ORIGINAL RESEARCH ARTICLE OPEN ACCESS Seed Germination In .

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ORIGINAL RESEARCH ARTICLEINTERNATIONAL JOURNAL OF BIOASSAYSISSN: 2278-778XCODEN: IJBNHYOPEN ACCESSSeed germination in Prunus cerasoides D. Don influenced by naturalseed desiccation and varying temperature in Central Himalayanregion of Uttarakhand, India.Bhawna Tewari*, Ashish TewariDepartment of Forestry & Environmental Science, Kumaun University, Nainital, Uttarakhand, India.Received: March 21, 2016; Revised: March 28, 2016; Accepted: April 11, 2016Abstract: Prunus cerasoides D. Don the Himalayan wild cherry is one lesser known multipurpose tree species ofHimalaya. The tree prefers to grow on sloping grounds between the altitudes of 1200-2400 m, on all types of soilsand rocks. The tree is used as a medicinal plant in Himalayan region. The fruit is edible and the pulp is used tomake a cherry brandy. The species has poor germination and seedling establishment in natural habitat. The overexploitation of seeds of the species coupled with relatively hard seed coat has adversely affects the germination ofseeds in their natural habitat. The information about the seed maturity and technique of germination enhancementis scanty. The present study was conducted to assess the exact maturity time and optimum temperature forenhancement of germination in seed of P. cerasoides. The fruit/seeds were collected from six sites covering thealtitudinal range of 1350 – 1810 m during the period (2003-2004). The colour change of fruit from dark green tored was a useful indicator of seed maturity. Maximum germination coincided with 50.24 0.19 % fruit and 30.11 0.57 % seed moisture content. Negative correlation existed between germination and seed moisture content (r 0.294; P 0.01). Significantly higher germination occurred when seeds were placed above the paper at 25º C.Key words: Seed moisture content; Maturation; Temperature; Germination percent; Germination capacityIntroductionThe lesser known woody perennials likeDiploknema butyracea, Myrica esculenta, Rhododendronarboreum, Prunus cerasoides, Grewia oppositifolia, Berberisspecies etc can play a paramount role not only inmeeting farm based fodder and fuel wood needsbut also in creating an income generating systemsat the village level in hilly regions (Tewari, 1997).Among the multipurpose tree species (MPT) ofHimalayas, Prunus cerasoides D. Don (FamilyRosaceae) commonly known as Paddam orHimalayan wild cherry is one lesser known andstudied MPT of Kumaun. This is the undercanopyspecies which commonly occurs in association ofQuercus leucotrichophora, Pinus roxburghii, Aesculusindica etc. in the rocky and sloping areas between1200 and 2400 m. (Troup, 1921). Because of itsmultipurpose value the species is very beneficialfor the upliftment of local people. It is one of 31multipurpose species which is used as a medicinalplant in Himalayan region (Samant et.al., 1998).The bark is used in psycho-medicines. The juicefrom the bark applied on body swelling andcontusions. Kernels used as a remedy for stonesand leaves are crushed with twigs and bark andsoaked in water, taken internally to stop abortionand other female disorders (Tewari, 2005). Tree ismainly used as rootstock for cultivation ofcherries, apart from being of medicinal value. Theplant is known to exude gums. The gum possessesantioxidant property (Malsawmtluangi et al., 2014).The tree contains 83 % Moisture, 3.11 % Ash, 7.32% Fiber, 0.319 % Vitamin C and 0.133 mg/gChlorophylls (Sundariyal and Sundariyal, 2001). InKumaun region it is highly used for ethnobotanicalpurposes. The well-seasoned timber of this speciesused to make ornamental furniture, walking sticksetc. which is durable and liable to either fungus or*Corresponding Author:Bhawna Tewari,Department of Forestry & Environmental ScienceKumaun University, Nainital, Uttarakhand, India.insect attack. Bark is used for tannin (Troup,1921).The fruit is edible and the pulp is used to make acherry brandy. The kernels contain oil similar tothat of bitter almond. The off season autumn /winter flowering in the species is also very usefulfor beekeeping. Prunus cerasoides has been identifiedas an excellent framework tree species forrestoring evergreen forest in seasonally dry tropicalforestlands (Pakkad et al., 2003).Seed maturation has been related to the physicalattributes in many species (Pandit et al., 2002). Inmany pine species generally maturity is reachedwhen moisture content of cone is below 50%(Tewari, 2005). Shah (2005) also observed thatseed maturity in Myrica esculenta is attained whenmoisture content of seed is 30%. Knowledge ofexact stage of seed collection can be feasible andof immense important to avoid the collection ofimmature and nonviable seeds which result innursery and plantation failures. However,dormancy in seeds can severely limit germination(Tewari et al., 2011).The regeneration of Prunus cerasoides is very poor inits natural habitat (Tewari et al., 2011). Pakkad etal., (2004 b) has reported around 40% germinationin Prunus cerasoides in the nursery. Literature revealsthat the regeneration of wildest edible speciesBaccaurua sapida (Sundariyal and Sundariyal., 2001)and Myrica esculenta (Pandey, 2002) is poor innatural habitat. Seeds of Prunus cerasoides have hardseed coat and possess mechanical dormancy(Baskin and Baskin, 2001). Shah (2005) has alsoreported mechanical dormancy in Myrica esculenta.4567

Bhawna and Tewari,This adversely affects the germination of thisspecies.The effect of environment on seed germination isvery complex because of interaction of externaland internal factors which modify the rate andmagnitude of germination. However, among thevarious environmental factors that influence theseed germination, water, temperature and light areof paramount importance (Rao, 1984).Temperature plays a significant role during theprocess of seed germination because variousbiochemical reactions in the food reserves of theseed depend on temperature (Kumar andBhatnagar, 1976). Combined effect of temperatureand media was also studied by Pandit, (2002) inseeds of Cupressus torulosa. Maximum and minimumtemperatures vary with the species. Variation intemperatures for seed germination within orbetween species has been reported in most of theHimalayan species (Thapaliyal and Gupta, 1980;Semwal and Purohit, 1980; Thapaliyal et al., 1985).Thapaliyal et al., (1991) has reported that 20º C and25 º C temperature on Top of the paper (TP) andbetween the papers (BP) enhances germination ofAlnus nitida.In Prunus cerasoides there has been little or nofocused studies on propagation techniques. Thestudies on wild edible plant are very limited(Tewari and Dhar, 1997). Tewari (2005) hasworked on nursery techniques of Prunus cerasoides.Pakkad et al., (2004 a) also worked on the geneticvariations and gene flow in Prunus cerasoides. Thepresent study was designed for assessing the exactmaturity time and optimum temperature forattaining the maximum germination in the seeds ofselected species.Material and MethodsStudy areaThe study area, lies between 1350 - 1810 m on theSouthern extremity of the lesser Himalayan zonein Kumaun, it lies at 29º24' N latitude and 79º28' Elongitude. The climate of this area is subtropicalmonsoon type with warmer temperatures towardslower elevation and cool temperature towardshigher elevation. Rainfall is governed by southwestmonsoon and the average annual rainfall duringthe study ranged from 2000-2200 mm. Theclimatic data were taken from Aryabhatt ResearchCentre for Observational Sciences, Nainital. Themean maximum temperature ranged from 12.3º C(January) and 27º C (May) and mean minimumtemperature from 4.5º C (January) to 16.7º C(August) during the study period. After a thoroughsurvey of Nainital district, six sites with suitablerepresentation of Prunus cerasoides were selectedbetween 1350 m and 1810 m altitude (Table 1).www.ijbio.comInternational Journal of Bioassays 5.5 (2016): 4567-4572Table 1: Site characteristics of Prunus cerasoides inNainital district.SitesAltitude(m)AspectShyamkhet I (S1) 1760NorthernShyamkhet I (S2) 1810NortheasternShyamkhet I (S3) 1730NorthernKhamari (S4)1350Mangoli (S5)1375Khurpatal (S6)1425NortheasternNorthernNortheasternAssociate speciesMyrica esculenta, Acercaesium, Pyrus communisMyrica esculenta, Acercaesium, Pyrus communisMyrica esculenta, Acercaesium, Pyrus communisAcer caesium, Pyrus communisPyrus communisAcer caesium, Pyrus communisSeed MaturityTen medium sized healthy trees of Prunus cerasoideswere marked with paint at each selected site atdistance varying between 80 to 100 m from eachother. The selected trees had a clear bole, diseasefree, had good number of flower/fruit andcompact crown. The height, girth and crown ofeach selected tree were measured. The height wasmeasured with Ravi multimeter and girth andcrown area (Length x Width) of each selected treewas measured with meter tape. The fruits werecollected from all selected sites subsequently at 10days interval (during last week of February – MidApril). The seeds collected from each tree, mixedand five replicates (100 fruits/seeds in eachreplicate) were taken and depulped in thelaboratory and dried under shade for 48 hours. Allthe fruit/seed parameters [length (mm), width(mm)] were measured with a digital vernier (Modelno. CD -1206” CS, accuracy 0.02 mm MitutoyoCo.) and weight of 100 fruits/seeds (g) wasmeasured using electronic balance (WENSAR).Moisture content of fruit/seed was determined onthe fresh weight basis by drying the material at 103 2º C for 16 1 h (ISTA, 1993) and thenreweighed. Seeds were then surface sterilized with0.1% HgCl2 and rinsed thoroughly under runningtap water. The Petri-dishes and germination paperwere sterilized at high temperature (130º C) for 4hours to make it free from fungal infection. Fivereplicates of 100 seeds were used. The petri-disheswere lined with germination paper and 100 seedswere then placed on them. The petri-dishes werekept in a seed germinator (20º C) under darkcondition for each collection date. Dailyobservation was taken and germination wascounted when visible protrusion of radical (1mm)occurred. The germination was monitored for 90days with water being added at regular interval.After completion of experiment germinationpercent and germination capacity was calculated(Shah et. al., 2010).Germination testThe experiment was carried out on fully ripenedfruits which had started to fall down. Afterdepulping, sterilization and washing threereplicates each of 100 seeds were used.4568

Bhawna and Tewari,International Journal of Bioassays 5.5 (2016): 4567-4572The Petri-dishes were lined with germinationpaper. To enhance the germination percent (as itwas found low in the test of maturity indices) theseeds were placed on two conditions i.e. abovepaper (AP) and between papers (BP) and allowedto germinate at different temperatures i.e. Roomtemperature (16 º - 22 º C T1), 20º C (T2) and 25ºC (T3) in dual chamber seed germinator. Dailyobservations were made for germination at varyingtemperatures following the procedure used instudying maturity indices.Statistical test:The data of maturity indices was statisticallyanalyzed for multiple analyses of variance(ANOVA) to show the significant differencebetween sites and dates. In germination testANOVA showed significant difference betweensites, temperature and conditions (Snedecor andCochran, 1967).CD was calculated asCD S.Em* t0.05 (t0.05 is t value at 5% level ofsignificance)Where S. Em is the standard error of differencecalculated as S.Em. Me / r (Lavania, 2004).ResultsTree characteristicsAmong all the six sites the mean tree height variedbetween 17.6 0.62 m (S3) and 22.2 3.04 m(S6). Mean tree diameter at breast height (dbh)varied between 50 0 cm (S5) and 72 5.6 cm(S1). Mean crown cover varied between 20.6 1.02 m2 (S2) and 39.3 1.76 m2 (S5) (Table 2).Table 2: Tree characteristics for different sites ( SE)S.No.Sites123456Shyamkhet (S1)Shyamkhet (S2)Shyamkhet (S3)Khamari (S4)Mangoli (S5)Khurpatal (S6)Mean treeMean treeheight (m) diameter (cm)20.7 0.8472 5.617.6 0.65 55.6 12.3317.6 0.6256 12.2218.9 1.6360 1018.1 1.5750 022.2 3.04 53.3 13.33Crowncover (m2)36.8 2.520.6 1.0234 3.0530.3 3.1739.3 1.7624 2.30Fruit/Seed characteristicsThe green colour of fruit changed with eachcollection date and pale red to red at finalcollection in second week of April. Across all thesites the fruit/seed length, width and weight of 100fruits increases gradually with each collection date.The mean fruit length ranged between 4.21 0.19mm and 15.69 0.29 mm, mean fruit widthbetween 5.57 0.12 mm and 11.53 0.2 mm andmean weight of 100 fruits ranged between 18.76 1.03 g and 114.57 2.1 g. The fruit parameters(length, width and weight of 100 fruits) variedsignificantly (P 0.01) across sites and dates ofcollection. The mean seed length ranged between7.46 0.1 mm and 12.87 0.29 mm, mean seedwidth between 4.31 0.09 mm and 8.91 0.08www.ijbio.commm and mean weight of 100 seeds ranged between20.37 0.18 g and 48.08 0.68 g. The seedlength/width varied significantly (P 0.01) acrosssites and dates of collection. The weight of 100seeds was varied significantly (P 0.01) across datesof collection. The fruit moisture content 50.24 0.19 % and seed moisture content 30.11 0.57 %at fifth collection in S6 site coincide withmaximum germination (20 8.8 %). Negativecorrelation existed between germination and seedmoisture content (r 0.294; P 0.01) (Fig. 1).There was no relation between other physicalparameters and maturity.Germination testAt room temperature the mean germination inseeds placed on AP, varied between 6.66 2.66%and 53.33 13.33 %. On BP germination variedbetween 0 0% and 7.49 4.16 % across all thesites (Table 3).Table 3: Variation in germination of Prunuscerasoides seeds subjected to three differenttemperatures (T1 room temperature), T2 20ºC and T3 25º C) and two conditions, abovepaper (AP) and between paper (BP). The valuesare mean of two years. MG Mean germination;GC Germination capacity.Site Temperature Condition M.G %S1 T1 (room)AP16.66 16.66BP0 0T 2 (20º C)AP6.3 0.3BP9.33 4T 3 (25 º C)AP59.16 0.83BP30.83 2.5S2 T 1 (room)AP25 15BP0 0T 2 (20º C)AP17.5 2.5BP9.5 0.5T 3 (25 º C)AP63.33 6.67BP46.66 3.33S3 T 1 (room)AP53.33 13.33BP0 0T 2 (20º C)AP1 1BP2.6 0.35T 3 (25 º C)AP63.33 6.67BP41.33 2S4 T 1 (room)AP6.66 2.66BP0.33 0.33T 2 (20º C)AP8.83 1.17BP2.33 2.33T 3 (25 º C)AP46.66 0BP22.83 3.83S5 T 1 (room)AP33.33 33.33BP10.66 4.33T 2 (20º C)AP5.99 1.66BP1.83 1.83T 3 (25 º C)AP54.99 1.66BP29.99 3.33S6 T 1 (room)AP30.99 17.66BP7.49 4.16T 2 (20º C)AP5 5BP6.5 0.5T 3 (25 º C)AP33.33 13.33BP29.99 3.33G.C 360.8352.5055.0057.5063.3355.834569

Germination %Bhawna and Tewari,International Journal of Bioassays 5.5 (2016): 4567-4572y -0.6651x 45.584R2 0.0868706050403020100significantly (P 0.01) across all thetemperatures and conditions (Table 4).sites,DiscussionPhysical indices have been widely used, particularlysince the seed collectors lend themselves to fieldestimation. Several workers throughout the worldhave investigated colour as a workable indicator0204060for several species (Shah, 2005). Distinct colourMoisture content (seed)changes have been associated with seed maturity inhard wood fruits. The physical characters ofFig. 1: Relationship between germination andfruit/seed have played a significant role in maturitymoisture content of seed in P.cerasoides across allindices (Tewari, 2005). Various mature andthe sites.immature fruits and seeds can be distinguished invarious ways e.g. by colour difference, increasedAt 20º C the mean germination in seeds placed onfirmness, brittleness, decreased moisture content,AP, varied between 1 1 % and 17.5 2.5 %. Onspecific gravity and by change in physicalBP germination varied between 1.83 1.83 % anddimensions (Shah et al., 2010). The fruit maturation9.5 0.5 % across all the sites (Table 3).of P.cerasoides became apparent with the change inits colour from green to pale green and finally redTable 4: Analysis of variance (ANOVA) forwith maturity. The germination was maximumvariations in germination on Prunus cerasoides seedswhen the colour of fruit turned pale green.of different sites subjected to differentUpadhyay et al., (2006) also found colour change totemperatures (T 1 room temperature), T 2 20ºbe one of the best criteria for determining maturityC and T 3 25 º C) and conditions (AP Abovein Bauhinia retusa. Moisture content and specificpaper, BP Between paper).Sig. / gravity are other two physical parameters that areSource ofMeanCharactersdfF. value non interrelated and more objective. Both have beenvariationsquaresig.reported as reliable maturity indices by numerousGerminationSite5731.4374.823.001* researchers (Shah, 2005).(Year 1)Germination(Year 2)TemperatureConditionSite xTemperatureSite x ConditionTemperature xConditionSite xTemperature xCondition.Site2116035.954 105.7322241.333 14.778.000*.000*10430.4982.8385204.1781.346 .255 NS2495.5833.268 .044 NS10373.4612.462 .014 NS563.52612.451.000*15295.009 337.94210800.000 238.625.000*.000*5Temperature2Condition1Site x10TemperatureSite x Condition5Temperature x2ConditionSite xTemperature x10Condition.(Germination Year 1)Site*CD at 5 %10.53Temperature*CD at 5 %35.26NS non-significant* Significant at 1% (P 0.01)** Significant at 5% (P 0.05).005**533. 83111.795.000*247. (Germination Year 2)*5.73*19.27At 25º C the mean germination in seeds placed onAP, varied between 33.33 13.33 % and 63.33 6.67 %. On BP the germination varied between22.23 3.83 % and 46.66 3.33 % across all thesites (Table 3). Across all sites the maximumgermination occurred at 25º C when seeds hadbeen placed on above paper. Germination variedwww.ijbio.comDecline in moisture content appears to be a goodindicator of seed maturity in P.cerasoides. Themoisture content of the seeds declined as fruitsmatured (Tewari, 2005). Decline in fresh weightmoisture content percent from maturing seeds isclosely related to seed maturity (Pandit et al., 2002).The maximum germination (20 0 %) at 30.11 0.57 % seed moisture content was observed inP.cerasoides. Shah et al., (2006) have reported thatmoisture content of 23.4 - 36.1 % can beassociated with optimum germination in Pyracanthacrenulata seeds.Negative correlation existed between germinationand seed moisture content (r 0.294; P 0.01) inP.cerasoides. There was no relation between otherphysical parameters and maturity in P.cerasoides.Edwards (1980) reported significant correlationbetween maturity and physical parameters butseveral studies have reported no correlationbetween the two parameters (Tewari, 2011).Edwards (1969) could not find any relationshipbetween physical parameters and maturity.The absorption rate is dependent upon degree ofseed coat permeability as the seed coat indirectlyinhibit rate of absorption by mechanicallypreventing seed tissue from expanding in pacewith moisture uptake. The presence of animpermeable seed coat may prevent water uptakeby seeds and so prevent germination, as seed do4570

Bhawna and Tewari,not resume physiological activity until they imbibea certain amount of water. Prunus cerasoides is onesuch species having hard seed coat dormancy.Pakkad et al., (2004 b) found around 40%germination in seeds of Prunus cerasoides in thenursery. The hard seed coat mechanical dormancyis usually found in Prunus genera due to stonyendocarp (Heit, 1967) (Baskin and Baskin, 2001)occurs in several other species like Acacia, Prosopis,Ceranotia, Robinia and Cassia etc. (Pant, 2002). Shahet al., (2010) has reported this kind of dormancy inMyrica esculenta. Seeds of some species germinatebetter at constant temperature and others onalter

Kumaun University, Nainital, Uttarakhand, India. 4567 Seed germination in Prunus cerasoides D. Don influenced by natural seed desiccation and varying temperature in Central Himalayan region of Uttarakhand, India. Bhawna Tewari*, Ashish Tewari Department of Forestry & Environmental Science, Kumaun University, Nainital, Uttarakhand, India.

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