Germplasm development of Vernonia galamensis as a new industrial oilseed crop

INDUSTRIALCROPS ANDPRODUCTS AN INTERNATIONAL

ELSEVIER

JOURNAL

Industrial Crops and Products 3 (1994) 185-200

Germplasm development of knonia galamensis as a new industrial oilseed crop A.E. Thompson a,*, D.A. Dierig a, E.R. Johnson a, G.H. Dahlquist a, R. Kleiman b a USDA, Agricultural Research Service, U.S. Water Conservation Laboratory, Phoenix, AZ 8.5040, USA b USDA, Agricultuml Research Service, National Center for Agricultuml Utilization Research, Peoria, IL 61604, USA Received 27 April 1994; accepted 13 June 1994

Abstract Genetic transfer of the day-neutral flowering habit found in Emonia galamensis ssp. galamensis var. petitiana [A 20295 (V 029)] has been successfully accomplished by intraspecific hybridization. Crosses were made in 1990-91 utilizing var. petitiana as the female parent and five other accessions from the V. galamensis complex as male parents. The F1 progenies of the crosses were grown in the greenhouse in 1991-92. Single plant selections were made within Fz and F3 field-grown populations in 1992 and 1993, respectively, in Arizona and various locations throughout the continental United States. Seed increase of selected F3 plants was made in Puerto Rico in 1993-94 for wide-scale evaluation, further selection, and initiation of agronomic crop production research. Selection for rapid germination in the seedling stage in Fz and F3 populations was effective in minimizing seed dormancy. Early germination, nine days after planting, increased from 10.9 f 1.6% for the Fz’s, to 34.3 f 1.8% for the Fj’s. Among the 59 Fz crosses grown under long-day conditions in the field in 1992, the percentage of Fz plants flowering ranged from 31% to lOO%, with a mean of 77.3 f 2.5%. Flowering percentage of F3 progenies from within 38 crosses increased to 94.2 f 1.6% with a range of 50% to 100%. Mean seed weights of greenhouse-grown S’s was 5.73 f 0.12 g/1000. Field-grown Fz’s and F~‘s had smaller seed weights, 2.50 f 0.05 and 2.11 f 0.06 g/1000, respectively. The FI’S also had higher seed oil and vernolic acid contents (39.8 f 0.4% and 80.8 f 0.4%) as compared to the F2’s (33.2 f 0.4% and 62.0 f 1.0%) and the F~‘s (32.1 f 0.6% and 64.9 f 0.8%). The relatively wide range in seed weight, and in oil and venlolic acid contents in both Fz’s and Fj’s demonstrate that directed selection for these yield factors should be effective. High genotype x environment interaction suggests that plant selection and evaluation should be conducted over a range of geographic and climatic conditions within the temperate zone to identify the most favorable production sites. The rapid progress made within the past five years indicates that commercialization of vemonia as a new industrial oilseed could be a reality within the next seven to ten years. Keywords:

Epoxy fatty acid; Natural epoxidized oil; Industrial oilseed; Domestication;

1. Introduction Substantial quantities compounds are utilized * Corresponding

author

Elsevier Science B.V. SSDI 0926-6690(94)00027-V

of epoxidized chemical by industry for manufac-

New Crops; Commercialization

turing adhesives, plastics, paints, and other coatings. The occurrence of natural epoxy fatty acids, which are found in the seed oils of a limited number of plant species world-wide, has stimulated industrial interest. Attention has recently been focused on the domestication and commer-

186

A.E. Thompson et al. I IndustrialCrops and Products3 (1994) 185-200

cialization of Kv-nonia galamensis (Cass.) Less. as a new industrial oilseed crop for the production of epoxidized oil and the associated 18 : 1 epoxy fatty acid, vemolic acid (cis-12,13-epoxycz&octadecenoic acid). However, most currently available germplasm of this species, which is primarily native to equatorial Africa, is not adapted for culture in the temperate zone since it requires exposure to short daylength for initiation of flowering and subsequent seed development. Vernolic acid was first discovered in the seed oil of Ernoniu anthelmintica, which is native to India and Pakistan (Gunstone, 1954). The plant chemical screening program of the USDA-Agricultural Research Service (ARS) National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois also identified and characterized the seed oil and vernolic acid contents of this species (Smith et al., 1959; Earle et al., 1960). The agronomic and chemical utilization research initiated in the 1960’s was reviewed by Perdue et al. (1986). V. anthelmintica flowered readily and produced seeds during the long daylengths of the growing season in various locations throughout the temperate zone in the United States. Unfortunately, lack of seed retention and other agronomic deficiencies contributed to the termination of domestication and commercialization efforts with this species. In 1964, Dr. Robert E. Perdue, Jr. discovered and collected an annual species of %noniu with good seed retention in a semi-arid area near Harar, Ethiopia. This plant, initially classified taxonomically as V. paucijba (Willd.) Less. is now identified as Vemonia galamensis ssp. galamensis var. ethiopica M. Gilbert (Perdue et al., 1986; Perdue 1988). Earle (1970) reported that this accession contained about 42% oil and 73% vernolic acid, substantially higher than the best selections of V. anthelmintica. Subsequent plant exploration efforts throughout Africa resulted in the collection of an array of subspecies and varieties of the I/emonia galamensis complex. Utilization research was initiated by USDAARS at the NCAUR on the original collection of V. galamends from Ethiopia (Carlson et al., 1981; Carlson and Chang, 1985). Research activities of Dr. EO. Ayorinde and coworkers at the Depart-

ment of Chemistry, Howard University, Washington, D.C., and Dr. S.K. Dirlikov and coworkers at the Coatings Research Institute, Eastern Michigan University, Ypsilanti, Michigan (references cited by Thompson et al., 1994a, b), which further demonstrated the feasibility of new uses for and commercialization of vernonia oil, have significantly heightened industrial interest. The V. galamensis collection, which now numbers 37 accessions representing the six subspecies in the complex, is now maintained in the USDA-ARS Working Vernonia Germplasm Collection at the U.S. Water Conservation Laboratory (USWCL), Phoenix, Arizona, and in the North Central Regional Plant Introduction Station, Ames, Iowa (Thompson et al., 1992). This collection has been characterized recently for oil and fatty acid contents, and flowering response (Thompson et al., 1994a, b). Additionally, over 25 other species of Wmonia have been collected recently throughout Africa, and are currently undergoing chemical evaluation at the NCAUR. In 1989, research was initiated at the USWCL to evaluate Vemoniu gdamensis as a potential new industrial oilseed crop for the commercial production of epoxidized fatty acids. Initial studies were focused on germplasm evaluation and enhancement. A significant finding was the identification of day-neutral germplasm in one accession of V. galamensis ssp. galamensis var. petitiana [A 20295 (V 029)], which flowered freely and produced seeds during the normal growing season throughout the United States (Carlson et al., 1992; Thompson et al., 1992, 1994a, b; Dierig and Thompson, 1993). Unfortunately, our evaluation demonstrated that var. petitiuna lacked important agronomic characteristics, which limited its usefulness in its present form. In late 1990, research was initiated at the USWCL to determine the feasibility of genetically recombining the day-neutral flowering response of var. petitiana with the desirable plant growth characteristics of var. ethiopica and other accessions of V. galamensis. Emasculation of individual perfectflowered florets in the capitula or seed heads of the composite is very difficult and time-consuming, and markedly reduces the number of hybrid seeds expected per pollination. Previous observation of

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29

68 358

288 24 17 28 1

(no.1

(no.1 52 4 7 4 1

plants grown

crosses made

Ft population

ga1amensi.v

59

43 4 7 4 1

(no.1

crosses

plants grown

F2 population

2074

1503 99 75 353 44

(no.)

plants

38

25 3 5 4 1

(no.1

crosses

62

43 5 5 8 1

(no.1

plants

plants selected

105

72 6 12 14 1

(no.1

selections b

plants grown

F3 population

2185

1551 128 237 257 12

(no.)

plants

23

16 2 2 2 1

(no.1

crosses

56

42 3 3 7 1

(no.)

plants

plants selected

intraspecific hybrids evaluated in Arizona in 1991-92, 1992, and 1993, respectively

a Refer to Table 1 for identification of parental lines and intraspecific Ft hybrids. b A total of 62 selected in Arizona, 28 in lbxas, 11 in Oregon, and 4 in Iowa. c Five additional “P” female parental plants not utilized in crosses with “E” were used in crosses with other male parents.

3oc

Total

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(no.1

(no.1

25 4 5 4 1

male

female

Plants used in crosses

PxE P x G1 P x G2 PXU PxM

designation a

cross

lhble 2 Composition of Fr, F2, and F3 populations of Vemonia

A.E. Thompson et aL I IndustrialCrops and Products3 (1994) 185-200

189

Fig. 1. Female parent Vemonia galamensisssp. galamensisvar. petitiana [A 20295 (V 029)], left hvo heads; F1 intraspecific hybrid, center two heads; and male parent K galamensisssp. galamensisvar. ethiopica [PI 312852 (V OOl)], right hvo heads. Note relative size difference in seed heads, and lack of seed set in var. petitiana.Seeds of F1 hybrid are at a less mature stage than those of the var. ethiopica parent.

and seed production (Fig. 1). The number of seed heads/plant and seeds/head were measured on 60 plants of the six parental lines, and from the 139 individual Fi plants. Seed weight/plant and lOOOseed weight were also determined for the 139 Fi plants. Seed weights (g/1000) were made from duplicate samples of the six parental lines. One hundred seeds of each of the 139 selected Fi’s and each of the six parental lines were planted in the greenhouse on 23 March, 1992 in seed flats containing a standard potting soil mix. The daylength at this time was about 12 h and 22 min. Seedling germination or emergence of the F2 and parental plants was recorded daily. The percentage of germination by nine days after planting (DAP) was designated as early, and after 39 DAP as total. Seedling plants were planted into trays for growth in the greenhouse prior to transplanting into the field. On 1 May, 1992, 39 DAP,

over 2100 F2 and 230 parental plants were transplanted into the field at the University of Arizona Maricopa Agricultural Center (MAC). The number of F2 populations from any one cross varied from one to nine, and the number of individual Fz plants per cross ranged from only one to 60. Those F2’s having at least 20 plants were divided into two lots and planted in duplicate plots. Plants were spaced 45 cm apart in a single row on a 1 m wide, raised planting bed. Plants were furrow irrigated as needed with no supplemental nutrients. F2 seeds of 34 hybrids were selected and distributed to cooperating federal, state, and industry scientists for evaluation and selection at nine locations throughout the United States. Seeds of the parental lines were also distributed to serve as a control. Information in regard to six of these locations were given by Thompson et al. (1994b).

190

A.E. Thompson et al. llndustrial Crops and Products3 (1994) 185-200

Seeds were planted and grown at the various locations following locally accepted cultural practices. Forty-three single plant FZ selections were made at three of the locations (Iowa 4, Oregon 11, and Texas 28), and aliquots of the seeds were returned to the USWCL for oil and fatty acid analysis, and further progeny evaluation. Plants grown in Arizona were observed periodically for evidence of visible flower bud formation up until 12 July or 115 DAI? Plants flowering by 20 May, 1992 or 58 DAP were classified as early. The percentages of plants flowering both early and in total were recorded. Mean days to flower were calculated by multiplying the appropriate number of DAP by the numbers of plants that had been recorded as flowering at 48, 58, 70, 85, 99, and 115 DAP. The sum of these six dates was then divided by the number of flowering plants in each F2 population. In addition to earliness of flowering, plants were periodically observed for other desirable growth characteristics such as determinate flowering and growth habit, numbers of flowering seed heads, size of seed heads, concentration of flowering and seed set, seed retention at maturity, plant vigor, and tolerance to soilborne diseases. Sixty-two single plant Fz selections were made throughout the populations in Arizona for favorable combinations of these desirable characteristics, and seeds were harvested on an individual plant basis. In 1993, F3 populations from the 105 (62 + 43) F2 single plant selections from 38 crosses were grown for further evaluation and selection in the field at MAC, utilizing essentially the same methods as for the 1992 planting (Table 2). One hundred seeds from each of the F2 selections were planted in flats on 16 March, 1993. The daylength at this time was about 12 h and 8 min. The percentage of early germination was recorded at eight DAP, and total germination after 20 DAI? Twenty-four seedling plants (12 + 12) of each selection were planted into trays for subsequent transplanting into the field in duplicate plots. On 12 May, 1993, about 2300 F3 and 50 parental plants, plus F2 plants for use as border rows, were transplanted into the field at MAC, utilizing the same planting methods, spacing, and cultural procedures as in the 1992 F2 planting.

Seeds of 23 Fs populations derived from selections from 17 crosses made in Arizona were distributed to cooperators in nine geographic locations throughout the country. As with the Fz’s, these populations were grown at the various locations following their locally accepted cultural practices. Early flowering data were collected on 8 June, 1993 (84 DAP), while total flowering response was recorded on 15 June (91 DAP), 29 June (105 DAP), and 12 August (118 DAP). Percentages of early and total flowering and mean days to flower were calculated by the same method as for the previous year. Individual plants were observed and rated for desirable recombinations of agronomic characteristics. Fifty-six Fs single plant selections were made from among 23 crosses (Table 2). Where adequate amounts of Fi, F2, and Fs seeds of individual crosses and selections were available, they were sent to NCAUR for oil and fatty acid analysis. This included seeds from 49 F1 plants from among 31 crosses; 85 F2 plants (including 11 from Oregon, 15 from Texas, and 1 from Iowa) from among 36 crosses; and 51 Fs plants from 22 crosses. Seed oil contents were determined by pulsed NMR, and fatty acid compositions by trans-esterification of the seed oil and GC of the resulting esters. Specific methods were the same as those previously reported (Thompson et al., 1994a). 3. Results and discussion 3.1. Seedset of parental lines and hybrids Mean number of seed heads/plant of the parental lines grown in the greenhouse varied from about 3 to 27 (Table 3). Var. petitiana, the female parental line in the five intraspecific hybrids, had the highest number, which was essentially the same as the two var. galamensis parental lines. Var. ethiopica was intermediate in seed head numbers. Numbers of seeds/head among the six parental lines varied greatly. As previously indicated, var. petitiana flowers are essentially autosterile, and none of the observed plants contained more than one seed/head (Fig. 1). The two accessions of var. galamensis and the accession of ssp. mutomoensis

A.E. Thompson et al. I Industrial Crops and Products 3 (1994) 185-200

191

‘Ihble 3 Flowering and seed set on greenhouse-grown, open pollinated parental and Ft hybrid plants a, utilizing the day-neutral, autosterile timonia galamensis ssp. galamensis var. petit&a [A 20295(V 029)] as the female parent in intraspecific crosses with five other varieties or subspecies of the complex (fall and winter months, 1991-92, Phoenix, Arizona) Parental line plant performance

P (n = 9)b

E (n = 20)

GI

‘32

(n = 4)

(n = 10)

U (n = 12)

M (n =5)

Parental lines l Seed heads/plant:

Mean (?i) k SE. Range

27 f 6 5-63

13 f 2 l-22

26 zt 6 14-44

25 f 6 9-17

6zk2 l-25

3 f 0.4 2-6

Number of seeds/head:

Mean (9 f SE. Range

0.1 * 0.1 o-1

80.0 f 4.1 40-112

0.2 f 0.1 o-1

6.2 zk 2.3 O-19

4.42

4.44

15.4 f 3.3 2-43

0.6 f 0.6 o-3

Seed weight (g/1000): c

Mean (n = 2) Ft hybrid plant performance

4.96 PxE (n = 87) b

5.36 P x G1 (n = 15)

P x G2 (n = 14)

PXU (n = 22)

6.30

2.73

PxM (n = 1)

‘ljpes of intraspecifric F1 hybrids 1 Seed heaaWplant:

Mean (Z) * SE. Range

17 f 5 17-171

66f 11 18-187

77 f 12 20-205

62 f 5 2-201

86

Number of seeds/head:

Mean (Y) SE. Range

27.4 f 1.5 1.1-62.8

2.9 f 0.6 0.6-9.4

5.7 f 1.1 0.7-13.9

17.3 f 3.7 0.5-50.8

3.5 -

9.7 & 0.6 0.7-29.2

0.8 f 0.1

0.3-1.7

2.1 f 0.4 0.3-5.2

5.8 f 1.2 0.2-14.7

2.5 -

5.44 zk 0.10 2.51-7.69

5.62 f 0.22 4.48-7.61

5.73 f 0.20 4.38-6.73

6.28 f 0.27 3.34-8.61

8.30 -

Seed weight/plant (g):

Mean (Z) f SE. Range Seed weight (g/1000):

Mean (Y) f SE. Range

a Refer to Table 1 for identification of parental lines and intraspecific Ft hybrids. b n = number of plants evaluated. c Seeds from self and interplant pollinations within parental lines. Mean of two samples.

were also essentially autosterile. However, some plants within one accession (Gz) of var. gulamensis appeared to exhibit some degree of autofertility. Individual plants of the taxonomically undetermined variety of ssp. galamensis appear to be segregating for autofertility, since some plants had seed heads that contained as many as 43 viable seeds. Seed weights (g/1000) were determined from either self-pollinated or from interplant-pollinated parental plants grown in the greenhouse (Table 3). The mean seed weight of the greenhouse-grown seed of the six parental lines was nearly two grams heavier than that previously reported for 1989

field-grown seed of these six parental lines: 4.70 g vs. 2.73 g (Thompson et al., 1994a). The number of seed heads/plant of the generally more vigorous Fi plants varied little among the five intraspecific hybrids, and each had nearly three times the number of seed heads produced by the parental plants (Table 3). All Fi plants produced some seeds without hand pollination. However, the numbers of seeds/head among the five hybrids varied markedly. The maternal parents that exhibited autofertility, appeared to have conferred some degree of autofertility to the two Fi hybrids (P x E and P x U). The increased number of seeds/head of P x E and P x U

192

A.E. Thompson et aL /Industrial Crops and Products 3 (1994) 185-200

also translated into heavier seed weights/plant for these two first-generation hybrids. Some differences were noted in lOOO-seed weight among the five hybrids (Table 3). The magnitude of the differences among the means of the five hybrids was measured with a t-test. Only the difference between the means of P x E vs. P x U, 0.84 g/1000 seeds, was statistically significant [t = 3.59 with 107 (86 + 21) degrees of freedom, with P > 0.011. Disregarding the seed weights of var. mutomoensis and its one hybrid, the mean seed weights of the four hybrids were about 0.7 g heavier than that of the five parental lines. This increase in seed size along with increased seed heads/plant, and generally more vigorous growth of the Ft plants is attributed to heterosis. A large range in seed weight was observed among the individual hybrid plants - from about 2.5 g to over 8.5 g/1000 (Table 3). Estimation of the heritable component of this variation is not possible from these data. However, it is reasonable to assume that seed weight should be negatively correlated with numbers of seeds/head. The calculated correlation coefficient of r = -0.136 for the total population of 139 plants was not significant. Only within the 22 plants of the hybrid population, P x U, was the correlation (r = -0.799) significant with P > 0.01. Part of the lack of association may be attributed to the apparent segregation of seed head size in the Fi population. The range in size of seed heads of the Fi plants was nearly as large as that of the smallest sized parent (P = & 1 cm) to the largest sized parent (E = f 2.5 cm). Not only were there differences in seed head size among the various types of crosses, there were also marked differences among individual crosses within a type of cross, and among the different individual plants within a given cross. Size of the individual seed head is an important economic character, and selection is being practised within the segregating generations to obtain large seed heads with good mature seed retention. 3.2. Seed germination and dormancy of F2 and F3 populations With most wild species, seed dormancy is a problem that must be overcome before it can be

successfully developed as a new crop. Vernonia is no exception to the general rule, but the level of dormancy in most of the available germplasm has not been seen as a serious problem. Previous observations had indicated that accessions of var. galamensis tended to have the highest rate of dormancy and poor seed germination. Germination or emergence rates of F2 seeds of the 139 individual Fi’s from 59 different crosses were determined by planting in seed flats in the greenhouse (Table 4). Our original intent was to select and transplant into the field for further evaluation and selection only those seedlings that exhibited early germination. It was reasoned that such selection pressure would tend to eliminate those plants carrying genetic factors for seed dormancy. Accumulated seedling germination up to nine days after planting (DAP) was observed and recorded as “early” germination. However, less than 11% of the seeds in the total population had germinated early, and some crosses (P x Gt and P x G2) had germination rates as low as only 1 to 2%. A very large variation in germination percentage and rate among the various plant progenies was also observed, ranging from 0% to 57%. In contrast, seeds of four of the six parental lines that served as a control in 1992, had a much higher rate of early germination - from 58% to 70% (Table 4). The two var. galamensis accessions, Gt and G2, had a low rate of early germination. Undoubtedly, dormancy is under genetic control since the two crosses in which these parental accessions were involved, also had low early germination. Since the low rate of early germination was limiting the number of seedlings we wished to transplant into the field for further evaluation, we continued to observe germination for a total of 39 DAP Even after this longer germination period, we recovered and transplanted seedlings into the field from only about 16% of the seeds planted. The range in germination rate of the F1 plants at 39 DAP had extended from 0% to 65%. A concurrent increase in the germination of the parental lines was also exhibited over the period of time from 9 DAP to 39 DAI? However, most of the increase is attributed to the two var. galamensis lines, Gt and especially Gz (Table 4). The overall rate and percentage of germination

A.E. Thompson et al. lIndustria1 Crops and Products 3 (1994) 185-200

Table 4 Early and total seed germination (%) of Fz and F3 intraspecific hybrid populations planted in seedling flats in greenhouse on 3123192and 3/16/93, respectively Crosses a

PxE

F2 F3

P x G1 F2 F3

P x G2 F2 F3

PxU

F2 F3

PxM

F2 F3

Total

Fz F3

Parental lines a 1992 P 1993 1992 E 1993 1992 GI 1992 G2 1992 U 1992 M

and parental lines of Vernonia galamensis

Seed germination (%) number

type

193

mean (?) III SE.

range

early b

total b

early

total

44 25

13.8 rt 2.0 41.3 * 3.9

18.1 f 2.2 56.0 f 3.0

o-57 12-83

O-65 34-83

4 3

2.2 l 0.9 29.7 f 1.5

8.0 f 2.1 43.7 f 4.4

1-5 27-32

4-13 37-52

7 5

0.6 f 0.3 8.4 f 2.3

7.1 f 2.5 24.0 f 5.6

o-2 8-14

2-19 22-38

4 4

6.0 f 1.9 30.5 f 12.7

17.8 f 3.8 43.0 f 14.4

l-10 l-62

10-28 3-72

1 1

10.0 19.0

23.0 21.0

10.9 f 1.6 34.3 f 3.4

16.2 f 1.8 48.5 f 3.1

O-57 O-83

O-65 O-83

58 29 62 37 5 4 61 70

64 57 66 57 10 42 63 71

60 38

a Refer to Table 1 for identification of parental lines and intraspecific Ft hybrids. b FZ (1992) early = 9 days after planting (DAP), total = 39 DAP; F3 (1993) early = 8 DAP, total = 20 DAP.

of the Fs population was considerably higher than that of the Fz: 34.3% vs. 10.9% for early, and 48.5% vs. 16.2% for total germination (Table 4). Early and total germination of individual selections increased to a high of 83%. Considerable variation was again observed among the five types of crosses for both early and total germination. The selections made within hybrid P x G2 continued to have the lowest germination rate. With the possible exception of hybrid P x M, which is represented by only one cross, the heavy selection pressure applied to eliminate dormancy in the F2 populations was influential in minimizing dormancy in the Fs’s. Continued selection for rapid germination in the seedling stage of subsequent breeding populations should eliminate dormancy as a constraint to successful crop production.

3.3. Flowering response of Fz and F3 selections and populations Mean early and total flowering percentage and mean days to flower of the parental lines, and the F2 and F3 populations are compared in Tables 5 and 6, respectively. In 1992, all six of the parental lines were planted with the Fz population to serve as controls. In 1993, only the two parents (P and E) were planted. For both years, var. petitiana (P) clearly demonstrated its ability to flower under long-day conditions. In contrast, the two parental lines, var. ethiopica (E) and the undetermined variety (U) clearly are obligate short-day plants, and none had flowered even after 115 DAP. The other three parental lines exhibited a variable response, and some plants apparently received sufficient ex-

A.E. Thompson et al. IIndustrial Crops and Products 3 (1994) 185-200

194

Table 5 Flowering response of parental lines of the Vemonia galamensis complex used to create five intraspecific hybrids grown as controls for evaluation of Fz and Fs populations, respectively in 1992 and 1993 at Maricopa, Arizona Parental lines a

P E Gl GZ U M

Number of plants

Mean flowering percentage b (%)

1992

early

1993

93 36 10 10 71 12

22 24 0 0 0 0

Mean days to flower

total

1992

1993

1992

1993

100 0 50 0 0 33

100 0

100 0 70 20 0 50

100 0

-

-

1992

1993

58.0

84.0

67.9 99.0

-

71.7

a Refer to Table 1 for identification of parental lines. b Early = 58 and 84 days after planting (DAP), total = 115 and 118 DAP for 1992 and 1993, respectively. Table 6 Flowering response of Fz and Fs populations of intraspecific hybrids involving Vernonia galamensis ssp. galamensis var. petitiana and five other accessions from the Kgalamensb complex, grown respectively in 1992 and 1993 at Maricopa, Arizona Crosses a

Mean days to flower

Mean flowering percentage (%) early b

total b

F2

F3

93.5 +I 2.4 50.0-100

77.7 rt 1.7 58.0-100.4

95.2 f 1.0 84.9-103.9

74.5 f 10.5 50.0-100

96.3 Z!Z1.9 93.8-100

75.5 + 4.3 66.5-87.3

93.6 f 3.4 89.5-100.3

43.3 f 7.7 26.1-66.7

81.1 k 7.9 42.9-100

96.8 f 0.9 94.7-100

78.9 +I 5.2 64.0-100.0

95.4 f 1.8 89.9-99.8

41.0 f 6.5 33.3-60.2

56.6 k 5.1 41.9-64.4

94.1 f 3.1 85.4-100

84.3 ?c 4.4 71.0-89.7

95.4 f 1.0 92.8-97.6

72.7

91.7

80.6

99.3

77.3 k 2.5 30.8-100 25.2 59

94.2 f 1.6 50.0-100 10.7 38

78.2 =k 1.5 58.0-100.4 14.3 59

95.2 f 0.7 84.9-103.9 4.8 38

F2

F3

F2

F3

P x E: Mean (Z) k SE. Range

32.9 f 4.0 O-100

36.5 f 4.1 4.2-87.0

78.9 f 3.0 30.8-100

P x Gt: Mean (Y) +I SE. Range

25.4 f 6.8 13.0-44.4

37.8 f 12.1 14.1-54.2

P x G2: Mean (X) f SE. Range

30.0 f 10.0 O-71.4

P x u: Mean (X) ZIG SE. Range

12.2 f 7.1 3.2-33.3

P x M: Mean (X) Total hybrid population: Mean (X) f SE. Range C.V. (%) Number of crosses

6.8

8.3

30.2 f 3.2 O-100 82.3 59

37.2 f 3.1 4.2-87.0 51.7 38

a Refer to Tables 1 and 2 for identification of crosses, numbers of crosses, and plants involved for each population and generation. b Early = 58 and 84 days after planting (DAP); total = 115 and 118 DAP for 1992 and 1993, respectively.

posure to short daylength in the initiate flowering. It is possible lines may be heterogeneous for ify the timing and expression response.

seedling stage to that these three genes that modof the flowering

Considerable variation in the flowering response of the F2 plants was observed both within and among the five different crosses (Table 6). About 30% of the plants in the total population had flowered by 58 DAF’, but early flowering

A.E. Thompson et al. lIndustria1 Crops and Products 3 (1994) 185-200

of individual crosses ranged from as low as 0% to 100%. The percentage of plants flowering increased to about 77% at 115 DAP, and the range narrowed to about 31% to 100%. Early flowering percentages of Fz plants within crosses P x U and P x M were about 20% lower than those of the other crosses. The total percentage of plants flowering within the P x U crosses was also markedly reduced. Mean days to flower within the total Fz population was about 78, with a range of about 40 days (Table 6). At less than two weeks after transplanting into the field (50 DAP), about 1.1% of the plants were observed to have visible flower buds. Slightly over 22% of the plants in the total population were classified as flowering 10 days later (58 DAP). In addition to a reduced percentage of both early and total flowering, plants within hybrid P x U tended to require a longer time to initiate flowering. Although data were not taken on the number of flowering heads per plant, large variation in flowering intensity was observed. In some instances, plants that flowered early failed to increase in flowering intensity, continued to grow vegetatively, and produced few flowering heads. Flowering intensity and concentration of flower and seed set, along with earliness of flowering, are important factors utilized in making single plant selections. Plants also varied considerably relative to plant height and other plant growth characteristics. About 1% of the plants were classified as having a determinate or semi-determinate flowering and plant growth habit (Fig. 2). This growth characteristic had never been observed within any of the parental accessions, and most likely is conditioned by a recessive gene or genes. Intensity of flowering and seed set also greatly influence the determinate nature of the plant’s growth, and complicate visual classification. Sixty-two single plant Fz selections were made in the Arizona planting (Table 2). This represents about 3% of the total number of 2074 plants in the field. Actually, the selection pressure was much higher since the number of F2 plants in the field represents only about 15% of the total number of seeds planted in the greenhouse. Forty-three additional single plant F2 selections were made by

Fig. 2. Senior author with compact, determinate F2 single plant selection of Vbnonia galamensh ssp. galamensis var. petitiana x V. galamends ssp. galamensti var. ethiopica. Note non-flowering, and wilt-susceptible F2 plants in the background.

our cooperators: 28 in Texas, 11 in Oregon, and 4 in Iowa. Forty-five (72.6%) of the 62 selections made in Arizona had a mean flowering date of 58 days or less. The mean days to flower of all the 62 selections was 63.7. One hundred seeds of each of the 106 Fz selections were planted in the greenhouse in 1993. As indicated previously, the germination rate was significantly higher due to selection against seed dormancy (Table 4). However, the growth rate of the F3 seedlings was noticeably slower than that of the two parental lines (P and E), and some F2 plants grown for use as border rows. We attribute the reduced vigor of the Fs’s to inbreeding depression. Even though we had planted the seeds a

196

A.E. Thompson et al. /Industrial Crops and Products3 (1994) 185-200

week earlier than the Fz’s in 1992 (16 March, 1992 vs. 23 March, 1993), the Fs plants remained 18 days longer in the greenhouse before they could be transplanted into the field on 12 May. To stimulate normal seedling growth before transplanting, daily applications of fertilizer in the form of starter solution were made. Growth rates of plants after transplanting into the field appeared to be comparable to the parental lines and the F2 plants in the border rows. A total of 2185 Fs plants survived transplanting into the field (Table 2). For some reason, the onset of flowering seemed to be delayed, and the fairly high rate of earliness of flowering in the F2 populations was not observed. The delay in floral initiation may also be another manifestation of inbreeding depression, which we believe is responsible for the reduction in seedling growth and vigor. The mean percentage of flowering of the total Fs population at 84 DAP was only about 7% higher than that of the Fz population at 58 DAP (Table 6). In contrast, the mean flowering percentage and standard error of the Fz’s at 85 DAP was 61.3 f 3.2%, nearly 24% higher than the Fs’s at 84 DAP. For comparison in Table 6, the mean flowering percentages of the individual F2 crosses at 85 DAP were: P x E = 62.3 f 3.9%, range 6.7-100%; P x Gi = 62.8 f 9.5%, range 47.8-88.9%; P x G2 = 65.6 f 11.3%, range 28.6-100%; P x U = 41.4 & 5.5%, range 29.055.6%; and P x M = 55.4%. Another indication of the delayed flowering of the F3’s is that the mean days to flower was about 17 days higher than that recorded for the Fz’s: 95.2 vs. 78.2 days. The means of all five of the crosses were very similar to the population mean. However, in this instance, P x U was not significantly later in flowering than the other crosses observed in the F2 generation. Even though the onset of flowering was delayed, there was a significant increase in the total percentage of plants flowering in the Fs generation: 94.2% vs. 77.3% for the Fz’s (Table 6). Relatively small differences were observed among the means of the five types of crosses. Although a range from 50 to 100% was observed for individual crosses; the range of variability in flowering percentage was decreased within the F3 population. This observation is validated by the reduced

coefficient of variation (C.V.): 10.7% vs. 25.2% for the F2. Considerable plant-to-plant variation was observed both within and among the F3 progenies of the 105 F2 selections. The reduction in growth and vigor of the Fs seedlings was not apparent in the mature plant growth in the field. A majority of the 56 F3 single plant selections made were the earliest in flowering (Table 2). Forty-six or 82.1% of the selections had flowered at the first recorded flowering date. The mean days to flower for the 56 selections was 86. An additional number of single plant selections were made at various locations throughout the United States. In general, tolerance of the F3 plants for soilborne diseases was higher than that observed in the F2 population. In 1992, an estimated 40% of the F2 plants were dead or dying at midseason due to infestation of soilborne fungi (Fig. 2). Plant losses due to the wilt disease were estimated to be as high as 70% at the end of the season in early September. In 1993, only about 10 to 15% of the F3 plants succumbed by midseason, and the loss at the end of the season was about 45%. This difference is attributed to rigorous selection for healthy plants in the F2, which was also practised within the F3 population. The parental var. ethiopica had previously manifest its susceptibility to soilborne diseases, especially under conditions of heavy, poorly drained soils. The source of the tolerance to soilborne diseases in the hybrids clearly comes from the var. petitiana parent. Both parents were planted as checks in adjacent plots in 1993. All of the var. ethiopica plants died by midseason, and none of the var. petitiunu plants exhibited any signs of disease infestation even at the end of the season. To date, no definitive research has been conducted to determine the causal agent, although Fu.wium spp., Rhizoctoniu spp., and Phymutotnchum omnivorum have been isolated from diseased plant roots. A clear need exists for more research in this area so that seedling populations can be screened to select for increased tolerance or resistance. Seeds of 51 of the Arizona F3 selections were analyzed for oil and fatty acid contents at NCAUR. Five of the selections had only enough seeds for field planting and evaluation in 1994,

A.E. Thompsonet al. IIndustrial Crops and Products 3 (1994) 185-200

Fig. 3. Coauthor Gail Dahlquist holding high-yielding, uniform-maturity F4 plant grown for seed increase at the USDA-ARS Winter Nursery, Isabella, Puerto Rico.

and were not analyzed. Thirty-four of these selections with the best plant characteristics and seed set were planted in a winter nursery at Isabella, Puerto Rico for seed increase on 5-10 October, 1993. Most of the selections grew very well and a good seed increase was harvested the week of February 14th, 1994 (Fig. 3). Portions of the increased seeds were distributed to our cooperating network for evaluation, further selection, and the initiation of crop production systems research. 3.4. Seed weights, seed oil and vemolic acid contents of Fl, F2 and FJ populations

Seed weights (g/1000) of the Fi, and selected F2 and Fs populations of the five types of intraspecific hybrids are compared in Table 7. The means,

197

standard errors, and ranges of the seed weights, as well as for oil and vemolic acid contents, were calculated from the number of individual plants that came from the indicated number of crosses in each type. The seed weight means and ranges of the five F1 hybrids are comparable to, but not exactly the same as those reported in Table 3 for the full population of the Fl’s. The selected population summarized in Table 7 includes only those Fi’s that gave rise to F2 and subsequent single plant selections. The numbers were further limited in Table 7 to those Ft’s that had sufficient residual seed to permit oil and fatty acid analysis. Seed weights of the Fi’s grown in the greenhouse were over twice as heavy as those of the FZ’S and Fs’s that were from plants grown in the field (Table 7). Part of this difference may be due to heterosis, but a major portion is attributable to environmental factors. Little differences were measured among the means of the five types of crosses in both the F2 and Fs generations. The slight, but consistent reduction in seed weight among the Fs’s, when compared to that of the comparable Fz’s, may be due to inbreeding depression. However, since they were grown in different years, the differences may be totally attributable to environmental effects. The effects of environmental factors on vernonia seed weights have been previously documented (Thompson et al., 1994b). The seed oil and vernolic acid contents of the five F1 hybrid populations were considerably higher than those of the F2’s and Fs’s. The differences in the F2 and Fs population means for seed oil content were relatively small: 33.2 f 0.4% vs. 32.1 f 0.6%, respectively. In contrast, the mean vernolic acid content of the F3 population was slightly larger than that of the F2: 64.9 f 0.8% vs. 62.0 f 1.0%. A portion of these differences can be attributed to environmental factors, which were previously shown to be of significance (Thompson et al., 1994b). Although there are only small differences among the means of the five types of hybrids for oil and vernolic acid contents, there is a wide range among the individual selections. To date, no conscious effort has been made to select for either oil or vernolic acid contents on a single plant basis. The previously observed heterogeneity

314 5.53 l 0.37 4.48-6.13 517 5.66 xk 0.33 4.38-6.71 419 6.09 k 0.42 4.45-7.28 l/l 8.30 36157 5.73 * 0.12 3.70-8.30 16.1

P x Gr: Number of crosses/plants Mean (Z) f S.E. Range

P x G2: Number of crosses/plants Mean (?i) f S.E. Range

P x u: Number of crosses/plants Mean (Z) SE. Range

P x M: Number of crosses/plants Mean

Total hybrid population: Number of crosses/plants Mean (Z) f S.E. Range C.V. (%) 22149 2.12 f 0.06 1.27-3.14 20.2

1.81

36185 2.50 f 0.05 1.58-3.59 18.1

l/l

1.99

2.02 f 0.13 1.42-2.50

217

2.06 f 0.22 1.79-2.50

213

213 2.38 f 0.09 2.25-2.55

15135 2.12 f 0.06 1.27-3.14

F3

l/l

4112 2.46 f 0.13 1.73-3.22

5112 2.80 f 0.12 2.62-3.34

2.61 f 0.09 2.25-2.90

3/6

23154 2.44 f 0.06 1.58-3.59

F2

31149 39.8 f 0.4 29.7-44.7 7.6

l/l

317 37.4 f 1.1 33.3-40.8

314 39.6 zk 1.0 37.2-42.2

l/l 35.3

23136 40.4 f 0.5 29.7-44.7

FI

36185 33.2 f 0.4 23.5-40.8 10.6

l/l 33.5

4112 31.8 f 1.3 23.9-38.0

5112 35.1 f 1.0 28.5-40.8

316 34.9 k 0.5 33.6-36.8

23154 33.0-0.5 23.5-40.4

F2

22149 32.1 f 0.6 21.3-39.7 12.1

l/l 31.9

217 31.2 f 1.8 21.3-36.1

213 32.3 f 1.6 30.2-35.4

213 31.7 f 0.9 20.3-33.2

15135 32.3 f 0.7 24.9-39.7

F3

31149 80.8 f 0.4 73.5-88.0 3.3

l/l

317 80.6 f 1.0 75.1-83.8

314 83.5 f 2.0 78.8-88.0

l/l 82.3

23136 80.5 f 0.4 73.5-86.8

FI

36185 62.0 f 1.0 44.6-82.6 14.8

l/l 48.5

4112 61.4 f 2.9 45.7-78.7

5112 71.3 f 2.5 55.7-82.6

316 62.3 f 2.1 57.2-71.9

23154 60.3 f 1.1 44.6-76.7

F2

18 : 1 epoxy content (%)

a Refer to ‘Ihble 1 for identification of intraspecific Ft hybrids. b Means, standard errors, and ranges were calculated from the number of individual plants that came from the indicated number of crosses in each type.

23136 5.60 rt 0.13 3.70-7.03

FI

Seed oil content (%)

22149 64.9 f 0.8 49.9-74.7 9.0

l/l 64.0

217 63.3 f 2.4 49.9-68.9

213 68.2 f 2.7 63.0-71.9

213 70.4 f 0.7 69.2-71.5

15135 64.5 f 1.0 52.6-74.7

F3

epoxy) contents of Ft, Fz, and F3 populations of Vemonia galamensis intraspecific hybrids grown in 1991-92, 1992, and

Seed weight (g/1000)

PXE: Number of crosses/plants b Mean (?Z) k SE. Range

Crosses a

lhble 7 Seed weight, seed oil and vernolic acid (l&l 1993, respectively

A.E. Thompson et al. lhdustrial

of the parental plants used in making the crosses, and the observed variability among the individual selections would indicate that selection for higher oil and vernolic acid contents should be feasible. Examination of the data on seed weight and vernolic acid contents of the F2 single plant selections made in Oregon and Texas indicated that they were generally higher than those made in Arizona. Data on seed weight and oil and vemolic acid contents of the selections were summarized in regard to location (Table 8). The means of the three locations were statistically compared by

lkble 8 Comparison of seed weight, seed oil and vemolic acid (18 : 1 epoxy) contents of Fz single plant selections from timonia galamensb hybrids, made in four geographic locations Seed weight Seed oil 18:l epoxy content (%) content (%) (g/1000) Oregon (n = 11): Mean (X) f S.E. Range

2.87 k 0.11 2.32-3.52

33.2 f 0.6 28.4-35.6

79.9 f 0.9 70.8-79.1

Texas (n = 15): Mean (sr) IL SE. Range

2.76 f 0.07 2.35-3.35

34.3 zt 1.1 28.5-40.8

70.6 f 1.9 57.1-82.6

Arizona (n = 58): Mean (Y) f S.E. Range

2.37 f 0.06 1.58-3.59

33.1 f 0.5 23.5-39.8

57.0 I!Z0.7 44.6-67.9

Iowa (n = 1): Mean

2.00

25.5

76.7

Comparisons of means: Oregon vs. Texas 0.11 Mean difference 0.13 t

1.1 0.77

4.3 1.82

0.50 3.48**

0.1 0.13

17.9 10.73”

0.39 3.27**

1.2 1.14

13.6 8.09**

Oregon vs. Arizona Mean difference

t Texas vs. Arizona Mean difference

t

Correlation coefficients, total population (n = 85) Seed weight (g/1000) 0.361” 0.477** Seed oil content (%) 0.296** **Mean difference between populations measured by I-test (with 24, 67, and 71 degrees of freedom, respectively), and correlation coefficients with 83 degrees of freedom significant at P = 0.01.

Crops and Products 3 (1994) 185-200

199

means of a t-test. Mean seed weights from the selections made in Oregon and Texas were not different, but both were heavier than the mean of the Arizona selections. No significant differences were measured among the seed oil content means. Again, there were no differences detected between the vernolic acid contents in the Oregon and Texas samples. However, the Arizona selections were significantly lower in vernolic acid than either the Oregon or Texas selections. Whether these differences have any genetic basis has not yet been determined. The question arises whether there is any association of seed weight with either oil or vernolic acid content, or between oil and vernolic acid contents. The total population of 85 Fz’s was utilized to calculate simple correlations (Bble 8). All of the three possible correlations were highly significant, with P = 0.01. However, these correlations have relatively little predictive value. The relationships seed weight/seed oil, seed weight/vemolic acid, and seed oil/vemolic acid, as measured by r2, only account for about 13%, 22%, and 9% of the total observed variation. At this time it is not possible to determine if these differences are under genetic control and amenable to selection, or largely influenced by environmental factors. Research is under way to select specifically for high oil and vernolic acid contents, and to determine the extent of the heritable component of these important yield factors. Since all the parental lines have fairly high and comparable levels of seed oil and vernolic acid (Thompson et al., 1994a), we anticipate that it will not be difficult to select relatively high yielding, commercially useful varieties or hybrids. 4. Conclusions Good progress is being made in converting the germplasm of Emonia galamends to day-neutral flowering by breeding and selection. It would appear that the day-neutral flowering response we have found and utilized in var. petitiana [A 20295 (V 029)] is most probably conditioned by a major dominant gene. However, it is clear that various environmental factors and modifying, quantitative genes also contribute to the remarkable array of

200

A.E. Thompson et al. /Industrial Crops and Products 3 (1994) 185-200

flowering responses. Environmental factors also modify such important yield factors as seed weight, and oil and vernolic acid contents. However, the relatively large ranges of these characteristics in both the F2 and the Fs populations indicate that directed selection for these important yield characteristics should be effective. Other important components of yield such as plant architecture, seed set, and seed retention are also influenced by environment, but also appear to be amenable to selection. Since the flowering response and other important agronomic and plant yield characteristics appear to have a high genotype x environment interaction, it will be necessary to select plants and evaluate them for desirable performance under an array of geographic and climatic conditions to determine the best potential crop production sites. Commercialization of vemonia for production of epoxidized oil is being greatly facilitated by the interest of industry, the support of federal research funds, and the development of an extensive network of cooperating scientists throughout the country. Based upon the rapid progress that has been made within the past five years, we predict that commercialization of vernonia as a new epoxidized oilseed crop could be a reality within the next seven to ten years. Acknowledgements Special recognition and thanks is given to W.W. Roath (USDA-ARS, Ames, Iowa), R.J. Roseberg (Oregon State University, Medford, Oregon) and M.A. Foster (Texas A&M University, Fort Stockton, Texas) for their time and cooperative effort in evaluating performance of the intraspecific hybrids, making single plant selections, and providing seeds for further evaluation and selection. Thanks is also given to Bliss S. Phillips, NCAUR, Peoria, Illinois for oil and fatty acid determinations.

References Carlson, K.D. and Chang, S.P., 1985. Chemical epoxidation of a natural unsaturated epoxy seed oil from l&nonia galamensis and a look at epoxy oil markets. J. Am. Oil Chem. Sot., 62: 934-939. Carlson, K.D., Schneider, W.J., Chang, S.P and Princen, L.H., 1981. I/entonia galamensis seed oil: a new source for epoxy coatings. Am. Oil Chem. Sot. Monogr., 9: 297-318. Carlson, K.D., Knapp, S.A., Thompson, A.E., Brown, J.H. and Jolliff, G.D., 1992. Nature’s abundant variety: new oilseed crops on the horizon. In: New Crops, New Uses, New Markets, 1992 Yearbook of Agriculture. USDA, Washington, D.C., pp. 124-133. Dierig, D.A. and Thompson, A.E., 1993. Vernonia and lesquerella potential for commercialization. In: J. Janick and J.E. Simon (Editors), New Crops. John Wiley, New York, N.Y., pp. 362-367. Earle, ER., 1970. Epoxy oils from plant seeds. J. Am. Oil Chem. Sot., 47: 510-513. Earle, ER., Wolff, I.A. and Jones, Q., 1960. Search for new industrial oils, III. Oils from Compositae. J. Am. Oil Chem. Sot., 37: 254-256. Gunstone, ED., 1954. Fatty acids, Part II. The nature of the oxygenated acids present in Venonia anthelmintica (Willd.) seed oil. J. Chem. Sot. (London), May, pp. 16111616. Perdue, R.E., Jr., 1988. Systematic botany in the development of knonia galamensis as a new industrial oilseed crop for the semi-arid tropics and subtropics. Symb. Bot. Ups., 28(3): 125-135. Perdue, R.E., Jr., Carlson, K.D. and Gilbert, M.G., 1986. Vemonia galamensis, a potential new crop source of epoxy fatty acid. Econ. Bot., 40: 54-68. Smith, C.R., Jr., Koch, K.F. and Wolff, I.A. 1959. Isolation of vernolic acid from Vemonia anthelmintica oil. J. Am. Oil Chem. Sot., 36: 219-220. Thompson, A.E., Dierig, D.A. and White, G.A., 1992. Use of plant introductions to develop new industrial crop cultivars. In: H.L. Shands and L.E. Weisner (Editors), Use of Plant Introductions in Cultivar Development, Part 2. Crop Sci. Sot. Am. Spec. Publ., 20: 9-48. Thompson, A.E., Dierig, D.A. and Kleiman, R., 1994a. Characterization of Vemonia galamensb germplasm for seed oil content, fatty acid composition, seed weight, and chromosome number. Ind. Crops Prod., 2(4): 299-305. Thompson, A.E., Dierig, D.A. and Kleiman, R., 1994b. Variation in Vemonia galamensis flowering characteristics, seed oil, and vernolic acid contents. Ind. Crops Prod., 3(3): 175-183 (this issue).