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Seed dormancy, germination and seedling characteristics of Elaeocarpus prunifolius Wall. ex Müll. Berol.: a threatened tree species of north-eastern India

Seed dormancy, germination and seedling characteristics of Elaeocarpus prunifolius Wall. ex Müll.... Background: Elaeocarpus prunifolius Wall. ex Müll. Berol. is a threatened tree species of north-eastern India. The present study was undertaken to investigate the type of dormancy prevailing in seeds of E. prunifolius, explore seed dormancy breaking techniques and assess seedling fitness. Methods: Ripe fruits of E. prunifolius were harvested from Jaintia hills, and seeds were subjected to various physical, manual and chemical treatments. The effect of plant growth regulators, viz gibberellic acid (GA ) and potassium nitrate (KNO ), were tested. Seedling vigour and survival based on seed weight were examined. Results: Germination took 6 months to initiate after seed dispersal and natural germination percentage of fresh seeds was 24%. Physical pre-germination treatments such as surface and acid scarification failed to overcome dormancy. Cracked seeds promoted germination (46%) with a mean germination time of 146 days (time to 50% −1 germination, T = 144 days). Among the GA treatments, split seeds treated with GA (3000 mg/L ) yielded the 50 3 3 highest germination (24%) with a T of 55 days whereas KNO did not promote germination. A combination of 50 3 GA and KNO , however, increased the germination to 31%. Between the seed weight classes, the highest 3 3 percentage of germination was observed in heavy seeds (25%) and the lowest in light seeds (20%). There was no significant variation between seed weight and germination time (p > 0.05). Seed weight had a significant effect on the shoot height, number of leaves and dry weight of seedlings (p < 0.05). Conclusion: Based on the seed tests, E. prunifolius seeds exhibits ‘combined’ dormancy (physical and physiological) as splitting seed coat and application of GA effectively broke dormancy. Splitting the seed coat is a cost-effective method for accelerating germination of seeds. Heavy-weight seeds produced better performing seedlings compared to their counterparts which may be viewed as an important reproductive strategy of the species. Keywords: Conservation, Elaeocarpus, Germination, Seedling growth, Threatened Background 2000; Gill et al. 2013; Tanaka-Oda et al. 2017). Thus, In recent years, studies on germination have emerged as such studies enable the development of suitable proto- an important tool for conservation of many species. cols for species conservation and management. Such studies aid understanding of natural regeneration The genus Elaeocarpus have ca. 360 species distributed processes as well as identifying possible causes of species across East Asia, Australia, Malaysia and the Pacific decline, persistence or spread in changing landscapes Islands. The International Union for Conservation of and their response to global climate change (Schütz Nature and Natural Resources (IUCN) have identified 38 Elaeocarpus species under various threat categories. Of these, 3 species are critically endangered, 4 endangered, * Correspondence: upkri@yahoo.com 20 vulnerable, 2 near threatened, 8 conservation Department of Basic Sciences and Social Sciences, School of Technology, North-Eastern Hill University, Shillong 793 022, India dependent and 1 data deficient (IUCN 2017). In India, Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 2 of 10 30 species occur (Murti 1993a, b), the majority of which broad-leaved forests with only a few matured individuals are confined to the northeast (Assam, Arunachal Pra- (Walter and Gillett 1998; Nayar and Sastry 1990). The desh, Sikkim, Manipur, Meghalaya, Mizoram, Nagaland, species is on the verge of extinction due to habitat degrad- Tripura) and south (Tamil Nadu and Kerela) India ation (Jain and Rao 1983; Haridasan and Rao 1985), which (Khan et al. 2003; Singh et al. 2013). Out of the six is further exacerbated by its on-going exploitation for tim- threatened Elaeocarpus species reported from the country, ber by local communities (Nayar and Sastry 1990). The one species (E. gaussenii Weibel) is critically endangered fruits of the species are also edible. and one endangered (E. blascoi Weibel), three are Vulner- Propagation of Elaeocarpus species from seed is chal- able (viz. E. prunifolius Wall. ex Müll. Berol, E. recurvatus lenging, as the seeds exhibit dormancy (Khan et al. Corner and E. venustus Bedd.) and one (E. munroii Mast.) 2003). The fruits of E. prunifolius mature in early Sep- falls under lower risk/near threatened category. Of these, tember and remain dormant throughout the winter (No- E. prunifolius is restricted to north-eastern India and the vember–February). The seeds germinate in April with others are confined to southern India. the onset of rain so moisture may be a requirement for Research on Elaeocarpus species has gained momen- germination. The inherent stony endocarp in many tum with the discovery of indolizidine alkaloid com- members of the genus seems to be a plausible factor for pounds in members such as E. fuscoides Knuth., E. poor germination (Bhuyan et al. 2002; Khan et al. 2003; grandis F. Muell., E. polydactylus Schltr. and E. densi- Ramasubbu and Irudhyaraj 2016). Some studies have re- florus Knuth (Katavic 2005) for their potential treatment vealed low (1–3%) and erratic seed germination in of diseases such as AIDS, diabetes and cancer (Wiart Elaeocarpus ganitrus Roxb. ex G.Don (Khan et al. 2003) 2006). In addition, fruits of species, viz E. floribundus and E. japonicus Siebold (Yang et al. 2001). Natural re- Blume, E. lanceifolius Roxb., E. braceanus Watt ex C.B. generation of the threatened E. blascoi was only 5% in Clarke and E. sikkimensis Mast, are edible (Andola et al. the wild (Ramasubbu and Irudhyaraj 2016) while in E. 2011;Das 2014) and species such as E. grandis, E. rumi- venustus, E. serratus Land E. williamsianus Guymer, natus F.Muell., E. foviolatus F. Muell. and E. coorangoo- there was no germination (Saravanan et al. 2011; Daha- loo J.F. Bailey & C.T. White are known for their good nayake et al. 2013; Rossetto et al. 2004). Contrastingly, timber value (Bristow et al. 2005). some species such as E. floribundus had fairly high ger- Elaeocarpus prunifolius is a middle-sized tree that at- mination (47%) (Das 2014). However, there is a dearth of tains a height of ca. 20 m at maturity (Nayar and Sastry knowledge (particularly on germination) of threatened 1990). The phenological calendar of the species is pre- species often targeted for conservation (Baskin and Bas- sented in Table 1. The inflorescences are 3–9 cm long, kin 2001). Khan et al. (2003) studied the germination and the flowers are pale yellow in colour. The ovoid capacity of E. ganitrus by subjecting the seeds to various fruits range from 0.8 to 1.2 cm in diameter. They change treatments and found that seeds cracked with a vice re- from green to bluish-black on maturation. There is only sulted in highest germination percentage (40%) followed one seed per endocarp. The ripe fleshy mesocarp is pre- by seeds soaked in hot water for 24 h and seeds fermen- dated by worms, insects and birds, and seeds are pre- ted for 20 days (37%). A mixture of silt and sand media dated by small rodents, thus, dispersing the seeds in the resulted in 40% germination in E. venustus whereas process (epizoochory) (field observation). seeds sown in a media of sand, silt and cow dung germi- Elaeocarpus prunifolius is a rare and threatened tree nated to about 8% (Irwin et al. 2013). In E. serratus, species distributed in the state of Manipur and Meghalaya seeds treated with 50% HNO germinated to 15% (Daha- in India and Bangladesh (Murti 1993a, b;World Conser- nayake et al. 2013). However, there is lack of information vation Monitoring Centre 1998). In Meghalaya, the spe- on the regeneration capacity of E. prunifolius. The poor cies is restricted to fragmented pockets of sub-tropical regeneration of the species in nature could be due to Table 1 Phenological calendar of E. prunifolius LF leaf flushing, LM leaf maturation, LFA leaf fall, FL flowering, FR fruiting, FM fruit maturation Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 3 of 10 hard seed coat and/or prolonged dormancy period. The Seed treatments results presented here form part of a larger study to as- sess the causes of the species rarity in nature by under- (a) Seed coat impermeability is often associated with standing the species phenology, mode of dispersion, the presence of impermeable palisade layers of predation, seed physiology, germination pattern, seedling lignified cells (Vazquez-Yanes and Perez-Garcia characteristics and factors debilitating seedling establish- 1976). Therefore, the following physical or chemical ment. The objectives of the current work were to: (i) treatments were applied in an attempt to disrupt understand the type of dormancy present in the seeds of the seed coat. For each experiment, three replicates E. prunifolius by subjecting the seeds to a set of physical of 50 seeds (irrespective of weight) were main- and chemical treatments; (ii) test if total germination tained. Seeds were: and germination rate were influenced by seed weight; 1a. Soaked in cool water (20 °C) for 24 h and (iii) examine if the seedling growth and survival 2a. Soaked in hot water (80 °C) for 24 h till the were influenced by seed weight. The results are dis- water cooled cussed in relation to possible factors responsible for low 3a. Soaked in boiling water (5 min in 100 °C and recruitment of this species in nature. immediately transferred to cold water) for 24 h 4a. Scarified by rubbing near the micropyle with Materials and methods sandpaper Seed source 5a. Cracked lengthwise with a vice Ripe fruits of E. prunifolius were harvested during 6a. Scarified near the micropyle using sandpaper mid-September 2013 from Jarain (25° 19.05′ N, 92° and soaked in warm water (45 °C) for 2 h 08.34′ E, alt 1200 m asl), the known location of the spe- 7a. Soaked in 95% H SO for 5 min 2 4 cies in Meghalaya, Northeast India (Haridasan and Rao 8a. Soaked in 95% H SO for 10 min 2 4 1985). As the species is rare, all mature trees were 9a. Soaked in 95% H SO for 15 min 2 4 marked wherever encountered and monitored for fruit 10a. Soaked in 95% H SO for 20 min 2 4 maturation. Fruits were collected randomly from ten 11a. A control was maintained by sowing seeds randomly selected trees and bulked together to form a (also irrespective of seed weight) without any of composite sample (ca. 4500 fruits). The fruits were the above-mentioned treatments. brought to the laboratory and soaked in normal tap water (18 °C ± 1 °C for 24 h) to soften the pulp and also The treated seeds were transferred to plastic trays to separate healthy seeds from damaged ones (Pipinis et filled with a mixture of garden soil and sand in the ratio al. 2011). Empty or damaged seeds are lighter and float of 3:1. Each tray was labelled and kept under laboratory to the surface and were thus separated from healthy conditions having an average temperature of 24 (± 1 °C) seeds. The fruits were hand crushed and the seeds col- and light (> 700 lx) duration of 8 h. The trays were lected. The seeds were washed thoroughly in running watered at 3-day intervals and monitored for germin- water to remove any adhering substance from the endo- ation for a period of 12 months. carp and disinfected in 0.2% potassium permanganate (KMnO ) solution for 2 h following the method of Zuo (b) The effect of plant growth regulators (PGRs) on (1994). The seeds were placed on paper to remove ex- breaking seed dormancy and the rate of cess moisture and were subjected to various germination was examined by performing the dormancy-breaking treatments within 5 days of seed col- following tests on seeds cracked lengthwise. Three lection. The remaining seeds were stored in an airtight replicates of 25 seeds were maintained for each container filled with a substrate (moist sand) and stored treatment. Cracked seeds were: −1 at a constant temperature of 5 °C (±1 °C) in a refrigerator 1b. Soaked in 200 mg L gibberellic acid (GA ) for further studies. for 48 h −1 2b. Soaked in 500 mg L gibberellic acid (GA ) Water imbibition test for 48 h −1 Twenty-five seeds were cracked with a vice, and the ini- 3b. Soaked in 1000 mg L gibberellic acid (GA ) tial weight of each seed was measured. The weights of for 48 h −1 25 uncracked seeds were also determined. Both sets of 4b. Soaked in 2000 mg L gibberellic acid (GA ) seed were then placed on Petri dishes (90 mm × 20 mm) for 48 h −1 lined with moist cotton and kept covered under identical 5b. Soaked in 3000 mg L gibberellic acid (GA ) laboratory conditions. Each seed was reweighed after 2, for 48 h 4, 6, 8, 10, 20, 30 and 40 h, and the percentage change in 6b. Soaked in 0.5% potassium nitrate (KNO ) for mass was averaged from the 25 seeds from each set. 48 h Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 4 of 10 7b. Soaked in 1% KNO for 48 h months after transplantation. To assess the effect of light 8b. Soaked in 1.5% KNO for 48 h on seedling growth, a threefold contrast in light level 9b. Soaked in 2% KNO for 48 h was created by covering the net houses with an increas- − 1 10b. Soaked in GA solution (200 mg L ) for 48 ing layer of shade netting. Each extra layer intercepted h and transferred to 0.5% KNO solution for an additional 25% (approximately) of the incoming radi- another 48 h ation, thus, creating three light regimes of approximately − 1 −2 −1 11b. Soaked in GA solution (500 mg L ) for 48 70–75% (16 ± 1.8 mol m day ), 25–30% (4 ± 0.46 mol −2 −1 −2 −1 h and transferred to 1% KNO solution for m day ) and 5–10% (1 ± 0.05 mol m day ) desig- another 48 h nated as high, intermediate and low light treatments re- 12b. Soaked in distilled water for 48 h spectively. Light intensity was measured with digital lux meter (Lutron LX-101A) at three different times during The control and treated seeds were placed in Petri the day (9.00 a.m., 12 noon and 3 p.m.) each month and dishes (90 mm × 20 mm) lined with moist filter paper the seasonal means were calculated. and transferred to growth chambers fitted with an LED To assess the effect of seed weight and light on growth light source (> 4000 lx) with a photoperiod of 8 h. A con- performance of the seedlings, ten randomly selected stant temperature of 25 °C (± 1 °C) was maintained for seedlings under each weight category and light condition germination. The germination of the seeds in the growth were harvested after one year of transplantation (May chambers was monitored at 3 d intervals for a period of 2015) and their shoot height, root length, the number of 4 months. leaves, leaf area and dry matter yield were determined. The leaf area was measured using a leaf area meter Seed viability (LICOR, Lincoln). Dry matter yield was determined by One hundred and fifty untreated seeds were randomly drying the plant material in an oven at 80 °C for 24 h to selected from those stored at 5 °C for 12 months. Each a constant weight. seed was cracked lengthwise and the embryo excised. Germination percentage was calculated using the Each embryo was tested for viability following the tetra- formula: zolium assay of Wang et al. (2005). The presence of liv- ing cells in the seeds converts the TTZ (2,3,5 triphenyl GðÞ % ¼  100; tetrazolium chloride) to formazan (red colour) through the hydrogen transfer reaction catalysed by the cellular where n is the number of germinated seeds and N is the dehydrogenase, thus, staining the viable seeds red. total number of seeds. The method of Coolbear et al. (1984) further modified by Farooq et al. (2006) was used Seed germination and seedling growth to calculate the time to reach 50% germination (T ): A subsample of 1500 seeds was used to examine the ef- fect of seed weight on germination and seedling growth. T ¼ t þ −n t −t =n −n ; 50 1 i j i j i Each seed was examined by the floatation method and 60 seeds were found to be damaged. The remaining 1440 healthy seeds were weighed individually. The num- where N is the final number of germinated seeds, n and ber of seeds in each of three weight categories (light (< n are the cumulative numbers of seeds germinated at 320 mg), intermediate (320–480 mg) and heavy (> 480 times t and t , respectively, when n < N/2 < n . i j i j mg)) was determined. Seeds from each category were sown under laboratory conditions in plastic trays (26 cm Seed coat thickness diameter, 8 cm height) within 10 days from the time of Seed-coat thickness was determined in each seed weight seed collection. A mixture of garden soil and sand media category to test the assumption that: (i) heavier seeds in the ratio of 3:1 was used for planting. Twenty-five had thicker coats; and (ii) thicker seed coats may delay seeds were sown per tray. Germination was recorded at germination. Seeds were cut across and seed coat thick- 3-day intervals up to 6 months and weekly up to 12 ness was measured using a calliper and the values aver- months. The number of germinated seeds was recorded aged. For each weight category, 30 seeds each were on each occasion. Seeds were considered germinated considered. when the radicles protruded out of the soil surface. Germinated seedlings (about 2 months old) were Data analysis transplanted into 20 × 17 cm poly bags and transferred To determine the effect of different treatments on ger- to the net house during May 2014. The moisture level mination, analysis of variance (ANOVA) was used was maintained by watering every 3 days. Seedling sur- followed by Tukey and Scheffe’s least significant differ- vival and growth were monitored for a period of 12 ence (p < 0.05). Assumptions of ANOVA were met Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 5 of 10 through a test for normality of variables (Shapiro-Wilk control seeds was 213 ± 5 days (T = 174 ± 9 days). No test) and homogeneity of group variances (Levene’s test). germination of seeds occurred after 230 days. Only treat- The relation between seed weight and seedling growth ments 1a, 5a and 6a resulted in any germinated seed- was analysed by regression. All the statistical analysis lings. Of these, treatment 1a (cold-water soak) reduced was performed using SPSS software (version 20). germination to 10.67% (T = 210 days) compared with the control (24%) while 14.67% of seeds (T = 216 days) Results germinated following treatment with a combination of Imbibition test scarification and warm water treatment (Treatment 6a). The initial moisture content of scarified seeds was 10.6% The germination percentage of seeds from these two and non-scarified seeds was 10.8%. After 40 h, both sets treatments was significantly lower (p < 0.05) than the of seeds had a moisture content of 11.2% so the increase control. In contrast, 45.83% of seeds cracked with a vice in the mass of scarified and non-scarified seeds was 6% germinated and this was significantly higher (p < 0.05) and 4% respectively. This difference was not significant than for all other treatments. The mean germination (p > 0.05) so uncracked seeds imbibe water at a similar time and T was also significantly reduced under this rate as cracked seeds. treatment (146 and 144 days respectively) (Table 2). Treatments (1b-11b) involving soaking cracked seeds Dormancy-breaking treatments in solutions of various plant growth regulators were Physical and chemical treatments 1a - 10a were com- compared with germination of cracked seeds soaked in pared to germination of untreated seeds (11a). water (Treatment 12b), Table 2. Cracking seeds (Treat- The germination percentage of control seeds was 24%. ment 5a) resulted in better germination than no treat- The mean number of days required for germination of ment (11a) but soaking seeds in water (Treatment 12b) Table 2 Germination of E. prunifolius seeds subjected to (a) physical, mechanical and chemical scarification and (b) plant growth regulators Treatment No. Treatment details Mean germination (days) Germination (%) T (days) a a 1a Cold water (20 °C) 210 ± 1 10.67 ± 1.33 210 ± 2 2a Hot water (80 °C) 0 0 0 3a Boiling water (100 °C) 0 0 0 4a Scarification near micropyle 0 0 0 5a Cracked seeds with vice 146 ± 1 45.83 ± 1 144 ± 2 a a 6a Scarified+warm water (2 h) 212 ± 1 14.67 ± 1.33 216 ± 2 7a 95% H SO for 5 min 0 0 0 2 4 8a 95% H SO for 10 min 0 0 0 2 4 9a 95% H SO for 15 min 0 0 0 2 4 10a 95% H SO for 20 min 0 0 0 2 4 11a Untreated control 213 ± 5 24.67 ± 3 174 ± 9 −1 1b 200 mg L GA 00 0 −1 a a 2b 500 mg L GA 78 ± 1 15.56 ± 2 77 ± 1 −1 b ad 3b 1000 mg L GA 63 ± 1 17.78 ± 2 64 ± 2 −1 c ae 4b 2000 mg L GA 70 ± 2 20 ± 0 71 ± 2 −1 d ce 5b 3000 mg L GA 54 ± 1 24.44 ± 2 55 ± 1 6b 0.5% KNO 00 0 7b 1% KNO 00 0 b b 8b 1.5% KNO 63 ± 3 8.89 ± 2 62 ± 3 9b 2.0% KNO 00 0 −1 b de 10b 200 mg L GA + 0.5%KNO 63 ± 3 22.22 ± 2 61 ± 3 3 3 −1 de f 11b 500 mg L GA + 1% KNO 56 ± 1 31 ± 2 56 ± 2 3 3 12b Water 0 0 0 T : time required to attain 50% germination, ±SEM (standard error of mean) Note: For each treatment, means followed by the same letter in each column do not differ significantly at p < 0.05 Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 6 of 10 −1 produced no germination. Seeds treated with 200 mg L Seed weight and seedling growth GA (Treatment 1b) did not germinate either but there Seed weight (n = 1440) ranged from 160 to 630 mg (mean was a positive linear relationship (Y = 0.0076x + 4.4304, weight 385.58 ± 1.90 mg). The distribution was normal R = 0.726, p = 0.03) between germination percentage (Shapiro-Wilk test), with intermediate seeds constituting and increasing GA concentration with a maximum 74.93% of the total seed population followed by light and germination of 24.44% for seeds treated with 3000 mg heavy that accounted for 16.31% and 8.75% respectively −1 L GA (T = 55 days), Table 2. Increasing concen- (Table 3). More heavy seeds (24.60%) germinated than 3 50 tration of GA decreased the time required to attain intermediate (22.80%) or light seeds (19.57%), Table 3. 50% germination (T )(Fig. 1), more so than the pot- There was no significant relationship between the seed ted experiments (treatment 1a-11a). No seeds germi- weight and germination time (p > 0.05). The time to 50% nated following treatment with 0.5%, 1% or 2% KNO germination (T ) was longest in intermediate seed weight 3 50 (Treatment 6b, 7b and 9b) but, surprisingly, a small (218 days) and shortest for light seeds (188 days) (Table 3). percentage of seeds (8.89%) did germinate following One year after transplantation, 100% of seedlings emer- −1 Treatment 8b (1.5% KNO ). Neither 200 mg L nor ging from heavy- and intermediate-weight seeds had sur- 0.5% KNO alone (Treatments 1b and 6b respectively) vived but only 85% of those from light-weight seeds. Seeds produced any germination yet the combination of the < 320 mg had an average coat thickness of 0.66 mm com- two (Treatment 10b) did result in 22% germination. pared with 1.42 mm for seeds > 480 mg. Light seeds had The highest germination percentage of 31% (T =56 an average length of 12 mm whereas seed length in inter- −1 days) was obtained using a combination of 500 mg L mediate and heavy seeds measured an average 13 mm and GA and 1.0% KNO . Results from multiple compari- 15 mm respectively. Similarly, seed width was lower in 3 3 son tests between the various treatments showed a light weight seeds (7 mm) as compared to heavy seeds (8 significant difference in the mean germination per- mm), Table 3. centage of seeds treated with a combination of 500 Significant differences (p < 0.05) were observed in the −1 mg L GA +1% KNO with all concentrations of shoot height, leaf area and dry biomass of seedlings 3 3 GA and KNO (p < 0.05) except for seeds treated in emerging from light seeds compared with those of inter- 3 3 −1 −1 3000 mg L GA and 200 mg L +0.5% KNO . mediate and heavy seeds (Table 4). However, there was 3 3 no significant difference between the intermediate and heavy seeds (p > 0.05). Seed viability Regression analysis showed that seed weight had a Embryos stained with tetrazolium indicated viability. Of positive impact on seedling vigour with heavier seeds the 150 seeds tested, 111 embryos (74%) were stained. producing larger and heavier seedlings (Table 5). Visual inspection of the embryos showed that they were curved with narrow cotyledons characteristic of 40% of Discussion Malesian Elaeocarpus species (Weibel 1968; Flora Mal- Seeds stored at 5 °C remained viable even after 12 esiana Symposium 1989). months of storage indicating ‘orthodox’ (i.e. will survive Fig. 1 Mean (±SEM) germination percentage (G%) and time to 50% germination (T ) under GA and KNO treatments at 25 °C. 50 3 3 −1 −1 −1 −1 −1 (Treatments: 2b = 500 mg L , 3b = 1000 mg L , 4b = 2000 mg L , 5b = 3000 mg L , 8b = 1.5% KNO , 10b = 200 mg L GA +0.5% KNO , 3 3 3 −1 11b = 500 mg L GA +1% KNO ) 3 3 Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 7 of 10 Table 3 Germination characteristics of E. prunifolius seeds Parameters Seed weight class(mg) Light (< 320) Intermediate (320–480) Heavy (> 480) Number of seeds sown (n) 235 1079 126 Average seed length (mm) 11.92 ± .064 13.47 ± .028 14.52 ± .059 Average seed width (mm) 6.80 ± .027 7.37 ± .017 8.02 ± .034 Number of seeds germinated 46 246 31 Proportion of Seed Contribution to total seed lot (%) 16.31 74.93 8.75 a a a Germination (%) 19.57 22.8 24.6 Mean germination (days) 211 ± 4 212 ± 1 213 ± 3 T (days) 188 ± 9 218 ± 7 204 ± 6 Seed coat thickness (mm) 0.66 ± 0.12 1.07 ± 0.06 1.42 ± 0.06 Weight of cotyledon (g) 0.077 ± 0.005 0.102 ± 0.002 0.117 ± 0.002 Note: For each treatment, means followed by the same letter in each row do not differ significantly at p < 0.05 drying and/or freezing during ex situ conservation) char- still remained dormant for ca. 3 months and the germin- acteristics (Chin et al. 1989). However, intact untreated ation time decreased further only by soaking in gibberel- −1 seeds took an average of 213 days to germinate under lic acid solution (≥ 500 mg L ) or a combination of favourable conditions, which clearly indicates the pres- gibberellic acid and potassium nitrate. Similar results ence of dormancy (Baskin and Baskin 1998, 2004). Phys- have been reported in species like Ramonda serbica ical and mechanical dormancy is the most common Pančić, R. nathaliae Pančić & Petrovič, Magnolia yunna- inhibitor of germination in many species. Threatened nensis (Hu) Noot. and M. punduana (Hk. f & Th.) Figlar tree species such as Cupressus atlantica (Youssef et al. (Gashi et al. 2012; Han et al. 2010; Iralu and Upadhaya 2012), Elaeocarpus blascoi Weibel (Ramasubbu and 2016). The presence of abscisic acid (ABA) in mature Irudhyaraj 2016) and Intsia bijuga (Colebr.) Kuntze seeds has been associated with seed dormancy (Matakia- (Thaman et al. 2006) exhibit exogenous dormancy where dis et al. 2009; Hilhorst and Karssen 1992), and studies the hard seed coats inhibit water penetration that is re- have established that the application of nitrate ions to a quired for embryo growth and development. germination medium leads to rapid decline of ABA. Reducing germination time and increasing germin- However, three of the four concentrations of potassium ation percentage are both important pre-requisites of a nitrate tested alone in the current study prevented ger- successful propagation initiative. Cracked E. prunifolius mination. The positive effect of some PGAs in the seeds germinated faster and in greater numbers than in- current study indicates that (in addition to physical dor- tact seeds, as expected by disrupting the hard coat sur- mancy) seeds may also have ‘physiological’ dormancy rounding the seed. Similar results have been reported in also called ‘combined’ dormancy (Baskin and Baskin E. ganitrus (Khan et al. 2003). However, cracked seeds 1998; Nikolaeva 1969), which was broken by the Table 4 Shoot height and root length (cm), number of leaves/plant, leaf area (cm ) and dry weight/plant (g) of seedlings of E. prunifolius (±SEM, n = 10) in light-, intermediate-and heavy-seed weight classes grown under three different contrasting light regimes after 1 year of transplantation Day length (h)/light intensity (mol Seed weight Shoot height Root length Number of leaves/ Leaf area/seedling Dry weight/plant −2 −1 2 m d ) (mg) (cm) (cm) plant (cm ) (g) a a a a a 10/16 < 320 20.57 ± .39 16.63 ± 0.54 7.2 ± 0.48 9.93 ± 0.18 0.56 ± 0.08 320–480 39.25 ± 0.90 26.80 ± 1.10 15.4 ± 0.52 21.09 ± 0.69 2.76 ± 0.67 a a a a a > 480 26.46 ± 0.17 17.28 ± 0.35 11.4 ± 0.39 13.72 ± 0.74 1.43 ± 0.33 b b 10/4 < 320 31.27 ± 1.07 25.84 ± 2.13 12.40 ± 0.25 15.81 ± 0.63 1.74 ± 0.24 b b c b b 320–480 46.68 ± 2.66 26.22 ± 0.72 15.6 ± 1.28 22.61 ± 1.03 2.95 ± 1.12 b b bc b b > 480 42.81 ± 0.71 30.63 ± 1.42 13.06 ± 0.68 24.48 ± 1.37 3.31 ± 0.48 c c c c 10/1 < 320 9.70 ± 1.18 5.82 ± 2.70 3 ± 0.40 3.63 ± 1.15 0.06 ± 0.15 d d d d c 320–480 18.96 ± 0.42 21.52 ± 1.19 7.4 ± 0.76 10.42 ± 0.83 0.27 ± 0.19 cd cd d cd c > 480 15.15 ± 0.46 13.80 ± 1.83 6.8 ± 0.17 7.44 ± 0.53 0.19 ± 0.22 Note: For each treatment, means followed by the same letter do not differ significantly at p< 0.05 (Tukey’s multiple range test) Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 8 of 10 Table 5 Seed weight influence on seedling characteristics in E. positively correlated with seed weight. This finding is simi- prunifolius. Regression Eq. (Y = c + mx) for the relationship lar to that reported in Artocarpus heterophyllus L., Alan- between initial seed weight (n = 10), shoot and root length gium lamarckii Thwaites, Quercus semiserrata Roxb. and (cm), number of leaves/plant, leaf area (cm ) and dry weight/ other oak species (Khan 2004;Ahirwar 2012;Barik et al. plant (g) 1996;Bonfil 1998;Tripathiand Khan 1990;Khanand Variables Regression equation rp value Shankar 2001). Heavy seeds are associated with greater Shoot height Y = 19.722 + 51.963 0.531 0.002 stocks of food and energy reserves and provide readily Root length Y = 16.401 + 28.830 0.530 0.002 available energy and resources to stimulate germination (Flint and Palmblad 1978). Number of leaves/plant Y = 10.814 + 7.838 0.313 0.091 The seedlings from heavy seeds survived better and Leaf area Y = 6.276 + 37.577 0.791 0.000 exhibited greater biomass but the amount of heavy seed Dry biomass Y = −0.540 + 8.284 0.763 0.000 in the sample tested was low. Similar results have been Significant at p < 0.05 observed in Quercus species (Tripathi and Khan 1990; Khan and Shankar 2001). The current study was part of a application of gibberellic acid (Baskin and Baskin 1998, larger project to examine issues with E. prunifolius ger- 2004). Physiological dormancy has also been reported mination in the wild (data not shown). The main con- from other species of Elaeocarpus such as E. floribiindus, straint of germination under natural conditions was E. petiolatm and E. stipularis (Ng 1978, 1980; Beniwal premature seed fall caused by strong winds and rain. Also, and Singh 1989). mature fruits were predated by rodents, birds, worms and The fruits of E. prunifolius mature in early September ants before the seeds germinated. A substantial number of and remain dormant throughout the winter (November– seeds did germinate but, young seedlings suffered mortal- February). The seeds germinate in April with the rise in ity due to desiccation during winter and also due to temperature and onset of rain indicating that these en- trampling. Similar observations have also been reported in vironmental cues may be a requirement for germination. other Elaeocarpus species (Matthew 1999). Similar moisture-aided germination has been reported in Prunus jenkinsii Hook. f. & Th. and other endemic spe- Conclusion cies of the region (Upadhaya et al. 2007; Upadhaya et al. Elaeocarpus prunifolius seed exhibits both physical and 2017). However, uncracked seeds imbibed only a small physiological dormancy. Splitting the seed coats with a amount of water during soaking and this was not im- vice is a prerequisite for propagation and is also a proved by pre-cracking the seeds. Soaking either cracked cost-effective method. In addition, treating cracked seeds or uncracked seeds in water before sowing might be ex- with a combination of GA and KNO accelerates the 3 3 pected to improve the speed and extent of germination germination rate, which may enable mass propagation but, surprisingly, resulted in no germination. In contrast, for reintroduction programmes. Heavy seeds showed Khan et al. (2003) found that soaking E. ganitrus seeds better survival and growth than lighter seeds so heavy in hot water for 24 h then fermenting them for 20 days seeds should be separated out wherever practical and led to 37% germination. All the sulphuric acid treat- used for propagation. ments test in the current study prevented germination Abbreviations of E. prunifolius seeds yet 15% of E. serratus seeds ABA: Abscisic acid; GA : Gibberellic acid; H SO : Sulphuric acid; 3 2 4 treated with 50% nitric acid germinated (Dahanayake et KMnO : Potassium permanganate; KNO : Potassium nitrate; T : Time to 50% 4 3 50 germination al. 2013). Seed-coat impermeability is usually associated with the Acknowledgements presence of one or more layers of impermeable palisade We thank the Headman of Jarain Village for permitting us to work in the forests. The critical comments received from the two anonymous reviewers layers of lignified cells (Corner 1976; Vazquez-Yanes and are also acknowledged. We also thank the editor for thoroughly editing and Perez-Garcia 1976). However, seed coat thickness did improving the manuscript. not impede or delay germination as germination per- Funding centage was higher in heavy seeds (which had thicker The first author acknowledges the financial support received from University coats) and the mean germination time was not signifi- Grants Commission (UGC) in the form of RGNF-JRF (F1-17.1/2013-14/RGNF- cantly different across the seed weight classes. Similar 2013-14-ST-NAG-43868/ (SA-III/Website). Dated: 06-Feb-2014). findings have been observed in Oenothera biennis L Availability of data and materials (Gross and Kromer 1986), Diplotaxis erucoides (L.) DC. Not applicable and D. virgata (Cav.) DC. (Perez-Garcia et al. 1995). Barnett Authors’ contributions (1997) observed that 69% of the variation in the speed of VI and KU conceived and designed the study. Experimental works were germination in five pine species was related to the seed carried out by VI. Data analysis was done by KU and manuscript was drafted coat. In the present study, germination percentage was by VI and KU. Both authors read and approved the final manuscript. Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 9 of 10 Ethics approval and consent to participate Farooq, M., Basra, S. M. A., Afzal, I., & Khaliq, A. (2006). Optimization of hydropriming Not applicable techniques for rice seed invigoration. Seed Science Technology, 34,507–512. Flint, S. D., & Palmblad, I. G. (1978). Germination dimorphism and development flexibility in the ruderal weed Heterotheca grandiflora. Oecologia, 36,33–43. Consent for publication Flora Malesiana Symposium. (1989). In P. Baas, K. Kalkman, & R. Geesink (Eds.), The Not applicable plant diversity of Malesia: Proceedings of the Flora Malesiana Symposium commemorating Professor Dr. C.G.G.J. van Steenis. Leiden: doi: 10.1007/978-94- Competing interests 009-2107-8. The authors declare that they have no competing interests. Gashi, B., Abdullai, K., Mata, V., & Kongjika, E. (2012). Effect of gibberellic acid and potassium nitrate on seed germination of the resurrection plants Ramonda serbica and Ramonda nathaliae. African Journal of Biotechnology, 11(20), Publisher’sNote 4537–4542. Springer Nature remains neutral with regard to jurisdictional claims in Gill, D., Magin, G., & Bertram, E. (2013). Trees and climate change. A guide to the published maps and institutional affiliations. factors that influence species vulnerability and a summary of adaptation options. Fauna and Flora International www.fauna-flora.org. Author details Gross, K. L., & Kromer, M. L. (1986). Seed weight effects on growth and Department of Environmental Studies, School of Human and Environmental reproduction in Oenothera biennis L. Bulletin of the Torrey Botanical Society, Sciences, North-Eastern Hill University, Shillong 793 022, India. Department 133, 252–258. of Basic Sciences and Social Sciences, School of Technology, North-Eastern Han, C.-Y., Welbaum, G., & Long, C.-L. (2010). 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Seed dormancy, germination and seedling characteristics of Elaeocarpus prunifolius Wall. ex Müll. Berol.: a threatened tree species of north-eastern India

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Abstract

Background: Elaeocarpus prunifolius Wall. ex Müll. Berol. is a threatened tree species of north-eastern India. The present study was undertaken to investigate the type of dormancy prevailing in seeds of E. prunifolius, explore seed dormancy breaking techniques and assess seedling fitness. Methods: Ripe fruits of E. prunifolius were harvested from Jaintia hills, and seeds were subjected to various physical, manual and chemical treatments. The effect of plant growth regulators, viz gibberellic acid (GA ) and potassium nitrate (KNO ), were tested. Seedling vigour and survival based on seed weight were examined. Results: Germination took 6 months to initiate after seed dispersal and natural germination percentage of fresh seeds was 24%. Physical pre-germination treatments such as surface and acid scarification failed to overcome dormancy. Cracked seeds promoted germination (46%) with a mean germination time of 146 days (time to 50% −1 germination, T = 144 days). Among the GA treatments, split seeds treated with GA (3000 mg/L ) yielded the 50 3 3 highest germination (24%) with a T of 55 days whereas KNO did not promote germination. A combination of 50 3 GA and KNO , however, increased the germination to 31%. Between the seed weight classes, the highest 3 3 percentage of germination was observed in heavy seeds (25%) and the lowest in light seeds (20%). There was no significant variation between seed weight and germination time (p > 0.05). Seed weight had a significant effect on the shoot height, number of leaves and dry weight of seedlings (p < 0.05). Conclusion: Based on the seed tests, E. prunifolius seeds exhibits ‘combined’ dormancy (physical and physiological) as splitting seed coat and application of GA effectively broke dormancy. Splitting the seed coat is a cost-effective method for accelerating germination of seeds. Heavy-weight seeds produced better performing seedlings compared to their counterparts which may be viewed as an important reproductive strategy of the species. Keywords: Conservation, Elaeocarpus, Germination, Seedling growth, Threatened Background 2000; Gill et al. 2013; Tanaka-Oda et al. 2017). Thus, In recent years, studies on germination have emerged as such studies enable the development of suitable proto- an important tool for conservation of many species. cols for species conservation and management. Such studies aid understanding of natural regeneration The genus Elaeocarpus have ca. 360 species distributed processes as well as identifying possible causes of species across East Asia, Australia, Malaysia and the Pacific decline, persistence or spread in changing landscapes Islands. The International Union for Conservation of and their response to global climate change (Schütz Nature and Natural Resources (IUCN) have identified 38 Elaeocarpus species under various threat categories. Of these, 3 species are critically endangered, 4 endangered, * Correspondence: upkri@yahoo.com 20 vulnerable, 2 near threatened, 8 conservation Department of Basic Sciences and Social Sciences, School of Technology, North-Eastern Hill University, Shillong 793 022, India dependent and 1 data deficient (IUCN 2017). In India, Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 2 of 10 30 species occur (Murti 1993a, b), the majority of which broad-leaved forests with only a few matured individuals are confined to the northeast (Assam, Arunachal Pra- (Walter and Gillett 1998; Nayar and Sastry 1990). The desh, Sikkim, Manipur, Meghalaya, Mizoram, Nagaland, species is on the verge of extinction due to habitat degrad- Tripura) and south (Tamil Nadu and Kerela) India ation (Jain and Rao 1983; Haridasan and Rao 1985), which (Khan et al. 2003; Singh et al. 2013). Out of the six is further exacerbated by its on-going exploitation for tim- threatened Elaeocarpus species reported from the country, ber by local communities (Nayar and Sastry 1990). The one species (E. gaussenii Weibel) is critically endangered fruits of the species are also edible. and one endangered (E. blascoi Weibel), three are Vulner- Propagation of Elaeocarpus species from seed is chal- able (viz. E. prunifolius Wall. ex Müll. Berol, E. recurvatus lenging, as the seeds exhibit dormancy (Khan et al. Corner and E. venustus Bedd.) and one (E. munroii Mast.) 2003). The fruits of E. prunifolius mature in early Sep- falls under lower risk/near threatened category. Of these, tember and remain dormant throughout the winter (No- E. prunifolius is restricted to north-eastern India and the vember–February). The seeds germinate in April with others are confined to southern India. the onset of rain so moisture may be a requirement for Research on Elaeocarpus species has gained momen- germination. The inherent stony endocarp in many tum with the discovery of indolizidine alkaloid com- members of the genus seems to be a plausible factor for pounds in members such as E. fuscoides Knuth., E. poor germination (Bhuyan et al. 2002; Khan et al. 2003; grandis F. Muell., E. polydactylus Schltr. and E. densi- Ramasubbu and Irudhyaraj 2016). Some studies have re- florus Knuth (Katavic 2005) for their potential treatment vealed low (1–3%) and erratic seed germination in of diseases such as AIDS, diabetes and cancer (Wiart Elaeocarpus ganitrus Roxb. ex G.Don (Khan et al. 2003) 2006). In addition, fruits of species, viz E. floribundus and E. japonicus Siebold (Yang et al. 2001). Natural re- Blume, E. lanceifolius Roxb., E. braceanus Watt ex C.B. generation of the threatened E. blascoi was only 5% in Clarke and E. sikkimensis Mast, are edible (Andola et al. the wild (Ramasubbu and Irudhyaraj 2016) while in E. 2011;Das 2014) and species such as E. grandis, E. rumi- venustus, E. serratus Land E. williamsianus Guymer, natus F.Muell., E. foviolatus F. Muell. and E. coorangoo- there was no germination (Saravanan et al. 2011; Daha- loo J.F. Bailey & C.T. White are known for their good nayake et al. 2013; Rossetto et al. 2004). Contrastingly, timber value (Bristow et al. 2005). some species such as E. floribundus had fairly high ger- Elaeocarpus prunifolius is a middle-sized tree that at- mination (47%) (Das 2014). However, there is a dearth of tains a height of ca. 20 m at maturity (Nayar and Sastry knowledge (particularly on germination) of threatened 1990). The phenological calendar of the species is pre- species often targeted for conservation (Baskin and Bas- sented in Table 1. The inflorescences are 3–9 cm long, kin 2001). Khan et al. (2003) studied the germination and the flowers are pale yellow in colour. The ovoid capacity of E. ganitrus by subjecting the seeds to various fruits range from 0.8 to 1.2 cm in diameter. They change treatments and found that seeds cracked with a vice re- from green to bluish-black on maturation. There is only sulted in highest germination percentage (40%) followed one seed per endocarp. The ripe fleshy mesocarp is pre- by seeds soaked in hot water for 24 h and seeds fermen- dated by worms, insects and birds, and seeds are pre- ted for 20 days (37%). A mixture of silt and sand media dated by small rodents, thus, dispersing the seeds in the resulted in 40% germination in E. venustus whereas process (epizoochory) (field observation). seeds sown in a media of sand, silt and cow dung germi- Elaeocarpus prunifolius is a rare and threatened tree nated to about 8% (Irwin et al. 2013). In E. serratus, species distributed in the state of Manipur and Meghalaya seeds treated with 50% HNO germinated to 15% (Daha- in India and Bangladesh (Murti 1993a, b;World Conser- nayake et al. 2013). However, there is lack of information vation Monitoring Centre 1998). In Meghalaya, the spe- on the regeneration capacity of E. prunifolius. The poor cies is restricted to fragmented pockets of sub-tropical regeneration of the species in nature could be due to Table 1 Phenological calendar of E. prunifolius LF leaf flushing, LM leaf maturation, LFA leaf fall, FL flowering, FR fruiting, FM fruit maturation Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 3 of 10 hard seed coat and/or prolonged dormancy period. The Seed treatments results presented here form part of a larger study to as- sess the causes of the species rarity in nature by under- (a) Seed coat impermeability is often associated with standing the species phenology, mode of dispersion, the presence of impermeable palisade layers of predation, seed physiology, germination pattern, seedling lignified cells (Vazquez-Yanes and Perez-Garcia characteristics and factors debilitating seedling establish- 1976). Therefore, the following physical or chemical ment. The objectives of the current work were to: (i) treatments were applied in an attempt to disrupt understand the type of dormancy present in the seeds of the seed coat. For each experiment, three replicates E. prunifolius by subjecting the seeds to a set of physical of 50 seeds (irrespective of weight) were main- and chemical treatments; (ii) test if total germination tained. Seeds were: and germination rate were influenced by seed weight; 1a. Soaked in cool water (20 °C) for 24 h and (iii) examine if the seedling growth and survival 2a. Soaked in hot water (80 °C) for 24 h till the were influenced by seed weight. The results are dis- water cooled cussed in relation to possible factors responsible for low 3a. Soaked in boiling water (5 min in 100 °C and recruitment of this species in nature. immediately transferred to cold water) for 24 h 4a. Scarified by rubbing near the micropyle with Materials and methods sandpaper Seed source 5a. Cracked lengthwise with a vice Ripe fruits of E. prunifolius were harvested during 6a. Scarified near the micropyle using sandpaper mid-September 2013 from Jarain (25° 19.05′ N, 92° and soaked in warm water (45 °C) for 2 h 08.34′ E, alt 1200 m asl), the known location of the spe- 7a. Soaked in 95% H SO for 5 min 2 4 cies in Meghalaya, Northeast India (Haridasan and Rao 8a. Soaked in 95% H SO for 10 min 2 4 1985). As the species is rare, all mature trees were 9a. Soaked in 95% H SO for 15 min 2 4 marked wherever encountered and monitored for fruit 10a. Soaked in 95% H SO for 20 min 2 4 maturation. Fruits were collected randomly from ten 11a. A control was maintained by sowing seeds randomly selected trees and bulked together to form a (also irrespective of seed weight) without any of composite sample (ca. 4500 fruits). The fruits were the above-mentioned treatments. brought to the laboratory and soaked in normal tap water (18 °C ± 1 °C for 24 h) to soften the pulp and also The treated seeds were transferred to plastic trays to separate healthy seeds from damaged ones (Pipinis et filled with a mixture of garden soil and sand in the ratio al. 2011). Empty or damaged seeds are lighter and float of 3:1. Each tray was labelled and kept under laboratory to the surface and were thus separated from healthy conditions having an average temperature of 24 (± 1 °C) seeds. The fruits were hand crushed and the seeds col- and light (> 700 lx) duration of 8 h. The trays were lected. The seeds were washed thoroughly in running watered at 3-day intervals and monitored for germin- water to remove any adhering substance from the endo- ation for a period of 12 months. carp and disinfected in 0.2% potassium permanganate (KMnO ) solution for 2 h following the method of Zuo (b) The effect of plant growth regulators (PGRs) on (1994). The seeds were placed on paper to remove ex- breaking seed dormancy and the rate of cess moisture and were subjected to various germination was examined by performing the dormancy-breaking treatments within 5 days of seed col- following tests on seeds cracked lengthwise. Three lection. The remaining seeds were stored in an airtight replicates of 25 seeds were maintained for each container filled with a substrate (moist sand) and stored treatment. Cracked seeds were: −1 at a constant temperature of 5 °C (±1 °C) in a refrigerator 1b. Soaked in 200 mg L gibberellic acid (GA ) for further studies. for 48 h −1 2b. Soaked in 500 mg L gibberellic acid (GA ) Water imbibition test for 48 h −1 Twenty-five seeds were cracked with a vice, and the ini- 3b. Soaked in 1000 mg L gibberellic acid (GA ) tial weight of each seed was measured. The weights of for 48 h −1 25 uncracked seeds were also determined. Both sets of 4b. Soaked in 2000 mg L gibberellic acid (GA ) seed were then placed on Petri dishes (90 mm × 20 mm) for 48 h −1 lined with moist cotton and kept covered under identical 5b. Soaked in 3000 mg L gibberellic acid (GA ) laboratory conditions. Each seed was reweighed after 2, for 48 h 4, 6, 8, 10, 20, 30 and 40 h, and the percentage change in 6b. Soaked in 0.5% potassium nitrate (KNO ) for mass was averaged from the 25 seeds from each set. 48 h Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 4 of 10 7b. Soaked in 1% KNO for 48 h months after transplantation. To assess the effect of light 8b. Soaked in 1.5% KNO for 48 h on seedling growth, a threefold contrast in light level 9b. Soaked in 2% KNO for 48 h was created by covering the net houses with an increas- − 1 10b. Soaked in GA solution (200 mg L ) for 48 ing layer of shade netting. Each extra layer intercepted h and transferred to 0.5% KNO solution for an additional 25% (approximately) of the incoming radi- another 48 h ation, thus, creating three light regimes of approximately − 1 −2 −1 11b. Soaked in GA solution (500 mg L ) for 48 70–75% (16 ± 1.8 mol m day ), 25–30% (4 ± 0.46 mol −2 −1 −2 −1 h and transferred to 1% KNO solution for m day ) and 5–10% (1 ± 0.05 mol m day ) desig- another 48 h nated as high, intermediate and low light treatments re- 12b. Soaked in distilled water for 48 h spectively. Light intensity was measured with digital lux meter (Lutron LX-101A) at three different times during The control and treated seeds were placed in Petri the day (9.00 a.m., 12 noon and 3 p.m.) each month and dishes (90 mm × 20 mm) lined with moist filter paper the seasonal means were calculated. and transferred to growth chambers fitted with an LED To assess the effect of seed weight and light on growth light source (> 4000 lx) with a photoperiod of 8 h. A con- performance of the seedlings, ten randomly selected stant temperature of 25 °C (± 1 °C) was maintained for seedlings under each weight category and light condition germination. The germination of the seeds in the growth were harvested after one year of transplantation (May chambers was monitored at 3 d intervals for a period of 2015) and their shoot height, root length, the number of 4 months. leaves, leaf area and dry matter yield were determined. The leaf area was measured using a leaf area meter Seed viability (LICOR, Lincoln). Dry matter yield was determined by One hundred and fifty untreated seeds were randomly drying the plant material in an oven at 80 °C for 24 h to selected from those stored at 5 °C for 12 months. Each a constant weight. seed was cracked lengthwise and the embryo excised. Germination percentage was calculated using the Each embryo was tested for viability following the tetra- formula: zolium assay of Wang et al. (2005). The presence of liv- ing cells in the seeds converts the TTZ (2,3,5 triphenyl GðÞ % ¼  100; tetrazolium chloride) to formazan (red colour) through the hydrogen transfer reaction catalysed by the cellular where n is the number of germinated seeds and N is the dehydrogenase, thus, staining the viable seeds red. total number of seeds. The method of Coolbear et al. (1984) further modified by Farooq et al. (2006) was used Seed germination and seedling growth to calculate the time to reach 50% germination (T ): A subsample of 1500 seeds was used to examine the ef- fect of seed weight on germination and seedling growth. T ¼ t þ −n t −t =n −n ; 50 1 i j i j i Each seed was examined by the floatation method and 60 seeds were found to be damaged. The remaining 1440 healthy seeds were weighed individually. The num- where N is the final number of germinated seeds, n and ber of seeds in each of three weight categories (light (< n are the cumulative numbers of seeds germinated at 320 mg), intermediate (320–480 mg) and heavy (> 480 times t and t , respectively, when n < N/2 < n . i j i j mg)) was determined. Seeds from each category were sown under laboratory conditions in plastic trays (26 cm Seed coat thickness diameter, 8 cm height) within 10 days from the time of Seed-coat thickness was determined in each seed weight seed collection. A mixture of garden soil and sand media category to test the assumption that: (i) heavier seeds in the ratio of 3:1 was used for planting. Twenty-five had thicker coats; and (ii) thicker seed coats may delay seeds were sown per tray. Germination was recorded at germination. Seeds were cut across and seed coat thick- 3-day intervals up to 6 months and weekly up to 12 ness was measured using a calliper and the values aver- months. The number of germinated seeds was recorded aged. For each weight category, 30 seeds each were on each occasion. Seeds were considered germinated considered. when the radicles protruded out of the soil surface. Germinated seedlings (about 2 months old) were Data analysis transplanted into 20 × 17 cm poly bags and transferred To determine the effect of different treatments on ger- to the net house during May 2014. The moisture level mination, analysis of variance (ANOVA) was used was maintained by watering every 3 days. Seedling sur- followed by Tukey and Scheffe’s least significant differ- vival and growth were monitored for a period of 12 ence (p < 0.05). Assumptions of ANOVA were met Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 5 of 10 through a test for normality of variables (Shapiro-Wilk control seeds was 213 ± 5 days (T = 174 ± 9 days). No test) and homogeneity of group variances (Levene’s test). germination of seeds occurred after 230 days. Only treat- The relation between seed weight and seedling growth ments 1a, 5a and 6a resulted in any germinated seed- was analysed by regression. All the statistical analysis lings. Of these, treatment 1a (cold-water soak) reduced was performed using SPSS software (version 20). germination to 10.67% (T = 210 days) compared with the control (24%) while 14.67% of seeds (T = 216 days) Results germinated following treatment with a combination of Imbibition test scarification and warm water treatment (Treatment 6a). The initial moisture content of scarified seeds was 10.6% The germination percentage of seeds from these two and non-scarified seeds was 10.8%. After 40 h, both sets treatments was significantly lower (p < 0.05) than the of seeds had a moisture content of 11.2% so the increase control. In contrast, 45.83% of seeds cracked with a vice in the mass of scarified and non-scarified seeds was 6% germinated and this was significantly higher (p < 0.05) and 4% respectively. This difference was not significant than for all other treatments. The mean germination (p > 0.05) so uncracked seeds imbibe water at a similar time and T was also significantly reduced under this rate as cracked seeds. treatment (146 and 144 days respectively) (Table 2). Treatments (1b-11b) involving soaking cracked seeds Dormancy-breaking treatments in solutions of various plant growth regulators were Physical and chemical treatments 1a - 10a were com- compared with germination of cracked seeds soaked in pared to germination of untreated seeds (11a). water (Treatment 12b), Table 2. Cracking seeds (Treat- The germination percentage of control seeds was 24%. ment 5a) resulted in better germination than no treat- The mean number of days required for germination of ment (11a) but soaking seeds in water (Treatment 12b) Table 2 Germination of E. prunifolius seeds subjected to (a) physical, mechanical and chemical scarification and (b) plant growth regulators Treatment No. Treatment details Mean germination (days) Germination (%) T (days) a a 1a Cold water (20 °C) 210 ± 1 10.67 ± 1.33 210 ± 2 2a Hot water (80 °C) 0 0 0 3a Boiling water (100 °C) 0 0 0 4a Scarification near micropyle 0 0 0 5a Cracked seeds with vice 146 ± 1 45.83 ± 1 144 ± 2 a a 6a Scarified+warm water (2 h) 212 ± 1 14.67 ± 1.33 216 ± 2 7a 95% H SO for 5 min 0 0 0 2 4 8a 95% H SO for 10 min 0 0 0 2 4 9a 95% H SO for 15 min 0 0 0 2 4 10a 95% H SO for 20 min 0 0 0 2 4 11a Untreated control 213 ± 5 24.67 ± 3 174 ± 9 −1 1b 200 mg L GA 00 0 −1 a a 2b 500 mg L GA 78 ± 1 15.56 ± 2 77 ± 1 −1 b ad 3b 1000 mg L GA 63 ± 1 17.78 ± 2 64 ± 2 −1 c ae 4b 2000 mg L GA 70 ± 2 20 ± 0 71 ± 2 −1 d ce 5b 3000 mg L GA 54 ± 1 24.44 ± 2 55 ± 1 6b 0.5% KNO 00 0 7b 1% KNO 00 0 b b 8b 1.5% KNO 63 ± 3 8.89 ± 2 62 ± 3 9b 2.0% KNO 00 0 −1 b de 10b 200 mg L GA + 0.5%KNO 63 ± 3 22.22 ± 2 61 ± 3 3 3 −1 de f 11b 500 mg L GA + 1% KNO 56 ± 1 31 ± 2 56 ± 2 3 3 12b Water 0 0 0 T : time required to attain 50% germination, ±SEM (standard error of mean) Note: For each treatment, means followed by the same letter in each column do not differ significantly at p < 0.05 Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 6 of 10 −1 produced no germination. Seeds treated with 200 mg L Seed weight and seedling growth GA (Treatment 1b) did not germinate either but there Seed weight (n = 1440) ranged from 160 to 630 mg (mean was a positive linear relationship (Y = 0.0076x + 4.4304, weight 385.58 ± 1.90 mg). The distribution was normal R = 0.726, p = 0.03) between germination percentage (Shapiro-Wilk test), with intermediate seeds constituting and increasing GA concentration with a maximum 74.93% of the total seed population followed by light and germination of 24.44% for seeds treated with 3000 mg heavy that accounted for 16.31% and 8.75% respectively −1 L GA (T = 55 days), Table 2. Increasing concen- (Table 3). More heavy seeds (24.60%) germinated than 3 50 tration of GA decreased the time required to attain intermediate (22.80%) or light seeds (19.57%), Table 3. 50% germination (T )(Fig. 1), more so than the pot- There was no significant relationship between the seed ted experiments (treatment 1a-11a). No seeds germi- weight and germination time (p > 0.05). The time to 50% nated following treatment with 0.5%, 1% or 2% KNO germination (T ) was longest in intermediate seed weight 3 50 (Treatment 6b, 7b and 9b) but, surprisingly, a small (218 days) and shortest for light seeds (188 days) (Table 3). percentage of seeds (8.89%) did germinate following One year after transplantation, 100% of seedlings emer- −1 Treatment 8b (1.5% KNO ). Neither 200 mg L nor ging from heavy- and intermediate-weight seeds had sur- 0.5% KNO alone (Treatments 1b and 6b respectively) vived but only 85% of those from light-weight seeds. Seeds produced any germination yet the combination of the < 320 mg had an average coat thickness of 0.66 mm com- two (Treatment 10b) did result in 22% germination. pared with 1.42 mm for seeds > 480 mg. Light seeds had The highest germination percentage of 31% (T =56 an average length of 12 mm whereas seed length in inter- −1 days) was obtained using a combination of 500 mg L mediate and heavy seeds measured an average 13 mm and GA and 1.0% KNO . Results from multiple compari- 15 mm respectively. Similarly, seed width was lower in 3 3 son tests between the various treatments showed a light weight seeds (7 mm) as compared to heavy seeds (8 significant difference in the mean germination per- mm), Table 3. centage of seeds treated with a combination of 500 Significant differences (p < 0.05) were observed in the −1 mg L GA +1% KNO with all concentrations of shoot height, leaf area and dry biomass of seedlings 3 3 GA and KNO (p < 0.05) except for seeds treated in emerging from light seeds compared with those of inter- 3 3 −1 −1 3000 mg L GA and 200 mg L +0.5% KNO . mediate and heavy seeds (Table 4). However, there was 3 3 no significant difference between the intermediate and heavy seeds (p > 0.05). Seed viability Regression analysis showed that seed weight had a Embryos stained with tetrazolium indicated viability. Of positive impact on seedling vigour with heavier seeds the 150 seeds tested, 111 embryos (74%) were stained. producing larger and heavier seedlings (Table 5). Visual inspection of the embryos showed that they were curved with narrow cotyledons characteristic of 40% of Discussion Malesian Elaeocarpus species (Weibel 1968; Flora Mal- Seeds stored at 5 °C remained viable even after 12 esiana Symposium 1989). months of storage indicating ‘orthodox’ (i.e. will survive Fig. 1 Mean (±SEM) germination percentage (G%) and time to 50% germination (T ) under GA and KNO treatments at 25 °C. 50 3 3 −1 −1 −1 −1 −1 (Treatments: 2b = 500 mg L , 3b = 1000 mg L , 4b = 2000 mg L , 5b = 3000 mg L , 8b = 1.5% KNO , 10b = 200 mg L GA +0.5% KNO , 3 3 3 −1 11b = 500 mg L GA +1% KNO ) 3 3 Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 7 of 10 Table 3 Germination characteristics of E. prunifolius seeds Parameters Seed weight class(mg) Light (< 320) Intermediate (320–480) Heavy (> 480) Number of seeds sown (n) 235 1079 126 Average seed length (mm) 11.92 ± .064 13.47 ± .028 14.52 ± .059 Average seed width (mm) 6.80 ± .027 7.37 ± .017 8.02 ± .034 Number of seeds germinated 46 246 31 Proportion of Seed Contribution to total seed lot (%) 16.31 74.93 8.75 a a a Germination (%) 19.57 22.8 24.6 Mean germination (days) 211 ± 4 212 ± 1 213 ± 3 T (days) 188 ± 9 218 ± 7 204 ± 6 Seed coat thickness (mm) 0.66 ± 0.12 1.07 ± 0.06 1.42 ± 0.06 Weight of cotyledon (g) 0.077 ± 0.005 0.102 ± 0.002 0.117 ± 0.002 Note: For each treatment, means followed by the same letter in each row do not differ significantly at p < 0.05 drying and/or freezing during ex situ conservation) char- still remained dormant for ca. 3 months and the germin- acteristics (Chin et al. 1989). However, intact untreated ation time decreased further only by soaking in gibberel- −1 seeds took an average of 213 days to germinate under lic acid solution (≥ 500 mg L ) or a combination of favourable conditions, which clearly indicates the pres- gibberellic acid and potassium nitrate. Similar results ence of dormancy (Baskin and Baskin 1998, 2004). Phys- have been reported in species like Ramonda serbica ical and mechanical dormancy is the most common Pančić, R. nathaliae Pančić & Petrovič, Magnolia yunna- inhibitor of germination in many species. Threatened nensis (Hu) Noot. and M. punduana (Hk. f & Th.) Figlar tree species such as Cupressus atlantica (Youssef et al. (Gashi et al. 2012; Han et al. 2010; Iralu and Upadhaya 2012), Elaeocarpus blascoi Weibel (Ramasubbu and 2016). The presence of abscisic acid (ABA) in mature Irudhyaraj 2016) and Intsia bijuga (Colebr.) Kuntze seeds has been associated with seed dormancy (Matakia- (Thaman et al. 2006) exhibit exogenous dormancy where dis et al. 2009; Hilhorst and Karssen 1992), and studies the hard seed coats inhibit water penetration that is re- have established that the application of nitrate ions to a quired for embryo growth and development. germination medium leads to rapid decline of ABA. Reducing germination time and increasing germin- However, three of the four concentrations of potassium ation percentage are both important pre-requisites of a nitrate tested alone in the current study prevented ger- successful propagation initiative. Cracked E. prunifolius mination. The positive effect of some PGAs in the seeds germinated faster and in greater numbers than in- current study indicates that (in addition to physical dor- tact seeds, as expected by disrupting the hard coat sur- mancy) seeds may also have ‘physiological’ dormancy rounding the seed. Similar results have been reported in also called ‘combined’ dormancy (Baskin and Baskin E. ganitrus (Khan et al. 2003). However, cracked seeds 1998; Nikolaeva 1969), which was broken by the Table 4 Shoot height and root length (cm), number of leaves/plant, leaf area (cm ) and dry weight/plant (g) of seedlings of E. prunifolius (±SEM, n = 10) in light-, intermediate-and heavy-seed weight classes grown under three different contrasting light regimes after 1 year of transplantation Day length (h)/light intensity (mol Seed weight Shoot height Root length Number of leaves/ Leaf area/seedling Dry weight/plant −2 −1 2 m d ) (mg) (cm) (cm) plant (cm ) (g) a a a a a 10/16 < 320 20.57 ± .39 16.63 ± 0.54 7.2 ± 0.48 9.93 ± 0.18 0.56 ± 0.08 320–480 39.25 ± 0.90 26.80 ± 1.10 15.4 ± 0.52 21.09 ± 0.69 2.76 ± 0.67 a a a a a > 480 26.46 ± 0.17 17.28 ± 0.35 11.4 ± 0.39 13.72 ± 0.74 1.43 ± 0.33 b b 10/4 < 320 31.27 ± 1.07 25.84 ± 2.13 12.40 ± 0.25 15.81 ± 0.63 1.74 ± 0.24 b b c b b 320–480 46.68 ± 2.66 26.22 ± 0.72 15.6 ± 1.28 22.61 ± 1.03 2.95 ± 1.12 b b bc b b > 480 42.81 ± 0.71 30.63 ± 1.42 13.06 ± 0.68 24.48 ± 1.37 3.31 ± 0.48 c c c c 10/1 < 320 9.70 ± 1.18 5.82 ± 2.70 3 ± 0.40 3.63 ± 1.15 0.06 ± 0.15 d d d d c 320–480 18.96 ± 0.42 21.52 ± 1.19 7.4 ± 0.76 10.42 ± 0.83 0.27 ± 0.19 cd cd d cd c > 480 15.15 ± 0.46 13.80 ± 1.83 6.8 ± 0.17 7.44 ± 0.53 0.19 ± 0.22 Note: For each treatment, means followed by the same letter do not differ significantly at p< 0.05 (Tukey’s multiple range test) Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 8 of 10 Table 5 Seed weight influence on seedling characteristics in E. positively correlated with seed weight. This finding is simi- prunifolius. Regression Eq. (Y = c + mx) for the relationship lar to that reported in Artocarpus heterophyllus L., Alan- between initial seed weight (n = 10), shoot and root length gium lamarckii Thwaites, Quercus semiserrata Roxb. and (cm), number of leaves/plant, leaf area (cm ) and dry weight/ other oak species (Khan 2004;Ahirwar 2012;Barik et al. plant (g) 1996;Bonfil 1998;Tripathiand Khan 1990;Khanand Variables Regression equation rp value Shankar 2001). Heavy seeds are associated with greater Shoot height Y = 19.722 + 51.963 0.531 0.002 stocks of food and energy reserves and provide readily Root length Y = 16.401 + 28.830 0.530 0.002 available energy and resources to stimulate germination (Flint and Palmblad 1978). Number of leaves/plant Y = 10.814 + 7.838 0.313 0.091 The seedlings from heavy seeds survived better and Leaf area Y = 6.276 + 37.577 0.791 0.000 exhibited greater biomass but the amount of heavy seed Dry biomass Y = −0.540 + 8.284 0.763 0.000 in the sample tested was low. Similar results have been Significant at p < 0.05 observed in Quercus species (Tripathi and Khan 1990; Khan and Shankar 2001). The current study was part of a application of gibberellic acid (Baskin and Baskin 1998, larger project to examine issues with E. prunifolius ger- 2004). Physiological dormancy has also been reported mination in the wild (data not shown). The main con- from other species of Elaeocarpus such as E. floribiindus, straint of germination under natural conditions was E. petiolatm and E. stipularis (Ng 1978, 1980; Beniwal premature seed fall caused by strong winds and rain. Also, and Singh 1989). mature fruits were predated by rodents, birds, worms and The fruits of E. prunifolius mature in early September ants before the seeds germinated. A substantial number of and remain dormant throughout the winter (November– seeds did germinate but, young seedlings suffered mortal- February). The seeds germinate in April with the rise in ity due to desiccation during winter and also due to temperature and onset of rain indicating that these en- trampling. Similar observations have also been reported in vironmental cues may be a requirement for germination. other Elaeocarpus species (Matthew 1999). Similar moisture-aided germination has been reported in Prunus jenkinsii Hook. f. & Th. and other endemic spe- Conclusion cies of the region (Upadhaya et al. 2007; Upadhaya et al. Elaeocarpus prunifolius seed exhibits both physical and 2017). However, uncracked seeds imbibed only a small physiological dormancy. Splitting the seed coats with a amount of water during soaking and this was not im- vice is a prerequisite for propagation and is also a proved by pre-cracking the seeds. Soaking either cracked cost-effective method. In addition, treating cracked seeds or uncracked seeds in water before sowing might be ex- with a combination of GA and KNO accelerates the 3 3 pected to improve the speed and extent of germination germination rate, which may enable mass propagation but, surprisingly, resulted in no germination. In contrast, for reintroduction programmes. Heavy seeds showed Khan et al. (2003) found that soaking E. ganitrus seeds better survival and growth than lighter seeds so heavy in hot water for 24 h then fermenting them for 20 days seeds should be separated out wherever practical and led to 37% germination. All the sulphuric acid treat- used for propagation. ments test in the current study prevented germination Abbreviations of E. prunifolius seeds yet 15% of E. serratus seeds ABA: Abscisic acid; GA : Gibberellic acid; H SO : Sulphuric acid; 3 2 4 treated with 50% nitric acid germinated (Dahanayake et KMnO : Potassium permanganate; KNO : Potassium nitrate; T : Time to 50% 4 3 50 germination al. 2013). Seed-coat impermeability is usually associated with the Acknowledgements presence of one or more layers of impermeable palisade We thank the Headman of Jarain Village for permitting us to work in the forests. The critical comments received from the two anonymous reviewers layers of lignified cells (Corner 1976; Vazquez-Yanes and are also acknowledged. We also thank the editor for thoroughly editing and Perez-Garcia 1976). However, seed coat thickness did improving the manuscript. not impede or delay germination as germination per- Funding centage was higher in heavy seeds (which had thicker The first author acknowledges the financial support received from University coats) and the mean germination time was not signifi- Grants Commission (UGC) in the form of RGNF-JRF (F1-17.1/2013-14/RGNF- cantly different across the seed weight classes. Similar 2013-14-ST-NAG-43868/ (SA-III/Website). Dated: 06-Feb-2014). findings have been observed in Oenothera biennis L Availability of data and materials (Gross and Kromer 1986), Diplotaxis erucoides (L.) DC. Not applicable and D. virgata (Cav.) DC. (Perez-Garcia et al. 1995). Barnett Authors’ contributions (1997) observed that 69% of the variation in the speed of VI and KU conceived and designed the study. Experimental works were germination in five pine species was related to the seed carried out by VI. Data analysis was done by KU and manuscript was drafted coat. In the present study, germination percentage was by VI and KU. Both authors read and approved the final manuscript. Iralu and Upadhaya New Zealand Journal of Forestry Science (2018) 48:16 Page 9 of 10 Ethics approval and consent to participate Farooq, M., Basra, S. M. A., Afzal, I., & Khaliq, A. (2006). Optimization of hydropriming Not applicable techniques for rice seed invigoration. Seed Science Technology, 34,507–512. Flint, S. D., & Palmblad, I. G. (1978). Germination dimorphism and development flexibility in the ruderal weed Heterotheca grandiflora. Oecologia, 36,33–43. Consent for publication Flora Malesiana Symposium. (1989). In P. Baas, K. Kalkman, & R. Geesink (Eds.), The Not applicable plant diversity of Malesia: Proceedings of the Flora Malesiana Symposium commemorating Professor Dr. C.G.G.J. van Steenis. Leiden: doi: 10.1007/978-94- Competing interests 009-2107-8. The authors declare that they have no competing interests. Gashi, B., Abdullai, K., Mata, V., & Kongjika, E. 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New Zealand Journal of Forestry ScienceSpringer Journals

Published: Dec 28, 2018

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