Evaluation of the Agronomic Performance of Vernonia (Vernonia galamensis) Germplasm

Vernonia [Vernonia galamensis (Cass.) Less.; 2n = 18] is a relatively new crop in many parts of Africa. Most of Vernonia species occur in South America but more than 300 species from Africa have been described with most occurring in Ethiopia and Madagascar. Apart from these two countries, vernonia is also grown in Cape Verde, Eritrea, Mozambique, northern Tanzania and Senegal (Fig. 1). The greatest diversity of vernonia is found in east Africa while a single variety occurs in West Africa. The genus vernonia comprises of more than a thousand species which vary from annual herbs and shrubs to perennial trees (Baye et al., 2001). There are six major subspecies namely afromontana, galamensis, gibbosa, nairobensis, lushotoensis and mutomonesis. Among these, galamensis shows the highest genetic diversity (Gilbert, 1986). It contains four botanical varieties namely australis, ethiopica, galamensis and petitiana (Gilbert, 1986).


Introduction
Vernonia [Vernonia galamensis (Cass.)Less.; 2n = 18] is a relatively new crop in many parts of Africa.Most of Vernonia species occur in South America but more than 300 species from Africa have been described with most occurring in Ethiopia and Madagascar.Apart from these two countries, vernonia is also grown in Cape Verde, Eritrea, Mozambique, northern Tanzania and Senegal (Fig. 1).The greatest diversity of vernonia is found in east Africa while a single variety occurs in West Africa.The genus vernonia comprises of more than a thousand species which vary from annual herbs and shrubs to perennial trees (Baye et al., 2001).There are six major subspecies namely afromontana, galamensis, gibbosa, nairobensis, lushotoensis and mutomonesis.Among these, galamensis shows the highest genetic diversity (Gilbert, 1986).It contains four botanical varieties namely australis, ethiopica, galamensis and petitiana (Gilbert, 1986).
The morphological characters of the Vernonia plant were described comprehensively by Perdue et al., (1986).The authors described Vernonia as herbaceous, usually annual, varying from small ephemerals 20 cm tall with a single flower head to robust rather diffusely branching somewhat shrubby plants which grow up to 5 m tall with many flower heads.The authors also noted that the stems branch only after the first flower head is formed and the inflorescence consists of a terminal flower head with lateral flower heads from the uppermost axils.The leaves alternate and are membraneous, 0.6-5.0cm wide, up to 25 cm long (Perdue et al., 1986).The classification of the species into six subspecies is based on characters of the phyllaries (Perdue et al., 1986).
Vernonia could potentially grow as a seed oil crop in tropical and subtropical environments with frost-free and short-day length for flower initiation and development.For instance, the crop was grown successfully in Zimbabwe where seed yields varied from 1.7 to 2.5 t/ha during 1986 to 1987.Thus far there are no released cultivars of V. galamensis.Development of improved varieties and production technologies are still in the early stages.The present study is based on germplasm collections from Ethiopia, the center of diversity for V. galamensis var.ethiopica.The study identified agronomically promising genetic resources useful in further development of more productive cultivars.Further agronomic and utilization research on V. galamensis needs to continue before it can be fully-established as a new crop.
In the US Vernonia domestication and large-scale production an oilseed crop was limited due to short-day length requirement for flower initiation and development.In these environments, frosts following flowering inhibits complete seed development and maturity.The crop also performs poorly in areas with excessive moisture, poor soil drainage and insufficient length of growing season.Dierig and Thompson (1993) indicated several barriers that limit full domestication, cultivation and production in the United States such as day-neutrality, autofertility, non-dormant seed germination, good seed retention, increased uniformity of seed maturity, and high oil and vernolic acid contents (Dierig and Thompson 1993).
The successful production of the crop requires well-drained and porous soils.In contrast, in poorly-drained soils, terminal growth is severely retarded and can stop before flowering.The upper portion of the plant dies and branches subsequently grow from the base of the plant, but also wither and die without flowering.Soil with intermediate drainage will produce plants that develop a few flower heads, but with low seed yields.Vernonia seed is planted directly into the soil at a depth of 1 to 2 cm and spaced at 60 cm (intra-row) x 60 cm (inter-row).A firm, level and weed free seedbed is necessary for enhancing rapid establishment and good stand.The seed is relatively small and often with poor germination.Therefore deeper planting is discouraged.
Weed control is essential in the early field establishment due to the poor seedling vigor of Vernonia.The weeds are manually controlled and recommendations on chemical weed controls are not available.No herbicides are currently registered for use in Vernonia.In our studies Vernonia has no major serious diseases and insect pests threatening the crop.Vernionia heads should be harvested when the plants show complete leaf senescence.The seed stays on the heads of the plants for 30 to 45 days after ripening.Immediately after harvesting, the heads are threshed manually in order to obtain the seed.
Although vernonia is cultivated in many tropical countries, the full potential of the crop as an oilseed is yet to be exploited in Africa and elsewhere.It is potentially a useful industrial oil seed crop for the production of natural epoxy oil (Thompson et al., 1994a;Mohamed et al., 1999).The seeds of vernonia produce naturally exposidized oil consisting of vernolic acid, palmitic acid, linoleic acid, arachidic acid, linolenic acid and steraic acid (Carlson et al., 1981;Ayorinde et al., 1988).Vernolic acid is the dominant fatty acid.Because of the production of the naturally exposidized oil, the industrialized processing of this oil is inexpensive.It is also friendly to the environment largely because it does not emit volatile organic compounds.In addition, the oil can be stored at sub-zero temperatures.
Vernonia oil is used in a variety of ways in the chemical industry.The seed of this crop produces useful natural epoxy fatty acids that are better than artificial epoxy oils.In addition, the vernonia oil from the seed contains a wide range of fatty acids such as vernolic acid, linoleic acid, oleic acid, palmitic acid and stearic acid which have industrial uses.The oil is useful in the manufacture of polyvinylchloride and structural polymers for the production of plastic materials (Mebrahtu et al., 2009) and petrochemicals.The cake formed after oil extraction is high (43.75%) in crude protein and is suitable for animal feed.
In spite of its multiple uses, the cultivation and commercialization of vernonia is limited by several factors.Firstly, the seed of vernonia does not attain maturity uniformly.Secondly, the pods shatter easily at maturity leading to significant yield loss. Thirdly, the vernonia plant is generally tall.To date, the crop has not been adapted to mechanized harvesting, seed threshing and cleaning.The harvesting and seed processing are carried out manually.
Due to the high oil and vernolic acid content and its relatively low shattering nature, subsp.galamensis var.ethiopica has been the major focal point of research aimed at domestication and commercialization of the crop.Viable production of vernonia as an alternative industrial crop in marginal tropical and subtropical areas by smallholder or commercial farmers hinges on the identification of suitable varieties that are adapted to the prevalent cropping systems.Alternatively, improved cultivars that are high yielding could also be adopted by farmers.However, there is a dearth of information on the agronomic performance of the current germplasm of vernonia.There are no improved cultivars adapted to the dryland tropical areas in southern Africa such as the Limpopo Province (South Africa).In such areas, vernonia could provide a source of raw materials for agro-processing industries.In addition, vernonia cultivation could provide a significant diversification of the existing cropping systems in the region.In the Limpopo Province of South Africa for instance, the climate is semi-arid and characterized by low mean annual rainfall (300 to 600 mm) with a predominantly sandy-loam soil with reduced fertility (Thomas, 2003).The rainfall pattern is highly variable in some ecotopes in the area (Mzezewa et al., 2010).These harsh agro-ecological conditions could be suitable for the domestication of vernonia and detailed investigations in the agronomic performance of the species would be necessary before large-scale production of the crop in the area is recommended to growers.Therefore objectives of this Chapter are three-fold.The first section focuses on the evaluation of the agronomic performance of V. galamensis var.ethiopica that was conducted in the in Limpopo Province of South Africa.The second section focuses on the selection of germplasm for high-quality and high-quantity oil that may be used further in strategic breeding of the crop aimed at developing an alternative industrial oil crop in the region and similar environments.The third segment of the Chapter examines the implications and recommendations for cropping systems particularly in the dry land areas in southern Africa.

Agronomic performance of Vernonia germplasm
In this field evaluation, 36 accessions of Vernonia galamensis subsp.galamensis var.ethiopica (Table 1) were obtained from the Biodiversity Institute (Ethiopia).The eastern and southeastern region of Ethiopia is considered as a natural habitat for V. galamensis subsp.galamensis var.ethiopica (Gilbert, 1986) in which this botanical variety is believed to be most diverse.The habitat is located at an altitude of 700 to 2400 m and receives little seasonal rainfall (about 200 mm).The soils are poor (infertile).Prior to the study, the homozygosity of each accession was maintained through three cycles of selections (Shimelis et al., 2008).In this study, the field experiments were established in Limpopo Province of South Africa under rain fed conditions during the 2006, 2007 and 2008 growing seasons.Experiments were established at two localities namely at Syferkuil (23˚ 84' S and 29˚ 71' E) and Gabaza.Syferkuil is situated at an altitude of 1261.6 m above sea level (asl).It has annual maximum temperature ranging from 28-30 °C and receives an average annual rainfall of 468 mm.This site has sandy loam soil, of the Hutton form, Glenrosa family, with the pH ranging from 6 -6.2.While Gabaza (23° 50′ S 30° 10′ E) has an altitude of 1100 m asl with an annual average rainfall of 600 mm.At Gabaza the annual temperature ranges from 15 -37 ºC and with clay-loam soil.In general, soil, climatic, and biological conditions of the two locations varies considerably.However, both locations are not prone to frost and hence suitable representative agro-ecologies conducive for Vernonia production in the Limpopo region.
Each experiment was laid out as a partially balanced lattice design with six incomplete blocks replicated three times.Each block was (21.6 m 2 ) 3 m wide x 7.2 m long and consisted of two rows spaced at 0.6m apart.Within the row, the seeds were planted at 0.6 m from each other.During planting and flower head initiation, fertilizer (12.5% N, 8.3% P, 4.2% K and 0.5% Zn) was split applied manually at a rate of 30 kg ha −1 (Shimelis et al., 2008).In literature, there are no reports on fertilizer response and nutrient requirement in V. galamensis.Preliminary observations in Ethiopia showed that the application of 150 kg/ha nitrogen enhances lodging.Thus only a maintenance amount of 20 kg/ha for N and P each was supplied (Baye et al. 2001).

Agronomic measurements
The agronomic traits that were measured included the duration (in days) to 50% flowering (50%DF), the number of productive primary heads (PPH) per plant, the number of productive secondary heads (PSH) per plant, the thousand seed weight (1000-SW) and seed yield (SY). Measurements for each accession were done on 10 plants that were selected randomly and tagged in each block within a replication.In both seasons (2005 and 2006) the field evaluation was conducted during the summer (January to June) cropping season.
Oil content (OC) (based on dry seed weight) was measured.Total lipid (TL) was extracted from ground seeds following the method of Folch et al. (1957), with a chloroform to methanol ratio of 2:1.An antioxidant, butylated hydroxytoluene, was added at a concentration of 0.001% to the chloroform-methanol mixture.Total extractable fat was determined gravimetrically and expressed as percent fat (%F) (w/w) per 100-g sample.The fatty acid composition was determined after transesterification of the extracted lipid by the addition of tri-methyl sulphonium hydroxide (Butte, 1983) and quantified using the gas chromatograph technique (Shimelis et al., 2008).Because of insignificant variations between the two seasons, the average of the data sets over the two seasons was used for statistical analysis.The data sets for each quantitative character were analysed using the SAS GLM procedure for a fixed model with the SAS software version 9.1.3(SAS 2004) followed by mean separation using Fisher's protected LSD.Phenotypic correlations between agronomic traits, oil content and fatty acids were determined using simple Pearson correlation.
The results of this evaluation showed significant differences among V. galamenisis var.ethiopica accessions for agronomical traits, oil and fatty acids content.The crop thrived at the location indicating its adaptation to the prevailing arid conditions (Fig. 2).Accessions Vge-10 and Vge-19 were relatively early flowering requiring 88 d to attain 50% flowering.In contrast, the accessions Vge-15 and Vge-36 flowered late (>140 d) (data not shown).In terms of plant height, four accessions namely Vge-18, Vge-19, Vge-25 and Vge-30 were relatively short, averaging about 133.6 cm.This indicated that the Vge-19, which was collected from eastern region of Ethiopia (Table 1), could be selected for both earliness and reduced height.In similar studies involving V. galamenisis, Angelini et al. (1997) and Bhardwaj et al., (2000) reported variations in duration to flowering and plant height.In comparison with the other accessions, Vge-19 obtained a high number of PPH (72).On the other hand, accession Vge-11 showed a relatively high number of PSH (35)  Under these agro-ecological conditions at the testing location, some of the germplasm was more productive in terms of the number of mature seed heads than reported by Bhardwaj et al,.(2000) who found twice as many immature seed heads (60 -80) compared to mature seed heads (20 -43) per plant.The seed yield was relatively high in accession, Vge-17, Vge-18, and Vge-19 averaging about 3.0 t ha -1 (Table 2).This was consistent with findings from other researchers working with germplasm of vernonia originating from east Africa (Thompson et al., 1994a;Mohamed et al., 1999;Baye et al., 2001).The duration to flowering as measured by 50%DF showed a poor association with 1000-SW but the number of PPH showed significant positive correlations with both seed yield and the number of productive secondary seed heads suggesting that improved seed yield in this species could be achieved through simultaneous selection of increased number of productive heads.
The oil analysis indicated considerable variation in the seed oil content with accession Vge-4 attaining the (35.86%) (Table 3).The observed variation in oil content in this study concurred with observations from other similar studies (Mohamed et al., 1999;Angelini et al., 1997).The fatty acid profiles showed vernolic acid (VA) content ranging from 72.21 to 77.06% (Table 3).Four accessions from the eastern region of Ethiopia (Vge-6, Vge-8, Vge-9, and Vge-11) and two from the south (Vge-25 and Vge-35) obtained superior yield of vernolic acid.In addition, there was considerable variation in the proportion of individual fatty acids among the accessions.For instance, the stearic acid in accession Vge-33 was about 73% of the palmitic acid but was almost equal (95%) to quantity of stearic acid in the accession (Fig. 3).
In similar studies, VA content was low (Angelini et al., 1997;Mohamed et al., 1999;Bhardwaj et al., 2000).Linoleic acid ranged from 12.05 to 14.73%.The highest oil yield (966.58 kg ha −1 ) was observed for accession Vge-4 which jointly with accession Vge-18 produced good seed yield.A significant positive correlation between 1000-SW and seed oil content was found indicating that the accessions with relatively heavier seed contained increased levels of oil content.However, there were poor associations between oil yield and other traits.
While the information regarding the heritability of these traits in vernonia is fragmentary at best, the variation and association between agronomic traits with seed oil and fatty acids suggested that there is merit in exerting effort aimed at the genetic improvement of these traits in vernonia.This could help in diversifying the existing African cropping systems since vernonia can be used as a cash crop in the production of natural epoxy oils.Industries produce epoxy oils by modification of petrochemicals and epoxidation of oils from seeds of soybean [Glycine max (L.) Merr.] and linseed (Linum ustitatissimum L.).However, artificially epoxidized oil is expensive and contains volatile organic solvents with high emission to the environment during processing and use.The release of volatile organic solvents into the air causes environmental pollution.In addition, the oil is highly viscous and cannot be stored below 0°C.The natural oil from vernonia has unique properties due to its non-volatility and low viscosity.According to Ray (1994), when used as a solvent in alkyd-resin paint, the natural oil becomes part of the dry paint surface due to its reactive dilutant properties, preventing emission of volatile organic solvents.Vernolic acid from plants was first discovered and isolated from V. anthelmintica (L.) Wild., native to India (Gunstone, 1954).This species had excessive seed shattering, which prevented its further development.The collection and subsequent evaluation of V. galamensis was found promising because of the quantity and quality of the seed oil and seed retention (Carlson et al., 1981;Thompson et al., 1994b;Perdue, 1988).The germplasm identified in this evaluation is currently being used in the strategic improvement of vernonia to maximize seed yield and oil content as an alternative crop in the province and similar environments.

Environmental influence on agronomic performance
In its natural habitat, vernonia thrives as a weed under marginal low rainfall and poor soil fertility (Gilbert, 1986).There are no major pests and diseases that limit the production of this crop.Therefore, vernonia is an alternative industrial crop particularly in low input tropical cropping systems such as prevalent in the semi-arid environments in southern Africa.Typically, the marginal areas are inhabited by smallholder growers who have limited resources for crop production.Crop failure due to moisture deficits in these areas is common.However, diversification of the cropping systems often reduces the risks of crop failure.
This component of the study examined the genotype x environment (Helgadóttir and Kristjánsdóttir, 1991; Lin and B inns, 1988) interaction of seed and oil yield in vernonia using 10 selected lines.Field experiments were established as described above (see Section 2).At maturity, both primary and secondary heads were harvested per plot.The seed yield was measured in three replicates over three years and at two locations.The oil content, total lipids and extractable fat content were determined and analysed as described above.
The results showed significant interaction (P≤0.05) between genotype x location for seed yield, seed oil content and oil yield (Table 4).Differential responses of the genotypes for the traits were detected across locations or years.At Gabaza, genotype Vge-18 obtained the highest seed yield (3337 kg/ha) during the 2008 cropping season (Table 5).Similarly, Vge-18 and Vge-17 attained relatively high seed yield at Syferkuil.The presence of genotypic variability and genotype x environment interaction for seed yield in vernonia was reported in other studies (Thompson et al., 1994a;Mohammed et al., 1999;Baye et al., 2001).These authors found variations in seed yield among Vernonia galamensis collections from East Africa that ranged from 60 to 2800 kg/ha.In this study, accession Vge-17 also showed stable performance across locations and years (Table 5).At Gabaza, accession Vge-4 obtained higher yield than at Syferkuil (Table 6).The relatively high oil yield in Vge-18 was attributed partly to the high seed yield.The study found significant variations in seed yield (1990-3337 kg/ha), oil content (25-43%) and oil yield (675-1370 kg/ha) among V. galamensis var.ethiopica selections when tested across two the locations over a three year period under the agro-ecological conditions in Limpopo province (South Africa).indicated that selecting for increased number of primary heads would tend to reduce simultaneously the duration to maturity as well as plant height in contrast with selecting directly for 50%DF and plant height.The analysis also showed that improvement in oil content in vernonia can be obtained through the simultaneous selection of lines that display high seed yield and seed weight.If only genotypic correlations were considered, early flowering, short plant stature and high number of productive heads could be useful selection criteria for achieving high seed yield in V. galamensis var.ethiopica.
There is no adequate information regarding associational studies on the agronomic traits in vernonia.Bhardwaj et al. (2000) reported non-significant correlations among seed yield and oil content while Baye and Becker (2005) reported a positive correlation between seed yield and seed weight in vernonia.In summary, selection for increased number of PPH is recommended as the principal selection criterion for improving seed yield in this species.The selection for 1000-SW and increased seed yield can be regarded as major selection criteria for enhancing oil content in V. galemanesis.

Prospects for genetic improvement of Vernonia
The main findings from the research work done so far suggest that some of the production and commercialization of the species can be addressed through a combination of conventional and molecular breeding approaches.For instance, plant height can be reduced routinely using standard modern plant breeding approaches.Similarly, the dehiscence of the heads can also be improved through breeding.This has been demonstrated in several other field crops (Kadkol et al., 1989;Morgan et al., 2000).Moreover, source of shattering resistance were identified in V. subsp.galamensis var.ethiopica.According to Morgan et al., (1988), in rapeseed, increased shatter resistance is desirable partly because it can delay harvesting in order to allow more even maturing of seeds and decrease the incidence of chlorophyll contamination from immature seeds in extracted oil.The variability in the traits of economic interest such as seed yield and oil yield of vernonia indicated that there is potential to select for increased levels of these traits.In other species, oil yield was manipulated through plant breeding approaches (Cahoon. 2003;Cahoon et al., 2007;Wittkop et al., 2009).
The various modern molecular tools that are applied in plant breeding (Li et  ) also offer exciting prospects for the genetic improvement of vernonia.While the development and use of transgenic field crops for food remains controversial in many parts of the world partly because of the perceived risks on human and animal health, transgenic cultivars of vernonia that are high yielding in industrial grade oils should probably find some support even among the critics of the technology.Shimelis et al., (2006) showed the feasibility of raising vernonia in a sheltered environment such as a greenhouse where, to all intents and purposes, undesirable gene flow (for instance from transgenic vernonia) can be prevented.High value crops, particularly horticultural and ornamental species, are routinely produced in controlled environments in many parts of the world.
The demand in the petro-chemical industries for the high quality of oils from this crop is likely to attract capital investment into the marketing and commercial production of vernonia in the marginal areas in Africa.Value addition industries for vernonia such as those for producing bio-based chemicals (Hatti-Kaul et al., 2007) in the region, would be useful for economic development.
In conclusion, we believe that there is merit in investing in the genetic enhancement of vernonia in Africa since the oils from the seed of this crop have numerous advantages over comparative by-products from other crops.There is ample evidence that the crop is adapted to the harsh agro-ecological conditions prevalent in Limpopo Province and beyond.Likely, improved cultivars that are non-shattering, high yielding and mature uniformly will be adopted more widely by growers in the region.

Figure 1 .
Figure 1.The general distribution of V. galamensis.a = West Africa region (Extending from Guinea through Ivory Coast, Mali, Bukino Faso, Ghana to Nigeria) b = Sudan, Kenya and Tanzania region; c = central-southern Ethiopia; d = 4 Malawi, Mozambique and eastern Zimbababwe region).(Source: Adapted from Perdue et al., 1986).

Table 3 .
Mean response of oil content and fatty acid composition (%) among the best 10 (based on seed oil) of vernonia that were evaluated during 2005 and 2006 in Limpopo Province (South Africa).(SO = seed oil; VA = Vernolic acid; VA = Linoleic acid; OA = oleic acid).

Figure 3 .
Figure 3. Palmitic and stearic acid levels among selected 10 accessions of vernonia that were evaluated during 2005 and 2006 in Limpopo Province (South Africa).

Table 2 .
per plant (Table2).Agronomic performance of the best 10 (based on seed yield) vernonia accessions that were evaluated for six traits during 2005 and 2006 in Limpopo Province (South Africa).(50%DF = number of days to 50% flowering; PHT = plant height; PPH = number of primary productive heads; PSH = number of secondary productive heads; 1000-SW = one thousand seed weight; SY = seed yield).

Table 4 .
Mean squares for seed yield, of oil and oil yield among 10 selected Vernonia accessions evaluated over three cropping seasons in in Limpopo Province (South Africa).

Table 5 .
Mean seed yield among 10 selected vernonia accessions that were evaluated over three cropping seasons at Gabaza and Syferkuil in Limpopo Province (South Africa).