Cindy Lowery³, Dick Auld¹, Efrem Bechere¹, Robert Wright ^1,3, Eric Hequet^2,3, Noureddine Abidi², and Wayne C. Smith⁴

¹Plant & Soil Science Dept.; Texas Tech University; Box 42122; Lubbock, TX 79409

²International Textile Center; Texas Tech University; Box 5019; Lubbock, TX 79409

³ TAMU Research & Extension Center; Rt. 3 Box 219; Lubbock, TX 79409

⁴ Soil and Crop Sciences, Texas A&M University, Box 2474, College Station, TX 77843

ABSTRACT

In 2002, six cotton (Gossypium spp.) genotypes were treated with three differential concentrations of Na Azide and Ethyl MethaneSulfonate (EMS) as well as a distilled H₂O control to determine effective dosages for chemical mutagenesis in cotton. Chemical treatment, genotype and the interaction of chemical treatment X genotype had a significant impact on germination and field emergence. Both 3% v/v EMS and 0.05% v/v Na Azide reduced seed viability to less than 50%. It also appeared that the diploid genotypes generally had a higher mortality that the tetraploid genotypes. Geneticists using mutagenesis should conduct initial studies to determine appropriate rates for specific mutagens prior to treating selected genotypes. It was also noted that use of seed lots with good germination would enhance the recovery of useful mutants.

Several mutant lines selected for the naked-tufted phenotype phenotype from the cultivars Atlas, SC 9023, and Tejas were grown in replicated plots at Lubbock, TX to determine lint yield and lint percent in both 2005 and 2006. The fibers harvested from these plots were analyzed at the Texas Tech University – International Textile Center using High Volume Instruments (HVI), Advanced Fiber Information Systems (AFIS), and standard yarn quality analyses (2005 fiber only). These trials indicate that selected naked-tufted mutants had comparable lint yields and lint percent of both the parental cultivars and commercial check cultivars. Several of the mutants also had very good HVI and AFIS fiber quality a significant reduction in seed coat neps.

Introduction

Cotton is the world’s leading textile fiber and it is grown on over six million hectares in the United States (US) (NASS, 2006). Cotton has historically been grown on more acres in this country than any other crop except for corn, wheat, and soybeans. Cotton fiber generates in exceeds of $6 billionyr^-1, while cottonseed oil and meal add another $500 millionyr^-1 to the total US agricultural economy. More than 440,000 domestic jobs are created by cotton production and processing which has a total impact of over $40 billion on the U.S. gross domestic product (National Cotton Council, 2005). United States cCotton exports also contribute about $2 billion yr^-1 to our the U.S. trade balance.

United States USUnited States cotton production is under increasing competition from synthetic fibers for the manufacture of yarns and textiles. In addition, the continuing decline of the US textile industry requires that over a third of U.S. produced cotton lint must be sold to international markets (National Cotton Council, 2005). Increasing the length, durability and strength of cotton fiber through genetic improvement should help U.S. grown cotton to compete both with synthetic fibers and in International markets.

There have been several genetic bottlenecks which severely restrict the genetic variability available in cultivated cotton. Upland cotton, Gossypium hirsutum, is thought to have arisen as a the result of an interspecific hybridization between an A- and D-genome diploid species about 1-2 million years ago (Wendel,1989). This single polyploidization event was the first genetic bottleneck. that constrains genetic variation available for cotton improvement. A second bottleneck was associated with occurred during the domestication of G. hirsutum from using only a small subset of the wild genotypes. A third bottleneck resulted from occurred when only limited sampling of only a few semi-domesticated progenitors from the Mexican-Guatemalan border which provided border were used to generate the germplasm base of our modern elite cotton gene pool. These These initial selections lines were brought northward into the U.S. and eventually into China, India, Australia and other countries (Hutchinson et al., 1947),. Finally, intensive breeding for narrowly-defined fiber quality parameters required for processing of this industrial crop has further narrowed the gene pool of commercial cotton germplasm that is commercially acceptable (May, et al.,1995)

The use of chemically-induced mutants has been highly successful in most major crops grown across the world but has only occasionally been used in improving cotton (Auld, et al., 1998). However, the relatively low level of genetic variability currently available in cotton would indicate this would be an ideal tool to increase genetic variability in this species. Mutagenesis has been shown to be an effective tool to create a wide range of phenotypic variation in both diploid and tetraploid Gossypium populations (Auld, et al., 2000; Larik, et al., 1983; Gaibullaev, et al., 1976; Hussein, et al., 1982 and Shattuck and Katterman, 1982). Some of these mutants such as the ‘naked and tufted’ trait have immediate application to cotton improvement by reducing seed coat neps as well as the energy and time required in cotton ginning. Other mutants created by the process will provide The creation of these new mutants is a powerful tools critical for both functional genetics and conventional genetic improvement studies in of cotton.

MATERIALS AND METHODS

Mutation Rate Study: In the spring of 2002, seed of six cotton lines were exposed to a distilled H₂0 Control, three rates of Ethyl MethaneSulfonate (EMS), and three rates of Na Azide at Texas Tech University. The six lines included three tetraploid - G. hirsutum lines (FiberMax 958, ACALA 1517-99, and TAM 94L-25), one tetraploid - G. barbadense (Pima S-7) and three diploid lines – G. arboreum (GA-TAMU, A2-60T, and A2-120W). Four samples of 250 what? and four samples of 50 untreated cotton seed (M₀ generation) from each of the six lines were imbibed for 16 to 20 hours in aerated distilled water.  The seed were then rinsed and placed in aerated distilled water.  The EMS and Na Azide treatments were pipetted into the solution under where it is allowed to mix with the seed for two hours to produce the M₁ generation.  The seeds were then removed and rinsed several times to remove any residual mutagen prior to hand planting the larger samples of M₁ seed in the field or conducting laboratory germination trial on the smaller seed samples.  The remaining solutions were treated with a strong base to denature the mutagens before disposal as potentially hazardous chemicals. The four ­50 seed samples of the treated M₁ seed were tested to determine germination 4 and 10 days after treatment in the growth chamber. Counts were made on the four 250 field planted seed samples 7, 14, and 21 days after planting to determine the rate of emergence under field conditions. Means of all treatments were subject to analyses of variance and the means separated using a Fisher’s Protected LSD at the 0.05 level of probability (SAS, 1992).

In the fall of 2002, the M₂ seed was harvested by removing one boll per plant. The M₂ generation was increased to the M₃ in 2004, the M₄ in 2005, and the M₅ using this same process. Because the M₁ generation is often highly chimeric, single plant selections were not made until the M₃ or later generations. Many of these populations are now being screened for fiber quality, growth habit, and physiological mutations.

Naked and Tufted Mutant Study: In 1996, six commercial varieties of cotton were treated with 2.45% v/v ethyl methanesulfonate. In 1999, three M₃ plants were identified identified from single mutant lines from the cultivars Atlas, SC 9023 and Tejas that had partially naked seed coats similar to those previously described as “naked-tufted” (Endrizzi and Ray, 1991). From 2001 to 2004, selections from the three M₃ naked-tufted seed coat mutants were evaluated in replicated trials at Lubbock, Texas. These trials were conducted to stabilize this trait and to evaluate the impact of the naked-tufted phenotype on lint yield, lint percent, fiber quality, and yarn spinning performance. In 2001, the initial three mutants were visually screened for the expression of naked-tufted seed coats. Fifteen individual plants exhibiting the naked-tufted phenotype were selected and grown in the field in 2002. This process was repeated in 2003 and 2004 to generated 40 individual plant selections with naked-tufted seed coats. In 2005 and 2006, these 40 selections, three parental lines and five check varieties were evaluated for expression of the naked-tufted seed coat trait, lint yield and lint percent. The fibers harvested from these plots were analyzed at the Texas Tech University – International Textile Center using High Volume Instruments (HVI), Advanced Fiber Information Systems (AFIS), and standard yarn quality analyses

RESULTS AND DISCUSSIONS

Mutation Rate Study: Mutagen treatment, genotype and the interaction of mutagen treatment X genotype had a significant impact on both germination and field emergence. When measured with either laboratory germination or field emergence, the 0.0005 v/v Na Azide, 0.005 v/v Na Azide and 0.01% v/v EMS treatments did not decrease the viability of the seed (Table 1). However, the 0.05 v/v rate of EMS almost completely destroyed seed viability. The 3% v/v EMS and 0.05% v/v Na Azide treatments reduced seed viability to less than 50%. Many geneticists conducting chemical mutagenesis like to burn the population so that seed viability is reduced by approximately 50% (Auld, et al., 1998).

The laboratory seed germination of the six lots treated with just H₂0 of seed ranged from 12.1% to 63.6%. The germination and field emergence data of the mutagenic were adjusted to a pure live seed basis to allow appropriate comparisons (Table 2). The diploid genotypes of G. arboreum and the single genotype of G. barbadense generally had a higher mortality rate when exposed to the mutagens than the G. hirsutum genotypes in both laboratory germination and field emergence (Tables 3 and 4). Geneticists using mutagenesis should conduct initial studies to determine appropriate rates for specific mutagens prior to treating selected genotypes. Use of seed lots with good germination and seedling vigor would enhance the recovery of useful mutants with chemical mutagenesis.

Even though this study had relatively small populations in the field, counts were made to determine the number of plants in each treatment which had chlorophyll chimeric plants. This index is often indicative of the relative effectiveness of different mutagenic treatments. It was interesting that seeds exposed to the water control and two lower rates of Na Azide did not produce chimeric plants (Table 5). However, the highest rate of EMS (0.05% v/v) produced 16 chimeric plants despite an average rate of field emergence of 3.1%. The other 5 chimeric plants were found in seedlings exposed to the highest rate of Na Azide and the other two rates of EMS.

Naked and Tufted Mutant Study: The naked-tufted phenotype was correlated with a reduction in seed coat neps (r = -0.47**) and an increase in seed oil content (r = 0.68**). One of the mutants selected from SC 9023 (SC 9023-NS-57-13-3) produced yarn that provided exceptional performance for ring spinning applications. Future studies are being conducted to will be needed to determine the inheritance and potential impact of the naked-tufted phenotype on other agronomic production characteristics of upland cotton such as the energy & time required in ginning.

These mutants appear to reduce or eliminate the occurrence of fuzz or linters which are short fibers tightly attached to the seed coat in most upland cotton varieties. These mutants appear to be phenotypically similar to the “naked-tufted” mutant initially described by Endrizzi and Ray in 1991. They concluded that the N^t₂ marker was simple dominant gene that was allelic to n₂ (Endrizzi and Ray, 1991) Their studies also showed that this gene was located on the long arm of chromosome 26. Our initial interest in this phenotype was based on the hypothesis that the removal of linters during saw ginning contributed significantly to increased short fiber content in upland cotton. Consequently, genetic elimination of the linters would reduce the short fiber content while simplifying ginning, oil recovery, and delinting of cottonseed.

Turley and Kloth had earlier reported on the impact of the N₁/n₁, N₂/n₂, and the N₃/n₃ genes on lint percent in cotton (Turley and Kloth, 2002). They proposed that only the genotype, n₁n₁N₂N₂N₃N₃, would generate a normal lint percent of 40.5%. A second study by Lee, et al., reported on the gene expression of the N₁N₁ genotype and its impact on fuzz development (Lee, et al. 2006). Both of these studies did not include analyses of the N^t₂ allele or an estimate of its impact on lint percent.

Based on data collected on 2005 fiber, it appeared that a selected line with a partially naked seed coat (SC 9023-NS-57-13-3) did have reduced short fiber content, enhanced maturity, and higher quality yarn in ring spinning. However, not all partially naked or naked seed coat lines showed similar performance. This would indicate that other factors in addition to seed coat characteristic impart short fiber content and fiber maturity. Therefore, these trials are being repeated in 2006. HVI analyses did not identify this line as superior, but AFIS analyses showed that this line had very low neps/gram, low short fiber content (weight), and a very high maturity ratio. This study showed that cotton breeders need to increasing rely on AFIS analyses in late generation screening for fiber quality. Future studies will be needed to determine if the enhanced yarn characteristics of SC 9023-NS-57-13-3 are the result of the partially naked seed coat characteristic or other traits inherent in this line.

Repeated from previous paragraph

We now believe the “naked and tufted” seed mutant may be critical to the development of ELS (Extra Long Staple) stripper cotton varieties in Texas. It also appears this mutation could significantly reduce both the time and the energy required for ginning, oil extraction, and delinting of cottonseed. The superior fiber quality and improved energy efficiency obtained with the naked and tufted mutant will drive its rapid incorporation into new varieties.

LITERATURE CITED

Auld, D.L., M.D. Ethridge, J.K. Dever and P.D. Dotray. 1998. Chemical mutagenesis as a tool in cotton improvement. P. 550-551. In: Proc. Beltwide Cotton Conf., San Diego, CA. 5-9 Jan. Natl. Cotton Counc. Am., Memphis, TN.

Auld, D.L., Efrem Bechere, M.D. Ethridge, W.D. Becker, E. Hequet and R.G. Cantrell. 2000. Registration of TTU 202-1107B and TTU 271-2155C mutant germplasm lines of upland cotton with improved fiber quality. Crop Sci., 40:1835-1836.

Du, X.M., J.J. Pan, R.H. Wang, T. ZH. Zhang, and Y. Zh. Shi. 2001. Genetic Analysis of Presence and Absence of Lint and Fuzz on Cotton. Plant Breeding 120:519

Endrizzi, J.E. and D.T. Ray. 1991. Monosomic and Monotelodisomic Analysis of 34 Mutant Loci in Cotton. Journal of Heredity 82:53-57.

Gaibullaev, I.K.H., M.K.H. Avazkhodzhaev, and A.E. Egamberdiev. 1976. Reaction of Chemomutants of Cotton to Infection by the Pathogen of Verticillium Wilt. Soviet Genetics 11(1);34-36.

Hussein, H.A.S., F.A. Al-enani, and M. El-Moghazi. 1982. Histological and Morphological Characteristics of a Glandless Cotton Mutant Induced with Sodium Azide. Egypt J. Genet. 11:167-173.

Hutchinson, J.B., Silow, R.A., and Stephens, S.G. 1947.  In "The evolution of Gossypium and the

differentiation of the cultivated cottons". Oxford University Press, London.

Larik, A.S., H.M.I. Hafiz, and Y.A. Al-Saheal. 1983. Azide Mutagenesis in Cotton (Gossypium hirsutum). Science and Environment 5: 33-42.

Lee, J.J., O.S.S. Hassan, W. Gao, N.E. Wei, R.J. Kohel, X.Y. Chen, P. Payton, S.H. Sze, D. M. Stelly, and Z. J. Chen. 2006. Developmental and gene expression analyses of a cotton naked seed mutant. Planta 223: 418-432.

May, O.L., D.T. Bowman, and D.S. Calhoun. 1995. Genetic diversity of U.S. Upland Cotton Cultivars Released between 1980? and 1990. Crop Sci. 35:1570-1574.

NASS, National Agricultural Statistics Service, USDA, 2006. [On Line]. Available at http://www.nass.usda.gov/index.asp (Verified 28 January 2007).

National Cotton Council. 2005. Economic Impact of US Cotton. http://www.cotton.org/econ/cropinfo/supply-demand.cfm

SAS Institute, Inc. 1992. SAS/STAS Users Guide of Version 7-Fifth Edition. SAS Institute, Inc. Gary, NC.

Shattuck, V. and F.R. Katterman. Enhanced Unscheduled DNA Synthesis if the Cotyledons of Gossypium barbadense L. by Ethyl Methansulfonate (EMS). Biochemical and Biophysical Research Communications 109(3):1017-1025.

Turley, R.B. and R.H. Kloth. 2002. Identification of a Third Fuzzless Seed Locus in Upland Cotton (Gossypium hirsutum L.). Journal of Heredity 93(5);359-364.

Wendel, J.F. 1989. New World Tetraploid Cottons Contain Old-World Cytoplasm. Proc Natl Acad Sci USA 86: 4132-4136.

Table 1. Germination and field emergence of cotton exposed to seven mutagenic agents average over six genotypes at Lubbock, Texas in 2002.

Mutagen	Germination			Field Emergence
Dose	Day 4	Day 10		Day 7	Day 14	Day 21
	- - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - -
Water:
(1) Control	100.0 a†	100.0 a†		39.8 b†	50.0 b†	46.4 b†
Na Azide:
(5) 0.0005 v/v	58.8 b	78.2 c		42.7 b	46.4 bc	47.0 b
(6) 0.005 v/v	47.7 c	90.5 b		51.1 a	58.5 a	58.3 a
(7) 0.05 v/v	11.0 d	46.5 d		27.3 c	46.4 bc	48.3 b
Ethyl MethaneSulfonate:
(2) 0.01 v/v	61.7 b	77.3 c		30.5 c	40.0 c	40.1 b
(3) 0.03 v/v	4.4 e	25.0 e		9.5 d	21.5 d	20.8 c
(4) 0.05 v/v	0.2 f	0.4 f		0.1 e	1.5 e	1.6 d
Coefficient of Variation	18.9%	15.3%		51.1%	36.1%	38.8%

^† Means within a column not followed by the same letter differ at the 0.05 level of probability by Fisher’s Protected Least Significance Test at the 0.05 level of probability.

Table 2. Germination and field emergence of six genotypes of cotton averaged over seven mutagenic treatments at Lubbock, TX in 2002.

Species

Non-Adjusted

Germination

Field Emergence

Genotype

Germination

Day 4

Day 10

Day 7

Day 14

Day 21

- - - - - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - -

G. hirsutum

(4) TAMU 942-25

49.9 c†

48.5 b†

73.3 a†

41.9 a†

54.0 a†

52.2 ab†

(5) ACALA 1517-99

63.6 a

51.1 b

67.7 b

46.1 a

60.4 a

59.0 a

G. barbadense

(3) Pima S-7

36.6 d

43.6 c

62.6 c

21.2 bc

14.6 d

15.3 d

G. arboreum

(7) G.a (TAMU)

59.3 b

58.9 a

70.1 ab

28.0 b

44.9 b

44.5 b

(2) A2 60-T

14.0 e

24.2 d

45.2 d

17.9 c

22.3 c

25.0 c

(1) A2 120-W

12.1 e

17.0 e

39.1 e

17.2 c

27.5 c

28.9 c

F Test

Geno x Trmt

28.0**

10.5**

2.4**

4.5**

4.1**

Coefficient of Variation

18.9%

15.8%

51.1%

36.1%

38.8%

^† Means within a column not followed by the same letter differ at the 0.05 level of probability by Fisher’s Protected Least Significance Test at the 0.05 level of probability.

Table 3. Germination after 10 days of six genotypes of cotton exposed to seven mutagenic treatments in Lubbock, TX in 2002.

Mutagen	TAMU		ACALA		Pima		TAMU		A2		A2
Dose	946-25		1517-99		S-7		GA		60-T		120-W
	- - - - - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - - - - -
Water
(1) Control	100.0	100.0		100.0		100.0		100.0		100.0
Na Azide
(5) 0.0005 v/v	93.3	93.0		85.3		98.8		44.0		43.8
(6) 0.005 v/v	95.5	93.0		91.8		100.0		79.0		83.5
(7) 0.05 v/v	52.3	52.0		52.0		73.8		19.5		22.5
Ethyl MethaneSulfonate
(2) 0.01 v/v	97.0	90.5		97.5		91.5		63.0		24.3
(3) 0.03 v/v	75.3	38.3		11.8		24.5		0.0		0.0
(4) 0.05 v/v	0.0	0.0		0.0		2.3		0.0		0.0
LSD (0.05) = 5.2%

Table 4. Field emergence after 21 days of six genotypes of cotton exposed to seven mutagenic treatments in Lubbock, TX in 2002.

Mutagen	TAMU		ACALA		Pima		TAMU		A2		A2
Dose	946-25		1517-99		S-7		GA		60-T		120-W
	- - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - - - -
Water
(1) Control	63.0	62.8		28.0		51.5		40.3		32.8
Na Azide
(5) 0.0005 v/v	66.5	81.5		20.0		58.5		30.5		25.3
(6) 0.005 v/v	67.3	84.0		14.8		53.8		54.0		76.3
(7) 0.05 v/v	59.0	73.8		19.3		65.5		25.3		46.8
Ethyl MethaneSulfonate
(2) 0.01 v/v	60.0	79.3		5.0		69.0		18.3		9.0
(3) 0.03 v/v	49.0	31.3		11.3		13.3		7.3		12.5
(4) 0.05 v/v	0.3	0.3		9.0		0.3		0.0		0.0
LSD (0.05) = 7.6%

Table 5. Total chlorophyll chimeric plants observed in six genotypes of cotton exposed to seven mutagenic treatments in Lubbock, TX in 2002.

Mutagen	TAMU		ACALA		Pima		TAMU		A2		A2
Dose	946-25		1517-99		S-7		GA		60-T		120-W
	- - - - - - - - - - - - - - - Total Seedlings - - - - - - - - - - - - - - - - - - -
Water
(1) Control	0	0		0		0		0		0
Na Azide
(5) 0.0005 v/v	0	0		0		0		0		0
(6) 0.005 v/v	0	0		0		0		0		0
(7) 0.05 v/v	0	1		1		0		0		0
Ethyl MethaneSulfonate
(2) 0.01 v/v	0	0		0		1		0		0
(3) 0.03 v/v	0	0		1		0		1		0
(4) 0.05 v/v	0	0		4		3		6		3

Naked Partially Partially Fuzzy

Seed Naked Seed Fuzzy Seed Seed

Figure 1. Seed of four cotton lines demonstrating differential expression of the “naked and tufted” phenotype after saw ginning.