Plant Cell Reports (1998) 17: 273–278
© Springer-Verlag 1998
J. K. Hemphill · C. G. A. Maier · K. D. Chapman
Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)
Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997
Abstract A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 µM) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitrogrown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 µM) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 µM BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other highfiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R1 seeds. Key words Cotton · Gossypium hirsutum L. · Pre-existing meristems · Clonal propagation · Plant regeneration Communicated by G. Phillips J. K. Hemphill · C. G. A. Maier 1 · K. D. Chapman (½) Cottonseed Development Group, Department of Biological Sciences, University of North Texas, Denton, TX 76203-5220, USA Fax no.: +1-940-565-4136 E-mail:
[email protected] Present address: Samuel R. Noble Foundation, Plant Biology, Ardmore, OK 73402, USA
1
Abbreviations AC Activated charcoal · BA 6-Benzylaminopurine · IBA Indole-3-butyric Acid · MS Murashige and Skoog
Introduction
Besides producing spinnable fibers, cotton (Gossypium) plants produce seeds with a potential multiproduct base such as hulls, oil, linters, and meal. Per ton of seed crushed, cottonseed yields 540 lb of hulls (27%), 320 lb of crude oil (16%), 160 lb of linters (8%), and 900 lb of meal (45%). These cottonseed products enter markets that are highly competitive (National Cottonseed Products Association 1990). This economic environment indicates the need for a highly efficient regeneration-transformation procedure for cotton to effectively address changes within the marketplace. Currently, transgenes are delivered to cultured plant tissues of cotton by two methodologies: particle bombardment (McCabe and Martinell 1993) and cocultivation with Agrobacterium tumefaciens (Firoozabady et al. 1987; Umbeck et al. 1987). Both methodologies produced transgenic plants, with different degrees of efficiency. The former method provides a means to introduce foreign genes into any elite cotton variety; however, the transformation efficiency was reported as 1 transgenic plant per 1,000 bombarded explants (McCabe and Martinell 1993). The latter method requires regeneration via somatic embryogenesis which has been successfully applied to only a few regenerable cotton cultivars (e.g., the Coker lines). Nearly 100 cotton cultivars are under cultivation in the United States and they are, in general, not as amenable to tissue culture techniques as the Coker lines (Trolinder and Xhixian 1989; Firoozabady and DeBoer 1993; Koonce et al. 1996). Therefore, this methodology involves the following: transformation of regenerable cells or callus tissues that were derived from the Coker lines; regeneration of putative transgenic plants through somatic embryogenesis; collection of transgenic T1 seeds and advancement of the desired trait(s) into
274
an agronomic background by plant breeding techniques. This strategy requires 10–14 months to obtain mature transgenic plants of the Coker lines and an additional 6–10 years are necessary to backcross the added-value traits into more agronomic cultivars. Moreover, plants regenerated from an embryogenic callus phase are sometimes sterile and/or show signs of somaclonal variation which affect both the phenotype and genotype of the plant (Stelly et al. 1989; Firoozabady and DeBoer 1993). Currently, cotton plant regeneration through somatic embryogenesis remains germplasm dependent (Koonce et al. 1996). Our initial strategy, therefore, has been to develop a rapid, germplasm-independent, plant regeneration procedure that can be coupled with efficient gene transfer methods which targets cottonseed gene expression for specific added-value products. In this paper, a procedure is reported that utilizes explants (pre-existing meristems) from three different in-vitro-grown tissue systems derived from diverse cultivars to regenerate mature R0 plants with viable R1 seeds.
Materials and methods Plant material and tissue culture media Explants or pre-existing meristems excised from three different invitro-grown tissue systems were utilized in this study. For experiments utilizing apices (pre-existing meristems) from in-vitro-grown 1-day-old seedlings, seeds were surface sterilized by the procedure described below. After a 24-h germination period, seeds with an extended radicle (>2 mm) were selected and their embryonic axes were isolated. The shoot meristems or apices were excised (approximately 2–3 mm from the apex) and placed vertically on the shoot induction medium (MS.3BA, Table 1). For experiments utilizing explants (pre-existing meristems) from in-vitro-grown 14- to 28-day-old seedlings, seeds were sterilized and germinated as described below and the following explants were isolated: shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes. These explants were placed vertically on the shoot induction medium (MS.3BA) and in the same orientation as in the intact seedling.
Table 1 Tissue culture media utilized to regenerate elongated shoots (2–3 cm) and developing plants of cotton. MS medium = Murashige and Skoog (1962) (MS) major and minor salts, MS vitamins, sucrose (Sigma) at 15 g/l, Phytagel (Sigma) at 2.2 g/l, pH 6.0. Modifications of MS.3BA = (1) B5 vitamins (Gamborg et al. 1968) and (2) modified Nitsch and Nitsch (1969) vitamins – thiamine-HCL at 10 mg/l and nicotinic acid at 0.5 mg/l. Plant growth regulators and activated charcoal were supplied by Sigma Stage
Medium
Composition
Days
Seed germination
MSGERM
14–28
Shoot induction Shoot maturation Root induction
MS.3BA
1/2 MS medium with sucrose (10 g/l), Phytagel (2.25 g/l), without hormones (pH 7.0) MS medium with 0.3 µ M BA MS medium with 3 g/l AC MS medium with 1 µ M/l IBA
MS3AC MS1IBA
21 14–21 28–42
For experiments utilizing explants (pre-existing meristems) from established in-vitro-grown shoots (3–5 leaf stage), apices and nodal meristems were isolated. For a continuous source of established invitro-grown shoots, apices and nodal meristems were excised periodically and placed on one of three shoot induction media (MS.3BA and two vitamin modifications of MS.3BA). Initially, when cultured on MS.3BA, these established shoots derived from diverse germlines (Paymaster HS26, CA-3076, Stoneville 7A, and Stoneville 474) senesced after approximately 4 weeks. Subsequently, other vitamin sources were tested to improve the culturability of in-vitro-grown shoots for an extended period of time (5–6 weeks). The vitamin sources that supported the best extended growth were: (1) B5 vitamins (Gamborg et al. 1968) and (2) modified Nitsch and Nitsch (1969) vitamins – thiamine-HCL at 10 mg/l and nicotinic acid at 0.5 mg/l (Table 1).
Seed sterilization and germination For seed sterilization, seeds (approx. 150) were placed in distilled water with Tween-20 (two drops per 100 ml) and washed with gentle brushing for several minutes. Following three rinsing steps with distilled water, the seeds were wrapped in several layers of cheesecloth and submerged in running distilled water for 3 h. Following this soaking step and under sterile conditions, the seeds were placed in 70% ETOH containing Tween-20 (two drops per 100 ml) for 1 min. The seeds were collected in a sterile strainer and rinsed with sterile ultrapure water (Milli-Q plus UF) for 3 min. After decanting the rinsing water, the seeds were placed in sterile 20% commercial bleach plus Tween-20 (two drops per 100 ml) for 20 min with continual rotation. The seeds were then thoroughly rinsed (3×3 min) with sterile Milli-Q plus UF water. This seed sterilization procedure required approximately 4 h to complete. Following the rinsing steps, the seeds were placed on sterile, moistened (2 ml of Milli-Q plus UF water) filter paper within a Petri plate and allowed to germinate in the dark (30°C). Germinating seeds were harvested from the Petri plates on a daily basis for 3 days. After germination, seed coats were removed and seeds were placed on seed germination medium (MSGERM, Table 1) in Magenta boxes and allowed to grow for 2 days under dark conditions (30°C) and then under growth chamber conditions (30°C; 85 µmol m–2 s–1; 16 h photoperiod) for a designated period (14–28 days). After 7–10 days, inverted sterile Magenta boxes and couplers were added to the existing Magenta boxes containing growing seedlings to allow for their continual growth. After a designated period (14–28 days), apices, secondary leaf nodes, primary leaf nodes, cotyledonary nodes, and, in preliminary experiments, other seedling explants (e.g., hypocotyls) were excised from in-vitro-grown cotton seedlings. Plant regeneration This plant regeneration procedure was designed to generate elongated shoots 2–3 cm in length from explants within 21 days. This was accomplished by culturing explants (containing pre-existing meristems) from in-vitro-grown tissues as described above in an upright position on a defined medium: MS major and minor salts, MS vitamins, sucrose (15 g/l), and Phytagel (2.2 g/l) (herein referred to as MS medium, Table 1). Growth hormones, 6-benzylaminopurine (BA; shoot induction–MS.3BA) and indole-3-butyric acid (IBA; root induction – MS1IBA), were added individually to the MS medium before autoclaving (Table 1). For elongated shoots 2–3 cm or greater, a shoot maturation step (MS3AC) (Table 1) was used to reduce endogenous levels of BA and to prepare shoots for rooting (14–21 days). For elongated shoots less than 2–3 cm, the tissues were transferred first to MS.3BA (14–21 days), and then to a modified MS3AC (plus BA at 0.3 µM) to support a continual elongating response (14–21 days), and next to MS3AC for shoot maturation. For rooting of elongated shoots, two methods were studied: an invitro culture step with IBA and an in-vivo step with Rootone. The former step consisted of a root induction medium where IBA at 1 µM was added to the MS medium (MS1IBA). The latter step consisted
275 of dipping the basal end of the shoot into Rootone and then transferring the shoot to autoclaved potted soil as described below. Except when noted, the culture medium used was adjusted to pH 6.0 with 1 N NaOH or 1 N HCl prior to adding Phytagel, and autoclaved at 120°C for 18 min. Petri dishes (100×25 mm) were sealed with Parafilm and Magenta boxes were covered with polypropylene enclosures and sealed with Parafilm. All cultures were maintained at 30°C and at constant light intensity (85 µmol m–2 s–1) under a 16-h photoperiod. The light source consisted of cool white fluorescent lamps. To initiate the rooting process, the potted shoots were hardened by enclosure within plastic bags to generate a humid environment. After 2–3 weeks, the plants were progressively removed from within the enclosed bags. For plant development, the regenerants were maintained at 30°C and at a light intensity of 155 µmol m–2 s–1 under a 16-h photoperiod. The light source consisted of cool white fluorescent and incandescent lamps. Shoots/plants were watered daily and a nutritional solution of Miracle-Gro (0.75 g/gallon, Stern’s Miracle Grow, Port Washington, NY) was used every 3rd day. Rooting was accomplished in 3/4 potting soil and 1/4 vermiculite. For plant maturation and flowering, 4–5 leaf stage plants were moved to a greenroom and maintained under the following environmental conditions: temperature, Hi (32–37°C) to Low (20–24°C) (seasonal range); light intensity depended upon the location within the room (66–134 µmol m–2 s–1) and a 16-h photoperiod. The light source consisted of high-pressure sodium (140 µmol m–2 s–1) and metal halide (100 µmol m–2 s–1) lamps.
Results and discussion
Table 2 Shoot formation of excised primary leaf nodes and cotyledonary nodes derived from in-vitro-grown 14-day-old cotton seedlings cultured on MS medium containing 0.3 µM BA (MS.3BA) Cultivar
Explants (n)
Shoots/ Explants explant forming (n) shoots (%)
Time required for shoot formation (days)
Stoneville 7 A Paymaster HS 26
46 37
2–5 2–5
7–21 a 7–21 a
a
94 97
Elongated shoots (2 – 3 cm) after a 3-week culture period
Table 3 Comparison of different cotton explants for their capacity to form shoots when cultured on MS.3BA. Explants were excised from three different in-vitro-grown tissue systems Explants
Response
Apices a,b,c Secondary leaf nodes b,c Primary leaf nodes b Cotyledonary nodes b Meristems from established shoots c Foliar leaves b Epicotyl segments b Hypocotyl segments b Cotyledon pieces b
Shoots Shoots Shoots Shoots Shoots Callus Callus Callus Callus
a
In-vitro 1-day-old germinating seeds (Stoneville 7A) In-vitro-grown 14-day-old seedlings (Stoneville 7A) Established in-vitro-grown shoots (Stoneville 7A, Paymaster HS 26, Stoneville 474, and CA-3076)
b c
The cotton plant regeneration procedure as described in this paper was first observed in a preliminary experiment where BA was tested for its ability to promote shoot formation from excised cotyledonary nodes that were derived from in-vitro-grown 14-day-old seedlings (Stoneville 7A; data not shown). This pilot experiment suggested that 0.3 µM BA when added to MS medium promoted shoot formation from excised cotyledonary nodes (pre-existing meristems). In contrast, higher concentrations of BA (3.0 µM and greater) suppressed shoot formation during the same 3-week culture period. In a follow-up experiment, 0.3 µM BA promoted a high rate of shoot formation from cultured primary leaf nodes and cotyledonary nodes that were derived from in-vitro-grown 14-day-old seedlings of both Stoneville 7A (94%) and Paymaster HS26 (97%) (Table 2). These shoots began to emerge at 7 days and elongated shoots were formed after 21 days. Using MS.3BA as a screening system, different explants derived from in-vitro-grown 1-day-old seedlings (Stoneville 7A), in-vitro-grown 14-day-old seedlings (Stoneville 7A) and from established in-vitro-grown shoots (Stoneville 7A) were tested for their ability to form shoots (Table 3). After a 3-week culture period, explants with preexisting meristems such as apices, secondary leaf nodes, primary leaf nodes, and cotyledonary nodes formed shoots; the remaining explants formed crystalline-type callus mostly at the cut ends of the explant. Occasionally, a hypocotyl segment formed a shoot which presumably developed from the pericycle region. Using a BA concentration screen (0.0–1.0 µM), excised apices from secondary leaf nodes, primary leaf nodes, and cotyledonary nodes which were derived from in-vitro-
Table 4 Effect of BA on shooting efficiencies (number of shoots/ number of pre-existing meristems) of secondary leaf nodes, primary leaf nodes, and the cotyledonary nodes excised from in-vitro-grown 28-day-old seedlings of cotton (Stoneville 7A) BA (µ M)
Week 2 Week 3 a
0.0
0.15
0.3
0.5
1.0
3/9 5/9
14/17 15/17
18/24 19 a/24
12/15 15 a/15
5/8 7/8
Elongated shoots (2–3 cm) after a 3-week culture period
grown 28-day-old cotton seedlings (Stoneville 7A) were stimulated to form shoots (Table 4). However, the optimal BA concentrations based on the development of elongated shoots (2–3 cm in height) after a 3-week culture period were 0.3 or 0.5 µM BA in MS medium (Table 4, Fig. 1). Similar results were found for Paymaster HS26 (Table 5). Shoot meristems (apices) from both Stoneville 7A and Paymaster HS26 were initiated to form elongated shoots when cultured on MS medium plus 1.0 µM BA (data not shown). When tested in a BA screen (0.0, 0.3, and 1.0 µM) for shoot formation, secondary leaf nodes, primary leaf nodes, and the cotyledonary nodes (pre-existing meristems) that were excised from in-vitro-grown 28-day-old seedlings of diverse genotypes formed elongated shoots at 0.3 µM BA (Table 6). In most cases, hormone-free medium and MS medium plus 1.0 µM BA supported shoot formation; how-
276
Fig. 1 a – f Plant regeneration scheme for cotton (Stoneville 7A). Explants such as secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 14-day-old seedlings were used as starting material. As viewed, primary leaf nodes a were cultured on MS.3BA for 3 weeks to initiate elongated shoots b; elongated shoots were induced to form roots on MS1IBA c and then transferred to soil d to develop flowers and set viable R1 seeds e. Multishoot formation was observed when elongated shoots were maintained on MS.3BA for 4 weeks or more f
ever, these media combinations failed to yield elongated shoots (2–3 cm or greater) during the 3-week culture period (Tables 4–6). Apices isolated from in-vitro-grown 1-day-old seedlings (Stoneville 7A and Paymaster HS26) formed shoots when cultured on MS medium plus a BA concentration screen (0.0, 0.3, 0.5, 1.0, and 10 µM). After a 1-week culture period, a range of BA levels (0.3 to 1.0 µM) supported normal growth with green-leaf development and hypocotyl elongation. The concentration of 10 µM BA, however, sup-
277
ported only small, light-green leaves without hypocotyl elongation. The control (hormone-free) in this experiment supported normal growth with green-leaf development, hypocotyl elongation, and root formation at the cut end of the elongated hypocotyl (data not shown). Shoot apices and nodal meristems isolated from established in-vitro-grown shoots (3–5 leaf stage) which were maintained on MS.3BA (or its vitamin modifications) formed elongated shoots within 3 weeks. Nodal meristems require 0.3 µM BA (MS.3BA). Shoot apices required a higher level of BA (1.0 µM). These meristems, however, failed to form elongated shoots on hormone-free medium during this 3-week culture period (data not shown). In this study, shoots were rooted by one of two methods: in-vitro culture on MS1IBA, or by transferring the shoot (+Rootone) directly to soil following a shoot maturation step (MS3AC; Table 7). Elongated shoots of both Stoneville 7A and Paymaster HS26 demonstrated a low efficiency of root formation when cultured on MS1IBA. However, both cultivars demonstrated a high efficiency of rooting when placed directly into soil (+Rootone) (Table 7). All cultivars utilized in this study are handled in this latter manner. It was noted that shoots greater than 3 cm rooted more quickly (about 2 weeks) than shoots that were less than 3 cm (about 3 weeks). After a hardening period (enclosed bags) of 2–3 weeks, the potted regenerants rooted, grew in height, and developed new leaves. All regenerated shoots that were advanced to soil were phenotypically normal and all of the matured plants regenerated to date have initiated flowers and set viable R1 seeds under greenroom conditions (as previously described). Selected stages in our regeneration procedure for cotton (Stoneville 7A) are shown in Fig. 1. Primary leaf nodes containing pre-existing meristems were cultured on MS.3BA for 3 weeks to initiate elongated shoots; these shoots were induced to form roots on MS1IBA and then transferred to soil to develop flowers and set viable R1 seeds. Multiple shoot formation was observed when elongated shoots were maintained on MS.3BA for 4 weeks or more. Similar developmental stages were found for Paymaster HS26 and the other cultivars used in this study (data not shown). The maturation step (MS3AC) was developed during experimentation with Paymaster HS26; however, Stoneville 7A and the other cultivars were also responsive to this phase of the regeneration system. The plant regeneration procedure described in this report provides a method to obtain elongated shoots from pre-existing meristems that were excised from three different in-vitro-grown tissue systems of diverse germplasm. When cultured on MS medium plus 0.3 or 0.5 µM BA, secondary leaf nodes, primary leaf nodes, and cotyledonary nodes were more responsive than apices. However, apices isolated from 28-day-old seedlings of Stoneville 7A, Paymaster HS26, CA-Series, and Stovepipe developed elongated shoots when cultured on 1.0 µM BA. Based on these results, pre-existing meristems from different explant sources appear to possess different degrees of dormancy due to apical dominance and meristem location. Similar results were found for intact, light-grown seedlings of Pisum
Table 5 Effect of BA on shooting efficiencies (number of shoots/ number of pre-existing meristems) of secondary leaf nodes, primary leaf nodes, and the cotyledonary nodes excised from in-vitro-grown 28-day-old seedlings of cotton (Paymaster HS26) BA (µ M)
Week 2 Week 3 a
0.0
0.15
0.3
0.5
1.0
10.0
3/35 16/35
16/22 15/22
29/35 29 a/35
23/26 24 a/26
19/28 22/28
1/13 0/13
Elongated shoots (2–3 cm) after a 3-week culture period
Table 6 Effect of BA on shoot formation (number of shoots/number of pre-existing meristems after a 3-week culture period) from secondary leaf nodes, primary leaf nodes, and the cotyledonary nodes excised from in-vitro-grown 28-day-old seedlings of different cotton cultivars Cultivar
BA (µ M) 0.0
Paymaster HS 26 Stoneville 474 CA-3050 CA-3066 CA-3076 CA-3084 Stovepipe a
16/35 20/26 4/5 8/11 2/7 4/5 10/14
0.3
1.0
a
29 /35 44 a/62 3 a/4 15 a/17 15 a/14 10 a/10 12a/17
22/28 40/66 6/6 11/11 5/7 6/10 13/21
Elongated shoots (2–3 cm) after a 3-week culture period
Table 7 Comparison of root induction methods for elongated cotton shoots. Shoots were transferred to medium containing 1 µM IBA (MS1IBA) or rooted directly by transferring to soil (+Rootone). The direct method was preceded by the shoot maturation step (MS3AC). Results are given as the number of shoots that rooted/total number of shoots
Cultivar
In vitro culture period on MS + 1 µ M IBA
Direct transfer to soil (+ Rootone)
Stoneville 7 A Paymaster HS 26
6/23 6/27
12/23 25/28
sativum L., where lower axillary buds formed elongated, leafy shoots after a single application of either BA or zeatin (Pillay and Railton 1983). In comparison, using our regeneration system, apices from 1-day-old seedlings did not show a cytokinin requirement for the formation of elongated shoots. Similar results have been reported by Gould et al. (1991) for apices isolated from 3-day-old seedlings of cotton; a low level of kinetin (0.1 mg/l) was also shown to initiate shoots. Following a 6-month culture period, a shoot tip culture study of cotton (G. hirsutum and G. arboreum) showed that MS medium containing indoleacetic acid (0.5 mg/l) and kinetin at different concentrations (1– 6 mg/l) initiated callus, adventitious buds, and shoots. In some cases, multiple shoots were formed from shoot tip cultures when the meristems were cultured on medium con-
278
taining naphthalene acetic acid (0.5 mg/l) and BA (2 mg/l) or kinetin (6 mg/l) (Bajaj and Gill 1986). In most cases, shoots were matured by transfer to either MS.3BA, MS3AC or a combination of MS3AC+0.3 µM BA and then MS3AC as determined by the height of the elongated shoots. This series of transfers was important for shoot elongation and maturation before the shoots were transferred to soil. Following the maturation step, rooting of elongated shoots was accomplished by direct transfer to soil following a basal dip into Rootone. Using this method, high rooting efficiencies were obtained for Stoneville 7A and Paymaster HS26 in comparison with an in-vitro step utilizing MS1IBA. Gould et al. (1991) were unsuccessful in obtaining in-vitro root induction for cotton shoots derived from isolated apices; however, a high frequency (30%) was reported when cotton shoots were cultured on medium containing charcoal and then transferred to potting soil (+Rootone). To date, this BA plant regeneration procedure has been germplasm independent; however, this should be substantiated in a more extensive cultivar screen. Generally, this cloning procedure resulted in rooted plants in approximately 6 weeks–3 months. In all cases, shoot regeneration occurred directly without a callus phase. All potted plants appeared morphologically similar to seed-derived cotton plants; they flowered and produced viable R1 seeds (e.g., 137 bolls from 32 Paymaster HS26 regenerants produced 2,852 seeds of which 2,203 seeds germinated). Recently, Agrawal et al. (1997) demonstrated that excised cotyledonary nodes derived from 35-day-old cotton seedlings (cv. Anjali-LRK 516) yielded multiple shoots when cultured on MS medium plus BA and kinetin (2.5 mg/l each). Elongation of these shoots required an additional step of liquid or agar MS medium without phytohormones. The work reported in the present paper demonstrated that pre-existing meristems (apices, nodal meristems, primary leaf nodes, and cotyledonary leaf nodes) derived from three in-vitro-grown tissue systems of diverse cotton germlines could be clonally propagated by a BA plant regeneration procedure. Efforts are underway to couple this BA plant regeneration procedure with a gene transfer method for the rapid introduction of added-value traits directly into highfiber-yielding cotton germplasm. Acknowledgements We gratefully acknowledge the following contributions to this work: A. Vasanawala for technical assistance and M. Wenske for the plant regeneration data for Stoneville 474. This research was supported by grants from the National Cottonseed Products Association, The Cotton Foundation, the University of North Texas, and the Texas Higher Education Coordinating Board
Advanced Technology Program (no. 003594-014). The authors express their appreciation to the following: Dr. Rick B. Turley, USDAARS, Stoneville (Miss.) for providing Stoneville 7A seeds; Dr. John J. Burke, USDA-ARS, Lubbock (Tex.) for providing Paymaster HS26 seeds; Dr. John Gannaway, Texas Agricultural Experiment Station, The Texas A&M University System, Lubbock (Tex.) for providing CA-Series and Stovepipe seeds, and Dr. Catherine Houck, Calgene, Davis (Calif.) for providing Stoneville 474 seeds.
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