In the callus induction phase, mature caryopses were cultured on MS medium ... Callus growth and plant regeneration, however, were significantly influenced by.
In VttmCell.Dev.Biol.--Plant32:227-232,October-December1996 9 1996Societyfor In VitroBiology 1054-5476/96 $05.00+0.00
CALLUS INDUCTION AND PLANT REGENERATION OF U.S. RICE GENOTYPES AS A F F E C T E D B Y M E D I U M C O N S T I T U E N T S JAMEEL M. AL-KHAYRI, CHRISTINE E. SHAMBLIN, RONALD W. McNEW,1 ANDEDWIN J. ANDERSON~ Departrrv~ntof Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701 (Received 21 February 1996; accepted 31 May 1996; editor B. V. Conger)
SUMMARY This study was conducted to establish and optimize a regeneration system for adapted U.S. rice genotypes including three commercial rice cultivars (LaGrue, Katy, and Alan) and two Arkansas breeding lines. Factors evaluated in the study were genotype, sugar type, and phytohormone concentration. The system consisted of two phases, callus induction and plant regeneration. In the callus induction phase, mature caryopses were cultured on MS medium containing either 1% sucrose combined with 3% sorhitol or 4% sucrose alone, and 0.5 to 4 mg'L -~ (2.26 to 18.10 pM) 2,4-D with or without 0.5 tug'L-1 (2.32 p.M) kinetin. In the plant regeneration phase, callus was transferred to 2,4-D-free MS medium containing 0 or 2 mg'L -~ (9.29 ~tM) kinetin combined with 0 or 0.1 mg'L -~ (0.54 ~tM) NAA. Callus induction commenced within a week, independent of the treatments. Callus growth and plant regeneration, however, were significantly influenced by interactions among experimental factors. Generally, the greatest callus growth and plant regeneration were obtained with 0.5 mg'L -t (2.26 pM) 2,4-D and decreased with increasing 2,4-D concentrations. Kinetin enhanced callus growth only when combined with 0.5 mg-L- 1 (2.26 p3/) 2,4-D, and 4% sucrose. Inducing callus on kinetin-containing medium generally enhanced regeneration capacity in the presence of sucrose but not with a sucrose/sorbitol combination. Media containing sucrose alone generally supported more callus proliferation, but the sucrose/sorbitol combination improved regeneration of some cultivars. NAA and kinetin had little effect on regeneration. Key words: in vitro; micropropagation; Oryza sativa; seed explant; sorbitol. acterize the genotypic response and to examine (1) the effect of 2,4dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin) concentration in the callus induction medium on the proliferation and regenerative capacity of callus induced from mature seeds, (2) the effectiveness of sucrose versus a combination of sucrose and sorbitol on the proliferation of regenerative callus, and (3) the effect of kinetin and naphthaleneacetic acid (NAA) in the regeneration medium on plant development.
INTRODUCTION The importance of rice (Oryza sativa L.) as a food staple worldwide makes it a prime target for genetic manipulations through biotechnological approaches. In vitro regeneration and genetic transformation of rice have been the focus of numerous studies, and various procedures for rice regeneration that vary in the type and concentration of growth regulators, sugars, solidifying agents, basal salts, and vitamins have been described (Heyser et al., 1983; Raghava and Nabors, 1984; Kavi Kishor, 1987; Abe and Futsuhara, 1989; Bajaj, 1991; Ogawa et al., 1992; Tsukahara and Hirosawa, 1992a, 1992b; Kothari et al., 1993; Hiei et al., 1994; Rueb et al., 1994; Guo et al., 1995). However, only a few studies have been conducted on U.S. germplasm (Chen and Luthe, 1987; Oard and Rutger, 1988; Mirlohi et al., 1989). Furthermore, conditions that are optimal for regenerating one cultivar often fail to produce plants in cultures of other cultivars (Abe and Futsuhara, 1986; Bhaskaran and Smith, 1990). The diversity of the available rice regeneration systems, coupled with the specificity of the requirements dictated by genotypes, necessitates the determination of the culture conditions suitable for genotypes of interest. This study was conducted to develop a regeneration system for U.S. rice genotypes. Our objectives were to char-
MATERIALSAND METHODS Plant material and sterilization. Rice (Oryza sativa L.) caryopses of cultivars 'LaGme,' 'Katy,' and 'Alan,' and Arkansas breeding lines Ark Ru 9101001 (abbreviated Ru91) and Ark Ru 9201191 (abbreviated Ru92)were obtained from the Arkansas Agricultural Experiment Station, Stuttgart, Arkansas. Caryopses were dehusked manually and surface sterilized for I min in 70% ethanol followed by 30 rain in 2.6% (wt/val) sodium hypochlorite (50% vol/vol Clorox) containing 0.1% Tween 20. The caryopses were rinsed several times in sterile distilled water and cultured on callus induction medium. Callus induction. The callus induction medium consisted of MS salts (Murashige and Skoog, 1962) containing (in mg'L-l) 1.0 thiamine-HC1, 1.0 pyridoxine-HC1, 1.0 nicotinic acid, 2.0 glycine, 100.0 myo-inositol, and 10.0 g'L -~ agar (Agar-agar/Gum agar) (Sigma Chemical Co., St. Louis, MO). The medium was adjusted to pH 5.8 with 1 N KOH, autoclaved at 121~ C and 1 • l0 s Pa (1.1 kg/cm2) for 15 min, and dispensed in 100 • 15-ram petfi dishes (25 ml medium per dish). The medium was also supplemented with either 1% sucrose combined with 3% sorbitol (sucrose/sorbitol) or 4% sucrose alone, 2A-D at 0.5, 1.0, 2.0, or 4.0 mg'L -l, and kinetin at 0 or 0.5 mg.L -l.
~Agricuhure Statistics Laboratory, University of Arkansas, Fayetteville, Arkansas 72701. 2To whom correspondence should be addressed at Department of Plant Pathology, 217 PTSC, University of Arkansas, Fayetteville, Arkansas 72701. 227
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AL-KHAYRI ET AL.
To induce callus, caryopses (10 caryopses per dish and four dishes per treatment) were placed horizontally on the surface of the medium, and the plates were sealed with Parafilm. The cultures were incubated at 24 + 2~ C under a 12-h photoperiod of cool-white fluorescent light (40 Ilmol-m-2"s- 1). After 4 weeks, callus was separated from the explants, transferred to fresh callus induction medium, and cultured for an additional 4 weeks for further proliferation. Individual calluses were weighed, divided into approximately 0.5 cm3 segments, and randomly placed on regeneration medium. Plant regeneration. The regeneration media contained the same basal components as just described, including the corresponding sugar supplement. The hormonal content, however, was changed to kinetin at 0 or 2.0 mg'L-~ combined with NAA at 0 or 0.1 mg'L-L The cultures were maintained for 12 weeks under the same conditions as the callus induction stage, during which time they were transferred to fresh media at 4-week intervals. Plant establishment. Plantlets about 1 cm long were separated from the regenerating callus and transferred to a hormone-free medium dispensed in 150 x 25-mm culture tubes (15 ml medium per tube). When the plants reached 5 to 10 cm in length, they were removed from the tubes, the residual agar was washed from the roots with tap water, and the plantlets were planted in 5-cm pots filled with potting mix (Redi-Earth Peat-Lite Mix, Grace-Sierra Hort. Products Co., Milpitas, CA). The plants were watered with half-strength MS basal salts, grown in clear plastic (polystyrene) containers with lids, and misted with tap water. The humidity was reduced during a 2-week period by gradually opening the container's lids. The plants were then transferred to a greenhouse, and 2 weeks later they were transplanted into 15-cm pots. Statistical analysis. To evaluate the effect of callus induction treatments, the experiment was conducted as a completely randomized 5 X 4 • 2 X 2 factorial design. The main factors were genotype at five levels, 2,4-D concentration at four levels, kinetin concentration at two levels, and sugar at two levels. The analysis of the effects of treatments on callus weight was based on 20 calluses per treatment chosen at random from the culture plates. To evaluate the effect of the regeneration treatments, the experiment was set up as 5 X 2 x 2 x 2 factorial with main factors being genotype at five levels, kinetin, NAA, and sugar each at two levels. The analysis for the percentage of plant regeneration was based on 10 to 15 callus segments per treatment. Data were subjected to analysis of variance (ANOVA), and levels of a factor, with all other factor levels held constant, were compared with a least-significant-difference test (LSD) at the 5% significance level. Transformation of the proportion data was not necessary.
RESULTS AND DISCUSSION Callus Induction
TABLE 1 ANALYSIS OF VARIANCE OF THE EFFECTS OF CALLUS INDUCTION TREATMENTS AND REGENERATION TREATMENTSON CALLUS PROLIFERATION AND ON SUBSEQUENTPLANT REGENERATION IN RICE P-value Source
Callus weight
DF
% Plant
regeneration
Callus induction treatments Genotype Kinetin 2,4-D Sugar Genotype x kinetin Genotype • 2,4-D Genotype X sugar Kinetin • 2,4-D Kinetin • sugar 2,4-D • sugar Genotype x kinetin X 2,4-D Genotype X 2,4-D • sugar Genotype • kinetin X sugar Kinetin X 2,4-D X sugar Genotype X kinetin • 2,4-D X sugar
4 1 3 1 4 12 4 3 1 3 12 12 4 3 12
0.0001" 0.1550 0.0001" 0.0001" 0.0013" 0.0001" 0.0010" 0.0001" 0.0001" 0.0001" 0.2106 0.0018" 0.0034* 0.0001" 0.0619
Plant regeneration treatments Genotype 4 Kinetin 1 NAA 1 Sugar 1 Genotype X kinetin 4 Genotype X NAA 4 Genotype X sugar 4 Kinetin • NAA 1 Kinetin • sugar 1 NAA X sugar 1 Genotype X kinetin • NAA 4 Genotype X kinetin X sugar 4 Genotype • NAA X sugar 4 Kinetin X NAA • sugar 1 Genotype • kinetin • NAA X sugar 4
0.0001" 0.4240 0.0001" 0.0134" 0.0001" 0.0001' 0.0001" 0.0019' 0.0001' 0.0001" 0.0001" 0.0007* 0.0001" 0.0067* 0.0551 0.0001' 0.0460* 0.8743 0.0001" 0.0020* 0.2558 0.0001" 0.4033 0.0776 0.0546 0.0005* 0.0001" 0.0226* 0.0219" 0.0019"
Significant value (P < 0.05). Rice caryopses began to swell within 3 days after culturing. Initially, the caryopses germinated and produced roots and shoots. This occurred particularly on media containing low concentrations of 2,4D (0.5 and 1.0 mg'L-t). The germination process was inhibited, especially root emergence, as the 2,4-D concentration increased to 2.0 mg'L-L Within a week, growth of seedlings ceased, and callus proliferation commenced at the crown region. At the highest 2,4-D (4.0 mg'L- 0 concentration, germination was completely inhibited, resulting in callus proliferation directly from the embryo regions of the caryopses. A previous study showed that rice callus proliferation occurs from the scutellum of embryos (Brisibe et al., 1990). Brisibe et al. (1992) showed that culturing excised rice embryos did not enhance the frequency of callus proliferation. Therefore, only intact caryopses were used in the present study. Ninety to 100% of the caryopses produced callus on all treatments. However, the amount of callus proliferation was significantly affected by interactions of the experimental factors. Interactions of the medium components with rice genotypes were observed to significantly influence callus proliferation and subsequent plant regeneration, as indicated by three-way interactions resuiting from the analysis of variance (Table 1). Because of the com-
plexity of these interactions, the effect of each experimental factor will be discussed as it relates to the other factors. Effect ofgenotype. Intergenotypic differences in response to tissue culture factors have been observed in rice (Bhattacharya and Sen, 1980; Wernicke et al., 1981; Abe and Futsuhara, 1986; Oard and Rutger, 1988). In the present study, callus weight varied among genotypes and ranged from 95 to 180 mg with 4% sucrose (Fig. 1). With the sucrose/sorbitol combination, callus growth was less and ranged from 60 to 115 mg (Fig. 1). The variability among genotypes was less pronounced when callus induction medium contained a sucrese/sorbitol combination. Nevertheless, significant differences in callus weight were observed among genotypes with both sugar supplements. In ascending order, RU92 produced the least amount of callus, followed by Katy, Ru91, LaGrue, and Alan (Fig. 1). (This order is correct when all combinations are considered.) Effect ofkinetin. The effect of kinetin on callus growth was variable, depending on the genotype and the sugar supplement. When the medium was supplemented with the sucrose/sorbitol combina-
VARIABLESIN U.S. RICE REGENERATION
Fig. 1. Callus proliferation from mature rice caryopsesas influenced by an interaction of genotype,kinetin concentration,and sugar supplement.
tion, the addition of kinetin inhibited callus growth in all genotypes, but the inhibition was significant only in Katy and LaGrue (Fig. 1). Conversely, when the medium was augmented with sucrose alone, callus weight of Katy and Alan declined significantly, but callus weights of LaGrue, Ru91, and Ru92 significantly increased in response to kinetin (Fig. 1). These findings were consistent with those of Oard and Rutger (1988), who observed that the stimulation of callus growth by the addition of kinetin was genotype-dependent and that different concentrations were required to maximize callus growth for different rice genotypes. Considering all genotypes, kinetin generally significantly stimulated callus growth when added to a medium containing 4% sucrose and 0.5 mg.L- 1 2,4-D (Fig. 2). However, to greater or lesser degrees, kinetin inhibited callus growth on media containing the other sugar and 2,4-D treatments evaluated (Fig. 2). Effect of sugar supplements. Sucrose is generally used as the major carbohydrate source in tissue culture media. Sorbitol also has been reported to enhance plant regeneration of rice when applied to the regeneration medium (Ozawa and Komamine, 1989; Yoshida et al., 1994). However, information on the effect of sorbitol on rice callus induction is scarce. Therefore, in this study we included sorbitol in the culture initiation medium rather than limiting it to the regeneration phase. Less callus growth resulted on media containing the sucrose/sorbitoI suppIement at all concentrations of 2,4-D and kinetin, but the extent of the reduction varied significantly among genotypes (Fig. 2). As the concentration of 2,4-D increased, the difference in callus weight between sucrose and sucrose/sorbitol treatments became smaller or completely insignificant (Fig. 2). This suggests that the concentration of 2,4-D exerted greater control on callus growth than did the sugar supplement.
229
Effect of 2,4-D concentration. In previous studies, the concentration of 2,4-D used in the callus induction medium for rice differed among tissue culture systems. Previously reported 2,4-D concentrations varied from 0.5 mg'L -~ (Chen and Luthe, 1987), 2.0 mg'L -~ (Kavi Kishor, 1987; Abe and Futsuhara, 1989; Rueb et al., 1994), to 4.0 rag-L-t (Tsukahara and Hirowawa, 1992a, 1992b). Oard and Rutger (1988) observed that the optimum concentration of 2,4-D for callus proliferation ranged from 0.2 to 1.0 mg'L-1, depending upon genotype, and that a higher level (2.0 mg'L -~) was inhibitory for all five rice genotypes included in their study. In the present study, increasing the concentration of 2,4-D inhibited callus growth (Fig. 2), but the degree of inhibition varied among genotypes. Genotypes LaGrue, Alan, and Ru91 showed a marked decrease in callus weight as the 2,4-D level increased from 0.5 mg'L-~ to 1.0 mg'L-' when the medium was supplemented with sucrose alone, but only a slight decrease was observed in cultures containing sucrose/sorbitol, the sugar supplement that was associated with reduced callus weights (Fig. 2). For Katy and Ru92, no significant differences in callus weight were detected on sucrose/sorbitol medium in response to increasing the concentration of 2,4-D (data not shown). In summary, the most important factor affecting callus growth was the concentration of 2,4-D. Although callus growth was obtained with the sucrose/sorbitol combination, callus proliferation was inferior in comparison to the sucrose treatment. The effect of kinetin on callus growth was genotype-specific. The best medium for callus induction for these genotypes should contain 0.5 mg-L-~ (2.26 ~M) 2,4-D, 4% sucrose, and either no kinetin for culturing Katy and Alan or 0.5 mg-L-l (2.32 gM) kinetin for culturing LaGrue, Ru91, and Ru92. For long-term maintenance of callus from these rice genotypes the 2,4-D level should be increased to 1.0 mg'L-~ (4.52 gM) because
FIG.2. Callus proliferation frommature rice caryopsesas influencedby an interaction between concentrations of kinetin, 2,4-D, and sugar supplement.
230
AL-KHAYRIET AL.
FIG. 3. Effect of the interaction of 2,4-D concentration, sugar supplements, and kinetin added to the regeneration medium on the percentage of plant regeneration for rice.
ments (Table 2). Interestingly, for some genotypes, there appeared to be an inverse relationship between callus weight and plant regeneration capacity. For example, Alan produced the largest callus but exhibited the lowest regeneration frequency, whereas Ru92 produced the least amount of callus but had the highest regeneration frequency. Furthermore, plant regeneration percentage was significantly influenced by the treatments to which calluses were subjected during the induction phase. Based on an ANOVA (Table 1), three-way interactions among the callus induction factors were observed to influence regeneration. Effect of kinetin. The addition of kinetin to the callus induction medium appeared to exert the least influence of all factors studied for callus regeneration capacity (Table 1). Nevertheless, kinetin differentially affected subsequent plant regeneration of various genotypes. Kinetin at 0.5 mg'L -t affected some genotypes in a manner that was dependent on 2,4-D concentration (Table 2). For example, kinetin had no significant effect on plant regeneration of Alan but stimulated the regeneration of Katy and LaGrue when added to media containing 0.5 mg'L -a or 2.0 mg'L -t 2,4-D, respectively (Table 2). The inhibition by kinetin observed in Ru91 occurred with 0.5 mg'L-~ 2,4-D (Table 2). Kinetin also inhibited regeneration of Ru92 when added to media containing 0.5 rag-L-~ 2,4-D, but this hormone improved regeneration in the presence of higher concentrations of 2,4-D (Table 2). The influence of kinetin was also dependent on the sugar supplement and genotype. When added to media containing the sucrose/ sorbitol combination, kinetin exerted no significant effect on the re-
lower concentrations allowed redifferentiation of the callus after prolonged culture (data not shown). Plant Regeneration A common method used for the regeneration of rice and other monocotyledonous species depends on withdrawing auxin from the medium. Generally, regeneration capacity is influenced by genotype and the components of both the callus induction and regeneration media. Manipulation of medium components, particularly the phytohormones, dramatically alters regeneration capacity and may influence the mode of regeneration in rice (Yoshida et al., 1994). In the current study, calluses produced only roots, rhizogenesis, or complete plantlets with root and shoot systems when transferred to a 2,4D-free regeneration medium. Because rhizogenesis was irrelevant to the objective of this study, the following discussion is limited to data on plant regeneration. Plant regeneration was observed as early as 3 weeks and continued to occur up to 10 weeks after calluses were placed on regeneration media. The earliest plant formation occurred from callus induced on 0.5 mg'L -~ 2,4-D, the concentration of 2,4-D that supported the greatest callus proliferation. Since achieving plant regeneration requires the reduction or elimination of 2,4-D, the expedited regeneration from callus grown on a low concentration of 2,4-D may be due to a low endogenous level of 2,4-D in the callus tissue. Effect ofgenotype. Like callus proliferation, regenerative capacities vary considerably among rice genotypes (Abe and Futsuhara, 1986; Oard and Rutger, 1988). After 12 weeks of culturing, overall averages of plant regeneration for each genotype were: Alan 15%, LaGrue 22%, Katy 26%, Ru91 34%, and Ru92 49%. However, higher frequencies of regeneration were associated with certain treat-
TABLE 2 EFFECTS OF CALLUSINDUCTIONTREATMENTSON THE PERCENTAGE OF SUBSEQUENTPLANT REGENERATIONFOR SEVERAL RICE GENOTYPES % Plant regeneration 2,4-D Concentration (rag L-1) Genotype
0.5
1
2
4
0.5
No kinetin Alan Katy LaGrue Ru 91 Ru 92 LSDb (0.05)
24a 29 20 83 62
19 26 24 57 48
19 26 20 14 32
1
2
4
0.5 mg L-~ kinetin 2 4 13 18 36
13R 67 21 18 42
14 26 27 48 64
16 11 38 17 52
6 17 12 15 58
14 1% Sucrose and
3% sorbitol Alan Katy LaGrue Ru 91 Ru 92 LSDb (0.05)
29' 52 23 47 40
24 36 25 55 53
29 35 28 14 35
4% Sucrose 3 20 9 14 42
9" 45 19 51 61
9 17 27 48 59
7 2 31 9 45
5 2 17 17 46
17
Walues are averages of total plant regeneration when callus induction media contained either sugar supplement. SLeast-significant-differencetest at the 5% significance level. ~ are averages of total plant regeneration when callus induction media contained either kinetin treatment.
231
VARIABLES IN U.S. RICE REGENERATION TABLE 3 EFFECTS OF THE REGENERATIONMEDIUMTREATMENTS ON THE PERCENTAGEOF PLANT REGENERATIONFOR SEVERAL RICE GENOTYPES. % Plantregenerationwith: NoNAA Genotypr Alan Katy LaGrue Ru91 Ru92 LSDb (0.05)
0.1 mgI.-* NAA
Sugar"
kmetin
No
2mgL
kmetm
~
kinetm
No
Suc/sor Suc Suc/sor Suc Sue/sot Suc Sue/sot Suc Suc/sor Suc
17 l0 43 12 19 28 42 22 30 56
23 6 29 11 23 20 30 44 45 52
22 7 37 15 21 13 39 39 35 55
2mgL-
kinetin 22 6 34 29 23 33 24 36 44 64
15
"Sugar supplements: suc/sor = 1% sucrose + 3% sorbitol; suc = 4% sucrose. hLeast-significant-differencelest at the 0.05 significance level.
generation frequencies of any genotype except Ru91 (data not shown). However, when media contained sucrose alone, kinetin significantly stimulated subsequent plant regeneration in Katy and Ru92 but not other genotypes (data not shown). Because kinetin did not necessarily benefit both the quantity and regenerative capacity of callus, the decision to include kinetin is based on genotype and the culture purpose, i.e., massive callus or regenerative callus. Effect of sugar supplements. The sugar combination selected in this study was based on a previous report by Tsukahara and Hirosawa (1992a) in which this combination was shown to improve regeneration of rice ev. 'Sasanishiki'. Although callus formation generally was inhibited by the sucrose/sorbitol combination, this treatment generally improved regeneration of Alan and Katy and was as effective as sucrose alone for the regeneration of LaGrue and Ru91, but inhibited the regeneration of Ru92 (Table 2). These results demonstrate that the effect of the carbohydrate source in the regeneration medium is genotype dependent. Effect of2,4-D concentration. When responses of all genotypes were taken together, incnrasing concentrations of 2,4-D in the callus induction medium generally reduced the regeneration capacity of callus in a manner that was influenced by genotype, the level of kinetin, and the sugar supplement (Fig. 3; Table 2). On media containing a sucrose/sorbitol combination, the regeneration capacity of callus induced with 0.5 to 2.0 mg'L -~ 2,4-1) was inhibited in the presence of kinetin (Fig. 3). However, the regeneration capacity of callus induced on 4.0 mg.L -~ 2A-D was unaffected by kinetin or sugar treatments (Fig. 3). On media containing sucrose alone, the regenerative capacity of callus induced with 1.0 and 2.0 mg'L- ~2,4D was further enhanced by kinetin, but at 0.5 mg'L- ' 2,4-D, kinetin inhibited regeneration (Fig. 3). Effect of NAA and kinetin in the regeneration medium. The presence of 0.1 mg-I.-' NAA in the regeneration medium generally had no significant effect on the regeneration of Alan, LaGrue, or Ru92
(Table 3). NAA significantly stimulated the regeneration of Katy and Ru91 in the presence of 4% sucrose, with 2.0 mg'L-~ kinetin or without kinetin, respectively (Table 3). Yoshida et al. (1994), in a study with rice cv. 'Kamenoo', observed that increasing the NAA lew:l in the regeneration medium from 0.1 to 1.0 mg'L-' either increased regeneration rate or caused no difference, depending on the other growth regulators present in the regeneration medium. In another study, the addition of NAA alone in the regeneration medium was found to inhibit regeneration of the rice cv. 'Sasanishiki', but when kinetin was also included in the regeneration medium, a substantial increase in regeneration was obtained (Tsukahara and Hirosawa, 1992a). The current study showed that NAA had a very limited effect on the regeneration of the genotypes tested (Table 3). Yoshida et al. (1994) found that 0.1 and 0.5 mg'L -~ kinetin were equally effective in the regeneration of rice cv. 'Kamenoo'. In another study, up to 2.0 mg'L -~ kinetin stimulated the regeneration of cv. 'Sasanishiki' (Tsukahara and Hirosawa, 1992a). In the current study, the inclusion of 2.0 tug'L- ~kinetin in regeneration media containing 4% sucrose and NAA significantly enhanced the regeneration frequency of LaGrue. This combination of kinetin and sucrose also stimulated regeneration of Ru91 in the absence of NAA. The other genotypes were unaffected by kinetin (Table 3). These results further demonstrate the genotype-speeific requirements observed throughout these studies. Similarly, Bhattacharya and Sen (1980) indicated that regeneration of rice in response to kinetin was stimulated, inhibited, or unchanged depending upon the genotype and other growth regulators present in the medium. Nearly 100% of the regenerants survived in potting mix under greenhouse conditions, regardless of cuhivar or the in vitro treatment. Under greenhouse conditions, regenerants grew normally and produced viable seeds. In conclusion, we have developed and optimized protocols for regenerating five important U.S. rice cultivars and breeding lines. Our results further emphasize the importance of genotype and its interaction with medium componcnts in the development of in vitro systems. The rice genotypes selected for the current work are important commercial varieties and breeding lines which had not previously been regenerated, a prerequisite to our future efforts to genetically improve these rice varieties and breeding lines using genetic transformation techniques. ACKNOWLEDGMENTS Support for this research was provided by the Arkansas Rice Research and Promotion Board. The authors wish to thank K. A. K. Moldenhauer and K. A. Gravoisfor supplying rice seed and A. S. Kline for technical assistance. REFERENCES Abe, T.; Futsuhara, Y. Genotype variability for callus formation and plant regeneration in rice (Oryza saliva L.). Theor. App]. Genet. 72:3-10; 1986. Abe, T.; Futsuhara, V. Selection of higher regenerative callus and change in isozymepattern in rice (Oryzasaliva I,.). Theor. Appl. Genet. 78:648652; 1989. Bajaj, Y. P. S. Biotechnologyin agmcultureand forestry, Vol. ] 4, Rice. Berlin: Springer-Verlag; 1991:645 pp. Bhaskaran. S.; Smith, R. H. Regeneration in cereal tissue culture: A review. Crop Sci. 30:1328-1336:, 1990. Bhattacharya, P.~ Sen, S. K. Potentiality of leaf sheath cells for regeneration of rice (Oryza sativa L.). Theor. Appl. Genet. 58:87-90; 1980.
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Oard, J. H.; Rutger, J. N. Callus induction and plant regeneration in elite U.S. rice lines. Crop Sci. 28:565-567; 1988. Ogawa, T.; Hagio, T.; Ohkawa, Y. Plant regeneration from isolated pollen grains in Indica type rice (Oryza sativa L.). Japan. J. Breed. 42:657679; 1992. Ozawa, K.; Komamine, A. Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L). Theor. Appl. Genet. 73:205-211; 1989. Raghava, R.; Nabors, M. W. Cytokinin mediated long-term, high-frequency plant regeneration in rice tissue cultures. Z. Pflanzenphysiol. 113:315-323; 1984. Rueb, S.; Leneman, M.; Schilperoort, R. A., et al. Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryzasativa L.). Plant Cell Tissue Organ Cult. 36:259-264; 1994. Tsukahara, M.; Hirosawa, T. Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. But. Mag. 105:227233; 1992a. Tsukahara, M.; Hirosawa,T. Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus. Plant Cell Rep. 11:550553; 1992b. Wernicke, W.; Brettell, R.; Wakizuka, T., et al. Adventitious embryoid and root formationfrom rice leaves. Z. Pflanzenphysiol.Bd. 103:361-365; 1981. Yoshida, K. T.; Fujii, S.; Sakata, M., et al. Control of organogenesis and embryogenesis in rice calli. Breed. Sci. 44:355-360; 1994.
