PlantCell Reports

Plant Cell Reports (1993) 12:316-319

~: Springcr-Verlag 1993

Culturing peanut (Arachis hypogaea L.) zygotic embryos for transformation via microprojectile bombardement Jennifer A. Schnall and Arthur K. Weissinger Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA Received September 25, 1992/Revised version received January 27, 1993 -Communicatcd by G. C. Phillips

Abstract. A method for rapidly producing fertile peanut (Arachis hypogaea L.) plants from embryo axes was developed for use with microprojectile bombardment. Using this method, the apical meristem housing the germ line cells was easily exposed for bombardment without compromising the viability of the plant. Germination was rapid on MS based, hormone-free medium containing 2% agar, while medium containing 0.6% agar caused embryo axes to swell and to develop slowly. Representatives from the market classes virginia, spanish, and runner all responded well to this procedure, while UPL Pn4 (valencia) failed to form new leaflets. Microprojectile bombardment did not substantially impair embryo development. This method circumvents chromosomal and developmental abnormalities that could potentially occur with other peanut in vitro culture systems. A b b r e v i a t i o n s : MS: Murashige and Skoog medium; GUS: B-glucuronidase; X-gluc: 5-bromo-4chloro-3- indoly113-D-glucuronic acid Introduction Genetic engineering of peanut (Arachis hypogaea L.) could greatly facilitate improvement of this important crop, but the production of stably transformed peanuts has yet to be reported. Gene transfer via Agrobacterium has so far proven difficult, resulting only in the production of stably transformed callus (Lacorte et al., 1991; Franklin et al., 1992). Microprojectile bombardment provides a powerful method for the transformation of recalcitrant plant species (for review, see Birch and Franks, 1991). In order for this method to be effective, transformation techniques must be compatible with a reliable regeneration procedure. Embryonic leaflets can be induced to form fertile plants via shoot organogenesis (Mroginski et al., 1981; McKently et al., 1991) and by embryogenesis (Baker and Wetzstein, 1992), but

Correspondence to: A. K. Weissinger

transformation of leaflets has so far only resulted in the production of stably transformed callus (Clemente et al., 1992). Peanut plants can also be regenerated from cotyledons (McKently et al., 1990; Daimon and Mii, 1991) and somatic embryos (Hazra et al., 1989; Ozias-Akins, 1989; Sellars et al., 1990), but all of these methods are both time consuming and susceptible to somaclonal variation (Graybosch et al., 1987), which can lead to loss of regeneration ability or desired genetic traits. Microprojectile bombardment of zygotic embryos offers an attractive alternative for the stable transformation of peanut. The goal is to target apical meristems housing germ line cells, resulting in production of chimeric plants yielding stably transformed progeny. This method has resulted in the commercial production of transgenic soybean (Christou et al., 1990). Targeting peanut apical meristems is difficult, due to the presence of embryonic leaflets which limit access to the apical region. This paper describes a rapid, reliable method for the preparation of peanut zygotic embryos for microprojectile bombardment, and subsequent culture to produce fertile plants.

Materials and Methods Genotypes. Seeds ofArachis hypogaea L. were chosen to represent four market classes, including the cultivars NC 7 (virginia), Tamnut 74 (spanish), UPL Pn4 (valencia), and Florunner (runner).

Culture procedures. Embryo axes of each genotype were manually excised from non-imbibed seeds. Because the dry embryonic leaflets were quite fragile, the apical meristems could easily be exposed for subsequent bombardment by gentle scraping of the region with a scalpel under a dissecting microscope. Embryo axes were sterilized by soaking for 2 min in a solution of 20% commercial bleach containing 1 drop of a mild dish washing detergent (Ivory liquid rM) per 100 ml, and rinsed 2X in sterile distilled H20. Axes were then transferred, radicle side down, onto 40 ml of medium containing 0.5X MS salts (Murashige and Skoog, 1962), pH 5.8,

317

Fig. 1. The development of cultured peanut zygotic embryo axes. All embryos are NC 7. A: Freshly-prepared embryo axis (Day 0). Embryonic leaflets have been removed to expose the apical dome (arrow). B: Embryo axis on day 6. C: Embryo axis on day 9. Note the emergence of new leaflet (arrow). D: Plant on day 14, with expanding leaflet (arrow). E : Plant 1 week after transfer to soil. F: Fertile plant, approximately 3 months after culture initiation. Scale bars represent 2 mm. solidified with 20 g/L agar (or 6 g/L agar where noted), in a 100 x 20 mm petri plate, at a density of 10 axes per plate. Depressions to hold the embryos were made in the medium prior to embryo plating using hot forceps. Plates were incubated in a growth chamber under a 16 h photoperiod at 28 ~ at a light intensity of 30 ~tmol m2s-1. After 4 to 6 weeks, plants were transferred to 20 cm pots containing 2 parts soil: 2 parts sand: 1 part Metromix (WR Grace, Cambridge, MA), and grown to maturity in a greenhouse without supplemental light, initially under a short day cycle (December and January).

Embryo measurement. Embryo length was measured from the top of the exposed apical meristem to the base of the radicle. Width was measured across the top of the embryo axis, along a central line perpendicular to the axis of the lateral buds. To increase uniformity, NC 7 embryos 3 x 4 mm (in width x length) were chosen for culture. Twenty embryo axes were cultured per treatment. Microprojectile bombardment. To examine the effects of bombardment on regeneration, a total of 19 NC 7 embryos on 4 plates containing 2% agar medium was bombarded 3 d after preparation and subsequently observed over a 2 week period. Twenty untreated embryos were used as controls. Bombardment was performed using

the Bio-Rad BIOLISTIC | PDS-1000/He particle delivery system. Gold particles 1.0 btm in diameter were coated with 5 ~tg pRT99 (containing the GUS gene behind the 35S promoter; Ttpfer et al., 1988) per 50 Ixl particle prep, as described in the manufacturer's protocol. Plates were positioned 8 cm from the launch assembly, and bombarded using a rupture disk pressure of 276.8 kg/cm 2 (1550 psi). To assay transient GUS expression, a total of 8 plates containing 4 embryos/plate was bombarded as above. GUS expression was assayed 4 d after bombardment by staining the top half of each embryo in X-gluc for 12 h at 37 ~ and counting GUS-positive blue loci (Jefferson, 1987).

Results

and

Discussion

Development of the apical region The apical m e r i s t e m c o u l d easily be e x p o s e d for bombardment without compromising subsequent d e v e l o p m e n t (Fig. 1). The a p i c a l m e r i s t e m o f a f r e s h l y - p r e p a r e d N C 7 e m b r y o axis g r o w n on M S m e d i u m supplemented with 2% agar appeared quite smooth and pale (Fig. 1A). Several days later, the

318 embryo expanded significantly, and the apical meristem became more well-defined (Fig. 1B); greening occurred approximately 2 d after embryo plating. A new leaflet emerged between day 5 and day 14 (Fig. 1C). Older embryos with expanding leaflets (Fig. 1D) were transferred to the greenhouse, where their leaflets continued to expand (Fig. 1E), and the plants flowered and set seed (Fig. 1F). The average duration from culture initiation to flowering of NC 7 plants was 3 months, compared to approximately 6 weeks from planting to flowering for conventionallygrown plants. All 13 4- to 5-month-old NC 7 plants currently growing have set seed. 10 8 .~

6

~

42

~

0.6%

8.0 6.0 4.0 2.0 0.0 0.1

0.2 0.3 0.4 0.5 0.6 0.7 Width:Length Ratio Fig. 3. The effect of agar concentration on NC 7 peanut embryo width. For conditions, see Fig. 2 legend.

2%

Effect of genotype on development t ~ ~

0

Length (ram) Fig. 2. The effect of agar concentration on embryo length. Twenty NC 7 peanut embryo axes 3 x 4 mm in size were cultured per treatment. Values expressed were measured 7 d after culture initiation.

To test whether this culture procedure is applicable to genotypes in different peanut market classes, representatives from four market classes were cultured on 2% agar medium and the timing of first leaflet emergence for each plant was noted (Fig. 4). NC 7 (virginia) peanuts developed leaflets most rapidly, with 95% of the plants producing first leaflets by day 20. Florunner plants also developed rapidly, with 70% of the plants initiating first leaflets by day 20; no

10 Culture media

[] NC7 ~J

A minimal Murashige and Skoog (MS) based medium promoted rapid, normal growth of excised embryo axes. Knop's agar, used by Braverman (1975) in the culture of peanut embryo axes, was equally effective (not shown), but is not as readily available as MS salts. MS medium supplemented with 3% sucrose, utilized by Atreya et al. (1984) in peanut embryo axis culture, inhibited greening and hampered rapid development (not shown) Agar content greatly influenced embryo axis germination. Medium containing 0.6% agar (chosen for ease of embryo plating) was compared with a 2% agar medium (chosen to simulate the mechanical constraint that cotyledons place on embryo axes). After 7 d in culture, NC 7 embryos on 0.6% agar were considerably shorter than those on 2% agar medium (Fig. 2). Embryos on 0.6% agar ranged from 5 mm to 11 mm in length; almost half were 7 mm long. In contrast, embryos on 2% agar ranged from 5 mm to 26 mm long. Embryos on 0.6% agar were swollen and therefore relatively wide, with a median width to length ratio of 0.5 compared to only 0.4 for embryos grown on 2% agar (Fig. 3). Moreover, 70% of the embryos on 2% agar were green after 7 d, while only 15% of the embryo axes on 0.6% agar were green. Subsequent plant development on 2% agar was also relatively rapid.

r'] Florunner

gl

TA

ur

1,=11tJw i, ii Day Fig. 4. The effect of genotype on the emergence of first leaflets. A total of 20 embryo axes was cultured for each peanut genotype. Plants were scored daily for the presence of distinct, centrally located, newly emerged leaflets. additional Florunner meristems initiated first leaflets after day 20. Ninety-five percent of the spanish (Tamnut 74) plants produced first leaflets by day 28, while the valencia (UPL Pn4) plants failed to initiate any first leaflets over the observation period. Radicle and hypocotyl growth occurred in the UPL Pn4 plants, but the apices browned. Perhaps a more gentle procedure is required for the removal of embryonic leaflets or surface sterilization in this genotype. Care

319 should be taken to avoid abrading the apical meristems in these small embryo axes. Moreover, the addition of ascorbic acid to the medium may help to prevent browning.

Effect of microprojectile development

bombardment

on

W o u n d i n g associated with m i c r o p r o j e c t i l e bombardment can sometimes impair regenerative ability (J. Sanford, pers. comm.). To test whether this was the case with this system, 19 embryos were bombarded 3 d after preparation and compared with 20 control embryos (Fig. 5). No first leaflets emerged until 5 d after bombardment. On day 5, first leaflets were visible on 25% of the plants in both treatments. Two weeks after bombardment, 68% of the bombarded plants had formed leaflets, while 75% of the control plants had initiated leaflets. Therefore, t h e bombardment conditions used in this study did not substantially impair regenerative ability. Moreover, bombardment did serve to deliver DNA to the apical meristem region. Bombardment of 32 embryos yielded an average of 9.1 GUS positive foci/embryo (SE=6.7) in the apical meristem region. The bombardment conditions were not optimized for transient expression. Clemente et al. (1992) could not detect a correlation between transient and stable expression in bombarded peanut leaflet tissue.

1oo

[]

Bombarded

80-

40r 200 16 Day Fig. 5. The effect of microprojectile bombardment on the emergence of new leaflets. Nineteen bombarded NC 7 peanut embryo axes were compared with 20 unbombarded (control) axes. Plants were scored as described in Fig. 4 legend. In conclusion, zygotic embryo axes can quickly and easily be prepared for microprojectile bombardment. This method allows for rapid exposure of apical meristems and subsequent recovery of healthy plants, even when the meristems are exposed to microprojectile bombardment. All of these elements will be essential for the successful production of stably transformed plants.

Acknowledgements. We would like to thank Dr. T.G. IsleiD (NC State) for the seeds used in this study. We thank Drs. T.G. Isleib and H.T. Stalker (NC State) for critically reviewing this manuscript. This work was supported in part by USDA-ARS Grant 58-6435-1117 from the National Program for Food Safety and Health, and support provided by the Office of Agriculture, Bureau for Science and Technology, US Agency for International Development, under Grant No. DAN-4048-G-00-0041-00.

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