 1980)Table 5, Changes in root-shoot rato (dry weight bass) of sl seedings grown one year under field and greenhouse conditions Month Feld Greenhouse Tune 3.96 July 1.66 Aug 2.09 Sep 239 oct 2a Nov 3.02 2m Dee 371 Bus Jan ans 338 Feb sor 2a9 Mar os 250 Apr sas 241 May 5.13 2a June 397 24 Se ee In contrast, for seedlings moved in November to the greenhouse, where the {temperature ranged from 30-31°C, root-shoot ratios remained at about 3:1 over the next four months (Table $). Such high root-shoot ratios in fast growing sal seedlings is similar to that of seedlings of several other tree species, and has been considered to be a prerequisite of continuous growth (Borchert, 1991), Total chlorophyil content increased 3-fold between June and October in vigorously ‘growing sal seedlings, but chlorophyll content decreased almost 75% between October and March in the field (Table 6). In contrast, total chlorophyll increased 2 8 times in seedlings growing in the greenhouse during winter. In field-grown seedlings, Protein content more than doubled in leaves and shoots, and increased approximately 1.5 times in roots between June and October, but then decreased by almost two thirds in leaves and shoots between December and March, while a steady increase ‘occurred in the roots (Table 7). Total protein levels rose again in rejuvenated seedlings from April onwards. In greenhouse seedlings, protein level increased constantly (Table 7) A significant loss of chlorophyll and protein specifically due to their decreased synthesis during chilling injury has been reported in other chilling-sensitive plants (Levitt, 1972; MeWilliam and Naylor, 1967). The inerease in chlorophyll and protein contents in greenhouse seedlings during December to March indicates a stimulation in their synthesis similar to the stimulation/recovery reported in chilling-sensitive plants during re-warming after a short chilling period (Levitt, 1972). Protein content in the roots of field grown sal seedlings was not influenced by low temperature, further supporting the dominance and active growth of roots during the chilling period,269 “Table 6, Changes in the total chlorophyll content in the eaves of sal seedlings grown for ‘one year under field and greenhouse conditions. Values are means of 10 replications 4SE. Values indicated by * are significantly diferent at P=0.05 i ‘Month “Total Chlorophyll (me) Field Greenhouse Se sone 1793 + 006 aly 31.73 = 0.08 Ave 4281 + 0.06 sep SB61 = 006 oct 63.09 + 0.12 Nov 4027 4 0.14 7127+ 0.10 Dee 24.32 + 0.06 86.59 + 0.09 dan 1941 + 0.06 1272 + 008 Feb 1816+ 0.11 162.8 + 0.10 Mar 17.84 = 005 1794.4.006 Ape 2991 £0.09 1862 = 008, May 59.72 + 0.06 1919 + 009 June 7491 £0.07 1966 + 0.07 ee “Table 7. Total protein content in the leaves, shoots and roots of sl seedings grown for one year inderfield and greenhouse conditions. Values are means of 10 replications 4SE. Green house values indicated by * ae significantly diferent from field at P= 0.0. SSS Month Leaf (mgie FW) ‘Shoot (mes FW) Root (mele FW) Field Greenhouse Field Greenhouse ‘Feld’ Greenhouse Se June 12,600.08 08.4320.06 10.3620.09 uly 13.5880.11 11.0620.09 11,5920.07 ‘Aug 14.9840.08 15.0320 08 119140.10 Sep 17.6020.20 18.2420.07 14.9940.07 Oct 25.8640.08 18,380.06 17.2820.16 Nov 227340.09 26.9980,09" 16.762007 19.9840,08% 20,9420.10 23,840.06" Dec 20,600.11 27.0940,09" 15.8540,09 20.4120.11* 22.4680.06 26.8440.12" Jan 16.4120.10 27.8940.07* | 14.2140.10 22.4840.07¢ 24,8420.21. 27.9150.09" Feb 11.6140.06 28.7840.10" 11,070.11 23.6740.10* 24.8920.12 29,9180.09" Mar 09.0740.05 29.0880.08* 07.0120.06 26.8340.06" 25.4640.06 1.834007" ‘Ape 12.0140.06 32.8020,09* 08.7840,15 27.4840,09% 25.6980.16 32.0980.12° May 13.2120.07 3$2040.11" 10.3680.13 30.0620.08* 25.84#0,09 34,0050.16* June 17.6040.11 37.9040.10" 14.3140.08 33.4620.10" 26,0140,10 36.7120,09* ie20 ‘These date indicate that when sal seedlings encounter low temperatures, they ‘undergo profound alterations in growth leading initially to cessation of aerial growth ‘accompanied by symptoms of necrosis, which terminates in seedlings with/without desiccated shoots and leaves. Chilling injury leads to massive mortality, and only 12-17% ofthe seedlings resumed shoot growth when temperatures rose above 20°C. Growth resumption in chilling-injured seedlings is understood to be related to the coppicing potential of sal seedlings because the root stocks act as strong sinks, accumulating reserves that provide the coppicing potential ACKNOWLEDGEMENTS Prof. M. L. Naik, Head, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur, for providing the filed, greenhouse and laboratory facilities. Financial asistance ‘was provided to S. Keshavkant by the Department of Science and Technology, New Delhi, India (SP/SO/AO1/93). REFERENCES ‘Ammon, D. 1. 1949. Copper enzymes in isolated chloroplasts. Plant Physiol. 24: 1-15 Bhatnagar, H. P. 1958, Transpiration studies on sal (Shorea robusia) seedlings and some assovated weed species. Proc Ind. Sci. Cong., 46 Session, Deli Borchert, R. 1991. IN, Physiology of Trees. (A. S, Raghvendra, ed.) 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