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HAILESLASSIE GEBREMESKEL KIDANEMARIAM
CULTIVATION OF Pelargonium graveolens L. IN ETHIOPIA
ACKNOWLEDGMENTS I am greatly indebted to the Ethiopian Institute of Agricultural Research, Wondo Genet Agricultural Research Center for sponsoring my graduate study and allowing me to use all laboratory facilities with good working environments that facilitated my research work. My special thanks go to all members of the chemical analysis laboratory who helped me a lot while carrying out the laboratory work.
I wish to express my appreciation and sincere thanks to my wife Kiros Alem and our Son Nahom, for the love they offered me and the determination and patience they showed whenever they missed me while I was concentrating on my study.
ŝ
LIST OF ABBREVIATIONS AND ACRONYMS ANOVA
Analysis of Variance
CAM
Crassulacean Acid Metabolism
C:G
Citronellol to Geraniol ratio
DAT
Days after Transplanting
FYM
Farmyard Manure
GC-MS
Gas Chromatography-Mass Spectrometry
GLM
General Linear Model
HSD
Honesty Significant Difference
LAI
Leaf Area Index
RCBD
Randomized Complete Block Design
SANDA
South African National Department of Agriculture
SAS
Statistical Analysis Software
UIDEA
Uganda`s Investment in Development Expert Agriculture
WGARC
Wondo Genet Agricultural Research Center
ŝŝ
TABLE OF CONTENTS ACKNOWLEDGMENTS
i
LIST OF ABBREVIATIONS AND ACRONYMS
ii
TABLE OF CONTENTS
iii
LIST OF TABLES
v
LIST OF TABLES IN THE APPENDIX
xi
LIST OF FIGURES IN THE APPENDIX
vii
ABSTRACT
viii
1. INTRODUCTION
1
2. LITERATURE REVIEW
4
2.1. An Overview of Rose-Scented Geranium Plant
4
2.1.1. Botany, origin and distribution
4
2.1.2. Ecology and production status
5
2.1.3. Weed management, water and nutrient requirements
7
2.1.4. Diseases and pests
9
2.1.5. Chemotypes/Cultivars
10
2.1.6. Nature, composition and uses of essential oils
11
2.1.7. Oil distillation methods
14
2.2. Factors Influencing Growth, Biomass and Oil Yield
15
2.2.1. Environmental factors
16
2.2.2. Agronomic factors
18
3. MATERIALS AND METHODS
24
3.1. Description of the Experimental Site
24
3.2. Treatments and Experimental Design
24
3.3. Cultural Practices
24
3.4. Data Collected
25
3.4.1. Growth component traits
25 ŝŝŝ
TABLE OF CONTENTS (CONTINUED)
3.4.2. Yield and yield component traits
26
3.5. Statistical Analysis
28
4. RESULTS AND DISCUSSION
29
4.1. Growth Parameters of Rose-scented Geranium 4.1.1. Plant height
29 29
4.1.2. Number of internodes per plant
30
4.1.3. Internodes length
31
4.1.4. Number of branches per plant
31
4.1.5. Number of leaves per plant
33
4.1.6. Leaf area
34
4.1.7. Leaf area index (LAI)
35
4.2. Agronomic and Chemical Traits of Rose-scented Geranium
37
4.2.1. Fresh leaf weight per plant
37
4.2.2. Dry leaf weight per plant
38
4.2.3. Leaf to stem ratio
39
4.2.4. Aboveground biomass
40
4.2.5. Fresh leaf yield
41
4.2.6. Dry leaf yield
43
4.2.7. Dry stem yield
44
4.2.8. Harvest index (%)
45
4.2.9. Essential oil content (%)
46
4.2.10. Essential oil yield
48
5. SUMMARY AND CONCLUSION
50
6. REFERENCES
52
7. APPENDIX
65
ŝǀ
LIST OF TABLES Tables
Page
1. Main effect of plant spacing and harvesting age on some plant growth characters of rosescented geranium
30
2. Interaction effect of plant spacing and harvesting age on branches number of rose-scented geranium
33
3. Interaction effect of plant spacing and harvesting age on leaves number of rose-scented geranium
34
4. Main effect of plant spacing and harvesting age on leaf area, dry leaf weight and leaf to stem ratio of rose-scented geranium
35
5. Interaction effect of plant spacing and harvesting age on leaf area index (LAI) of rosescented geranium
36
6. Interaction effect of plant spacing and harvesting age on fresh leaf weight of rose-scented geranium
38
7. Interaction effect of plant spacing and harvesting age on aboveground bimass of rosescented geranium
41
8. Interaction effect of plant spacing and harvesting age on fresh leaf yield of rose-scented geranium
43
9. Interaction effect of plant spacing and harvesting age on dry leaf yield of rose-scented geranium
44
10. Interaction effect of plant spacing and harvesting age on dry stem yield rose-scented geranium
45
11. Interaction effect of plant spacing and harvesting age on harvest index (%) of rose-scented geranium
46
12. Interaction effect of plant spacing and harvesting age on essential oil content (%) of rose-scented geranium
48
13. Interaction effect of plant spacing and harvesting age on essential oil yield of rose-scented geranium
49
ǀ
LIST OF TABLES IN THE APPENDIX Appendix Tables
Page
1. Monthly rain fall (mm) at Wondogenet
65
2. Monthly minimum temperature (oC) at Wondogenet
66
o
3. Monthly maximum temperature ( C) at Wondogenet
67
4. Treatment descriptions
68
5. Analysis of variance for effect of plant spacing and harvesting age on impotant parameters of rose-scented geranium
69
6. Analysis of variance for effect of plant spacing and harvesting age on agronomic and chemical traits of rose-scented geranium
69
7. Association among yield and yield components of rose-scented geranium
ǀŝ
70
LIST OF FIGURES IN THE APPENDIX Appendix Figure
Page
1. Hydro-distillation set-up
71
ǀŝŝ
EFFECT OF PLANT SPACING AND HARVESTING AGE ON GROWTH, BIOMASS AND OIL YIELD OF ROSE-SCENTED GERANIUM (Pelargonium graveolens L. HERIT)
ABSTRACT In order to investigate the effect of plant spacing and harvesting age on growth, biomass and oil yield of rose-scented geranium; a study was carried out at the research field of Wondo Genet Agricultural Research Center in the 2013/14 cropping season. The study was a 4 x 5 factorial combination based on randomized complete block design (RCBD) with three replications. The experiment was consisted of four levels of plant spacing (30 x 30, 30 x 40, 30 x 50 and 30 x 60 cm, which result in 111111, 83333, 66666 and 55555 plants/ha, respectively) and five levels of harvesting age (90, 105, 120, 135 and 150 days after planting (DAP)). Main effects of plant spacing and harvesting age significantly influenced plant height, number of internodes, internodes length, leaf area and dry leaf weight/plant. While interaction effect of the two factors significantly influenced number of branches/plant, number of leaves/plant, leaf area index, fresh leaf weight/plant, aboveground biomass/ha, fresh leaf yield/ha, dry leaf yield/ha, dry stem yield/ha, harvest index, essential oil content and essential oil yield/ha. The finding revealed that higher number of branches and leaves were recorded at 30 x 60 cm spacing combined with 90 and 135 DAT and higher leaf area, dry leaf weight and fresh leaf weight were recorded at 30 x 60 cm along with 120, 135 and 120 DAT, respectively. At 30 x 30 cm spacing combined with 150, 120 and 150 DAT, highest plant height, leaf area index and number of internodes were obtained; while, the highest aboveground biomass, fresh leaf yield, dry stem yield and dry leaf yield were produced at 30 x 30 cm spacing with 120, 135, 150 and 135 DAT, respectively. Significantly, higher essential oil content and harvest index were produced at the treatment combination of 30 x 40 cm spacing when harvested at 90 DAT. On the other hand, the essential oil yield (21.01kg/ha) at 30 x 30 cm plant spacing when harvested at 135 DAT was relatively higher than those of all treatment combinations, which, however it did not statistically different with that of 30 x 30 cm combined with 120 DAT (20.87 kg/ha). In total, it can be recommended to use 30 x 30 cm spacing level with harvesting age of 120 to 135 DAT for essential oil yield production at Wondo Genet area.
ǀŝŝŝ
1. INTRODUCTION Rose-scented geranium (Pelargonium graveolens L. Herit) is an important high value perennial, aromatic shrub which belongs to the family Geraniaceae (Shawl et al., 2006). It is originated from South Africa and it is widely cultivated in Egypt, India, China, and to a lesser extent in Central Africa, Madagascar, Japan, Central America, Belgium, Reunion Islands, Congo and Europe (Shawl et al., 2006; Singh et al., 2011). There are about 700 different species in the Geraniaceae family (Lis-Balchin, 2002; Shawl et al., 2006) out of which rose-scented geranium (P. graveolens) grows for production of essential oil from its leaves, tender shoots and flowers by using steam- and/or hydro-distillation (Shawl et al., 2006; Verma et al., 2011).
The essential oil of rose-scented geranium is widely used in soaps, perfumery and cosmetic industries (Lis-Balchin, 2002; Singh et al., 2008; Verma et al., 2010), pharmaceutical industries (Rao, 2002), aromatherapy (Verma et al., 2011), flavoring agent in major food categories, alcohol and soft drinks (Shawl et al., 2006). It is used in skin care oil because it has the ability of opening skin pores and cleaning complexions (Miller, 2002; Peterson et al., 2005). It is also used in the treatment of dysentery, diarrhea and colic (Shawl et al., 2006), in heavy menstrual flow and menopause problems (Verma et al., 2010), hemorrhoid, inflammation, cancer, diabetes, gallbladder problems, gastric ulcers, jaundice and liver problems (Mosta, 2006; Shawl et al., 2006), in reducing pain due to the post-herpetic neuralgia, sterility and urinary stones (Greenman et al., 2003).
Traditionally rose-scented geranium is used to staunch bleeding, healing of wounds, ulcer, skin disorders, antibacterial and insecticidal properties (Shawl et al., 2006), mite control, eczema and athletes foot problems (Jalali-Heravi et al., 2006; Jeon et al., 2008). The leaves are used as a form of herbal tea to de-stress, fight anxiety, ease tension, improve circulation and to cure tonsillitis (Peterson et al., 2005).
The main constituents of rose-scented geranium oil are citronellol (19.28-40.23%), geraniol (6.4518.40%), linalool (3.96-12.90%), iso-menthone (5.20-7.20%), citronellyl formate (1.92-7.55%), Guaia6,9-diene (0.15-4.40%) and traces of over hundred compounds (Joy et al., 2001; Boukhris et al., 2012).
ϭ
The oil yield and content of aromatic plants are affected by numerous factors which are difficult to segregate from each other, since many of these factors are interdependent and influence one another. Among these are geographical condition and genetic variation (Hussain, 2009), harvesting interval (Blank et al., 2012; Kumar et al., 2013), plant spacing (Yasin et al., 2003; Khazaie et al., 2007) and postharvest drying and storage (Hussain, 2009). Reports also indicated that the yield and quality of geranium was affected by harvesting frequency and plant shoot age (Motsa, 2006), population density and seasonal changes (Demarne, 2002), plant part distilled (Mallavarapu et al., 1997), temperature (Motsa et al., 2006; Kumar et al., 2013), light and humidity, length of exposure to sunlight, availability of water, altitude and the presence of fungal diseases and insects (Ramakrishna and Ravishankar, 2011). The oil content and yield may also change as a result of the harvesting methods used, the isolation techniques employed, the moisture content of the plants at the time of harvest and the prevailing steam distillation conditions (Hussain, 2009).
Plant spacing is one of the most important factors affecting growth, biomass, essential oil content and yield in aromatic plants (Falzaria et al., 2006; Khorshidi et al., 2010). Establishment of optimum plant population per unit area in the field is essential to get maximum oil content and yield. Under conditions of sufficient soil moisture and nutrient, higher plant population is necessary to utilize all the growth factors efficiently. Optimum plant density avoids competition between plants for growth factors such as water, nutrient, light and enables for efficient use of crop land without wastage (Singh and Singh, 2002; Ozer, 2003).
In aromatic crops, it is clearly indicated that the essential oil content and composition is related to the age of the leaves, thus emphasizing the importance of the growth stage at which harvesting takes place (Motsa, 2006). The right time, age and frequency of harvesting rose-scented geranium plants has always been a controversy. It is not clear when and at what growth stage the herbage should be harvested or the stage at which it can produce the greatest essential oil. Another controversy that surrounds the production of geranium essential oil is the variation in oil composition which is speculated to be influenced by environmental factors (Motsa, 2006). According to Rao (2000), first harvest is carried out in three to six months after transplanting; with good agricultural practices harvesting in this range of months helps to avoid losses of oil yield due to leaf senescence.
Ϯ
Despite the diverse advantages rose-scented geranium have, research works in Ethiopia on this plant has been limited. Preliminary population density trials on geranium were conducted to identify optimal population required for production of this plant under different agro-ecologies of Ethiopia (Beemnet et al., 2012); however, the trails did not consider the effect of plant density on the oil contents of the crop. In general, lack of information on appropriate agronomic practices is considered to be among the major obstruction to embark on mass production and utilization of this valuable plant in the country. Thus, it is believed necessary to assess appropriate production technologies that would enable to maximize biomass and essential oil yield in order to exploit this economically important plant as a cash crop. Therefore, the objective of this study was to assess the effect of plant spacing and harvesting age on growth, biomass and oil yield of rose-scented geranium.
ϯ
2. LITERATURE REVIEW 2.1. An Overview of Rose-Scented Geranium Plant
2.1.1. Botany, origin and distribution
The genus Pelargonium, to which rose-scented geranium (Pelargonium graveolens) belongs, is one of the five genera that are classified in the Geraniaceae family (Miller, 2002; Rao, 2009). The basic chromosome number of Pelargonium is x=11 and the somatic number for P. graveolens is 2n=88. The Reunion cultivar is heptaploid (2n=77) suffering to some degree of male sterility (Weiss, 1997). Approximately 80% of the 270 distinct and so far discovered Pelargonium species are found in the Western Cape Provinence of South Africa (Lis-Balchin, 2002; Miller, 2002; Saraswathi et al., 2011). Taxonomically revised Pelargonium contains a total of 24 species and among these only P. asperum, P. graveolens, P. radens, P. capitatum, P. roseus, P. tomentosum, P. zonale and P. roseum are used in cultivation for geranium oil production (Verma et al., 2006; Lalli et al., 2008; Saraswathi et al., 2011).
Members of the genus Pelargonium include annuals and perennials of various anatomic and morphological features such as bulbs and tuberous roots, which could have contributed to the survival of the plants in harsh environmental conditions (Miller, 2002; Lewu et al., 2007). In addition some Pelargonium species are characterizes by succulent stems that possibly enable them to undergo crassulacean acid metabolism (CAM) in water stressed conditions (Johns et al., 2003), thereby improving its water use efficiency (Lambers et al., 1998).
Pelargonium distributed to other counties by European colonials and nowadays it is widely cultivated in Algeria, Egypt, China, France, Morocco, Russia, South Africa, Central America, Belgium, Spain, Madagascar, Reunion Islands, Congo and India (Joy et al., 2001; Shawl et al., 2006; Charles, 2013). Geranium oil has been produced in several East and West African countries, principally in Kenya and Nigeria (Charles, 2013). Rose-scented geranium was introduced to Ethiopia from South Africa around 1951 by French investors. Nowadays it is a prioritized crop at the National Agricultural Research Institute, Wondo Genet Agricultural Research Center under the aromatic and medicinal plants research commodity.
ϰ
It is an erect, branching perennial shrub that grows up to 1.3 m in height, forming compact clumps to about 1 m in diameter. The root system grows up to 30 cm deep and can spread extensively. It has multiple soft green or green to grey stems becoming woody with age. The stems are covered with two kinds of bristles; some are long and fine while others are short and scarcely visible and the number, length and degree of branching vary from cultivar to cultivar (Weiss, 1997; Demarne, 2002; Miller, 2002).
The whole plant of rose-scented geranium is very aromatic but leaves, stalks and flowers are the most economical parts for essential oil distillation. The essential oil is extracted from leaves, stalks and flowers mainly by using hydro-and/or steam distillation. The oil is yellowish to green, greenish-olive, brownish green mobile liquid (Charles, 2013).
Rose-scented geranium is a hybrid flowering plant that suffers from some degree of male sterility making it difficult to propagate through seeds. Male sterile genes inhibit the development of viable pollen and prevent normal self-fertilization, resulting in infertile seeds. As a result these plants are mainly propagated by stem cuttings from healthy mother plant material, but root cuttings and suckers are equally effective, although they require more time to produce. Application of tissue culture is possible, though more expensive than the current methods (Saxena et al., 2008). Cuttings are made from young top shoots and healthy plants grown outdoors. Organic natural rooting hormones can be used to encourage rooting. Cuttings of 10 to 15 cm in length are made from young top shoots and propagated in trays or seedbeds. A mixture of 30% fine compost and 70% sand are used for planting of the cutting on bed. After the onset of roots from 2 to 6 weeks, the plants can be replanted in the field. Cuttings can also be made successfully from older wood having 15 to 30 cm in length and can be planted directly in the field if these are strong and healthy (DAFF, 2009).
2.1.2. Ecology and production status
Rose-scented geranium grows well in temperate, subtropical and tropical climate with a long growing season without extreme weather conditions. Mild climate with low humidity is ideal for its growth (Joy et al., 2001; Kritika et al., 2012). The plant is evergreen when cultivated but die back in nature during drought and winter season. The crop is grown as rain fed in hilly areas and under irrigation in an altitude range of 1000-2100 m.a.s.l. (Joy et al., 2001). Climatic condition having warm winter and mild ϱ
summer with well distributed annual rain fall is considered to be ideal for growth; although the plant is able to survive even short night chills below 0oC without permanent physiological damage. Temperature in the range of 10-30oC during the growing season is indicated to give maximum leaf growth and high essential oil content (Weiss, 1997; DAFF, 2009).
Warm climate increase herbage growth and total essential oil yield (Motsa et al., 2006). Kumar et al. (2001) indicated that P. graveolens gave a higher yield in sub-tropical (hot) areas than in temperate regions. High humidity, heavy rainfall coupled with mist or fog, frost and water logging conditions are determinant for the crop growth and favor disease development (Maiti et al., 2006). The favorable rainfall for dry land growing of rose-scented geranium is in the range from 1000 to 1500 mm that is uniformly distributed throughout the season. In areas where rainfall is less, it can be grown with supplementary irrigation (Joy et al., 2001; Eiasu et al., 2008).
Rose-scented geranium is mostly grown on a wide variety of soils, but prefers well drained sandy to loam soils which is rich in organic matter with soil pH range of 5.5 to 6.5 (UIDEA, 1998). To avoid iron and manganese toxicity, SANDA (2006) advised to keep soil pH between 5.8 and 6.2. Ram et al. (1997) also reported that both herbage growth and essential oil yield were slightly higher at soil pH of 8.4 than at a soil pH between 4.5 and 5.1 in calcareous sandy loam soil. Moreover, many hybrid geraniums failed to flower well and leaves developed brown spots at pH below 5.5. Ideal soil types are rich in organic matter and have clay content of not more than 40% and also be calcium rich for good growth and development. Furthermore, soils with good drainage are required to prevent water logging and reduce incidence of root diseases (Maiti et al., 2006).
Generally in high rain fall areas, a plant population between 50,000 to 80,000 plants/ha is recommended whereas for low rain fall plant population of 20,000 to 30,000 plants/ha is recommended. A lower plant density will leave too many open spaces which will result in more weeds (DAFF, 2009). Reports on rose-scented geranium have shown that yield is highly dependent on management, fertilization, moisture and climate (Charles, 2013). The expected plant mass of harvested Pelargonium is 15 to 50 metric tonnes of fresh material/ha at a density of 30,000 to 60,000 plants/ha. Under extreme dry land conditions between 5 to 22.50 kg essential oil/ha at 0.10 to 0.45% oil recoveries from steam distillation out of herbage yield of 5 metric tonnes/ha are reported (DAFF,
ϲ
2009). On the other hand, yields of more than 70 tonnes have been realized with good management (Shawl et al., 2006; Charles, 2013). Cultivation of Pelargonium species for oil was started in the 19th century in the Grasse region of France (Weiss, 1997). It was started in an effort to substitute for the real `Rose of the Lavant` an essential oil obtained from Rosa damascena with a similar odour as geranium oil (Demarne, 2002). Annually, about 600 tonnes of geranium oil, estimated at 12.5 million US dollars, is delivered to the international markets (Lubbe and Verpoorte, 2011). China is the world leading geranium oil producer (80-110 tonnes/year) followed by Egypt (50-55 tonnes/year), Reunion Islands (6 tonnes/year) and India (2 tonnes/year). To satisfy the world essential oil demand, the need for an additional 20 to 25 tonnes of high quality geranium oil is indicated (Sedibe, 2012). The chief importers of rose-scented geranium oil are the USA, France, Germany, UK, other European countries and Japan; importing about 230 metric tonnes/year. France is a major re-importer of geranium oil which is often further distilled and reblended to client specifications (Demarne, 2002).
2.1.3. Weed management, water and nutrient requirements
The rooted cuttings of field grown rose-scented geranium require a minimum of 30-35 days for establishment and a further 45-50 days for the canopy to close and thus enable the crop to compete successfully with weeds. It is susceptible to weed competition during the first 90 days after planting and the field must be kept weed free during this period to minimize yield losses. This long critical period was suggested to include four manual weeding operations (Kothari et al., 2002; Rao and Bhatacharya, 1997).
Unrestricted weed competition causes a reduction in oil yield of up to 70% with losses being directly attributed to the reduction in plant spread, reduced number of branches/plant and reduced leaf area (Kothari et al., 2002). The authors reported that the application of the pre-emergence herbicides pendimethalin or oxyfluorfen is more effective method of controlling weeds in rose-scented geraniums than three hands weeding, hoeing and mulching done 45 days after transplanting. On the other hand, oil quality (citronellol, geraniol, linalool, isomenthone, 10-epi-Ȗ-eudesmol, geranyl formate, citronellyl formate, cis-Roseoxide and trans-Roseoxide contents) was indicted not to be affected by weeds.
ϳ
Good harvests are obtained with an evenly distributed annual rainfall ranging between 1000 to 1500 mm in summer rainfall regions. Weiss (1997) reported low herbage yield with high oil concentrations of P. graveolens in Kenya after a three month dry period, compared to a three month wet period. Reports indicated that geranium can tolerate drought but growth can be severely retarded, also changing the oil characteristics and reducing oil yield (Brown et al., 2008).
Singh et al. (1996) reported that 50 mm cumulative pan evaporation applied at a depth of 30 mm optimized herbage and oil yield of rose-scented geranium. The foliage and oil yield recorded was 23 t/ha and 18.80 kg/ha respectively. The data obtained was compared to the foliage yield of 19.50 and 17.30 that obtained at 75 and 100 mm cumulative pan evaporation respectively. The oil yield at 75 and 100 mm cumulative pan evaporation was 15.60 and 13.90 kg/ha, respectively. Similarly, Singh (1999) reported that rose-scented geranium foliar and oil yield increased when the moisture regime was raised from 0.3 to 0.6 irrigation water to cumulative pan evaporation ratios on red sandy loam soils (alfisols). According to the author foliage and oil yield obtained at these moisture regimes (0.3 to 0.6 irrigation water to cumulative pan evaporation ratio) were 34 t/ha and 102 kg/ha compared to herb yield of 27.7 t/ha and an oil yield of only 72 kg/ha. Geraniol and citronellol were not affected by the changes in the moisture regime.
Application of 160 kg nitrogen/ha in association with a rice straw mulch optimized geranium oil yield in India (Ram et al., 2003). They also reported that simultaneous application of paddy mulch straw and 160 kg nitrogen/ha fertilizer increased the citronellol concentration while geraniol decreased with this application combination.
Geranium oil crops respond linearly to nitrogen application when in a balanced fertilizer. Well decomposed farmyard manure (FYM) at 10-15 tonnes/ha is applied in the soil at the time of land preparation. High nitrogen levels can increase herbage yield, however, it could result in lower oil yield per mass. Application of nitrogen at the rate of 120 kg/ha (in 3-4 splits, one as basal and the rest after at each harvest), 60 kg P2O5, 165 K2O, 250 kg CaO, 28 kg MgO, 15 kg Na and 10 kg S depending up on the soil status increase the herbage yield and oil quality (Maiti et al., 2006).
Studies have confirmed that total oil yield positively respond to fertility particularly to nitrogen level. Singh (1999) treated rose-scented geranium with 0, 100 and 200 kg/ha nitrogen. The author did not ϴ
show any change in oil composition but the highest plant growth and essential oil yield were obtained from the plots that receive 200kg/ha nitrogen. Ram et al. (2003) also studied response of rose-scented geranium to 0, 80, 160 and 240 kg/ha nitrogen with and without organic mulching. The result indicated that both fresh biomass and essential oil yield were improved by 160 kg/ha nitrogen application with organic mulching but major essential oil constituents (Citronellol and geraniol) was no affected by nitrogen level.
According to Araya et al. (2006) the application of organic nitrogen at a rate of 100 kg/ha increased fresh foliage and oil yield by 57.5%. When organic nitrogen application was increased to 300 kg/ha, the oil yield increased by 180.7%, compared to the zero nitrogen application. Generally citronellol percentages tend to increase with increasing nitrogen levels but the higher organic nitrogen levels reduced the guaia-6,9-diene content. However, it can be concluded that the application of nitrogen will in most cases increase both foliage and oil yields of geraniums.
2.1.4. Diseases and pests
Rose-scented geranium cultivation is subject to numerous and very virulent fungal and bacterial diseases. Several lists were established by different plant pathologists who have studied and referenced the pathogens in the producing countries (Rao, 2002). According to DAFF (2009) wilt, dieback, leaf blight, leaf spot, root and stem rot, anthracnose are frequent and can be ascribed to several species of fungus (Botrytis, Septoria, Cercospora, Armillaria, Rosellinia, Phomopsis, Pythium, Fusarium) and bacteria (Pseudomonas solanacaerum). Some of these pathogens cause severe damage, sometimes leading to total destruction of the crop and the impossibility of growing rose geranium again on the same plot.
The suggested control measures for these pathogens are only partially satisfactory, even if anthracnose and botrytis can be controlled by spraying specific fungicides. Despite the need for resistant varieties, genetic improvement program does not seem reasonable or economically viable regarding the economic importance of the crop in the individual oil-producing countries (Demarne, 2002).
Rose-scented geranium is attacked by many different species of pests belonging mainly to the Hemiptera, Coleoptera and Lepidoptera families. Among the most important pests are the white grubs, ϵ
thrips, cutworms, cockchafers, whiteflies, aphids, mites, termites and white peach scale (DAFF, 2009). According to Rao (2002), from the research done in India, there are several species of nematodes that can damage the crop and inflict yield losses up to 75.8 %. Chemical control seems possible as well as biological control with a nematophagous fungus and/or companion cropping with nematicidal plants (periwinkle and marigold).
2.1.5. Chemotypes/Cultivars
Geranium oil is obtained from various cultivars grown under distinct environment. The hybrids are mainly derived from crosses among Pelargonium graveolens, P. capitatum and P. radens which are commonly known as rose-scented geranium (Lis-Balchin, 2002). The commercially available rosescented geranium chemotypes are distinguished by the country of origin; and accordingly the Bourbon, Egyptian, Moroccan, Algerian and Chinese cultivars are the major ones (Weiss, 1997; Williams and Harborne, 2002).
The essential oil composition of rose-scented geranium varies with chemotype (Demarne, 2002); for instance, the Bourbon type is characterized by 1:1 citronellol to geraniol ratio (C:G), lower citronellol and citronellyl ester levels and high content of geranyl esters, linalool, guaia-6,9-diene and isomenthone (Gupta et al., 2001; Williams and Harborne, 2002). Oil from the Egyptian type has citronellol to geraniol ratio similar to Bourbon type but with lower guaia-6,9-diene content (Gupta et al., 2001). Oil from the Chinese and Algerian types is known for high citronellol to geraniol ratio ranging between 3 and 4 (Kulkarni et al., 1998). Geranium oil produced in South Africa is said to have a composition similar to that of the Bourbon type (SANDA, 2006).
From the market point of view the Bourbon type oil is regarded as the best quality and is priced higher than the other oils (Lubbe and Verpoorte, 2011). Qualitatively oils from the Moroccan, Algerian and Egyptian types ranked next to the Bourbon type and presumably earn a premium over the oil from the Chinese type which has highly variable odour and is the cheapest in price (Weiss, 1997). Apart from the commercially renowned rose-scented geranium chemotypes, several essential oil rich members of the genus Pelargonium and their hybrids have been reported. Essential oil of Pelargonium graveolens cv. Kunti (grown in India) is rich in geraniol (40-50%) whereas essential oil of somaclonal mutant of the same cultivar was found to contain isomenthone (71%) as its major constituent (Gupta et al., 2001). ϭϬ
2.1.6. Nature, composition and uses of essential oils
An essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from plants. Essential oil is defined as the volatile oil stored in extracellular spaces in the epidermis or mesophyll cells of plants (Sedibe, 2012). Essential oils are also known as volatile or ethereal oils due to the low boiling characteristic of most of the compounds that constitute them, or simply as the "oil of" the plant from which they were extracted. Oil is "essential" in a sense that it carries a distinctive scent, or essence, of the plant (Olle and Bender, 2010).
Essential oils are plant secondary metabolites that impart the aroma and flavour characteristic to the plant. They are classified under secondary metabolites because of lack of sufficient evidence that shows they are directly involved in the normal plant metabolic process such as growth and viability (Lambers et al., 1998). Essential oil is contained in specialized structures in all or some plant parts; cavities or ducts in the epidermis of eucalyptus leaves or citrus fruit peels; and glands or hairs originating from epidermal cells of the modified leaf hairs of geranium (Salyh, 2013).
Why plants secrete oils or waxes has yet to be fully explained, although certain cavities can reasonably be attributed to their presence; the oil might be produced to act as protect plants against being eaten by herbivores and against being infected by microbial pathogens, attractants (odor, color, taste) for pollinators and seed-dispersing animals and function as agents of plant-plant competition and plant-microbe symbioses (Weiss, 1997; Taiz and Zeiger, 2002). While some terpenes from eucalyptus leaves are known to contribute allelopathic effects on the forest floor which inhibits germination and growth of competitors. Highly scented oil contained in flowers of essential oil crops is generally accepted as an aid to attract pollinators for the reproduction process o plants (Weiss, 1997; Rao, 2000).
Essential oils are complex mixtures of large number of individual compounds with a variety of highly functionalized chemical entities (Kayser et al., 1998). Plant volatile oils are variable mixtures of principally terpenoids, specifically monoterpenes (C10) and sesquiterpenes (C15) although diterpenes (C20) may also be present, and a variety of low molecular weight aliphatic hydrocarbons (linear, ramied, saturated and unsaturated), acids, alcohols, aldehydes, acyclic esters or lactones; and
ϭϭ
exceptionally
nitrogen- and sulphur- containing compounds, coumarins and homologue of
phenylpropanoids are present (Dorman and Deans, 2000; Iijima et al., 2004).
According to Viljoen et al. (2005) essential oil composition may show dramatic variation among chemotypes/cultivars of the same plant species. The authors identified that essential oil of five chemotypes namely, myrcenone rich type (36-62%), carvone rich type (61-73%), piperitenone rich type (32-48%), ipsenone rich type (42-62%) and linalool rich type (>65%) of Lippia javanica (Verbenaceae) in South Africa. Composition and amount of volatile oils also vary among parts/organs of the same plant (Kuiate et al., 2006) and plant morphological stage (Kothari, 2004a). In several plant species, the non-woody plant materials are the major source of essential oils (Dorman and Deans, 2000) which are synthesized and/or stored within the grandular trichomes that develop on the surface of leaves and other organs of the plants (Gaspar et al., 2003; Iijima et al., 2004).
Rose-scented geranium oil is among the top 20 available plant volatile oils (Williams and Harborne, 2002). The hydro- and/or steam-distillation of the leaves generated pale yellow coloured oil (yield of 0.19%, v/w), which possesses a tenacious rose like odour such as citrus and minty undertones (Motsa, 2006). The chemical composition of the oil is complex in nature and comprises a wide array of compounds. The composition of these chemical compounds varies among geranium that originated from different counties (Rana et al., 2002). The authors identified thirty compounds that contribute up to 99.10% of the oil that is produced by rose-scented geranium. For commercial purpose only six compounds are determined; linalool, citronellol, geraniol, citronelly formate, geranyl formate and guaia-6,9-diene. The relative proportion of these compounds determines the odour quality of the oil. The citronellol to geraniol (C:G) ratio is used by the perfume industry to determine oil quality. A citronellol to geraniol ratio greater than 3 signifies oil of low odour quality. In contrast, a citronellol to geraniol ratio ranging from 1 to 3 is associated with a better odour quality of the oil (Saxena et al., 2000).
Upon GC-MS analysis, the essential oil of rose-scented geranium was found to contain 47 constituents; the volatile oil contained 67.39% monoterpenoids, 25.40% sesquiterpenoids and 7.21% other compounds (Boukhris et al., 2012). The main constituents of the oil is citronellol 19.28-40.23%, geraniol 6.45-18.40%, iso-menthone 5.20-7.20%, linalool 3.96-12.90%, citronellyl formate 1.927.55%, cis- and trans-rose oxide 1.00-2.50%, menthone 0.78-1.50%, caryophyllene 0.74-1.04%, nerol ϭϮ
0.67-1.24%, cis- and trans-linalool oxide 0.36-0.92%, cis- and trans-ocimene 0.10-0.36%, pinene 0.280.86%, Guaia-6,9-diene 0.15-4.40%, phellandrene + limonene 0.12-0.21%, geranyl acetate 0.101.08%, myrcene 0.06-0.19%, ß-pinene 0.04-0.16% and traces of over hundred compounds (Joy et al., 2001).
Rose-scented geranium essential oil is used as an important ingredient in the perfumery and cosmetic industries all over the world. It is one of the best skincare oils because it is good in opening skin pores and cleaning oily complexions (Miller, 2002; Peterson et al., 2005). It also used for treatment dysentery, hemorrhoids, inflammation, heavy menstrual flows and cancer. The French medicinal community is currently treating diabetes, gallbladder problems, gastric ulcers, liver problems, sterility and urinary stones with this oil (Peterson et al., 2005). The leaves are used as a form of herbal tea to de-stress, fight anxiety, improve circulation and to cure tonsillitis (Peterson et al., 2005). It is helpful in relieving tension, eczema, dermatitis, anxiety, circulation, nausea, tonsils, flu, burns, rheumatism, nervousness, stress and fatigue (Motsa, 2006).
The Chinese homeopathies, on the other hand, believe that the essential oil opens up the liver charka and promote the expulsion of toxins that prohibit the achievement of balance within the body (Higley and Higley, 2001). In recent studies between the essential oil of geranium and tropical capsaicin, a commonly prescribed conventional remedy for shingles pain, it was discovered that geranium essential oil was extremely useful in reducing pain due to post-herpetic neuralgia followed by shingles (Greenman et al., 2003). It blends well with all kinds of scents, floral and oriental bouquets and is extensively used in perfumery, cosmetics, food industry (Eiasu et al., 2008). It is widely used for scenting soaps due to its stability in the slightly alkaline medium and also used in the manufacture of perfume compounds. Rose-scented geranium employed as a flavouring agent in major food categories, alcoholic, beverage, soft drinks and especially with pink coloured products (Gough, 2002; Kumar et al., 2001).
The volatile oil is well documented for its antimicrobial, haemostatic, astringent, diuretic, deodorant and vermifuge properties and also used to treat acne, depression, anger and other respiratory disorders. On the skin, oil helps to balance the secretion of sebum and clears sluggish and oily skins, while the antiseptic properties make this oil an effective aid to help with burns, wounds, ulcers and other skin problems. It is useful for treating jaundice and can also be used for restraining nose bleeds and other ϭϯ
hemorrhages (Kritika et al., 2012). Gomes et al. (2006) explained that in masculine perfumes, the oil is used as heart note, adding a floral character to green compositions. Fresh leaves of the plant can be used to impart as rose flavor to dessert and jellies (Gough, 2002).
2.1.7. Oil distillation methods
The extraction of essential oils from plant material can be achieved using a number of methods such as expression, hydro-distillation, steam-distillation and solvent extraction. In each method there may be some variation and refinements; and extraction may be conducted under reduced pressure (vacuum), ambient pressure or excess pressure. The choice of extraction method depends on the nature of the material extracted; stability of the chemical components and specification of the targeted product (Sedibe, 2012).
The most common method of essential oil extraction from plant materials is by distillation with hydro- and/or steam- distillation (Coleman, 2003). Other methods of oil extraction that exist in the perfumery and food industries include cold pressing or scarification, which is used to release the volatile substances from peels of fruits, especially citrus peels, effleurage which is used to extract oil from delicate plant materials such as flowers, solvent extraction and the latest being supercritical carbon dioxide extraction (Anitescu et al., 1997; Coleman, 2003).
Since plant essential oils are a mixture of several compounds with a wide range of chemical and physical properties (Deans, 2002); different distillation methods and distillation phases are expected to have different effects on the chemical as well as physical state of the compounds (Amin et al., 2001; Peterson et al., 2005; Babu et al., 2007). Babu and Kaul (2005) investigated the impact of different hydro-distillation techniques (water distillation, water-steam distillation and steam distillation) with or without recycling the hydrosol on the amount and composition of essential oil of rose-scented geranium. The result showed that hydrolysis of some constituents resulted in changes in essential oil composition. The author also realized that the amount and composition of recovered oil depend on the solubility of essential oil constituents. Therefore, using of steam distillation in combination with water distillation (in the latter distillation phase) would give the desired essential oil yield and quality. This also supported reports of Rao et al. (2002), which indicate that an average 7% of the total essential oil yield could be recovered from hydrosol by hexane extraction. ϭϰ
Peterson et al. (2005) also investigated the effect of supercritical fluid extraction technique on the amount and composition of rose-scented geranium oil by using supercritical carbon dioxide as a solvent in combination with different pressure, temperature, carbon dioxide flowing rate and extraction durations. The result indicated that pressure and extraction time significantly affected essential oil composition. The author highlighted that, an optimum temperature, extracting time and carbon dioxide flow rate supercritical fluid extraction technique improved essential oil recovery to nearly 17 times (2.53%) that of the essential oil extracted by the steam-distillation techniques (0.15%). The overall results confirm that the composition of essential oils obtained by the different extracting techniques could vary in amount and composition, which may result in products that would misrepresent the essential oil yield, composition and the natural aromatic characteristics of the oil in the source plant (Amin et al., 2001).
2.2. Factors Influencing Growth, Biomass and Oil Yield
In general, factors that influence the yield and chemical composition of rose-scented geranium oil are location, age of the leaves, application of growth regulators, drying of the biomass prior to distillation, method of distillation and improper storage of the extracted volatile oil (Rao and Bhattacharya, 1997). Climatic parameters such as temperature, rainfall and photoperiod are also among the major role players in the growth and biosynthesis processes in plants (Lis-Balchin, 2002). Harvesting of such plants is done in such a way that there will be maximum production of biomass and preservation of subsequent shoot development (Weiss, 1997).
The ability of plant development governs the frequency and yield of subsequent harvest. This also depends on the compatibility of the plant to environmental conditions. Harvesting of plant may be determined by weather conditions at harvest, but to obtain maximum yield plant should be routinely sampled to determined oil content. The moment of harvesting/sampling may also influence the oil composition not only related to the time of the day but also to the growth period per stage, location and growing season (Motsa, 2006). Essential oil content depends upon external and internal factors affecting the plant such as: environmental and climate conditions, season of collection, age of plants, the stage of harvesting or genetic data. Plasticity in the chemical composition of essential oil in response to climate factors, either in nature or under controlled conditions is less known (Aprotosoaie et al., 2010). Isolation of essential ϭϱ
oil (volatile oils) was obtained from fresh leaves and to certain extent from stalks and flowers upon hydro-distillation in Clevenger apparatus for three to four hours. The oil was separated, dried over anhydrous sodium sulfate and kept in a dark glass bottle at 4°C for the analysis (Eiasu et al., 2008; Aprotosoaie et al., 2010).
According to Weiss (1997) rose-scented geranium produce maximum leaf growth with high oil content under warm, sunny condition and oil content normally increases from the onset of flowering and reduces after full blooming. According to Kumar et al. (2001) the highest oil yield from geranium was related to better vegetative growth per unit time in hotter environment. Harvesting of rose-scented geranium is usually conducted at three to six months interval using sickle. The main determining factors considered before harvesting are plant developmental stage, weather condition, crop management and labor availability. Labor availability is the main determinant factor because rosescented geranium is harvested manually due mechanical harvesting result in low biomass yield sent to distillation (Demarne, 2002; Rao, 2002).
2.2.1. Environmental factors
Crop plants depend largely on temperature, solar radiation, moisture and soil fertility for their growth and nutritional requirements. Temperature plays an important role in plant growth and yield as a whole. Plants respond to temperature change in most of their metabolic activities such as photosynthesis respiration and transpiration (Murtagh, 1996). According to Weiss (1997) rose-scented geranium as plant produces maximum leaf growth with high oil content under warm sunny conditions.
Kumar et al. (2001) reported that warm environment favored vegetative growth of rose-scented geranium per unit time. But in contrary to this Rao (1996) reported during summer rose-scented geranium subjected thermal (atmospheric as well as soil) and moisture stress ended up producing low biomass yield. Other essential oil crops such as peppermint were strikingly influenced by temperature. Plant dry matter, morphological development and oil yield respond positively to higher temperature. Maximum leaf and stem was produced less than 30oC day temperature and the leaf mass ratio increases with increasing day temperature. The combination of high day and low night temperature produced the greatest leaf mass ratio (Motsa, 2006).
ϭϲ
Moisture is one of the common phenomenons that in water stressed environments plant growth is negatively affected (Turtola et al., 2003). It is one of the most important environmental factors required for plant growth and development. The negative effects are expressed in the rate of vegetative growth, reproduction, flowering, biomass and oil yield. The reverse may be true in the case of biosynthesis of secondary metabolites such as essential oils depending on species, degree of water stress and plant shoot age (Murtagh, 1996; Turtola et al., 2003).
Pelargonium species are characterized as drought tolerant, but in prolonged drought condition they show poor vegetative growth. Areas with sharply defined wet and dry seasons were able to produce good biomass and oil yield provided there was no extended period of water logging. For instance, low biomass (21.80 tonnes/ha) and high oil yield (23.30 kg/ha); high biomass (28.20 tonnes/ha) and low oil yield (20.00) were recorded in rose-scented geranium after three dry months as compared to three wet seasons respectively in Kenya (Weiss, 1997).
Photoperiod has a strong effect on plant performance, with respect to vegetative g rowth and reproductive behavior (Farooqi et al., 1999). Runkle (2002) proposed that photoperiod may also influence plant height, branching and other plant growth characteristics. It may also exert a direct influence through modulation of relevant metabolic pathways, from photosynthetic carbon production and its partitioning to the Rohmer route (non-mevalonate pyruvate-glyceraldehyde-3phosphate driven isopentenyl pyrophosphate synthesis), further leading to generation of essential oil terpenoids (Farooqi, et al., 1999).
Leaf area and leaf greenness increased under long-day photoperiods in geranium. Plant dry mass was also reported to increase as a consequence of increased chlorophyll content (Langton et al., 2003). Adams and Langton (2006) took the studies of photoperiod further by observing the effect of long-day lighting on plant growth in winter. The authors reported that geranium plants increased in fresh shoot mass and the marketing stage was reached earlier than normal. These findings indicated that geranium grows better under long-day photoperiods, which was contrary to the studies of Runkle (2002) who suggested that geranium was a day neutral plant.
Photosynthetic light responses of thyme and related it to physiological traits, dry matter, shoot formation and essential oil accumulation (Motsa, 2006). The author found that variability in ϭϳ
shoot yield and essential oil was indeed associated with photosynthetic activities. All plants grown under supplemental light had an upright growth with relatively deep green coloured leaves and were aesthetically more appealing, while those grown under natural light showed a prostrate, open type of growth, an adaptation that would improve light penetration to interior leaves. Shorter, stouter and thicker leaves with more tillers and branches were developed by plants grown under supplemental light (Letchamo and Xu, 1996).
Metabolic processes have a direct relevance to essential oils obtained from the foliage of certain plants such as mints. This was evident in the three different Mentha species (M. arvensis, M. citrate, M. cardiaca) subjected to different photoperiodic treatments where it was discovered that the three species were long-day plants, exhibiting substantially higher vegetative proliferation under long day conditions. Shorter-day conditions resulted in slower growth and reduced herbage yield (Farooqi et al., 1999). Non-availability or restricted availability of photosynthates adversely affects essential oil synthesis and accumulation since it is biosynthesized as a secondary plant metabolite, i.e. product of photosynthesis (Rao et al., 1996). The environment under which these plants are produced should allow high photosynthetic rates since the oil yield is a product of leaf yield and oil content. Both parameters are affected by the foliage density within a plot (Badi et al., 2003).
2.2. 2. Agronomic factors
Plant spacing
The major restraint in aromatic crop production is improper crop spacing in the field. Depending on the environment, production system and cultivar, plant density is an important factor affecting aromatic plants (Ozer, 2003). Plant density or plant population is defined as the number of main stems within a unit area of land (Ball et al., 2000). It is therefore, necessary to determine the optimum density of plant populations per unit area in order to obtain maximum yield. The optimum use of plant spacing has dual advantage. It avoids the strong competition between plants for growth factors such as water, nutrient and light. Conversely optimum plant density enables for efficient use of crop land without wastage (Wajid et al., 2004; Falzaria et al., 2006; Khorshidi et al., 2010). The effect of plant spacing on growth and secondary metabolites is largely due to change in the interception of radiant energy. Plant spacing is one of the most important factors affecting yield, yield ϭϴ
components, oil content and essential oil yield in aromatic plants (Falzaria et al., 2006; Khorshidi et al., 2010; Ismail et al., 2013). The optimization of this factor can lead to a higher yield in the crop by favorably affecting the absorption of nutrients and exposure of the plant to the light (Khorshidi et al., 2009). In suitable plant spacing, plants completely use environmental conditions or macronutrients and inter- or intra-specific competition becomes less. Plant spacing could have significant impact on plant disease incidence (Morteza et al., 2009).
The yield potential of the individual plant is fully exploited when planting under wider spacing because wider spaced plants have more nutrition, water and air; however, in the narrower spacing they are overcrowded and have restricted conditions for development because they have limitations in the maximum availability of these factors (Ball et al., 2000; Ozer, 2003).Yield per plant decrease gradually as plant population increase in particular area. As plant density increases, the amount of dry matter in vegetative parts also increases. Both biological and economical yield increased with increasing plant population up to certain point and subsequently no addition in biological yield can be obtained and economic yield decrease (Singh and Singh, 2002).
Yield of Black Cumin was reduced as plant densities reduced due to shortening of the growing cycle decreased the amount of radiation intercepted during the growing season which leads to decrease total dry weight of plant (Rahnavard et al., 2010). According to Khorshidi et al. (2009) significant effect of plant density on essential oil percentage of Foeniculum vulgare, the higher essential oil percentage was obtained with the lowest densities of planting while the minimum essential oil percentage was obtained at the highest density of planting. The higher percentage of anethole (83.07%), estragol (3.47%), fenchone (8.04%), p-cymene (4.45%), terpinene (0.54%), sabinene (0.51%), and Pinene (0.48%) were obtained with space between plants 25, 10, 20, 20, 15, 20, and 25cm, respectively. Effect of three planting density (4, 8 and 12 plants/m2) on the vegetative and reproductive characteristics of valerian (Valeriana officinalis L.) indicated that lower spacing influenced the growth, quantity and quality characteristics of valerian (Taleie et al., 2012). Plant spacing exerts an effect on plant height at flowering, number of stem and branches/plant, yield/plant and yield/ha in thymus (Khorshidi et al., 2009). The authors showed that the higher dry matter yield of thyme was obtained with smaller densities of planting. Similarly, the effect of three plant densities in Sweet Basil (20, 40
ϭϵ
and 60 plants/m2) indicated that highest amount of dry matter, percentage and the yield of effective substances were produced in the lower plant density (Arabasi and Bayran, 2004).
On the other hand, studies on the effects of different plant population densities, ranging from 20-130 plants/m2 in rapeseed, showed that pods/plant, seed weights and dry matter/plant decreased as plant population density increased. Plants grown at high density had fewer pod bearing branches/plant but produced more branches and that with an increase in density 1000 seed weight increased (Ozer, 2003). Variation of density in stands would affect inter-branch competition and hence offer scope for increasing seed yields that affect harvest index of individual branches (Pirzad et al., 2011). In cultivated thyme, plants grown using 15cm planting had the highest fresh and dry weight of shoots than at the spacing of 25 cm (Al-Dein and Al-Ramamneh, 2009). The higher plant population density in Origanum syriacum, produce higher fresh yield and resulted increase in fresh weight of branches, number of leaves and fresh weight of leaves produced per unit area (Al-Kiyyam et al., 2008).
According to Berimavandi et al. (2011) in Calendula officinalis maximum of plant dry weight, flower number/plant, branch number/plant, flower dry weight and amount of essential oil 100/g dried flower were obtained at the 20 plants/m2, while maximum of flower dry weight and amount of essential oil, both per unit area were obtained at the 60 plants/m2. Plant spacing significantly affected forage yield of Mott elephantgrass; the highest yield per hectare was obtained at narrow spacing than wider spacing (Yasin et al., 2003).
Essential oil content and oil yield of Tagetes lucida significantly responded to plant spacing in both seasons during the three cuts. The higher essential oil percentage was obtained with the lowest densities of planting (Ismail et al., 2013). Optimum plant population densities of 60,000 plants/ha gave the highest essential oil content in valerian (Morteza et al., 2009). In three different plant spacing (50 x 50, 50 x 60 and 50 x 80 cm) of rose-scented geranium, highest values of essential oil yield (7.56 ml/m2), that are characteristics of direct interest for the market, were obtained at a spacing of 50 x 50 cm of geranium plant (Blank et al., 2012). According to Beemnet et al. (2012) the highest values of fresh leaf yield/ha and essential oil yield/ha were obtained at plant population of 111,111 plants/ha (30 x 30 cm of plant and row spacing).
Harvesting age ϮϬ
The main object in aromatic crop production for essential oil is to optimize the biomass production and to harvest the crop before any deterioration on biomass, oil content and quality occurs. It is clearly indicated that in aromatic crops, the chemical composition of the essential oil is related to the age of the leaves, thus emphasizing the importance of the growth stage at which harvesting takes place (Motsa, 2006). Moreover, essential oil content changes according to harvest time (hour) of the day; but, the most critical time for harvesting of rose-scented geranium is on sunny days usually in the morning (Maiti et al., 2006).
According to Telci and Hisil (2008) considerable variations in growth of coriander herb oil are based on differences in genetic structure of plants, environment, growth conditions and harvest stage. The authors reported that growing seasons having different rainfall, temperature and light intensity from early spring periods to late autumn influence the crop yield and quality of coriander. Harvesting stage of plant has an influence on quantity and quality of essential oil in most essential oil bearing plants (Ramezani et al., 2009). Moreover, optimum harvesting age depends on the target compound desired. For instance, in Artemisia, if the artemisilin is the main target, the maximum yield occurs at early blooming while, if the main target is artemisinic acid, the maximum yield is obtained at full blooming (Laughlin et al., 2002)
Essential oil yield and composition vary with developmental stage of the whole plant, plant organs and cells (Sangwan et al., 2001; Gora et al., 2002). In some Lamiaceae species (Mentha piperata and Saliva officinalis) the secretion and synthesis of secondary metabolites in to the sub-cuticular space in the peltate glandular hairs start in very young leaves. In this regard, repeated harvesting can be beneficial or determinant for oil production depending on the environmental factors (Aflatuni, 2005). In Erogeron canadensis, the content of limonene in leaves declined with advance in leaf age, while the opposite was true in flower oil but positive correlation between essential oil yield and dry mater accumulation were observed (Gora et al., 2002).
According to Kothari et al. (2004a) biomass yield was greater in the first harvest and gradually decline in subsequent harvest of Ocimum tenuiflorem but the methods of harvesting have no significant effect on biomass yield. Contrary to the decrease in biomass yield the essential oil content is lower in the first harvest increased gradually in the subsequent harvests to reach maximum in the fourth harvest. Rosescented geranium is normally harvested by cutting at 15-20 cm above ground to allow re-establishment Ϯϭ
of new leaves for the process of photosynthesis (Weiss, 1997; Demarne, 2002). According to Motsa (2006) rose-scented geranium biomass yield is usually high at first harvest become constant and decline with subsequent harvest. However, in Ethiopia after the first harvest most of the harvested plants fail to sufficiently regenerate new shoot growth which makes subsequent harvesting uneconomical (Beemnet et al., 2009).
The oil content and yield of aromatic plants are often altered during harvesting and post harvesting processes (Motsa, 2006). This alteration occurs due to spontaneous conversions occurring continuously, which changes the essential oil composition (Jose et al., 2006). Studies with effect of various harvesting age on agronomic, quality and quantity of thyme (Thymus critriodorus) showed that the highest essence yield was obtained in beginning of blooming (Golparvar and Bahari, 2011). According to Zheljazkov et al. (2011) lemongrass biomass yields were highest in second harvest, lower in the first harvest and lowest in harvest three. According to the authors the combined biomass yields of harvest one and harvest three were lower but oil yields were higher compared to harvest two alone.
In spearmint (Mentha spicata L.) at 60 days after transplanting higher leaf weight and leaf to stem ratio were obtained but harvesting after 180 days after transplanting resulted in lower fresh leaf weight and leaf to stem ratio (Solomon and Beemnet, 2011b). According to the authors the essential oil content and essential oil yield of spearmint were significantly increased when harvesting were made 180 and 150 days after transplanting.
According to Mighri et al. (2009) essential oil yield of Artemisia herba-alba was significantly higher for plants harvested at flowering stage compared to other stages. In Davana (Artimisia pallens W.) essential oil content was higher at the full emergence of flower heads than at anhesis and initiation of seed set stages (Mallavarapu et al., 1999). Maximum essential oil content of thyme (Thymus vulgaris) was obtained at the beginning of blooming stage (Badi et al., 2003). Sellami et al. (2009) also reported that essential oil yield were higher during early vegetative growth, decreased significantly during the late vegetative stage and again increased at flowering stage. Harvesting time significantly influenced the essential oil in lavender (Lavandula angustifolia) were essential oil content decreased from first harvest to the last harvest (Baydar and Erbas, 2005). According to Weiss (1997) information on rose-scented geranium oil yield at different harvest ages is limited in literature. The author indicated harvesting age varies with leaf age, with young leaves having ϮϮ
greater content than older leaves. Motsa et al. (2006) investigated the pathway of oil biosynthesis, metabolism and impact of plant shoot age on essential oil yield and composition. They observed that total oil yield per harvest had an increasing tendency and reached maximum around time 19 th week of each re-growth cycle. In three different harvest stages (8, 12 and 16 weeks) of rose-scented geranium, highest values of essential oil yield (7.56 ml/m2) were obtained at harvest stage of eight weeks. Long periods of harvest were less productive because decreases of values of all variables were observed in the last harvests (Blank et al., 2012).
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3. MATERIALS AND METHODS 3.1. Description of the Experimental Site
The field experiment was carried out at Wondo Genet Agricultural Research Center (WGARC) in 2013/14 main cropping season. The center is situated at about 264 km south of the capital, Addis Ababa. Geographically it is located at 07° 19' North latitude and 38° 38' East longitude with an altitude of 1876 m.a.s.l. According to the records from 1983 to 2012, the site receives mean annual rainfall of 1182 mm (Appendix Table 1) with an average minimum and maximum temperature of 9.86 and 28.28 °C, respectively (Appendix Table 2 and 3). The soil textural class of the experimental area is sandy clay loam (Nitosol) with pH of 6.4 (Abayneh et al., 2006).
3.2. Treatments and Experimental Design
The experiment consisted of four levels of plant spacing (30 x 30, 30 x 40, 30 x 50 and 30 x 60 cm, which resulted in 111111, 83333, 66666 and 55555 plants/ha, respectively) and five levels of harvesting ages (90, 105, 120, 135 and 150 days after planting (DAP). The experiment was arranged in a 4 x 5 factorial combination using randomized complete block design (RCBD) with three replications. Thus, there were 20 treatment combinations in triplicates.
The treatments were randomly allotted to each plot and the details of the treatment combination are given in Appendix Table 4. The experimental plot had an area of 7.2 m2 (6 m length x 1.2 m width). The space between replications and plots was 1.5 m and 1 m respectively. Plants in the two middle rows out of the four rows per plot constituted the net plot used as the sampling unit. Five plants from the middle rows were taken for sampling and data analysis.
3.3. Cultural Practices
On year old fresh soft wood cuttings, having 10-15 cm length, were taken from the top parts of diseasefree plants of rose-scented geranium cv. Shito. The cuttings were planted in 10 cm polyethylene bags filled with fine mixtures of top soil, sand and compost (3:1:2 ratio, respectively) at the nursery site of WGARC. The seedlings were grown in a nursery for about 90 days at the center. The experimental area Ϯϰ
was ploughed and harrowed to provide a fine and pulverized soil. Uniformly grown seedlings were selected, hardened and transplanted to the experimental field after 90 days of planting in the nursery site. Nitrogen (in the form of urea) at a rate of 120 kg/ha (Maiti et al., 2006) was applied uniformly in three splits, one during transplanting and the other 30 and 60 days from the date of transplanting. All appropriate agronomic practices such as weeding, watering and hoeing were conducted manually both at the nursery and experimental field uniformly.
3.4. Data Collected
Data were collected randomly from the two middle rows of each plot. A total of 17 quantitative traits were recorded to evaluate the growth, biomass and oil yield of rose-scented geranium as follows:
3.4.1. Growth component traits
The following growth component traits were collected from five randomly sampled plants of each plot at each harvest stages (90, 105, 120, 135 and 150 DAT).
Plant height (cm): Plant height was measured in centimeter from the base of the plant to tip of the main stem; the mean height was worked out and expressed in cm.
Number of internodes per plant: The total numbers of internodes found in the randomly sampled plants were counted and averaged to get number of internodes per plant.
Internodes length (cm): Three branches per plant were randomly selected, then three internodes (one each from top, middle and lower portion of the internodes) were selected and the length of internodes found between two nodes were measured, the mean internodes length was worked out and expressed in cm.
Number of branches per plant: The total numbers of branches arising from the main stem were counted manually and mean value was worked out.
Ϯϱ
Number of leaves per plant: The total numbers of leaves found in the randomly sampled plants were counted and averaged to get number of leaves per plant. Leaf area/plant (cm2): The leaf area was worked out by disc method at the different growth stages as per the procedure of Edje and Osiru (1988) and Edje and Ossom (2009). Leaf lets were separated from petiole and fifteen leaf discs of known size were taken through a cork borer from randomly selected fifteen leaves of five sampled plants for dry matter distribution. The discs and the remaining parts of the fifteen leaves as well as the leaves collected from the sampled plant were oven dried at 68°C for 72 h until a constant weight and the leaf area was calculated by the following formulas and the mean of five plants was expressed in cm2 per plant. ͳͷ
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Leaf area index (LAI): The leaf area index was calculated as the relationship between leaf area/plant divided by land area covered by individual plant, calculated according to the following formula (Sestak et al., 1971).
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3.4.2. Yield and yield component traits
The following yield and yield component traits were collected from five randomly sampled plants of each plot at each harvest stages. All yield and yield component traits were collected in the morning hours (9:00-10:00 A.M).
Fresh leaf weight/plant (g): The average fresh leaf weight of the randomly sampled plants was immediately recorded after the leaves were separated from stem.
Dry leaf weight/plant (g): The mean oven leaf dry weight of the sampled plants from each plot after drying 100g leaf sample from each sampled plants at 68°C for 72 h until constant weight was reached. Ϯϲ
The sum of the dry leaf weight of sampled plants was divided by the number of sampled plants to work out the mean.
Leaf to stem ratio: At harvest, the fresh weight of randomly selected plants was separately recorded then dried leaf to dried stem weight was noted. The dried leaf to dried stem ratio was calculated by divided the dried leaf to dried stem weight.
Aboveground biomass (t/ha): All plants in the central rows of each plot were harvested and aboveground biomass per net plot was estimated. The aboveground biomass per hectare was estimated from the aboveground biomass per net plot to net area of the plot.
Fresh leaf yield (t/ha): All plants in the central rows of each plot were harvested and fresh leaf yield per net plot was estimated. The fresh leaf yield per hectare was estimated from the fresh leaf yield per net plot to net area of the plot.
Dry leaf yield (t/ha): Dry leaf yield per hectare was obtained from the harvested plot and converted in to yield/ha. All plants in the central rows of each plot were harvested and dry leaf yield per plot was estimated by taking composite sample of the leaves and dried in hot oven at 68°C for 72 h until constant weight was reached. The dry leaf yield per hectare was estimated by dividing the dry leaf yield per net plot to net area of the plot.
Dry stem yield (t/ha): Dry stem yield per hectare was obtained from the harvestable plot and converted in to yield/ha. All plants in the central rows of each plot were harvested and dry stem yield per plot was estimated by taking composite sample of the leaves and dried in hot oven at 68°C for 72 h until constant weight was reached. The dry stem yield per hectare was estimated from the dry stem yield per net plot to net area of the plot. Harvest index: Harvest index (dimensionless) is a ratio of the marketable oil yield (kg/ha) on dry weight basis to the harvested dry hay yield (aboveground dry biomass yield (kg/ha)) (Okwany, 2011). It is an indication of the oil concentration in the harvested green rose-scented geranium hay.
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Essential oil content (%): Essential oil content was obtained by hydro-distillation, according to the procedure described by Daniel et al. (2008). For this experiment, dry leaves of rose-scented geranium were placed in round bottom flask and subjected to hydro-distillation in a Clevenger apparatus (Appendix figure 1).
Harvested plants were separated into leaf and stem then according to Guenther (1972); dry leaves having biomass of 300 g/composite sample was charged in the Clevenger apparatus along with 700 ml of water and trapped for 3 h. Water was poured in to the flask until the plant part submersed completely. The round bottom flask was placed on the boiling heating mantle and the water and plant sample were allowed to boil for 3 h and the essential oil were collected and measured by using pipette reading. The percentage of essential oil content was determined according to the following formula (Rao et al., 2005).
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Essential oil yield (kg/ha): The volume of the oil collected in the collecting tube of the apparatus, dehydrated, measured and expressed on weight by weight (%w/w) dry basis. Then According to Badawy et al. (2009) the essential oil yield/ha was determined by the following formula.
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3.5. Statistical Analysis
Data were subjected to analysis of variance (ANOVA) using General Linear Model (GLM), statistical analysis software program (SAS inst., 2004). The Tukey's Studentized Range (HSD) Test was used to compare the mean separations at 5% probability level and for the interpretation of the correlation coefficient analysis.
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4. RESULTS AND DISCUSSION 4.1. Growth Parameters of Rose-scented Geranium
4.1.1. Plant height
The results from the analysis of variance revealed that interaction effect of plant spacing and harvesting age had no significant effect on plant height. However, the effect of plant spacing was highly significant (P
