Genet Resour Crop Evol DOl 1O.IOO7/sl0722-01Q..9538-8

Morphological and reproductive characterization of guar (Cyamopsis tetragonoloba) genetic resources regenerated in Georgia, USA

J. B. Morris

Received: 19 August 20091 Accepted: 25 January 2010 © Springer Science+Business Media B.V. 2010

Abstract Guar, Cyamopsis tetragonoloba is used (clusters) based on low, intermediate or high seed worldwide for food stabilization, fiber source, food, numbers. Guar accessions clustered in groups 2 and and industry. The United States Department of 3 appear to be more genetically related than those in Agriculture, Agricultural Research Service, Plant group 1. Genetic Resources Conservation Unit (USDA, ARS, PGRCU) conserves 1,298 accessions originating Keywords Guar· Cluster analysis . from India, Pakistan, and breeding lines from the Principal component analysis . Variability . USA. My objectives were to determine: (1) if these Dendrogram . Morphology . Reproduction guar accessions can successfully be regenerated in Georgia, USA, (2) which traits contribute the most variation, and (3) to provide estimates for genetic Introduction relatedness among these accessions. Guar accessions were directly seeded to the field in Griffin, GA Guar, Cyamopsis tetragonoloba (L.) Taub. is a self· between early May and mid June 2006-2008. At pollinated diploid (2n 14) annual legume grown 50% maturity, 73 accessions were characterized for during the summer season in India, Pakistan, and the morphological, phenological, and reproductive traits high plains of Texas USA region. Guar flowers are during the regeneration cycles. High quality plants purple to pink, approximately 8 mm long, and form regenerated from most of the accessions and in axillary racemes. The calyx has five unequal linear produced 80 to more than 9,300 seeds per accession. teeth. The standard is orbicular, and the wing petals Guar can be successfully grown and regenerated are oblong, while the keel petals are as long and in Griffin, GA. Coefficients of variation and princi­ broad as the wings. The ten stamens are all fertile, pal component analysis revealed variability among and the filaments form a tube while the anthers are accessions for these traits evaluated. Cluster analy­ apiculate. The pollen grains are circular and 40-43 Jl sis separated guar accessions into three groups in diameter. The stigma expands into a head-shape while, the style is short and slender (Chandrasekbaran and Ramakrishnan 1928; Gillett 1958; Jam 1966; Menon et al. 1968). The primary use is for galacto­ mannan gum (also known as guar gum) which is J. B. Morris ([8]) extracted from guar seed and used as a stabilizer in Plant Genetic Resources Conservation Unit, USDA, ARS, 1109 Experiment St., Griffin, GA 30223, USA ice cream and other frozen deserts (Morris et al. e-rnall: [email protected] 2004). Currently guar gum is used in numerous

Published online: 18 February 2010 ~ Springer Genet Resour Crop Evol

nutraceutical and pharmaceutical additives (Morris row plot with 3--6 m between rows. Seed regenera­ 2004) as well as laxatives, paper, oil well drilling, in tion and characterization can be accomplished with­ the mining industry, processed cheese products, meat out replications for the conservation of guar binder, meat products and pet foods, dressings, gennplasm, based on recommendations of the sauces, and beverages (Whistler and Hymowitz USDA, ARS, NPGS, New and Industrial Crop 1979). Immature guar pods are used as a vegetable Gennplasm Committee (N1CGC). Plots were irri­ and additional uses include forage, cover crop, and as gated with sprinklers as necessary. Plant height and a green manure crop. Guar grows best in a well pod length were recorded in dm and mm, respec­ drained soil including sandy, sandy loam, and coarse tively. Leaf texture was based on a rating scale of 1 textured alluvial soils with a pH of 7 or higher and to 3 where 1 glabrous, 2 pubescent, and cannot tolerate excess moisture during the growing 3 = mix of glabrous and pubescent types. Stem type season (Whistler and Hymowitz 1979). was also based on a scale of 1 to 3 where Although guar seeds have been used in the 1 = branched, 2 = not branched, and 3 = mix of pharmaceutical and nutraceutical industries, the com­ branched and unbranched types. A visual observation position and content of fiber, isoftavonols and ftavo­ for the number of days from planting to 50% nols in guar seeds has largely been ignored. Thus maturity of plants within each plot (row) was significant genotypic variability for both total dietary recorded as well. Mature pods were harvested from fiber and soluble dietary fiber was found in seed from each guar accession as they matured, dried at 21°C, several guar accessions (Kays et al. 2006). Several 25% RH for about 1 week:, and threshed. Seeds were guar accessions also showed significant variability for then counted and weighed. seed derived daidzein, genistein, quercetin, and Principal component analysis and PC SAS proce­ kaempferol (Wang and Morris 2007). When com­ dure CLUSTER analysis were then used for multi­ pared to soybeans, guar seeds produced very low variate analysis of the data. PROC PRINCOMP (SAS amounts of daidzein and genistein, however guar Institute 2003) was perfonned for all traits. Eigen­ seeds produced significantly more kaempferol than values and the percentage of variances explained by soybeans (Wang and Morris 2007). each principal component were also determined. The The objectives of this study were to regenerate and similarity matrix was entered into PROC CLUSTER characterize guar accessions for plant height, leaf in SAS (SAS Institute 2003) for cluster analysis with texture, pod length, stem type, days to 50% maturity, both Ward's minimum variance method (SAS Insti­ seed number and 100 seed weight in Griffin, GA, tute 2003) and unweighted paired group method USA during the 2006, 2007, and 2008 regeneration using mathematic averages (upGMA) by specifying cycles using coefficients of variation, principal com­ the WARDS and AVERAGE options (SAS Institute ponents, and cluster analysis. 2003). Both clustering methods were used to verify which method was best at representing the true genetic relationships among guar accessions. Ward's Materials and methods and UPGMA are commonly used clustering algo­ rithms for gennplasm analysis. Ward's method uses 2 All guar accessions studied are conserved at the the semi partial R , s summed across cluster members USDA, ARS, Plant Genetic Resources Conservation as the distance between clusters (Ward 1963). Unit located in Griffin, GA, USA. Seed accessions Standard errors (SE) and coefficients of variation studied were collections from India, Pakistan, and (CV) were also determined to confinn variability breeding material from the USA (Table 1). Approx­ using principal component analysis (SAS Institute imately 50 seeds from each guar accession were 2003). Coefficients of variation were calculated directly seeded to field plots consisting of a clayey, automatically using SAS (SAS Institute 2003) refer­ kaolinitic, thermic typic kanhapludults soil series at ring to the ratio of a standard deviation over the Griffin, GA, between early May and the second week average. Standard errors were calculated by dividing of June 2006-2008. About 25~50 plants representing the standard deviation by the square root of the each accession per plot were regenerated in one 6 m sample size using SAS.

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Table 1 Morphological, phenological and reproductive traits of guar evaluated from 2006 to 2008 Acc. Origin Ht Leaf Pod Stem type DTM Sd. No. 100 sd. Wt texture length

2006 158121 Delhi, India 8 72 1 164 '700 3.3 164299 Tamil Nadn. India 9 58 1 185 484 3.0 164353 India 13 2 60 4 175 1023 2.9 164420 India 10 2 67 162 1200 4.5 164476 Rajasthan, India 17 2 65 173 3944 3.1 176377 Delhi, India 14 2 62 177 916 2.8 186477 Tamil Nadu, India 5 1 52 1 117 9358 3.0 212900 India 8 2 50 1 177 812 2.8 212987 India 13 2 76 127 5076 3.3 223685 India 9 2 78 140 2780 3.6 250358 Pakistan 10 1 60 162 2338 3.0 253187 12 2 57 137 3360 2.9 263877 Delhi, India 10 2 70 1 177 840 3.5 263883 Delhi, India 13 76 2 151 626 3.2 288351 India 13 2 66 149 5450 3.0 288377 India 12 2 57 177 1200 3.4 288415 Gonda!, Rajkot, India 11 61 102 528 3.1 288419 Umrala, Bhavnagar, India 7 74 1 158 530 3.5 288432 Bithura Kurd, Pali, India 11 55 1 157 1412 3.2 288435 Bandsa, Pali, India 14 2 52 158 2940 2.8 288442 Osian, Jodhpur, India 10 2 60 144 4000 2.8 288443 RampUl'3, India 10 2 60 117 1530 3.7 288751 India 13 2 65 2 144 4992 3.1 288760 India 18 2 60 133 1440 3.7 288762 India 15 2 67 149 5928 4.0 288763 India 9 2 55 142 2780 4.1 322844 Delhi, India 13 2 58 131 3686 2.9 323045 Delhi, India 12 2 60 1 185 520 3.6 338745 Delhi, India 17 2 55 2 149 3660 2.3 338847 India 9 2 67 137 2216 4.2 338856 India 8 2 60 131 568 3.1 338872 India 9 2 32 143 730 2.5 338877 India 8 2 60 147 538 3.3 340647 Delhi, India 9 2 50 166 600 3.4 426631 Pakistan 11 3 48 1 158 2750 3.6 593054 Texas US 6 1 55 1 110 7562 3.1 593058 Texas US 11 3 71 131 6683 3.2 2007 158116 Delhi, India 9 2 65 4 123 1000 2.7 164170 India 12 3 51 4 150 408 2.7 164386 India 13 2 50 4 153 80 2.6 180285 India 8 2 52 130 100 3.0 212986 India 11 3 75 4 105 1000 4.8

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Table 1 continued

Ace. Origin Ht. Leaf Pod Stem type DTM Sd. No. 100 sd. Wt texture length

217924 Delhi, India 12 50 4 130 1580 3.3 217925 Delhi, India 7 2 55 120 4200 2.8 250211 Pakistan 9 2 55 130 620 2.6 250360 Pakistan 8 2 54 120 3780 3.0 340601 Delhi, India 14 2 45 101 1042 3.3 340055 India 11 2 62 1 153 160 3.5 426630 Pakistan 14 2 60 1 110 1246 3.8 426636 Pakistan 9 3 55 130 1720 3.2 426638 Pakistan 13 1 48 1 135 2686 3.3 426639 Pakistan 14 1 50 1 96 1710 3.4 426643 Pakistan 14 9 55 1 101 2220 3.9 428579 Bhiwani, Haryana, India 12 9 51 1 118 1520 2.4 428580 Bhiwani, Haryana, India 8 3 60 1 130 680 3.1 428581 Bhiwani, Haryana, India 12 2 70 137 800 2.9 430376 Rajasthan, India 9 2 60 1 130 1380 3.1 537279 Pakistan 7 2 58 1 117 600 3.5 537281 Pakistan 8 2 52 117 4950 3.1 593048 Texas, US 8 80 1 152 100 2.9 593049 Texas, US 7 1 62 1 152 680 2.8 593059 Texas, US 7 1 52 1 127 3210 2.5 2008 179683 India 9 2 58 4 102 1401 2.8 288390 India 12 2 70 4 137 370 3.6 288414 Bedipura, Rajkot, India 10 1 78 137 548 4.5 288424 Bhrugupur, Surendranagar. India 11 1 75 137 792 3.8 288429 Santpura, Sirohi, India 12 3 110 102 4340 3.6 288737 India 15 2 65 106 691 3.6 288738 India 15 2 65 4 102 742 3.2 288742 India 12 3 75 4 102 695 3.6 288747 India 22 2 75 4 106 3076 3.7 288748 India 14 2 75 1 106 5587 3.9 288761 India 12 2 65 1 106 5142 3.8 SE 0.36 0.15 1.31 0.12 2.80 234.17 0.06 CV(%) 28 63 18 72 17 93 15

Results was observed from wide ranges for these character­ istics (Table 2). Successful plant regeneration occurred for all of the Plant height ranged from five to 22 dm with most guar accessions tested. Morphological, phenological, of the accessions producing short plants averaging and reproductive characteristics observed among 10 dm. However, 18 guar accessions produced plants accessions are reported in Table l.Variability in plant averaging 14 dm tall, while 4 accessions produced height, pod length, days to maturity and seed number tall plants averaging 19 dm across all years. The

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Table 2 Phenotypic traits Variable Maximum Minimum Range Mean SD in guar based on data from 2006 to 2008 field Plant ht. (cm) 22.0 5.0 17.0 11.0 3.1 regenerations Plant surface 3.0 1.0 2.0 1.9 0.60 Pod length (rnrn) 110.0 32.0 78.0 60.9 11.2 Stern type 4.0 1.0 4.0 1.5 1.0 No. of days to 50% maturity 185.0 96.0 89.0 137.7 23.9 Seed number 9358.0 80.0 9278.0 2141.0 2001.0 100 seed wt. (g) 4.8 2.3 2.5 3.3 0.47

coefficient of variation for plant height was 28% (9,358). However 9 guar accessions produced high indicating low variability between individuals within seed numbers as well including PI 212987, PI the same guar accession. Forty-six and 17 accessions 288351, PI 288748, PI 288751, PI 288761. produced pubescent and glabrous leaves, respectively PI 288762, PI 531281, and both breeding lines (PI while ten guar accessions produced plants showing a 593054 and PI 593058) averaging 5,708 seeds per mixture of pubescent and glabrous leaf texture. The accession. Eighteen intermediate seed producing coefficient of variation for leaf texture was 63% accessions averaged 3,220 seeds per accession and indicating a fairly high amount of variation for this 45 low seed producing guar accessions averaged 810 trait between individuals within the same guar seeds per accession. The coefficient of variation was accession. Pod length ranged from 32 to 110 mm 93% indicating higher variation between individuals with 59 accessions producing pods averaging 58 mm within the same guar accession for this trait. Seventy­ in length. Thirteen guar accessions produced an three accessions had an average 100 seed weight of average pod length of 75 mm, while only 1 accession 3.3 gm with a coefficient of variation of 15% (PI 288429) produced the longest pod reaching indicating very low variation between individuals 110 mm. The coefficient of variation for pod length within the same guar accession. Phenotypic variation was 18% indicating very low variability for this trait for the PGRCU guar collection can be explained on between individuals within the same guar accession. the basis of plant selection leading to the potential Fifty-nine accessions produced guar plants with more development of cultivated varieties or breeding than one branch and three accessions produced single material. branched plants. Eleven guar accessions produced plants with a mixture of multiple branching and single branched plants. The coefficient of variation Ranking of guar accessions was 12% which shows a fairly high degree of variation between individuals within the same guar Across all 3 years, eight accessions, including PI accession for stem type. 186477 (Tamil Nadu India), PI 212987 (India), PI The majority of the guar plants (36 accessions) 288351 (India), PI 288748 (India), PI 288761 (India), matured an average of 117 days after planting with PI 288762 (India), PI 593054 (breeding line), and PI fifteen of these maturing an average of 104 days after 593058 (breeding line) produced the most seeds, planting. The accession PI 426639 from Pakistan averaging 6,348 seeds per accession. Of these eight, produced the earliest maturing seed (96 DTM). the earliest maturing accessions at 106 DAP included Twenty-one goar accessions produced intermediate both PI 288748 and PI 288761 producing an average maturing plants averaging 145 days after planting of 13 dm tall, multiple-branched plants averaging while sixteen accessions produced the latest maturing 5,365 seeds. However, PI 186477 produced 5 dm tall plants averaging 169 days after planting. The coef­ and multiple-branched maturing 117 DAP and the ficient of variation for DTM was 17% indicating a highest number of seeds (9,358). The breeding line, very low amount of variation for this trait between PI 593054 produced 6 dm tall multiple-branched individuals within the same guar accession. The guar plants maturing 110 DAP, and producing 7,562 seeds. accession PI 186477 produced the most seeds An additional breeding line, PI 593058 produced

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taller (11 dIn) multiple-branched and early maturing accounted for 19% of the variation and was mostly plants (131 DAP) with 6,683 seeds. The early due to stem type and seed number. These traits are maturing (127 DAP) accession PI 212987 produced closely related because the number of branches on a 13 dIn tall, multiple-branched plants yielding 5,076 plant equates to seed yield. The third principal seeds. Both PI 288351 and PI 288762 produced component explained 17% of the variation and is multiple-branched plants averaging 14 dIn tall, composed primarily of DIM and seed number. The maturing 149 DAP, and yielding an average of fourth principal component explained 12% of the 5,689 seeds. These accessions outperformed the variation and consisted of plant height and DTM. cultivars (PI 537279 and PI 537281) from Pakistan These three traits in principal components 3 and 4 for seed production. relate to early or late maturity and yield. The fifth principal component accounted for 12% of the vari­ ation and was primarily due to leaf texture. Therefore, Principal component analysis potential exists to develop cultivars with improved early or late maturity, and high or low seed yield. Stem Principal component analysis accounted for 25% of type was significantly correlated with plant height the total variation at the first principal component (1 = 0.27*, n = 73) and seed number had a signif­ (Table 3). The amount of variation accounted for, icant negative correlation with DIM (1 = -0.24*, cumulatively, by adding principal components 2 n = 73). One hundred seed weight was significantly through 6 was 44,61,73,85, and 94%, respectively. correlated with pod length (1 = 0.43**, n = 73). The first principal component was most correlated Generally, variation in these morphological, with plant height, pod length, and l00-seed weight phenological, and reproductive traits among guar (Table 4). These three traits relate to plant architecture accessions was reflected by wide ranges for most of and seed yield. The second principal component these characteristics (Table 2). Since these traits studied are quantitative, the variability among acces­ sions is attributed to genetic differences and the Table 3 Eigenvalues and the proportion of total variability environment in which they were regenerated. among guar accessions (2006-2008) as explained by the principal components Principal Eigenvalue % Variability % Cumrnulative Discussion component

1 1.7666 25.24 25.24 Limited studies similar to this one have been 2 1.3191 18.85 44.08 conducted in the tribe Indigoferea, however several 3 1.1847 16.93 61.01 experiments have been conducted on species in the 4 0.8439 12.06 73.06 Phaseoleae tribe including butterfly pea (Morris 2009b), swordbean (Morris 2007), horsegram and 5 0.8153 11.65 84.71 perennial horsegram (Morris 2008a). Coefficients of 6 0.6340 9.06 93.77 variation, principal components, and cluster analysis

Table 4 Eigenvectors, Principal components principal components for seven morphological, 2 3 4 5 6 phenolOgical, and seed traits in guar accessions Plant ht (ern) 0.43 0.26 -0.03 0.69 -0.25 -0.37 (2006-2008) Plant surface 0.37 0.23 -0.29 0.Q7 0.81 0.23 Pod length (rnm) 0.44 -0.32 0.40 0.008 -0.14 0.59 Stem type 0.32 0.60 -0.007 -0.25 -0.38 0.30 No. of days to 50% maturity -0.38 0.13 0.49 0.57 0.18 0.30 Seed no. 0.08 -0.50 -0.58 0.33 -0.20 0.27 100 seed wt. (g) 0.45 -0.35 0.40 -0.12 0.18 -0.43

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provide quality characterization information about combined germplasm arranged the guar accessions regenerated genetic resources. Both coefficient of into 3 clusters yielding clusters of sizes 45, 18, and 10 variation and principal component analysis are useful accessions (Fig. 1). The cluster and multivariate parameters to determine an estimate of how much analysis conducted on the data were able to clearly each trait contributes to its variation and have separate low seed producing guar accessions from successfully been used to characterize regenerated those accessions producing intermediate to high seed Senna (Morris 2009a), and butterfly pea (Morris numbers. Cluster or group 1 consisted of 45 low seed 2009b) genetic resources. Each of these and this producing guar accessions. Cluster 2 consisted of 18 current study have shown that much of the variation intermediate seed producing accessions and cluster 3 was contributed primarily from days to 50% maturity, consisted of 10 accessions producing high seed plant height, and seed reproductive traits. Other numbers. Average linkage cluster analysis grouped investigations using coefficients of variation have the original 73 guar accessions into well defined also been successfully used to characterize regener­ phenotypes with three distinct seed number produc­ ated swordbean (Morris 2007), horsegram and peren­ ing groups (Fig. 2). Group 1 represents 45 low seed nial horsegram (Morris 2008a), as well as Rhynchosia producing guar accessions. Group 2 consisted of 18 minima (Morris 2008b) genetic resources. Studies intermediate seed producing guar accessions, while conducted on Lathyrus sativus (polignanol et al. group 3 represented 10 guar accessions producing 2005) and butterfly pea (Morris 2oo9b) suggested that high seed numbers. All three groups were represented seed yield characteristics explained the largest by collected or donated guar accessions from India amount of variation using principal component and Pakistan as well as USA developed breeding analysis. lines and cultivars. One outlier in group 3 (pI Cluster analysis using Ward's method of morpho­ 186417) with a phenotypic distance index of 1.3283 logical, phenological, and reproductive traits for the to the high seed producing accessions showed similar

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-ce III ::l 0­ W GroupS, ri: 0.4 high (ij seed 1: Group 2, ~ Intermediate 'E seed I1l numbers 00 0.2 /' I

Group 1, low seed nu nbers If 0.0 1452232122421322323232512115311231242241242441233222224422522341552225222 5298838716286288383645368689465146828837621226623518282258985326991883666 6838888623884388868300748403048204866809887664828078386636380866332887888 1507787398542044846862213320631964364736459664487394676614073664009772377 2843473707629411715841779884551802733678472337444622863383545447558658546 1097228707149559746371900658536610702063394696245059531875978737487111182 Ace

Fig. 1 Cluster dendrogram for morphological, phenological, and reproductive characters of the combined guar germplasm (73 accessions)

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2.0

Group 2, intennediate seed numbers

Group 1, low s numbers

1452232122421322323225312115311231242241242441233222224422522341222522255 S2938307'62862883936S34G8689465146B28B3782'~2G823518282~8598S32818838B899 883BS88~238843a88ee30704840304820486a809a8166482a0783B6693380866288188833 1507787398512044848822613320631964364736459664407394676641073864977231700 2843173707029411715817479884551802733678472337444622863338545447865954655 1097228701149559746319700658536610702063394696245059531857978731111118248 Ace Fig. 2 Dendrogram of distance between clusters based on baseline indicate average phenotypic distances between acces­ morphological, phenological, and seed reproductive differ­ sions. Three distinct clusters and one outlier (very high) for ences. Accession numbers are given (Acc). Values on the seed numbers can be distinguished

morphological and DTM resemhlance to these acces­ probable that these guar accessions also originate sions. The guar accessions clustered in groups 2 and 3 from northern or northwestern regions in India. The showed relatively closer genetic relationships than intennediate seed producing guar accessions included those in group 1. Using the distance values indicated six accessions originating from north central India in Fig. 2, the groupings at any similarity level can be while only one accession originated from northwest identified. For example, PI 426636 and PI 426639 India. Five accessions originated from Pakistan as originate from Pakistan with a phenotypic distance well, one breeding line from Texas and one from an index of 0.0128, which indicates their close morpho­ unknown origin were all included in the intennediate logical similarities. Likewise, two breeding lines seed producers also. Thus it is quite possible that developed in Texas (PI 593054 and PI 593058) have most of the other Indian accessions also originated a phenotypic distance index of 0.3119 indicating their from north central India. The high seed producers close relationship with Group 3, which consists of 10 included one accession from southern India (Tamil accessions. Nadu), one from Pakistan and two Texas breeding Ward's clustering method separated the guar lines. With such limited geographic infonnation accessions into several distinct clusters. Even though regarding the additional six high seed producers, it the guar collection consists of relatively minimal is also possible that these six accessions also had geographic infonnation, these clusters tended to southern Indian origin as well. This should be useful define accession groups with similar geographic when selecting accessions from the guar gennplasm origins. For example twelve of the low seed produc­ collection. In the future, molecular marker analysis ing accessions originated from northern or north­ should contribute to identifying variation in the rest western India while five originated from Pakistan. of the guar accessions curated in the USDA, ARS, Two breeding lines developed in Texas were also PGRCU collection. The analysis revealed tighter found in these low seed producing guar accessions. clustering among the intennediate and high seed Only one accession (PI 164299) originated from producing guar accessions which indicates greater southern India, Tamil Nadu. Since the additional 25 genetic variability in the low seed producing guar accessions were from unknown areas in India, it is accessions.

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