In Vitro Cell. Dev. Biol. 30P:187-191, October 1994 �9 1994 Society for In Vitro Biology 1054-5476/94 $02.50+0.00

SHOOT ORGANOGENESIS FROM CULTURED SEED EXPLANTS OF PEANUT (ARACHIS HYPOGAEA L.) USING THIDIAZURON

ZHIJIAN LI, ROBERT L. JARRET, ROY N. PITTMAN, ANn JAMES W. DEMSKI l

Department of Plant Pathology, Georgia Station, 1109 Experiment Street, Griffin, Georgia 30223 (Z. L., J. IV. D.); and USDA/ARS, Plant Genetic Resources Unit, 1109 Experiment Street, Griffin, Georgia 30223 (R. L. J., R. N. P.)

(Received 25 January 1994; accepted 10 June 1994; editor G. C. Phillips)

SUMMARY

Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed cxplants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A l-wk exposure to l0 ~RM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequendy developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.

Key words: Arachis species; peanut; organogenesis; plant regeneration; tissue culture; thidiazuron.

INTRODUCTION

The ability to regenerate plants from cultured cells, tissues, or organs provides a vehicle for plant transformation of crop species. To date, numerous protocols for efficient plant regeneration have been developed and are widely used in crop improvement efforts; examples include regeneration of haploid plants via anther culture, protoplast fusion and regeneration, and plant regeneration from cultured somatic tissues for propagation of elite genotypes. Many of these protocols have been successfully integrated into genetic engi- neering programs directed toward the use of plant tissue regenera- tion systems for the production of transgenic plants (Potrykus, 1991).

Since the initial work on plant regeneration from cultured so- matic tissues of peanut (Arachis hypogaea L.) in the early 1980s (Mroginski et al., 1981; Pittman, 1983), much attention has been focused on the refinement of procedures for the stimulation of so- matic embryogenesis in cultured peanut tissues and organs (Ozias- Akins, 1989; McKently et al., 1989; Sellars et al., 1990; Durham and Parrott, 1992; Ozias-Akins et al., 1992; Gill and Saxena, 1992). A somatic embryogenic pathway for plant regeneration has been considered desirable for plant transformation because it is assumed that somatic embryos originate from single cells and re- generated-transformed plan,ts are unlikely to be chimeric.

Organogenesis is an alternative regeneration pathway for use in plant transformation studies (Christou, 1993). Several investigators have reported plant regeneration from cultured peanut tissues via organogenesis (McKently et al., 1991; Cheng et al., 1992). How- ever, in these studies plant regeneration occurred at a low fre-

1 To whom correspondence should be addressed.

quency and was often limited to only a few genotypes. We report here a procedure for efficient shoot formation and plant regenera- tion from cultured hypocotyl tissues of peanut exposed to thidia- zuron (TDZ). This technique is effective across a wide range of peanut genotypes.

MATERIALS AND METHODS

Plant materials. Peanut (A. hypogaea cv. EC-5) seeds were harvested from greenhouse-grown plants, surface-sterilized in 70% ethanol for 1.5 min, 0.5% sodium hypochlorite solution for 10 min with vigorous agitation, and then rinsed 3 to 4 times in sterile water. The seed coat was removed and seeds were separated into the following groups: intact whole seed (IWS), whole seed without epicotyl/plumule (WSWE), whole seed without radicle (WSWR), intact half seed (IHS), half seed without epicotyl/plumule (HSWE), half seed without radicle (HSWR), half seed without epicotyl/plu- mule, and radicle (HSWER), embryo axis (EA), excised hypocotyl section (HCS), cotyledonary section (CS), and immature leaf (IL). Explants without radicle or epicotyl were prepared by removing the embryonic radicle and/or epicotyl with plumule, respectively, from the point of attachment to the cotyledons. All the half seed explants were made by longitudinally slicing the embryo axis of an intact seed so that each explant consisted of an intact cotyledon and an attached longitudinally split embryo axis.

Seed explants were incubated in an antibiotic solution of 50 mg/liter each of ampicillin and carbenicillin for 3 h at room temperature on a rotary shaker (100 rpm) before placement on culture medium. CS and IL explants were obtained from 12-day-old seedlings germinated in the presence of 10 #M TDZ following the procedures of Gill and Saxena (1992).

For experiments using immature seeds, peanut pods at various stages of development were harvested from greenhouse-grown plants. Pods were washed in tap water and surface-disinfected in 70% ethanol for 1.5 min, 0.5% sodium hypochlorite solution for 10 min with agitation, and then rinsed twice in sterile water. Intact seeds were removed from pods and soaked in a solution of 0.25% sodium hypochlorite for 10 min and rinsed 5 to 6 times in sterile water. Immature seeds were grouped into four develop- mental stages: I) cotyledon initiation stage where cotyledons were transpar-

187

188 LI ET AL.

TABLE 1

RESFONSE OF PEANUT (CV. EC-5) SEED EXPLANT TYPES TO TDZ FOR SHOOT INDUCTION

No. of Shoots per Explant,* Explant Type Mean (-+SE)

IWS 2.3 (0.3) WSWE 10.0 (1.6) WSWR 21.8 (4.5) InS 5.0 (0.9) HSWE 32.5 (10.7) HSWR 49.0 (5.3) HSWER 85.8 (3.2) EA 3,0 (0,4) ntis 13.3 (L3) CS 2.5 (1.0) IL 0

"Shoots with at least one expanded leaf were counted from each explant after 6 wk of culture on MS medium containing 10 #M TDZ. Each treat- ment contained 15 explants in 5 Magenta boxes. SE represents standard error.

ent and about 2 to 3 mm in length; II) mid-cotyledonary or torpedo stage where cotyledons were milky-white, soft, and one-third the size of mature seed cotyledons; ill) late eoty~edonary stage where cotyledons were apprcx~- mutely one-half the size of mature seed cotyledons, and IV) mature seed stage. All seeds were further processed into HSWER explants and treated in antibiotic solution before culture, as described previously.

Culture media and conditions. The basal medium used in all experi- ments, unless noted otherwise, was composed of Murashige and Skoog (MS) basal salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et at., 1968), 3% sucrose, and 0.8% agar (Sigma Chemical Co., St. Louis, MO). Media were adjusted to pH 5.8 with 0.5 M NaOH and autoclaved at 121 o C for 20 min before use- TDZ (technical grade, Nor-Am Chemical, Wilming- ton, DE) was prepared as a 1 mM stock solution in 0.01 N KOH and added to the media before autoclave sterilization.

Cultures were initiated in Magenta boxes containing 30 m[ of culture medium (three expiants/eulture vessel; five vessels/treatment}. All cultures were maintained at 26 - 2 ~ C under a 16-h photopedod wtth a light 'intensity of apFroximate~y 40 ttE - m -~ �9 C ~ .SabcuItures were made every 20 days. Shoot number was determined by counting the number of shoots with expanded leaves arising from each explant 6 wk after cullure initiation,

In experiments determining the effect of TDZ exposure time on shoot initiation, HSWER explants of mature seeds ofA. hypogueu cv, EC-5 were cultured on medium containing 10 #M of TDZ for a 1-, 2-, 3-, or 4-wk period, then transferred to hormone-free MS medium for further shoot growth and subsequent observation.

Plant regeneration- Individual shoots induced from HSWER explants ofev. EC-5 cultured in the presence of 10 #MTDZ for 1 wk were excised at their base and transferred to 25 • 150-ram culture tubes containing 15 ml of hormone-free MS medium solidified with 6 g/liter afar, When shoots reached a height of mare than 5 cm they were subeuhured to [5 ml of MS medium containing 1 rag/liter a-naphthaleneacetic acid (NAA} to induce root formation, Rooted plantlets were transplanted to 6-cm pots containing a standard poring mix (Promix BX, A. Y,. l'{ummert Seed Co., St. Louis, MO), placed under intermittent mist for 2 wk, and grown to malurity in the greenhouse.

RESULTS

Response of different seed explants to TDZ. In our preliminary efforts to achieve plant regeneration following the procedures de- scribed by Gill and Saxena (1992), we observed that shoot forma- tion occurred from the hypocotyl tissue of some seedlings that were cultured continuously in the presence of TDZ. To further investi- gate this phenomenon, we isolated various types of seed explants and cultured them for 6 wk on MS medium containing I0 ttMTDZ.

The shoot formation response of these explants is summarized in Table 1.

Seed explants turned green shortly after culture initiation, in the presence of 10 #M TDZ, cotyledons swelled up to twice their origi- nal size within 1 wk. IWS explants produced soft, short, and swollen roots. The epidermal layers of these roots frequently ruptured, and occasionally profuse callus formation was observed. Only a few adventitious shoots were initiated in the area of the axillary buds. Leaves developing on these shoots were greatly thickened and vitre- ous in appearance. Enlarged radicles and vitrified leaves were also observed on shoots developed from IHS explants. IllS explants produced approximately twice as many shoots per expiant as IWS explants.

Removal of either the epicotyl or radicle (WSWE or WSWR explants) resulted in a dramatic increase in the number of shoots produced per explant when compared to IWS explants (Table 1). This shoot-promoting effect was further accentuated in HSWE and HSWR explants. In each instance the excision of the radicle was more stimulatory to shoot production than the removal of the epico- tyl/plumule. Removal of both the radicle and the epicotyl/plumule (HSWER explants) resulted in a 17-fold increase in the number of shoots per explant when compared to IHS explants, or a 37-fold increase when compared to IWS explants.

During the first week of the culture period, the epidermal region of the hypocotyl of HSWER expiants expanded and fumed green. After an additional 2 to 3 wk, multiple shoot primordia emerged over the surface of the hypocotyl tissue (Fig. i a). Histologic exami- nation of thin sections of regenerating tissue indicated that shoot primordia were primarily derived from the subepidermal meriste- matic cells (Fig. 1 b) These shoots had morphologically normal leaves and developed rapidly, reaching a height of 2 to 3 cm after 6 wk (Fig. 1 c).

The proper positioning of half-seed explants on the culture media was critical to the stimulation of organogenesis and subsequent shoot formation. When the adaxial side of the cotyledon was placed downward in contact with the medium, organogenesis did not occur. Expiants remained white and were not responsive to the TDZ treat- ment throughout the cuhure period.

In the presence of 10 #M TDZ, the radicle of EA explanta swelled rapidly and subsequently produced a friable callus. Only a few shoots were produced from the original apical and axillary mer- istems of these explants. HCS explants produced more shoots per explant than EA explants. Few or no shoots were produced from CS or IL explants after 6 wk, although shoot primordia occasionally developed along the edge of IL explants.

Effects of TDZ exposure duration on shoot formation. We ob- served that adventitious shoots, from HSWER explants of cv. EC-5 induced by and cultured continuously in the presence of TDZ, be- came f, accd when they were excised {rum the primary explar~t and transferred to rooting medium. Histologic examination of these shoots revealed that the vascular bundles in the stem tissues had blackened and decayed, suggesting a toxic effect of TDZ up shoot development. These findings prompted us to investigate the effects of prolonged exposure of HSWER explants to TDZ on shoot initia- tion and development.

The effects of TDZ-exposure duration on the frequency of shoot initiation is presented in Fig. 2. HSWER explants exposed to TDZ for 1 wk produced approximately 70% of the shoots produced by HSWER explants exposed to TDZ for 4 wk. Shoots developing from HSWER explants after a 4-wk exposure to TDZ were densely dis-

ORGANOGENESIS FROM A. HYPOGAEA 189

25O

1 2 3 4 T D Z e x p o s u r e d u r a t i o n (wk)

Fro. 2. Effects of explant exposure time to TDZ on adventitious shoot initiation. HSWER explants of cv. EC-5 were cultured on MS medium containing l0 tiM TDZ for various lengths of time and then transferred to hormone-free MS medium. Data were collected 6 wk after culture initiation. Vertical lines represent standard errors.

transfer to hormone-free MS medium was used for subsequent ex- periments.

Effects of seed developmental stage on shoot initiation/ regeneration. Seed developmental stage was found to be a critical factor affecting the frequency of TDZ-induced shoot formation. Stage I and stage II explants produced callus growth from their cotyledonary tissues and only a few shoot primordia were produced from their hypocotyl tissues after exposure to TDZ (Fig. 3). In contrast, a significantly greater number of shoots were produced from the hypocotyl tissue of seed explants in developmental stages Ill and IV. Subsequent shoot development was more rapid from late cotyledonary (III) and mature seed (IV) explants.

Effects of TDZ concentration on shoot induction. The optimal

Fro. l . TDZ stimulation of shoot formation from cultured peanut seed explants, a, Formation of multiple shoots from hypocotyl tissue of HSWER explants of cv. EC-5. From left to right: original explant, explant 1 wk after exposure to TDZ, and explant 4 wk after culture initiation. Bar = 0.7 cm. b, Cross section of shoot-forming region of HSWER exptant cv. EC-5. Bar = 0.3 era. c, Multiple shoots with expanded leaves developing from hypocotyl tissue of HSWER explant 6 wk after culture initiation. Bar = 1 cm.

tributed around the hypocotyl region, and grew slowly. Most of these shoots continued to develop slowly and became flaccid when excised from the primary explant, as observed previously. In con- trast, shoots induced from HSWER explants cultured for 1 wk in the presence of TDZ followed by subculture to hormone-free MS medium were vigorous with normal stems and leaves, most reaching 2 cm in height within 6 wk. Rapidly growing plantlets were obtained within a period of 2 mo. from these shoots after excising them from the primary explant and rooting them in NAA-containing media. Based on these results, a 1-wk exposure to 10 ttM TDZ followed by

160

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120 . . . . . . . . . . . . . . . E l 0 0 . . . . . . . . . . . . . . . i

. . . . . . . . . . . . . . . i

60 . . . . . . . . . . . . . . . i

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20 . . . . . . . . . .

0 I II HI IV

Stage of seed development

FiG. 3. Effects of seed developmental stage on shoot initiation induced by TDZ. HSWER explants of ev. EC-5 were cultured on MS medium containing 10 #M TDZ for 1 wk and then transferred to hormone-free MS medium. Stages represent: I, cotyledon initiation; II, mid-eotyledonary; III, late cotyledonary; and IV, mature seed. Data were collected 6 wk after culture initiation. Vertical lines represent standard errors.

190 LI ET AL.

0 0 0.010.05 0.1 0.5 1 2 5 10 15

TDZ concentration (uM)

Fro. 4. Effects of TDZ concentration on shoot initiation from cultured peanut hypocotyl tissues. HSWER explants of cv. EC-5 were cultured on MS medium containing 10 ttM TDZ for 1 wk and then transferred to hormone-free MS medium. Data were collected 6 wk after culture initiation. Vertical bars represent standard errors.

concentration of TDZ stimulating shoot initiation was determined by culturing HSWER explants of cv. EC-5 on media containing TDZ at concentrations ranging from 0.01 to 15 ~tM. Increasingly larger numbers of shoots were produced as the TDZ concentration in- creased (Fig. 4). There was a sharp increase in the number of shoots produced from seed explants cultured on media containing 2 #M TDZ, as compared to explants cultured on media containing 1 /.tM TDZ. TDZ concentrations of 5 to 10 ~tM resulted in maximal stimulation of shoot production. Concentrations of TDZ greater than 10 ~tM seemed to inhibit shoot formation.

Morphologic variation among regenerated shoots was correlated with TDZ concentration. TDZ concentrations of 0 or 0.01 #M TDZ stimulated the production of friable callus formation at the base of the developing plantlets. Culture of HSWER explants on medium containing low concentrations of TDZ (< 1 ~M) resulted in the devel- opment of vigorous shoots that had larger leaves and that produced roots. TDZ concentrations greater than 2 #M resulted in the produc- tion of shoots that were smaller than those shoots that developed from explants cultured on lower concentrations of TDZ. Adven- titious shoot buds arose on the periphery of shoot stems of plantlets regenerated from explants cultured at higher concentrations of TDZ (>2 gM). Shoots with fused stems were occasionally observed at all TDZ concentrations. Flowers frequently developed directly from explants cultured on media containing 0 or 0.01 #M TDZ. Struc- tures similar to umbellate flower buds occasionally developed from explants cultured in the presence of 5 #M or greater TDZ.

Genotypic responses to TDZ. Genotypie effects on shoot induc- tion by TDZ were examined using nine U.S. peanut cultivars, repre- senting the four major peanut types, and one wild species (A. g/a- brata). All A. hypogaea genotypes responded similarly, producing from 56 to 174 shoots per explant (Table 2). Explants from A. glabrata produced fewer shoots than those from A. hypogaea. Among the peanut cultivars examined, cv. EC-5 produced a signifi- cantly greater number of shoots. The high in vitro regenerability of this cultivar has been observed previously in our studies on regener- ation from isolated peanut protoplasts (Li et al., unpublished).

Phenotypes of regenerated plants. Sixty regenerated plantlets of

cv. EC-5 were transferred to potting soil and grown in a greenhouse. Twenty percent of the surviving plants were noticeably more vigor- ous than the remaining plants. These 20% had larger and thicker leaves, and larger stems with shorter internodes than control (ger- mininated seed-derived) plants. However, all plants produced phe- notypically normal flowers and set viable seed at an average rate of 22.5 seed-bearing pods per plant.

DISCUSSION

Thidiazuron was originally developed in 1976 as a cotton defo- liant (Arndt et al., 1976). Subsequent studies indicated that TDZ has a strong eytokininlike activity exceeding that of zeatin (Mok et al., 1982). The promotion of cell division and tissue growth by TDZ has been attributed to its ability to stimulate rapid conversion of ribonueleotides to biologically active ribonueleosides (Capelle et al., 1983) or to cause the synthesis and accumulation of purine eytokin- ins (Thomas and Katterman, 1986), or both. TDZ has been used to induce somatic embryogenesis, adventitious shoot formation, and axiflary shoot proliferation in numerous plant species (Huetteman and Preece, 1993; Lu, 1993). The stimulation of somatic embryo- genesis and the subsequent regeneration of #ants from seedling explants of peanut (A. hypogaea) by TDZ have been reported (Gill and Saxena, 1992). Our results demonstrate that rapid shoot for- mation and plant regeneration of peanut via organogenesis, from hypocotyl explants with attached cotyledons, can be achieved after a short period of exposure to TDZ. Shoot formation frequencies ob- tained using this procedure are considerably higher than those re- ported previously (McKently et al., 1991; Cheng et al., 1992). In addition, the organogenetie response described here is less geno- type-specific than many previously described regeneration proto- cols for this species. Regenerated #ants are normal in appearance and produce seeds.

Thidiazuron is one of the most stable compounds used in plant tissue culture media. It is resistant to degradation by cytokinin oxi- dase (Mok et al., 1987). Also, it is more biologically active than most other cytokinins including benzylaminopurine and zeatin. Ow- ing to its unusual stability and tendency to accumulate in plant

TABLE 2

SHOOT REGENERATION RESPONSE OF NINE PEANUT (,4. HYPOGAEA) CULTIVARS AND A. GLABRATA TO TDZ

No. of Shoots per Explant, ~

Genotype Market Type/Species Mean (_+SE)

Georgia runner Runner 123.0 (7.6) AT-127 Runner 107.2 (7.0) Florigiant Virginia 55.9 (7.4) NC-6 Virginia/runner 76.4 (19.9) NC-7 Virginia/runner 58.1 (11.9) Pronto Spanish 113.0 (26.1) EC-5 Spanish 173.7 (14.9) Star Spanish 105.3 (11.9) Tennessee red Valencia 87.8 (16.2) P1468370 A. glabrata 38.3 (5.8)

HSWER explants were cultured on MS medium containing 10 #M TDZ for 1 wk and then subcuhured on hormone-free MS medium for 5 wk. Shoots with at least one expanded leaf were counted from each exp]ant. Each genotype contained 15 explants in five Magenta boxes. SE represents standard error.

ORGANOGENESIS FROM A. HYPOGAEA 191

tissues, TDZ has been found to be associated with many develop- mental abnormalities, including hyperhydricity (vitrification) of re- generated shoots, abnormal leaf morphology, stunted shoots, and the retardation of shoot elongation and rooting. These adverse ef- fects have been summarized by Lu (1993). Growth abnormalities may be attributed to the ability of TDZ to induce ethylene biosynthe- sis and the subsequent senescence of plant tissues (Suttle, 1985).

We observed that the regeneration of plants subsequently devel- oping deformed and thickened leaves was often associated with the culture of explants in the presence of TDZ for more than 4 wk. The enlarged leaves and thickened stems observed among some regen- erated shoots persisted to the mature plant stage, well after transfer to the greenhouse. Our data suggest that exposure of peanut ex- plants to TDZ for a short period (1 wk) reduces the frequency of morphologic abnormalities induced by TDZ while maintaining a high frequency of regeneration. Knowledge concerning the effects of TDZ on genetic stability may improve the utilization of this com- pound in plant regeneration and transformation studies.

The inclusion of intact cotyledons is essential in efforts to induce high frequency shoot formation from cultured hypocotyl tissues fol- lowing exposure to TDZ. A hormonal interaction between these two tissues may be necessary for the hydrolysis and utilization of cellu- lar reserves in the cotyledon that are required for the differentiation of hypocotyl cells (Metivier and Paulilo, 1980). Based on the obser- vation that the presence of an attached radicle prevents the uptake of exogenous cytokinin (Gepstein and Ilan, 1970), we believe that the removal of the radicle and shoot apex from the embryonic axis may disrupt endogenous hormone biosynthesis, enhance the uptake and accumulation of TDZ by the hypocotyl tissue, and thus provide a continuous source of cytokinin to the cotyledonary tissues to be used for starch hydrolysis (Hutton and Van Staden, 1982).

Thidiazuron has a tendency to be accumulated in toxic amounts in plant tissues (Mok et al., 1982; Huetteman and Preece, 1993). We suggest that the attached cotyledons in our explants serve as a reservoir for the deposition of TDZ and thus prevent the accumula- tion of toxic levels of this compound in regenerating hypocotyl tis- sues. The rapid enlargement of cotyledons, and the reduction of vascular bundle damage in tissues over-exposed to TDZ during the culture period, seems to support this hypothesis.

Plant regeneration in this study was achieved largely via somatic organogenesis. Histologic observation indicated that shoot primor- dia were derived from meristemoids in the epidermal and subepi- dermal cell layers of the hypocotyl, as described by others (Pittman et al., 1983). The protocol for plant regeneration from cultured hypocotyl tissue described here may provide an alternative experi- mental system for studies of genetic transformation using Agrobac- terium-mediated gene transfer as described by Mante et al. (1991), and other approaches that require numerous target cells capable of regenerating into plants.

ACK~OWLZ~MEr~rs

This research was supported in part by Peanut CRSP, U.S. AID grant DAN-4048-G-00-0041-00; in part by the Georgia Commodity Commis- sion for Peanuts; and in part by State and Hatch Funds allocated to the University of Georgia. A technical-grade sample of thidiazuron was kindly provided by Nor-AM Chemical Co., Wilmington, DE. We thank Drs. C. J. Chang, J. Latimer, C. Robacker, and M. C. Deom for critical review of the manuscript. We also acknowledge the technical assistance of Ms. Regina Estes and the EC-5 seed from Jim Kirby.

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