Protoplasma (1998) 204:71-83

PROTOPLASMA �9 Springer-Verlag 1998 Printed in Austria

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Adriana P. M. Rodriguez** and Hazel Y. Wetzstein*

Horticulture Department, University of Georgia, Athens, Georgia

Received March 19, 1998 Accepted July 9, 1998

Summary. Somatic embryos produced in vitro may exhibit structur- al abnormalities that affect their subsequent germination and conver- sion into plants. To assess the influence of auxin type on embryo ini- tiation and development, a morphological and histological compari- son was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4- dichlorophenoxyacetic acid (2,4-D), using light and scanning elec- tron microscopy. Both auxins promoted enhanced cell division, par- ticularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell pro- liferation, epidermal continuity, callus growth, and embryo morphol- ogy. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal mor- phology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heteroge- neous regions of cell division. Proliferating cell regions were com- posed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medi- um with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meri- stems were often lacking or partially obliterated due to callus prolif- eration. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.

Keywords: 2,4-Dichlorophenoxyacetic acid; Auxin; Carya illinoi- nensis; Embryo induction; Naphthaleneacetic acid; Somatic embryo- genesis.

*Correspondence and reprints: Horticulture Department, 1111 Plant Science Building, University of Georgia, Athens, GA 30602-7273, U.S.A. **Present address: CENA, Universidade de Silo Paulo, Piracicaba, SP, Brazil.

Abbreviations- 2,4-D 2,4-dichlorophenoxyacetic acid; BAP 6-ben- zylaminopurine; NAA naphthaleneacetic acid; SEM scanning elec- tron microscopy.

Introduction

Somatic embryogenesis can be induced in vitro in many plant species and has been of interest for its potential applications in clonal propagation, gene transformation, and embryo development studies. Generally, embryogenic cultures are initiated by exposing explant tissues to an auxin-containing medi- um, followed by transfer of tissues to a secondary medium with no or low auxin concentration to pro- mote embryo development. Ideally, somatic embryos exhibit morphological characteristics similar to the zygotic condition. However, abnormalities in somat- ic-embryo morphology have frequently been ob- served. Morphological quality of somatic embryos has been shown to affect the efficiency of conversion into plantlets, in that somatic embryos exhibiting clear bipolarity, well-defined cotyledons, and a well- developed shoot apex convert better than embryos showing morphological abnormalities (Lazzeri et al. 1987, Hartweck et al. 1988, Cruz et al. 1990, Wetz- stein and Baker 1993, Rodriguez and Wetzstein 1994). The general application of somatic embryo- genesis requires the development of uniform, high- efficiency regeneration systems that can produce plants that perform similarly to those derived from seed (Wetzstein and Baker 1993).

72 A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures

In a number of embryogenic systems, gross morpho- logical evaluations of somatic embryos have shown that abnormalities are associated with the use of 2,4- dichlorophenoxyacetic acid (2,4-D) in the induction medium. Somatic embryos induced by naphthalene- acetic acid (NAA) had more normal morphology than those induced by 2,4-D in Glycine max (Lazzeri et al. 1987, Hartweck et al. 1988), and Carya illinoinensis (Rodriguez and Wetzstein 1994). NAA-induced somatic embryos germinated at higher frequencies than those induced by 2,4-D (Lazzeri et al. 1987, Ozean et al. 1993, Sofiari et al. 1997). A somatic embryogenic system producing repetitive secondary embryos has been developed in C. illinoi- nensis which is efficient and can produce large num- bers of clonal plants suitable for the field (Mathews and Wetzstein 1993, Wetzstein et al. 1996). Potential applications in pecan include clonal rootstock pro- duction (which is unavailable by conventional propa- gation methods) and direct gene transfer of traits such as resistance to pests and environmental stresses. Because pecan somatic embryogenic cultures can be induced with either NAA or 2,4-D, they may be valu- able material to study auxin effects on initiation and development. We have previously observed that the type of auxin used during induction of these cultures affects the morphology and subsequent germination and conversion of embryos into plants (Rodriguez and Wetzstein 1994). Somatic embryos induced on medi- um with 2,4-D frequently lacked a well-developed shoot apex and/or exhibited varying degrees of fusion

of multiple embryos. This is in contrast to embryos induced on medium with NAA, which had more bipo- lar embryos with well-defined apices and cotyledons. The objectives of this work were to present a morpho- logical and histological study of the initiation and development of pecan somatic embryos, and to con- trast structural and developmental differences associ- ated with induction by NAA or 2,4-D. Also of interest was an assessment of the influence that auxin type has on the occurrence of structural abnormalities in cul- ture. Such information could provide insight into how auxins modify embryo developmental patterns.

Material and methods In vitro culture conditions

Immature pecan fruits, Carya illinoinensis (Wagenh.) C. Koch cv. Stuart, were explanted and placed in culture as previously described (Rodriguez and Wetzstein 1994). Briefly, fruits were collected at 15 weeks post pollination, i.e., at the stage when cotyledons are rapidly exPanding, endosperm is both liquid and gelatinous, and hardening had occurred for about 2/3 the length of the shell. Whole fruits were sterilized, then dissected under aseptic conditions. The immature embryos were excised and then cotyledons were cut into pieces of ca. 1 cm 2 and placed randomly on induction medium. The induction medium consisted of a modified Woody-Plant Medium (Lloyd and McCown 1980) minus glyeine and supplemented with 30 mg of sucrose, 1 g of casein hydrolysate, 0.1 g of inositol, 3 g of Gel-gro (ICN Biochemicals, Cleveland, Ohio, U.S.A.) per liter, 1.2 gM BAP, and either 32 or 64 gM NAA, or 9 or 27 gM 2,4-D. Media were dis- pensed into 15 • 100 mm petri dishes and cultures were sealed with parafilm. The choice of auxin type, concentrations, and other media amendments was based on previous results that showed 100% of explants formed somatic embryos under these conditions. Our previ-

Figs. 1-9. Explant and early-stage characteristics of pecan somatic embryos induced with NAA. Bar: Figs. 1-4 and 7-9, 100 gm; Fig 5, 20 gm; Fig. 6, 50 gin

Fig. 1. SEM micrograph of a cotyledon at time of explanting from immature zygotic embryo showing slightly undulated surface

Fig. 2. Zygotic cotyledon cross section (at explanting) stained with acid fuchsin/toluidine blue showing a uniseriate epidermis, parenchymatous mesophyll cells, and vascular strands (arrowheads)

Fig. 3. Zygotic-cotyledon cross section (at explanting) stained with IKI, showing a few scattered starch grains (arrowhead) in the mesophyll cells, e Epidermis

Fig. 4. Explant at day 7, showing an increase in thickness and number of cell layers

Fig. 5. Enlargement of boxed area in Fig. 4, showing meristematic nature of subepidermal cell layers. Cells are larger and more vacuolate in more internal cell layers

Fig. 6. Cross section of explant on day 14, stained with acid fuchsin/toluidine blue showing highly cytoplasmic cells with prominent nuclei and nucleoli in the subepidermal layers, e Epidermis

Fig. 7. Cross section of day 14 explant, stained with IKI; note intense accumulation of starch in subepidermal layers, and absence in internal mesophyll layers

Fig. 8. Cross section of an embryogenic protuberance (day 10) stained with acid fuchsin/toluidine blue; the embryogenic region is formed by intense proliferation of the more superficial layers of the explant tissue

Fig. 9. SEM micrograph (day 10) showing an embryogenic protuberance with an intact epidermis

A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures 73

74 A.P.M. Rodriguez and H. Y, Wetzstein: Pecan somatic embryogenic cultures

ous studies have shown that the auxin type during induction (i.e., NAA or 2,4-D) and not the concentration affects pecan somatic embryo morphology and conversion (Rodriguez and Wetzstein 1994). The explants were maintained on induction medium for one week, then transferred to basal medium which was the same medium without growth regulators. For developmental studies, tissue pieces were sampled at days 0, 7, 10, 14, 17, and 21. Ten explant pieces were randomly collected per sampling time, auxin type and concen- tration. Half the samples were prepared for light microscopy, and half were prepared for scanning electron microscopy (SEM). All media were adjusted to pH 5.7 before addition of Gel-gro and auto- claved at 121 ~ for 20 min. Cultures were maintained in the dark at 30 ~

Light microscopy

Samples were fixed in a mixture of 3% paraformaldehyde and 2% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2 at 4 ~ Tissues were dehydrated in a series of methyl cellosolve, ethanol, propanol, and n-butanol, then embedded in Historesin (Leica Instruments, Hei- delberg, Federal Republic of Germany), which consists of hydro- xyethylmethacrylate. Sections 5-6 gin thick were cut with a metal knife using an HM350 rotary microtome (Microm, Heidelberg, Fed- eral Republic of Germany). Serial sections aided in interpretation. Sections were stained with acid fuchsin/toluidine blue O (Feder and O'Brien 1968) or Coomassie brilliant blue R-250 (Fisher 1968), and mounted in Mount-quick (distributed by EMS, Fort Washington, Pa., U.S.A.). Starch was visualized by staining sections in 50% IKI solu- tion (Jensen 1962). All sections were examined under bright field with a Standard (Carl Zeiss, Thomwood, N.Y., U.S.A.) microscope and photographed on Plus-X pan film (Kodak, Rochester, N.Y., U.S.A.).

Scanning electron microscopy

Samples'were fixed in 4% (v/v) glutaraldehyde in 0.1 M cacodylate buffer at 4 ~ overnight, dehydrated ttn'ough an ethanol series, and critical-point dried through carbon dioxide. Samples were mounted on aluminum stubs, sputter coated with 60 nm gold/palladium, and

examined under a 505 SEM (Philips, Mawah, N.J., U.S,A.) at 20 kV. Photomicrographs were taken with Positive/Negative 665 black and white film (Polaroid Corporation, Cambridge, Mass., U.S.A.).

Results

Histological characterization of the zygotic tissue at the time of culture

The zygotic cotyledonary tissue at explanting was white, thin, and flattened, ca. 10 to 15 mm long and 0.3 mm thick. The surface was generally smooth and slightly undulated (Fig. 1). In previous studies we determined that embryogenesis was optimum in tis- sues that were at the stage of rapid cotyledon expan- sion and before deposition of storage material. Cotyledonary tissues had a single epidermal layer and eight to twelve layers of compactly arranged meso- phyll cells (Fig. 2). The mesophyll consisted primari- ly of parenchyma cells interspersed with vascular strands; few intercellular spaces were observed. Epi- dermal and mesophyll cells were generally isodiamet- ric, with a condensed nucleus and a thin layer of cyto- plasm, either forming cytoplasmic strands or ap- pressed against the cell membrane by a large central vacuole. Cell division in the epidermal layers was mainly anticlinal or oblique and in different planes in the mesophyll. Epidermal cells were smaller than mesophyll cells. Cells in the subepidermal layers were somewhat smaller than more internal mesophyll ceils (Fig. 2). A few starch granules were present scattered throughout the mesophyll cells, but only occasionally in epidermal cells (Fig. 3).

Figs. 10--17. Somatic embryo development in cultures induced with NAA. Bars: Figs. 10, 11, 13, 14, and 17, 200 gm; Figs. 12, 15, and 16, 100 gm

Fig. 10. LM micrograph of explant on day 17 showing several somatic embryos (point) forming from extensive embryogenic regions (er); part of the original explant is visible in the section, ca Callus

Fig. 11. SEM micrograph of raised embryogenic regions and developing embryos on day 21; epidermis is continuous over the embryogenic area

Fig. 12. SEM micrograph of globular (g) and early-cotyledonary-stage (c) embryos from tissue at day 10. Cotyledon enlargement often occurred without elongation of the embryo axis

Fig. 13. Somatic embryo development was asynchronous, with various stages within the same embryogenic area. Note raised embryogenic regions (arrowheads), heart-shaped (h) embryos, embryos with developing cotyledon primordia (arrow), and cotyledonary-stage embryos (c)

Fig. 14. Numerous somatic embryos often formed along cut edges and were associated with callus proliferation (ca); callus cells were large and loosely packed

Fig. 15. SEM micrograph showing heart-stage embryos (h)

Fig. 16. Section stained with coomassie blue, showing a globular embryo attached to an embryogenic protuberance through a broad base; epi- dermis is continuous (day 17)

Fig. 17. LM micrograph of a section from day 21, stained with coomassie blue showing a somatic embryo undergoing repetitive embryogene- sis (arrowhead). (The two circular structures are part of an adjacent primary somatic embryo in cross section)

A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures 75

76 A.P.M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenie cultures

Characterization of somatic embryogenesis induced by NAA

Explant enlargement and formation of embryogenic regions

During the first week in culture a uniform enlarge- ment was observed throughout the tissues of the explant. At day seven, when the explants were trans- ferred to basal medium, tissues were cream colored, and cotyledon thickness was about twice that of day zero (with ca. 18 to 25 cell layers) (Fig. 4). Parenchy- ma cells in the middle region of the cotyledons were about the same size compared with day zero but were more loosely packed and had more intercellular spaces. Cells had a large central vacuole, with mitotic figures observed infrequently. In contrast, the cells in the three or four subepidermal abaxial or adaxial lay- ers were smaller than the more internal cells (Fig. 5). These cell layers were highly mitotic with frequent periclinal and occasionally anticlinal and oblique division planes. The epidermal and subepidermal cells had prominent nuclei, with distinct nucleoli, higher cytoplasmic content and smaller vacuoles than con'esponding cells at day zero. The epidermis exhib- ited occasional anticlinal cell divisions. Starch con- tent increased from day zero and accumulation was primarily in the subepidermal layers. At day 14, the epidermis and cells in the first four to six subepidermal layers were densely cytoplasmic, had few and small vacuoles, and a large nucleus and prominent nucleolus (Fig. 6). In the mesophyll,

intense cell proliferation continued to increase the thickness and number of cell layers of the explant. The degree of vacuolization and size of the vacuoles gradually increased from the subepidermal cells toward the internal cell layers of the mesophyll, where cells with a large central vacuole were present. Mitotic activity became localized mainly to the subepidermal layers. Starch was evident and restrict- ed to the first eight to ten subepidermal cell layers (Fig. 7). Intense periclinal cell divisions of the more superficial cell layers gave rise to bulging, embryo- genic protrusions (Figs. 8 and 9). Starch granules dis- appeared during the formation of embryogenic regions and subsequent development of somatic embryos. The epidermis generally remained intact and exhibited anticlinal divisions. Random planes of division were observed in internal areas of the explant.

Proembryo formation

Proliferation of embryogenic protrusions gave rise to globular embryos (Figs. 10 and 11). Embryos had an epidermis continuous with the embryogenic area and explant. In some cases, embryos arose singly or in groups from cotyledonary tissue that exhibited limit- ed outward proliferative growth (Fig. 12). Alterna- tively, multiple embryos often differentiated from more extensive embryogenic protrusions (Fig. 11). These extensive embryogenic areas were especially prevalent next to cut edges of the cotyledonary

Figs. 18-27, Early development and embryogenesis of explants induced on medium with 2,4-D. Bars: Figs. 18 and 20-22, 200 gm; Figs. 19 and 27, 25 gm; Figs. 23 and 24, 100 gm; Figs. 25 and 26, 50 gm

Fig. 18. General view of the enlarged explant tissue on day 7 showing an increased number of cell layers and cells with high cytoplasmic con- tent

Fig. 19. Enlarged area from Fig. 18 showing the anticlinal and periclinal planes of cell division in the mitotically active subepidermal cell lay- ers of the explant

Fig. 20. Embryogenic protrusion developing from a highly mitotic area of the cotyledon (day 7). Extensive internal mesophyll layers con- tributed to the embryogenic protrusion; several localized globular proembryogenic structures (g) are evident. Protrusions were composed of het- erogeneous cell types; the epidermis was frequently ruptured (arrowheads)

Fig. 21. Embryogenic protrusion involving more superficial cotyledon cell layers (day 7)

Fig. 22. SEM micrograph of embryogenic protrusions showing intact and ruptured epidermal areas (day 10)

Fig. 23. SEM micrograph showing discontinuity of the epidermis associated with callus proliferation on the surface of proembryogenic protru- sions (day 10). c a Callus

Fig. 24. SEM micrograph of a nonembryogenic area showing callus proliferation and epidermal rupture (day 10)

Fig. 25. Cross section of part of the cotyledon showing a discrete region of callus proliferation; callus cells are large and highly vacuolated; some cells have accumulated phenolic compounds (p) which stained green with toluidine blue (day 7)

Fig. 26. LM micrograph of cotyledon showing trichome-like structures which accumulated phenolic (p) compounds (day 7)

Fig. 27. SEM micrograph of trichomeqike structures on the surface of the explant

A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures 77

78 A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures

explants, where embryo development was asynchro- nous, ranging from globular to cotyledonary stages (Figs. 13 and 14). Although callus proliferation was frequently associated with the embryogenic regions (Figs. 10 and 14), somatic embryos formed directly from embryogenic protuberances (Figs. 10 and 11) which developed on both abaxial and adaxial sides of the explant. The epidermal layer remained continuous over the entire embryogenic area and the embryos had a broad base of attachment (Figs. 12 and 16). Callus proliferation was observed at the wounded areas (Fig. 14), with callus cells being large, of various shapes, and loosely packed (Fig. 10).

Embryo development

Proembryos had a single epidermal layer and small isodiametric cells with large nuclei and darkly stain- ing nucleoli (Fig. 16). Cells were tightly aggregated, with few intercellular spaces. Heart-shaped embryos were evident (Fig. 15). Cotyledon primordia often developed before elongation of the embryo axis (Figs. 12 and 14). A distinct shoot apex was visible sur- rounded by developing cotyledon primordia (Fig. 14). Procambial differentiation was evident at this stage and no vascular continuity existed between the embryo and explant tissue. Globular and heart-shaped embryos contained little starch (Fig. 16), which was verified with IKI staining. However, some accumula- tion was observed in older somatic embryos, especial- ly in the cotyledonary regions. Starch granules were observed in some areas of the explant tissue, scattered beneath areas of embryo development and more inter- nal layers. Lateral protrusions on some primary somatic embryos gave rise to embryogenic regions that differentiated into secondary embryos. Sec- ondary embryos likewise formed without intervening callus directly from the primary embryo tissue (Fig. 17). Secondary embryogenesis was observed early in cultures. From day 17, cultures had different stages of embryogenesis, ranging from proembryogenic protru- sions on the primary explant to regions with prolific secondary embryogenesis.

Characterization of somatic embryogenesis induced by 2,4-D

Explant enlargement and formation of embryogenic regions

A general cellular proliferation was observed in cotyledons at the end of the induction period (7 days). Cell proliferation was variable within an explant and

thickness varied from about 12 to 30 layers of meso- phyll cells (Figs. 18 and 20). Mesophyll cells were more meristematic and had a higher cytoplasmic con- tent than at the time of explanting (Fig. 2). The first four subepidermal cell layers exhibited high mitotic activity and were densely cytoplasmic (Fig. 19). Cells in these subepidermal cell layers were smaller and had larger nuclei than more internal mesophyll cell layers. In some regions cell proliferations gave rise to extensive embryogenic regions (Figs. 20-22). These highly mitotic areas were characterized as having small, densely staining cells with a high nucleus-to- cytoplasm ratio. The extent of the cotyledon cells that made up the embryogenic regions varied. In all cases, internal mesophyll cell layers were involved, with the extensive proliferation of cells forming raised protru- sions. Occasionally, an embryogenic region incorpo- rated more than 75% of original cotyledonary cell layers (Fig. 20). In other cases fewer mesophyll cell layers were implicated (Fig. 21). Embryogenic regions varied in shape and size. Some protrusions had an intact epidermis, while others showed rupture of the epidermis, often with callus proliferation (Fig. 23). Callus proliferation was also associated with surface rupture in nonembryogenic areas, which could encompass extensive (Fig. 24) or discrete regions (Fig. 25). Callus cells were large, irregularly shaped, highly vacuolate and frequently contained green colored components when stained with acid fuchsin/toluidine blue. According to Feder and O'Brien (1968), cellular contents that are stained green with the metachromatic stain toluidine blue contain polyphenols or lignins. Trichome-like cell proliferations from epidermal cells, resembling somatic embryos described in other systems, were also occasionally observed on explants (Fig. 26 and 27).

Proembryo formation

Localized globular, proembryogenic structures were frequently observed within embryogenic protrusions (Fig. 20). These structures were composed of com- pactly arranged isodiametric cells which were mitoti- cally active and dividing in various planes. These proembryos were situated peripherally and often interspersed with more vacuolate cells and/or callus. Discontinuity of the epidermal layer of the explant was frequently observed on the surface of these embryogenic protrusions. Alternatively, embryos arose directly without intervening callus from subepi-

A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures 79

Figs, 28-33. Development of somatic embryos m cultures induced with 2,4-D. Bars: Figs, 28 and 30, 200 gin; Fig. 29, 100 gm; Figs. 31-33, 400 gm

Fig. 28. SEM micrograph showing proembryo (pe) and developing embryo with shoot apex (asterisk) encircled by multiple primordia (arrow- heads); embryos are developing close to extensive embryogenic areas (arrow) associated with proliferating callus (day 14)

Figs. 29 and 30. SEM and LM micrographs of barrel-shaped embryos with a ruptured epidermis in the shoot meristem (asterisk). Note the broad base of embryo attachment to the explant. Some cotyledon primordia (arrowhead) are evident (day 10). v s Vascular strand

Fig. 31. SEM micrograph of tissues at day 21, showing somatic embryos of variable types including more zygotic-like forms that had a promi- nent shoot primordium (asterisk) with laterally positioned cotyledon primordia (c), and fan-shaped (arrowheads) somatic embryos showing adaxial fusion of the cotyledons and absence of a defined shoot primordium (day 21)

Fig. 32. SEM micrograph showing tubular (arrow) and fan-shaped (arrowheads) embryos lacking a defined shoot primordium (day 21)

Fig. 33. SEM micrograph showing fasciated structures composed of multiple fused somatic embryos (day 21)

80 A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures

dermal cell layers of the explant and exhibited a con- tinuous epidermis. Embryos arose from both abaxial and adaxial surfaces. These isolated surface embryos occurred often close to more extensive embryogenic protrusions (Fig. 28).

Embryo development

Early embryo development proceeded with elonga- tion of the axis. The point of embryo attachment was broad, resulting in barrel-shaped somatic embryos (Figs. 29 and 30). A prominent shoot apical primordi- um was often evident. On occasions the shoot apical meristem exhibited rupture of the epidermis and cal- lus proliferation. Embryos showed differentiation of vascular tissue not connected to the explant (Fig. 30). Cotyledon primordia arose laterally on the embryo apex as rounded structures. In some cases multiple primordia developed concentrically around an apical meristem (Figs. 28 and 31). Some primordia were irregularly positioned around the apex or missing in some sectors (Fig. 29). Alternatively, development of laterally oriented meristematic areas resulted in expansion and enlargement of broad fan-shaped cotyledonary structures. This was usually associated with the adaxial fusion of the cotyledons and absence of a defined shoot apex (Figs. 31 and 32). In other cases, growth in height of a girdling cotyledonary pri-

mordium, encircling the embryo apex, resulted in tubularly shaped embryos (Fig. 32). In some regions, groups of multiple embryos emerged from extensive embryogenic areas that were often associated with callus. Embryos remained singular (Fig. 31) or devel- oped into fasciated structures resembling multiple embryos (Fig. 33).

Comparison of cultures induced on medium with NAA or 2,4-D

Significant differences were observed in the timing and pattern of initiation and development of somatic embryos induced on media supplemented with NAA or 2,4-D. Notable differences were mitotic activity, timing and organization of proembryogenic cell pro- liferation, epidermal continuity, callus growth, and somatic-embryo morphology. A comparison of gener- al characteristics is summarized in Table 1.

Discussion

This study presents a detailed comparative histologi- cal and morphological description of the initiation and development of pecan somatic embryos induced on medium with either NAA or 2,4-D. Cultures induced with 2,4-D exhibited enhanced mitotic activ- ity and more intense callus proliferation compared to cultures induced with NAA. A critical comparison of

Table 1. Summary of general characteristics observed in pecan somatic embryogenic cultures induced by NAA or 2,4-D

Characteristic NAA a 2,4-D b

Origin of proembryogenic protrusions

Cytology of proembryogenic protrusions

Formation of proembryogenic protrusions Appearance of developed embryos Embryo morphology

Epidermis of explant and somatic embryo Overall callus proliferation during first

21 days Effect of auxin concentration

Occurrence of repetitive embryogenesis

abaxial and adaxial subepidermal cell layers

homogeneous, mainly composed of isodiametric meristematic cells

by day 10 by day 14 generally single with a defined apical

meristem

continuous little, mainly on cut ends of explant

not apparent in this range

by day 17

more internal origin

heterogeneous, meristematic cells with discrete globular proembryos interspersed with callus

prior m day 7 by day 10 of variable forms; single or fused

embryos, with or without a discrete apical meristem, often fan-shaped with no distinct apical meristem

continuous or ruptured marked, no specific regions

at lower concentrations tissue response attenuated, slower rate of embryo development and trichomes

not observed at up to day 21

aNAA at 32 or 64 btM was used in the induction medium b2,4-D at 9 or 27 t.tM was used in the induction medium

A. P. M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures 81

early developmental stages was presented, which could be related to the occurrence of a higher rate of abnormal somatic embryo formation in 2,4-D- induced cultures. The embryogenic response in pecan is dependent on explant developmental stage (Wetzstein et al. 1989), with greatest embryogenesis obtained when cotyle- dons are mitotically active and rapidly expanding. In contrast to the results obtained by Canhoto and Cruz (1996), where mature zygotic embryos responded well, embryogenesis in pecan does not occur when cotyledons are more mature and accumulating reserves. With the current study the explant tissue was composed primarily of parenchymatous cells. A 100% embryogenic response was routinely obtained with either 2,4-D or NAA at the concentrations and conditions of this study. The embryogenic regions formed from the more mitotically active subepider- mal cell layers through rapid division and prolifera- tion of cells. No proliferation giving rise to somatic embryos was observed in more internal mesophyll cells, as described by Dubois et al. (1991) and Mo and Arnold (1991). In pecan, an increase in mitotic activity of cotyle- donary cells was observed when induction was with either NAA or 2,4-D. In the present work, most of the proliferating cells exhibited common features charac- teristic of meristematic cells. However, compared with NAA, mitotic activity was generally more intense in tissues induced with 2,4-D. Induction with 2,4-D appeared to promote diverse forms of cell pro- liferation, leading to heterogeneous cell types com- posed of embryogenic and callus cells. In contrast, cultures induced on NAA had embryogenic regions composed of homogeneous, isodiametric meristemat- ic cells. Callus proliferation was mainly restricted to cut or wounded areas of the explant. The amount of callus proliferation was similarly related to auxin type in soybean (Glycine max), where somatic embryo- genic cultures on media with NAA produced less cal- lus than those on media with 2,4-D (Lazzeri et al. 1987). This observation concurs with Dudits et al. (1991) who recognized that 2,4-D can induce cell division both as unorganized callus growth or in a well-coordinated pattern of polarization further lead- ing to embryo development. Tissues induced on medium with 2,4-D generally exhibited more pronounced cell proliferation than with NAA and had regions associated with epidermal rupture and callus growth. In addition, embryogenic cell protrusions and the appearance of embryos

occurred at least 3 to 4 days earlier. This observation is in accord with the idea that 2,4-D is a more active auxin than NAA (Jacobsen 1983, Gamburg 1988). The greater efficiency of 2,4-D compared with NAA has been attributed to its higher mobility and limited rate of oxidation and conjugation in tissues (Ingen- siep 1982, Jacobsen 1983). Such factors may result in a higher net accumulation of 2,4-D and may in part contribute to the greater cell proliferation and more internal origin of embryogenic cells observed here. In contrast, our earlier study (Rodriguez and Wetzstein 1994) suggested that when cultures were induced with NAA embryogenesis occurred faster than with 2,4-D. However, in that study cultures were rated weekly with only the aid of a dissecting microscope. The extensive proliferation of callus obtained with 2,4-D likely obscured early embryogenic events. Formation of embryogenic protuberances was preced- ed by pronounced accumulation of starch granules in the more superficial subepidermal cell layers of the explant. Starch was rapidly used during formation of embryogenic regions and was absent in globular and heart-shaped embryos. Some starch granules were seen again at early cotyledonary stages and we believe this could be related to initiation of secondary embryogenesis in these tissues. Late cotyledonary somatic embryos, which did not enter repetitive embryogenesis, did not show starch accumulation at this stage. This pattern of starch accumulation and use has been reported earlier in other embryogenic and organogenic systems (Arnold 1987, Hartweck et al. 1988, Barciela and Vieitez 1993). Starch is thought to serve as a prime source of energy for cell proliferation and growth. The results of this study strongly suggest a multicel- lular origin of embryos, as indicated by the large bulging meristematic areas observed and the broad attachment of embryos. A multicellular origin has similarly been observed in other systems including Camellia japonica (Barciela and Vieitez 1993), Helianthus annuus (Bronner et al. 1994), Feijoa sell- owiana (Canhoto and Cruz 1996), Trifolium repens (Maheswaran and Williams 1985), and Picea abies (Mo and Arnold 1991). This is in contrast to early concepts of somatic embryogenesis, where embryos were defined as having a single-cell origin (Street and Withers 1974) and no vascular connections with the maternal tissue (Haccius 1978). Trichome-like struc- tures similar to those characterized as early somatic embryos in Trifolium repens (Maheswaran and Williams 1985) were present. However, further devel-

82 A.P.M. Rodriguez and H. Y. Wetzstein: Pecan somatic embryogenic cultures

opment of these structures was never observed. Stain- ing with acid fuchsin/toluidine blue showed a high accumulation of phenolic compounds, which have been identified with trichomes of several species (Peterson and Vermeer 1984) including pecan (Wet- zstein and Sparks 1983). A greater diversity of embryo forms was observed in 2,4-D-induced cultures, with a higher incidence of abnormalities. Some embryos had defined shoot and cotyledon primordia, resembling zygotic morpholo- gy. Alternatively, different abnormalities were appar- ent during early stages of cotyledon enlargement including fused, fan-shaped, or trumpet-shaped cotyledons. Tissues were characteristically composed of highly proliferative, heterogeneous cell types including callus. Somatic embryos differentiated from extensive embryogenic protrusions that initiated from multiple cell layers of cotyledon explants. This intensive heterogeneous proliferation could interfere with normal patterns of embryo development. Canho- to and Cruz (1996) attributed a high incidence of abnormal embryos in Feijoa sellowiana to a multicel- lular origin of most embryos. Halperin and Wetherell (1964) observed that high concentrations of 2,4-D could cause abnormalities, or inhibit the development of apical meristems. In our studies with pecan cul- tures induced with 2,4-D, we observed somatic embryos with shoot apices disrupted due to callus proliferation. Polar auxin transport is critical to regulate the initia- tion of the embryonic axis and for the establishment of bilateral symmetry in early embryogenesis (Fry and Wangermann 1976, Schiavone and Cooke 1987, Liu et al. 1993). It should be noted that in cultures induced with 2,4-D, development of somatic em- bryos, including cotyledon expansion, was evident by day 7, when explants were still on induction medium with an auxin. These early and subsequently forming embryos had a greater incidence of abnormality than embryos developing from cultures induced with NAA. A poorly developed apex with fused cotyledons was characteristic of 2,4-D-induced somatic embryos. High exogenous auxin levels in the medium or pre- sent in the tissue through absorption could greatly modify synthesis and relative distribution levels of auxins within the embryo. Liu et al. (1993) proposed that the site of auxin synthesis was the shoot pri- mordium or surrounding area, and that auxin polar transport would allow normal cotyledon formation. In contrast, inhibition of polar transport resulted in ran- dom diffusion of the auxin to the surrounding cells

and formation of collar-like cotyledons. It may be possible that in addition, lower synthesis of auxin would occur in poorly formed apices, which were observed in the present study, and in turn affect avail- ability to cotyledon-forming areas. Interestingly, the divergences in embryo morphology observed in the two types of auxin used in induction persists for sev- eral months after transfer from induction medium. It is unknown if this could be explained by a possible residual effect of 2,4-D. This work shows that the type of auxin used for the induction of somatic embryogenic cultures can have a pronounced effect on the extent of cell proliferation and the cell types or populations that become mitoti- cally active. The mode of initiation and development of embryogenic regions can in turn influence the mor- phology, germination, and conversion of somatic embryos. In the pecan system, both 2,4-D and NAA can elicit high embryogenic responses. This work demonstrates that in the development of embryogenic protocols, a valuable consideration would be to opti- mize the selection of appropriate induction conditions to produce normal and functional embryos. Future studies of interest would be to determine how preva- lent the differences in action of these and other auxins are in other culture systems.

Acknowledgements We thank Conselho Nacional de Desenvolvimento Cientffico e Tec- nol6gico do Brasil (CNPq) for providing scholarship support for this project, and Hazel Magner and Cathy Kelloes for technical assis- tance.

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