Helianthemum lippii (L.) Dum. Cours. is a perennial shrubby plant 10-45 cm tall that belongs to the family Cistaceae. The effects of pretreatments on germination of Helianthemum lippii provide information regarding germination requirements of this species, which could be used for conservation studies. Five different pretreatment were applied to enhance seed germination. Scarification followed by germination at 6 different constant temperatures (10, 15, 20, 25, 30 and 35°C) with continuous light or dark, Scarification followed by GA3 soaking (100, 250,500,750 and 1000 ppm), Heat and moist stratification (only intact seeds), KNO₃ and H₃BO₃ soaking (only intact seeds). The results obtained from this study indicate that germination in H. lippii was promoted by scarification. Scarification of seeds resulted in high germination while increasing germination temperature (35°C) decreased the germination of scarified seeds. Exposure to light and dark had no effect on germination. Heat stratification, cold stratification, KNO₃ and H₃BO₃ treatments were ineffective in increasing germination of H.lippii seeds. Scarification yielded maximum germination without soaking in GA₃ It increased the germination of Helianthemum lippii seeds from 1 to 99%. Increase in GA₃ concentration decreased the germination of this species. Scarification succeeded in breaking dormancy of H.lippii seeds suggesting that this species exhibits seed coat dormancy and in nature it may happen due to the abrasion of seed coat by sand particles or other biotic and abiotic factors.Desert Plants is published by The University of Arizona for the Boyce Thompson Southwestern Arboretum. For more information about this unique botanical journal, please email the College of Agriculture and Life Sciences Publications Office  at pubs@cals.arizona.edu

Padmesh, S.

Tawfiq, H.

Zaman, S.

The University of Arizona

18 Desert Plants Effect of Pre-germination treat-ments on Seed Germination of Helianthemum lippii (L.)Dum.Cours. S. Zaman S. Padmesh H. Tawfiq Aridland Agriculture and Greenery Department Kuwait Institute for Scientific Research P.O. Box 24885 Safat, 131 09 Kuwait Tel: (00965)4989844; Fax: (00965)4989809 E-mail: szaman@safat.kisr.edu.kw Abstract Helianthemum lippii (L.) Dum. Cours. is a perennial shrubby plant 10-45 em tall that belongs to the family Cistaceae. The effects of pretreatments on germination of Helianthemum lippii provide information regarding germination requirements of this species, which could be used for conservation studies. Five different pre-treatment were applied to enhance seed germination. Scarification followed by germination at 6 different constant temperatures (10, 15, 20, 25, 30 and 35°C) with continuous light or dark, Scarifica-tion followed by GA3 soaking (100, 250,500,750 and 1000 ppm), Heat and moist stratification (only intact seeds), KN03 and H3B03 soaking (only intact seeds). The results obtained from this study in-dicate that germination in H. lippii was promoted by scarification. Scarification of seeds resulted in high germination while increas-ing germination temperature (35°C) decreased the germination of scarified seeds. Exposure to light and dark had no effect on ger-mination. Heat stratification, cold stratification, KN03 and H3B03 treatments were ineffective in increasing germination of H.lippii seeds. Scarification yielded maximum germination without soak-ing in GA3' It increased the germination of Helianthemum lippii seeds from 1 to 99%. Increase in GA3 concentration decreased the germination of this species. Scarification succeeded in breaking dormancy of H.lippii seeds suggesting that this species exhibits seed coat dormancy and in nature it may happen due to the abrasion of seed coat by sand particles or other biotic and abiotic factors. Keywords: Germination, scarification, dormancy, conservation, biotic, abiotic factors. Introduction The vegetation of Kuwait is under severe pressure due to mul-tiple interacting factors such as overgrazing, uprooting of woody shrubs, increased recreation, gravel quarrying, environmental fac-tors and natural processes. Additionally, the Gulf War increased the constraints and pressures on the desert ecosystem (Omar et al. 2000). Helianthemum lippii (L.) Dum. Cours. is distributed in small patches in northern central and southern part of Kuwait. He-lianthemum lippii is confined to the extreme desert, such as the southern Negev and the Arava Valley, where mean annual rainfall is less than 70 mm and the vegetation is restricted to wadis. The plant is between 10 and 45 em high with white stems (Fig. 1 ). Seeds are very tiny and brown in color (Fig. 2). Seeds of Helian-themum lippii mature during April and May and are dispersed dur-ing summer by wind. Seeds of different plant species have been found to require different conditions for germination. These condi-tions enable them to germinate in the right period of the right sea-son, and in the right place, and to survive in extremely hot deserts with completely unpredictable dates and distribution of rainfall (Gutterman, 1993). Omar et al. (2000) stated those desert truffles such as Tirmania and Terfezia are associated with the roots of He-lianthemum lippii. In Saudi Arabia, Helianthemum lippii and He-lianthemum ledifolium are the main host plant associates for desert truffles (Bokhary 1987). In the past, AI Dubdibah the southwest part of Kuwait was full of the desert truffle (Tirmania and Terfezia) locally known as Fuq'a or Kamaa. They are believed to be associ-ated with Helianthemum lippii, which was common in the area. However, Fuq'a cannot be seen nowadays due to land degradation problems in the area (Omar et al. 2000). Helianthemum lippii has high potential for use in conservation practices. Figure 1. Helianthemum lippii Zaman et al. Seed Treatment 19 Figure 2. Helianthemum lippii seeds Propagation from seeds is the most common method in many range plants. However many range plants have dormancy and vari-ous methods are used to overcome dormancy. Baskin and Baskin (1998) stated that dormancy is widespread among desert shrubs, with only a few species having non-dormant seeds. Scarification enhanced germination of many range species. Thanos et al. (1992) stated that seed coat hardness and impermeability to water might be the most important causes of the dormancy present in several spe-cies of Cistaceae. The germination studies in native desert plants are the preliminary step in its conservation and also for revegeta-tion program. This study was therefore designed to determine the effects of seed treatments on germination of H. lippii, seeds. The results of this research will help in the establishment of a protocol for seed multiplication of Helianthemum lippii to enhance the ser-vices of the desert ecosystems in Kuwait. Materials and Methods Environmental Conditions of Kuwait Kuwait is a small arid country extending between latitudes 28° 33'and 30° 05' N and longitudes 46° 33' and 48° 30' E in the northeastern part of the Arabian Peninsula. It has a surface area of 17,818 km2 covering the mainland and a number of offshore islands. Summer is hot and the temperature rises up to 51 oc with very low humidity. Winter is cool and the average temperature stays around 13°C with maximum 23°C. The rainfall is minimal, averaging about 115 mm y 1 (fluctuates between 25 and 250 mm), but evaporation is very high, ranging from 3.1 to 21.6 mm d-1• Rainfall occurs anytime between mid October and late April. Soil of Kuwait is mostly sandy in texture, has a high infiltration rate and is calcareous in nature (Omar et al. 2000). Underground water resources are limited and brackish in nature with total dissolved solids (TDS) concentrations ranging from 3.0 to 10.0 g L-1• Seed Collection Helianthemum lippii seeds were handpicked from natural range-land population in Sulaibiya, (N 29° 8' 42.3"; E 47° 40' 59.2") southern part of Kuwait (Fig. 3) during April2004. The fully dried seeds were collected, manually cleaned, and stored at room tem-perature (22±3°C and 30-40% RH). After 4 months under labora-tory conditions the germination rates were determined. Seed Treatments Germination Conditions Germination experiments were conducted in 9cm diameter dispos-able petri dishes lined with Whatmann filter paper. The filter paper was moistened with distilled water. Four replicates of 25 seeds were used for each treatment. Germination was recorded everyday and the seeds were considered germinated when the radicle pro-truded to the length of 2mm. IRAQ ! IIIII!U/f/f,/1//F~il .an]~~ Sulaibiya 0 sAUDI ARABIA UJJ.llilllillllll X.,. CJ SWJiila ..... ~tlx-oi& mmn ........ --f7lllJ ....... ~ ~ ~.....,...._ ~ ~---13!:1 ,..._...,.._ Figure 3. Location of Kuwait, the seed collection site and the major types of vegetation in Kuwait 20 Desert Plants Control- Intact seeds receiving no treatment Scarification - Intact seeds were scarified using sandpaper. Temperature and light - Intact seeds and scarified seeds were exposed to different combinations of temperature (10, 15, 20, 25, 30, and 35°C), light and dark. Darkness was obtained by wrapping the petri dishes with 2 layers of aluminium foil. Heat and moisture stratification - Intact seeds were ex-posed to periods of time at 50°C and different moisture levels at (4°C). KN03- Intact seeds were soaked in different concentra-tions of potassium nitrate. H3B03- Intact seeds were soaked in different concentra-tions of Boric acid. GA3 - Scarified seeds were soaked in different concentrations of Gibberellic acid. Statistical analysis The data analysis was conducted by using R 2.2.1 statistical pro-gram. The standard error of mean and the level of significance were obtained by analysis of variance (ANOVA). All reported values were used to determine if the difference were significant among treatments. Results Effect of Scarification, Light and Temperature In Helianthemum lippii light did not influence seed germination and did not present significant interactions with any of the other variance factors, temperature or pre germinative treatments (Fig. 4). Temperature significantly affected seed germination (P<0.001). Increase in temperature (35°C) decreased the germination of con-trol and mechanically scarified seeds. At 35°C, irrespective oflight conditions seed germination of intact seeds reached its lowest values (>3%). Scarification significantly promoted germination (P<0.001) and the seeds germinated faster within 24 hours and reached the maximum germination. The final germination percent-age of the control seeds (without any pretreatment) ranged from 1-13%. In intact seeds no significant difference in germination was found among the light and dark treatment. However the incubation temperatures significantly affected the final germination of scari-fied and intact seeds. Effect of Heat and Cold Stratification treatment Heat and cold stratification treatments had no significant effect on germination (Fig. 5). When comparing the germination percentag-es of control with 75 days heat stratified seeds there was no signifi-cant difference (P = 0.49). Germination of 10, 20, 30, 40, 60, 100 and 160 days heat stratified seeds were significantly lower than the control (1 0 days (P = 0. 78), 20 days ( P = 0.58), 30 days ( P = 0. 77), 40 days ( P = 0.12 ), 60 days ( P = 0.57 ), 100 days (P = 0.56) and 160 day ( P = 0. 79). Similarly cold stratification at 5°C for 10, 20, 30, 50, 75, 100, 135, 150 days did not improve seed germination percentages ( P = 0.53, P = 0.17, P = 0.72, P = 1, P = 0.26, P = 0.16, P = 0.73 and P = 0.69). Effect of H3B03 and KN03 treatment After 24 hours soaking, percentages of seed germination at dif-ferent concentration of H3B03 were summarized (Fig. 6). When comparing the germination of the control (without any treatment) with germination of 0.02, 0.04, 0.06, 0.08, 0.1 and 0.2% boric acid treatment there were no significant differences (0.02 (P=l), 0.04 (P=0.52), 0.06 (P=0.74), 0.08 (P=0.72). In potassium nitrate treatment (Fig. 4) average germination percentage of 10% in 0.2% KN03 (P<0.05) was higher than the 0.1, 0.3, 0.4 and 0.5% KN03 (P = 1 ). (Fig. 7) Effect of Scarification and GA3 The germination experiment in Hlippii revealed that the manual scarification was the most effective treatment to promote germina-tion (P<O.OOl) and 90- 100% of germination was achieved after 1 day. While in the same time the corresponding percentages for all other GA3 concentration immersed seeds ranged between 51% and 93%. Increasing GA3 concentration significantly reduced (P<O.Ol) the germination percentage (Fig. 8). Discussion The effects of pretreatments on seed germination are given in Fig. 4, 5, 6, 7, and 8 respectively. In Helianthemum lippii seeds the highest germination was obtained in manual scarification. Similar results were reported in H almeriense, H appeninum, H cinereum, H. hirtum and H squamatum (Perez-Garcia and Gonzalez-Benito, 2006). Light and dark had no significant effect on germination per-centage. However the incubation temperatures significantly affect-ed the germination percentage of scarified and unscarified seeds. Seeds of many shrubs germinate equally well in light and darkness and those of some species germinate to higher percentage in dark-ness than in light (Baskin and Baskin 1998). The complete ineffec-tiveness of light on Cistaceae seed germination could be explained (Thanos and Georghiou 1988) by either a very low Pfr threshold level for phytochrome action (satisfied even under Far-Red light) or an inconspicuous level of phytochrome action (minimized by evolution). The lowest germination percentage of scarified seeds was obtained at the highest constant temperature (35°C). Agami and Gutterman (1987) observed similar result in H. vesicarium and H. ventosum. The germination of seeds in light and dark suggest-ing that they can germinate if they come slightly buried in the soil where water may be available. In Helianthemum lippii dry heat and moist chilling had a signifi-cantly negative effect on the germination. However some pretreat-ments (H3B03, KN03) slightly increased germination percentage. In Helianthemum lippii heat treatment was not effective in increas-ing germination which agreed with results for other Helianthemum species. (Perez and Gonzalez 2006). Drake et al. (1998) reported only 9% of germination after weeks of cold pretreatment in Aster curtus. Stratification treatments were not effective in improving seed germination and this means that no physiological dormancy was involved. The boric acid treatment applied to the Helianthe-mum lippii seeds was not harmful to the seeds but did not signifi-cantly enhance germination compared to the control. A study of seed germination and growth of three populations of A triplex poly-carpa under greenhouse conditions using water cultures showed it to be very tolerant to high concentrations of boron in the growth medium. Germination was not affected by boron (Chatterton, 1969). In Helianthemum lippii 0.2% KN03 slightly increase the germination (10%). The International Seed Testing Association (1996) recommends 0.2% potassium nitrate (KN03) as a chemical that may break physiological dormancy in some species, including grasses (Pons 1989). Potassium nitrate is believed to penetrate the embryo and stimulate metabolic activity (Bradbeer 1988). Zaman et al. Seed Treatment 21 The germination experiments revealed that the mechanical scari-fication was the most effective treatment to promote germination and 90-100% of germination was achieved within 24 hours inHe-lianthemum lippii. While in the same time the corresponding per-centages for all other GA3 concentrations immersed seeds ranged between 51% and 95%. GA3 at higher concentration (1 OOOppm) highly inhibited germination. GA3 in high concentrations could be turned toxic for permeable seeds. Scarification treatment signifi-cantly improved germination of Helianthemum lippii seeds over water control. In the absence of GA3, scarification yielded higher germination than controls. In previous studies seed dormancy of Helianthemum squamatum has been broken by mechanical scarifi-cation (Escuedero et al. 1997). The highest germination percentag-es were obtained with manual scarification. Mechanical scarifica-tion resulted in dramatic increases in germination when completed in less than 2 days. On the other hand, heat treatment lowered the germination rate to levels similar to those of control seeds. In conclusion Helianthemum lippii seeds have low germination rate due to hardness and impermeability of the seed coat. The re-sults of the present study showed that scarification was the most effective method in breaking the dormancy of Helianthemum lip-pii seeds used in this study. This sandpaper scarification has the advantage of requiring less time, however, small amounts of seeds must be handled during scarification. An alternative scarification method should be explored for this species for most restoration and propagation needs. References Baskin, C.C. and J.M. Baskin 1998. Seeds, Ecology, Biogeogra-phy and Evolution of Dormancy and Germination. Academic Press, San Diego, pp. 666 Bradbeer, J .W. 1988. 'Seed dormancy and germination'. Blackie and Son, Glasgow and London. Bokhary, H.A. 1987. Desert truffles 'Al-Kamah' of the Kingdom of Saudi Arabia. Occurrence, identification and distribution, Arab Gulf Journal of Scientific Research B5 (1987), pp. 245-255. Chatterton, N. J., C. M. McKell, J.R. Goodin and F.T. Bingham. 1969. A triplex polycarpa: II. Germination and Growth in Water Cultures Containing High Levels of Boron. Agronomy Journal 61:451-453 (1969). Drake, D., K. Ewing, K. and P. Dunn. 1998. Techniques to pro-mote germination of seed from Puget Sound Prairies. Restora-tion and Management Notes 16: 33-40. Escudero, A., L. F. Carnes, and F. Perez-Garcia. 1997. Seed ger-mination of gypsophytes and gypsovags in semiarid central Spain, Journal of Arid Environments, 36 pp. 487-497. Gutterman, Y. and M. Agami. 1987. A comparative germination study of seeds of Helianthemum vesicarium Boiss. and He-lianthemum ventosum Boiss., perennial desert shrub species inhabiting two different neighbouring habitats in the Negev desert highlands, Israel. Journal of Arid Environments, 12 (3 ), pp. 215- 221. Gutterman, Y. 1993, Seed germination in Desert Plants. P. 23 Springer-Verlag, Berlin Heidelberg, New York. 253 pages International Seed Testing Association (1996). International Rules for Seed Testing. Seed Science and Technology, 24, Supple-ment. Perez-Garcia, F. and M.E. Gonzalez-Benito. 2006. Seed germina-tion of five Helianthemum species. Effect of temperature and presowing treatments. Journal of Arid Environments 65 ( 4) pp. 688 - 693. Pons, T. L. 1989. Breaking of seed dormancy by nitrate as a gap detection mechanism. Annals of Botany, 63,139-143. Omar, S., Y. Al-Mutawa, and S. Zaman. 2000. Vegetation of Ku-wait: AI Assriya Printing Press Publishing and Distribution Company and the Kuwait Institute for Scientific Research, Kuwait. Thanos, C.A. and K. Georghiou. 1988. Ecophysiology of fire-stimulated seed germination in Cistus incanus ssp. creticus (L.) Heywood and C. salvifolius L. Plant, Cell Environment. 11, 841-849. Thanos, C. A., K. Georghiou, C. Kadis, and C. Pantazi. 1992. Cistaceae : a plant family with hard seeds, Israel Journal of Botany 41 pp. 251-263. Ton~er, G. and S. Tans. 2000. The caper (Capparis ovata Desf. var. papaestina Zoh.) culture in Turkey. Pakistan Journal of Biological Science 3, 568-570. 22 9 8 7 6 '$. s:: 5 0 ·-a~ .s § 4 u C) 3 2 1 0 120 100 ~ i 80 ~ g., .j "' .s 60 6 0 40 20 0 Desert Plants Fig. 4 Effect ofscaliftcadou,ligllt aud tf'mpet·atol'e 011 ge1·miuadou of HelumtluiiiltmlqiJiii seeds( :1:: S.E ) • 10 l.S • • 20 25 OetminatWn tesn perature (OC) • Intact teed~ Scarified teed~ • 30 3.S I Fig. 5 Germination of Helialltll,,llltlltliJJPii seeds in •·e~'Ponse to stratift(ation t1·eatment (%±S.E) I I II I I I I I I I ; -,.- I I I 0 10 20 30 40 50 60 75 100 135 150 160 Storage duration (days) , Gennination% of heat stratified seeds Gennination% of cold stratified seeds '$. c Q -~ 'I t!) 120 I 00 80 '$. c 0 'i 60 'i t:l 40 20 9 8 7 6 I Zaman et al. Seed Treatment Fig. 6 Effect ofBot·ic add on HelinllliUJIUUitlippiiseed gt>nnination (%::!:S.E) ·-I~ ! I~ ~I~ -I--I- .--} Control 0.02 0.04 0.06 0.08 0.1 Boric acid Concentnlion (%) I 0.2 Fig. 7 Effect ofPotassimn nitrate on HtlilmtlumlUII.Iippiiseed gennhtation (%±S.E) 12 10 8 6 4 2 0 I Control 0.1 0.2 0.3 0.4 Potassium nitrate concentration(%) Fig. 8 Effect of snrification and GA3 ou genninadou of Heliantlte10mn lippii seeds (%:i::S.E) I I I I I 0.5 Control Scarification S + I OOppm S + 250ppm S + 500ppm S + 750ppm S + I OOOppm Scarification+ GA3 concentration 23 

Effect of Pre-germination Treatments on Seed Germination of Helianthemum lippii (L.) Dum.Cours.

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Effect of Pre-germination Treatments on Seed Germination of Helianthemum lippii (L.) Dum.Cours.

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