Plant Cell, ?issue and Organ Culture 44: 183-188, 1996. 183 (~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

K i r s t e n Vier te l & D i e t e r Hess* Institute of Plant Physiology, University of Hohenheim, Emil Wolff Str. 25, D-70593 Stuttgart, Germany (* requests for offprints)

Received 10 August 1993; accepted in revised form 29 March 1994

Key words: clonal propagation, somatic embryogenesis, shoot tip, Triticum aestivum L.

Abstract

Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 ~tM 2,4-dichlorophenoxyacetic acid (2,4-D) for culture initiation, and 5 ktM 2,4-D for subculturing, was optimal; 90% of 4 day-old 'Turbo' seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-D, but supplemented with 2.22 ~ 6-benzylaminopurine and 0.27 glVl naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic 'Turbo' calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using 'Nandu' were within the same range.

Abbreviations: BA - 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic acid; NAA - naphthaleneacetic acid

Introduction

The application of many biotechnological methods in cereal improvement involves development of regen- erable tissue cultures. Superior plant regeneration depends on the production of embryogenic callus, which is also used to initiate embryogenic suspensions.

The most commonly used source of embryo- genic callus cultures of wheat are immature embryos (reviewed by Scott etal., 1990), but other explants such as mature embryos (Mohmand & Nabors 1991 ; Kato et al., 1991), leaf segments (Wernicke & Milkovits 1984; Barcelo et al 1991; Rajyalakshmi et al., 1991), and longitudinally split shoot tips (Wernicke & Milkovits 1986) were used with varying success. The capability of wheat tissue cultures to regenerate plants is influ- enced not only by the starting material, but also by cul- ture media and genotype (reviewed by Mathias 1990).

In comparison with rice (Oryza sativa L.), wheat remains a species needing additional research regard- ing genetic transformation. Transgenic wheat could yet

be obtained only via particle bombardment of imma- ture embryo-derived callus (Vasil et al., 1992; Weeks et al., 1993). This method required highly embryogenic cultivars (Vasil et al., 1993; Weeks et al., 1993) or a period of 4-5 months to establish embryogenic Type C callus (Vasil et al., 1992). Therefore, the objec- tive of our work was to develop a genotype indepen- dent method that utilized shoot tip explants for rapid production of embryogenic callus, to limit the risk of somaclonal variation. Shoot tip-derived callus may be used in gene delivery by particle bombardment and/or A grobacterium.

Materials and methods

Preparation of plant material

Seeds of greenhouse-grown Triticum aestivum L., cvs. Turbo and Nandu plants, were used to initiate aseptic seedling cultures. Both varieties are common German

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summer wheat varieties. 'Turbo' has a strong winter wheat background. 'Nandu' is a hybrid of 'Cosir' x ('Janus' x 'Kolibri').

Grains were surface disinfected with 70% (v/v) ethanol for I min, followed by immersion in commer- cial bleach (10.5% [v/v] hypochlorite) with additional 0.01% (v/v) Tween 80 T M (Serva) and 0.01% [w/v] Captan T M (Merck) for 1 h, and subsequently washed three times with sterile deionized water. Germination and seedling culture were carded out at 25°C and 16-h photoperiod (cool white fluorescent light, 5000 1 x) on WH medium (Wagner & Hess, 1974) with- out plant growth regulators, supplemented with 2.0% (w/v) sucrose and 0.8% (w/v) agar (gum agar; Sig- ma). Prior to autoclaving, pH was adjusted at 5.8. Two grains per compartment were incubated on 2 ml medi- um in replica dishes (25 compartments, each 100 x 100 mm). After 4 days, seedlings were transferred to 1000 ml glass jars containing 100 ml WH medium (6 seedlings/jar) and either grown for another 6 days or directly taken for explant excission.

Explant preparation

Shoot tip and leaf base explants were excised from 4 and 10 day-old seedlings of 'Turbo' and 'Nandu', accordingly, they were called 4T and 4N, 10T and ION.

Shoot tip explants were obtained using a dissecting microscope by removing the grain, roots, coleoptile and leaves from the seedling. The leaf directly sur- rounding the apex was trimmed to a size of 1-2 mm. The size of the shoot tip enclosed by the leaf base was approximately 0.5 mm (Fig. la). After removal of roots and coleoptile leaf base, explants were excised from the first and second leaf of 4 and 10 day-old seedlings, respectively. Leaves ranged in length from 35-50 mm for first, and 45-60 mm for second leaves. Explants comprised of the basal 5 mm of the leaf base, omitting parts of the apical meristem.

Callus induction and maintenance

Two types of culture media were used to induce and maintain callus, the first MSB, consisted of MS salts (Murashige & Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 3% (w/v) maltose, 0.8% (w/v) agar (gum agar; Sigma), and pH 5.8. The second ML3, was L3 medium (Jahne et al., 1991) supplemented with 3% (w/v) maltose, 0.8% (w/v) agar (gum agar; Sigma), and pH 5.8. MSB was autoclaved, ML3 was prepared

double concentrated, filtersterilized (pore size 0.22 grn; Millipore) and mixed with a double concentrated, auto- claved agar solution. Both media were supplemented with two different concentrations of 2,4-D (5 and 10 IJaM, respectively). All cultures were kept at 25°C in darkness.

For callus induction shoot tip explants were incu- bated in 15 ml test tubes (2 explants/test tube) contain- ing 3 ml induction medium. Leaf base explants were incubated in petri dishes (60 × 15 mm, 5 explants/dish) containing 8 ml induction medium. After 4 weeks individual shoot tip- and leaf base-derived calli were transferred to 30 ml glass jars containing 5 ml of the respective maintenance medium with either unchanged or reduced 2,4-D content and incubated for another 4 weeks. Medium and 2,4-D content will be indicated as follows: 10/10, 10/5 or 5/5 preceeding MSB or ML3 indicate 2,4-D level in callus induction/maintenance media.

Regeneration

Eight weeks after culture initiation, shoot tip- and leaf base-derived calli were subcultured on MSB without 2,4-D, but ~upplemented with 2.22 IsM BA and 0.1 ~ NAA. Cultures were kept at 25°C. Light intensity was increased from darkness to a 16-h photoperiod (cool white fluorescent light) of 2500 1 x after one and 4000 1 × after another week.

Plantlets derived from germinating somatic embryos and shoots regenerated from leaf base cal- lus were exposed to 10000 1 x (cool white flourescent light). Reaching a length of 5-7 cm they were trans- ferred individually to 1000 ml glass jars containing 100 ml rooting medium 190-2 (Zhuang 1984) without plant growth regulators supplemented with 3% (w/v) sucrose, 1% (w/v) activated charcoal and 0.8% (w/v) agar (gum agar; Sigma). Prior to autoclaving pH was adjusted to 6.0.

Ex vitro culture of regenerant plants

Well rooted single plants were potted in autoclaved soil (70% white peat and 30% clay). For acclimatisa- tion, the plants were covered with a plastic bag, kept in a growth chamber at 25°C in a 16-h photoperiod as before and after 3 days transferred to the greenhouse. The plastic covers were retained for another three days and thereafter removed. Subsequently, plants were grown under greeenhouse conditions: during summer times the photoperiod of a middle European temperate

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Fig. 1. Regeneration of Triticum aestivurn L. cv. Turbo using shoot tip-derived embryogenic callus cultures. Erabryogenic callus was induced on medium ML3 10/5. The bars represent 500 ~tm. (a) Freshly isolated shoot tip explant excised from a 4 day-old seedling. The surrounding leaf base was partly removed for optical reasons. (b) Early somatic embryo structures formed after 2 weeks on regeneration medium, 10 weeks after explant excision. (c) Somatic embryo-derived plantlets after 3 weeks on regeneration medium, 11 weeks after explant excision. (d) Green regeneraat plants after 2 weeks culture on rooting medium 190-2, 15 weeks after explant excision (e) Fertile green regenerant plant under greenhouse conditions, about 24 weeks after explant excision.

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region, and heating up to 20°C, if the ambient tem- perature fell below 14°C, during winter times 20°C and a photoperiod of 16 h by additional illumination (mercury-discharge lamps, 15000 1 ×). Fertilizer was given at weekly intervals (N/P/K = 12/8/11, 2 ml per 1 I of water).

Statistics

Each experiment was repeated once with a number of explants between 20 and 27. As an exception, exper- iments concerning shoot tip cultures on MSB media were not repeated, but explant number ranged from 25 to 32.

For statistical evaluation of embryogenic callus induction frequencies and frequencies of regeneration, X2-tests were used. Mean numbers of regenerated plants per explant were analysed following Students T-test.

Results and discussion

Callus induction

Irrespective of explant type, medium and cultivar test- ed, 100% of the explants developed callus after 1-2 weeks of culture. After 4 weeks two types of callus had formed from shoot tip explants. Translucent, soft, and watery callus resulting from surrounding leaf base and node tissue, was removed. The remaining compact cal- lus subsequently gave rise to embryogenic callus. Leaf base explants produced, besides a translucent, soft, and watery type of callus, white and friable callus.

Formation of embryogenic callus and plant regenera- tion

Wheat shoot meristems are highly morphogenic. Wer- nicke & Milkovits (1986) longitudinally split shoot tips of 21 randomly selected cultivars and incubated the half-cylinders on nutrient media. In the presence of 2,4-D primordia were kept in a proliferating bud- ding state. Removal of 2,4-D resulted in an outgrowth of shoots and roots, but no data concerning the rate of organogenesis versus embryogenesis were given (Wer- nicke & Milkovits 1986).

In our investigations, using shoot tip-derived cul- tures, embryogenic callus was formed 1-2 weeks after transfer to regeneration medium. It was characterized by its white, compact, and nodular appearance, and

E x p l a n t s f o r m i n g e m b r y o g a n i c c a l l u s [%]

4 T 10"I" 4 N 1ON

MSB 10110 ~ MSB 5/5 E ~ ML3 10/10 ~ ML3 10/5 ~ ML3 515

Fig. 2. Induction of embryogenic callus in shoot tip explants of wheat. Four (4) and ten (10) day-old seedlings of cvs. Turbo (T) and Nandu (N) were used as explant sources. The explants were kept on the induction/maintenance media indicated below. Evaluation 10 weeks after explant excision. Explants showing embryogenic callus are indicated as percent of the total number of explants. Standard deviations are indicated for ML3 media. Values of ML3 media differ statistically significant (p <0.01) from those of MSB media using the X 2-test.

developed into somatic embryos (Fig. lb). Germina- tion of somatic embryos started some days after their development, and reached a maximum after 3, 5 weeks of culture on regeneration medium.

Embryogenic callus

Formation of embryogenic callus was influenced by induction/maintenance medium, 2,4-D content, gent- type and age of the explant source (Fig. 2). On MSB medium the induction frequency of embryogenic cal- lus was zero at a low 2,4-D content (MSB 5/5), and 4-12% at the higher 2,4-D concentration (MSB 10/10). On the contrary, up to 90% of the shoot tips produced embryogenic callus on ML3 media. The stimulation of embryogenesis by ML3 medium might be due to the composition of the nitrogen source. The NH4+: NO3- ratio of anorganic nitrogen in ML3 is 11:89 compared to 36:64 in MSB. Additionally ML3 supplies organ- ic nitrogen, which as well as a reduced ammonium content might be favourable for embryogenesis and regeneration (Barcelo et al., 1993).

Irrespective of the induction/maintenance medium no significant differences between cvs. Turbo and Nan- du were obtained, if 4 day-old seedlings were used for explant excision (4T and 4N). Likewise, the frequen- cy of embryogenic callus induction from 10 day-old seedlings of 'Turbo' are comparable to 4T and 4N.

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Explant8 regenerating plants I%] 1oo

4T lOT 4N 1ON

mmMaB 10110 ~ M S B 5/5 ~ M L a 10110 B~IML3 1015 I~ IML3 5/5

Fig. 3. Plant regeneration from embryogenic shoot tip callus. Four (4) and ten (10) day-old seedlings of cvs. Turbo (T) and Nandu (N) were used as explant sources. The explants were cultivated on the induction/maintenance media indicated below, and thereafter on regeneration medium (see materials and methods). Evaluation 13 weeks after explant excision. Explants showing regenerant plantlets are indicated as percent of the total number of explants. Standard deviations are indicated for ML3 media. Values of ML3 media differ statistically significant (p <0.01) from those of MSB media using the xZ-test.

Number of regenerated planta per explant

lO

9 -

7 -

6 -

5

4

4T

? jT T I~\\\\\\\\\\\\\\ ' ~

lOT 4N 1ON

leaf balell [ ~ =hoottips

Fig. 4. Mean number of plants per explant regenerated from leaf base and shoot tip calli, respectively. Four (4) and ten (10) day-old seedlings of evs. Turbo (T) and Nandu (N) were used as explant sources. In both types of explants, mean values of cultures incubated on ML3 10/5. Evaluation 15 weeks after leaf base and shoot tip excision, respectively. Standard deviations are indicated.

shoot per explant (Fig. 4). Only minor differences between 'Turbo' and 'Nandu' were observed.

Explants of older 'Nandu' seedlings (10N), however showed a significantly decreased embryogenic callus induction frequency, indicating a pronounced effect due to age and genotype (Fig. 2). Leaf base-derived callus never showed embryogenic characteristics.

Plant regeneration

Plant regeneration in shoot tip-derived cultures occured by germination of somatic embryos and devel- opment of plantlets (Fig. lc). Once more ML3 was superior to MSB, reaching maximal values (90% of explants with regenerafit plantlets) at a 2,4-D content of 10 p~I during callus induction, and 5 ~M during callus maintenance (ML3 10/5). Once more, different regeneration capacities revealed a dependence on the age (Fig. 3). Calli derived from 4 day-old seedlings (4T and 4N) exhibited higher regeneration rates compared to older explant material (10T and 10 N). Comparing explants of the same age (4T versus 4N, and 10T versus 10N) genotypic differences were less pronounced.

Leaf base-derived calli regenerated shoots exclu- sively via organogenesis, there was no evidence for embryogenesis. Using shoot tips up to 8 plantlets developed from callus induced in a single explant (Fig. ld), whereas leaf base callus resulted in less than 1

Ex vitro culture

Plantlets from shoot tip explants could be easily adapt- ed to soil. A very high percentage (95-100%) of the regenerants survived on soil and could be grown into phenotypical normal and fully fertile plants (Fig. le). Eight months were needed from excision of the shoot tips until harvesting the grains produced by the regen- erated plants. In contrast, survival of leaf base-derived regenerants was variable and ranged from 28 to 90% across experiments, mainly due to week rooting of the shoots regenerated.

Considering the possibility of gene ~ansf¢rs, the shoot tip derived embryogenic callus system described here offers advantages compared with immature embryos. Firstly shoot tip explants excised from seedlings of the same developmental stage repre- sent a reliable source for embryogenic callus pro- duction, without the need of growing parental plants under conlxolled environmental conditions until imma- ture embryos develop. Secondly, genotype dependent restrictions found with immature embryos (Chowd- hury et al., 1991; FelfOldi & Purnhauser 1992), limit- ing a successfull transformation to highly embryogenic cultivars (Vasil et al., 1993 ; Weeks et al., 1993), might be overcome, since shoot tip-derived callus of two rather different summerwheat varieties exhibited only

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minor genotypic differences concerning the formation of embryogenic callus and its regeneration capacity. This holds true for two South African wheat varieties as well (data not shown). Nevertheless, further investi- gations are required to prove definitely, whether shoot tips might provide a genotype independent source for embryogenic callus. Thirdly, the use of shoot tips likewise offers a chance to obtain highly regenerable embryogenic callus suitable for particle bombardment in a shorter period of time compared to Type C callus (Vasil et al., 1992). Time saving would minimize the risk of somaclonal variation, which is detrimental with regard to wheat improvement via genetic transforma- tion.

Acknowledgements

Part of this work was supported by the Vater und Sohn Eiselen Stiftung, Ulm. Seeds of T. aes t ivum were kind- ly provided by F. von Lochow-Petkus GmbH, Bergen and A. van der Westhuizen.

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