Plant regeneration at high frequency from mature sunflower cotyledons

Plant Science, 73 (1991) 219--226

219

Elsevier Scientific Publishers Ireland Ltd.

Plant regeneration at high frequency from mature sunflower cotyledons Nathalie Knittel, Alejandro S. Escand6n and Giinther Hahne Institut de Biologie Mol~culaire des Plantes du CNRS, 12 Rue du G~n~ral Zimmer, 67084 Strasbourg C~dex (France)

(Received July 1lth, 1990;revisionreceivedSeptember 12th, 1990;accepted September 14th, 1990)

Methods for sunflower (Helianthus annuus L.) tissue culture and transformation are to date characterized by a low degree of efficiency, reliability and reproducibility. The most widely used approach for regeneration of plants from somatic cells makes use of immature embryos,a material laborious to obtain. We describe here an efficient regeneration systemusingcotyledonsof youngplantlets, yielding up to ten shoots per cotyledon. Factors found to influence quantitative aspects (frequencyof response) and qualitative aspects (responsetype) include hormonal balance and nitrogen supply in the medium, as well as explant type, age and genotype. Physical culture conditions, while exerting a certain effect, appear to be of minor importance. A number of regenerated plants have been transferred to the greenhouse and seeds were obtained from each plant. The total time required from isolation of the explant to harvest of seeds was 4--6 months. Key words: cotyledon;Helianthus annuus; plant regeneration; sunflower

Introduction The application of biotechnological methods for the study and the improvement of sunflower ( H e l i a n t h u s a n n u u s L.) is mainly limited by the difficulty of regenerating plants in a reproducible and efficient fashion. Plant regeneration has been reported from a variety o f tissues, such as immature embryos [1--3], from callus cultures by either somatic embryogenesis or shoot morphogenesis [4 --6] and even from protoplasts [7]. Considering the difficulties generally encountered with regeneration o f sunflower plants, approaches involving callus cultures induced on tissues such as hypocotyl, or protoplast cultures, appear to be useful for plant regeneration under specific conditions only, or from certain genotypes. So far, the only explant allowing reproducible plant regeneration in many Correspondence to: G. Hahne, Institut de BiologieMol6culaire

des Plantes du CNRS, 12 Rue du G~n6ral Zimmer, 67084 Strasbourg Cedex, France.

laboratories with an appreciable variety of genotypes is the immature embryo. However, to obtain this explant demands a considerable effort, as the donor plants must be grown almost to maturity under controlled conditions and embryos isolated from immature seeds. In several plant species, cotyledons have been found to possess a high potential for plant regeneration [8m10]. Often the basal part of the cotyledon can give rise to a number o f shoots without an intervening callus phase. Whole plants are easily regenerated from these shoots. Cotyledons have the advantage of being easily and quickly available and do not demand much effort for their excision. We describe here an efficient protocol for the fast (direct) regeneration of sunflower plants from cotyledons of young plantlets. Some important factors influencing the regeneration efficiency have been identified and optimized and fertile plants regenerated. Seeds from regenerated plants can be harvested 4 - - 6 months after the beginning o f the experiment (germination of the donor seed).

0168-9452/91/$03.50 © 1991Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland

220 Methods Most experiments were performed using the inbred line H A 300B, generously provided by Sanofi Elf Biorecherches (Lab6ge, France). The other genotypes tested were obtained from the same source or were a kind gift of Cargill (Sauzet, France). Seeds were sterilized in 70070 ethanol (1 min) followed by 4070 sodium hypochlorite, diluted f r o m a 16°70 commercial solution ('eau de Javel'; 15 min under vacuum, then 15 min at atmospheric pressure with a drop o f detergent added). The sterile seeds were imbibed in water for 6 h in darkness (20°C), then germinated on solidified, half strength Murashige and Skoog (MS) medium [21], containing 10 g/l sucrose. Seeds were kept in darkness (20°C) for 3 days before transfer to light (25 °C with 16 h days at 20--30/~E m -2 s -~ provided by O s r a m Daylight fluorescent tubes L58W/19 and 8 h nights). The germinated seeds were maintained under these conditions for 1--5 days, depending upon the experiment. Cotyledons were excised f r o m the plantlet, taking great care to avoid including the axillary meristem. Each cotyledon was routinely divided into two halves (basal and distal). In some experiments, the basal (proximal) half was further divided and the abaxial and adaxial quarters were cultured separately. For most of the experiments, we used a modified version o f the medium described by Paterson and Everett [6]. If not indicated otherwise, it was supplemented with 1 rag/1 benzyladenine (BAP) and 0.5 mg/1 a-naphthalene acetic acid (NAA). This medium contains a very high amount of KNO 3 (a total o f 6.9 g/l). In order to maintain constant level o f K when varying the NO 3- content, an appropriate amount o f K2SO 4 was added. In the experiment testing different nitrogen sources, mannitol was added in varying amounts to keep the osmolarity constant at a value equiva-

lent to the medium o f Paterson and Everett [6]. The osmolarity has always been verified with a freezing-point osmometer (Gonotech, Berlin). Each experimental value is the mean of three independent experiments using 6 cotyledons each. Each culture was evaluated at 7 and 21 days after their initiation. Shoots having grown to a size o f approximately 5 m m could be excised and rooted on agar-solidified (Merck, 7 g/l) half strength MS medium containing 10 g/l o f sucrose. Plantlets with welldeveloped roots were transferred to vials containing vermiculite and the same (but liquid) medium for 1--2 weeks, and then transferred to soil and kept in the greenhouse with temperature set to 19°C; 16 h daylength (if necessary, the light level was maintained at around 10 000 Ix with supplementary lighting, provided by high-pressure sodium and mercury lamps at equal ratio), and 8 h nights. Results and Discussion Cotyledons of young sunflower plantlets represent convenient explants for the initiation of cultures and they also have a considerable potential for regenerating shoots which then quickly give rise to whole plants (Fig. 1). The expression of this potential is influenced by a number of diverse factors, some of them related to the plant material (such as age, or genotype), others to the culture conditions (such as medium composition, or physical culture conditions). We have investigated the influence of a number of these factors on quantitative and qualitative aspects o f the morphogenic response.

Explant age and physical culture conditions The morphogenic response declined rapidly with increasing age of the cotyledons. The seedlings shed their seed husk during the fourth day of

Fig. 1. Regenerationof plants from cotyledon explants. (a) and CO)Large, fast growing shoots originate on the proximal (basal) part of the cotyledon (type 1). In Co),note the small shoots surrounding the base of the large one. Their proliferation is encouraged by the removal of the predominant shoot. Bar = 3 mm. (c) Clusters of small shoots originate from the surface of the basal part of the cotyledon (type 2). Bar = 3 mm. (d) and (e) Proliferation of nodular tissue, breaking through the epidermisof the distal part of the cotyledon (type 3; (d) bar = 3 mm) and clusters of shoot primordia originating from such tissue ((e) bar = 3 mm). (f) and (g) Regenerated plants in greenhouse (bar = 5 cm). Seeds were obtained from each inflorescence.

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culture, this being the first possible moment for a controlled excision of the cotyledons. Multiple shoot formation was observed in up to 80°7o of the explants only 4--6 days after germination and decreased to 0 thereafter (Fig. 2). No difference was observed when seedlings were cultured entirely under long-day conditions (16/8 h), shortday (12/12 h) conditions, complete darkness, or continuous light (data not shown). However, the response was maximal under long-day conditions with a dark preculture period of 3 days. The other culture regimes yielded a 20°/0 lower frequency of responding explants. Since the highest percentage of responding explants was obtained at day 4, these conditions were used for all further experiments.

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Fig. 2. Influence of explant age on the regeneration frequency. Cotyledons were put into culture at various times after onset of germination. The seedlings were cultured for 3 days in darkness, then under long-day conditions (16 h/8 h). The explants were cultured on the medium after Paterson and Everett [6], containing 2 mg/l BAP as the only hormone. II, percentage of responding explants; r-I, number of shoots per cotyledon.

Hormones

Phytohormones are indispensible for induction of the morphogenic response in sunflower cotyledons. A relatively wide range of combinations of the auxin, NAA and the cytokinin, BAP, is capable of inducing the formation of a few shoots on a relatively large portion (20--80o7o) of explants (for details see Fig. 3) but in many cases this shoot

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Fig. 3. Influence of the hormonal composition of the culture medium on shoot induction frequency. Cotyledons (4 days) were cultured on PER medium supplemented with the indicated concentrations of NAA and BAP and number of shoots per cotyledon as well as the percentage of responding explants were determined. The range for efficient regeneration is quite large. The combination 0.5 mg/l NAA and 1.0 mg/l BAP was identified as the best compromise, because it induced the least callus development.

223 formation is accompanied by the formation of roots or callus. A high frequency of shoot induction with limited interference by callus or root formation is restricted to a more limited range of hormone combinations. BAP alone is sufficient for a satisfactory response but the addition of a low concentration of NAA proved beneficial (Fig. 3). In the presence of high NAA concentrations shoot induction is severely reduced or even abolished. The best compromise between shoot induction and callus/root formation was found to occur at 0.5 mg/1 NAA combined with 1 mg/l BAP. This combination of hormones induced three types of morphogenic response: (1) The formation of a few (1--10) fast growing, easily rooting shoots that could be excised 10--12 days after culture initiation. This response was limited to the basal part of the cotyledon and the upper part of the petiole (Figs. la and lb). (2) The second type is characterized by the induction of a relatively large number of small shoots, squeezed together in one or several small areas (a few mm 2) in the basal part of the cotyledon (Fig. lc). These shoots grew relatively slowly, probably as a result of strong competition between them, and were extremely difficult to root. (3) In contrast to the types 1 and 2, the third type of response observed was located preferentially on the distal part of the cotyledon and was accompanied by a proliferation of tissue (Figs. ld and le). A very high number of small shoots (up to 100) emerged from this tissue. These shoots were also slow-growing and difficult to root. All three responses have been observed mixed on the same cotyledon, although absence (or excision) of the large, fast-growing shoots appears to favor development of the other responses. Nitrogen source

In general, the responses of cultured sunflower tissues appear to be strongly influenced by the nitrogen source contained in the medium. A high concentration of NO 3- has been shown to favor callus growth and shoot regeneration from these callus cultures [6] whereas the culture of hypocotyl protoplasts is most efficient when glutamine is the sole nitrogen source [11[. The experiments described above were all performed using the nitrate-enriched medium described by Paterson

and Everett [6], which gave satisfactory results. In order to study the possible influence of the N source on the morphogenic response in sunflower cotyledonary explants the different N sources were supplied singly or in combination (Fig. 4). In the total absence of externally added nitrogen, the explants remained green and showed the usual increase in size. A low but significant percentage (< 20%) of cotyledons formed a small number of shoots (on average 0.1 shoot/cotyledon). With ammonium as the sole nitrogen source (supplied as (NH4)2SO4), the explants turned brown, degenerated quickly and no shoot formation was ever observed. The amino acids glutamine and asparagine, when supplied as sole N sources, stimulated shoot regeneration slightly above background (no N source) and a similar observation was made with casein hydrolysate as sole N source (for details, see Fig. 4). Nitrate stimulated shoot formation to a higher level with, in particular, the percentage of responding explants showing an increase with increasing nitrate concentrations while the number of shoots formed per explant remained relatively low. The highest frequencies, however, were obtained with combinations of several N sources (Fig. 4), the best being the original formulation [6]. It is clear from these experiments that the nitrogen supply has a decisive influence on the morphogenic response of sunflower cotyledons. The important factor is not a single compound but an appropriate balance of N sources, at sufficiently high concentrations, appears to be necessary for an optimal response. Orientation on the m e d i u m

As different parts of the cotyledon responded in different ways, we divided cotyledons into a distal half and a proximal half. Both were cultured with either the abaxial or the adaxial epidermis in contact with the medium. Some of the basal (proximal) parts were further subdivided to obtain the abaxial and adaxial basal quarters and these were cultured with either the epidermis or cut face towards the medium. The distal halves rarely regenerated shoots directly, regardless of orientation. However, 48°/0 of these explants responded with the tissue proliferation of type 3 (described above) on the adaxial

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Fig. 4. Influenceof the N source on shoot induction frequency. Cotyledons(4 days) were cultured on modifiedmedium after Paterson and Everett [6] (supplemented with 0.5 mg/l NAA and 1.0 mg/l BAP) containing the indicated N sources. The number of shoots per cotyledonand % of ¢xplants responding are shown. The best response was obtained with the original medium formulation, with organic supplements being of minor importance.

(upper) epidermis, provided this side was facing the medium. The other orientation and the abaxial (lower) epidermis in either orientation, never gave rise to any morphogenic response. The basal part, when left intact, regenerated a few shoots (mean, 1.5) on 60070 of the explants from the adaxial face when this was oriented away from the medium. The abaxial surface o f intact basal explants was never observed to give rise to shoots whatever the orientation. The simple operation o f further subdividing the basal half increased the regeneration frequency considerably on both the adaxial (7 shoots/cotyledon on 70°70 o f the explants) and the abaxial face (0.3 shoots/cotyledon on 10% of the explants), provided that the cut surface was oriented towards the medium.

Apparently, the nutrient supply to the cultured tissue is facilitated when a wounded surface is in contact with the medium, which increased the morphogenic response. The type o f response (1 or 2) does not seem to depend on these factors. Nevertheless, the adaxial face remains more responsive than the abaxial one.

Genotype A m o n g the factors having an influence on regeneration capacity, the genotype is usually ranked quite high. For this reason, we studied the frequency of shoot induction for a variety of sunflower lines: one hybrid (Mirasol), one inbred restorer line (RHA 274) and four inbred lines usually employed as female parent (HA 89, H A 401, H A

225 Table !. Genotype dependence of shoot induction. Explants were cultured on medium after Paterson and Everett [6], modified to contain 0.5 mg/! NAA and 1.0 mg/l BAP. Genotypes

Type

Shoots/cotyledon

Explants responding

(%) HA 300A HA 300B HA 89A HA 89B HA 699B HA 401B RHA 274 Mirasol

Inbred; cms Inbred; fertile Inbred; cms Inbred; fertile Inbred; fertile Inbred; fertile Inbred; restorer Hybrid

9.4 9.5 0.3 0.5 0.4 0.6 2.3 1.2

70 80 30 35 25 40 50 65

300, H A 699). Two of these lines (HA 89, H A 300) were used in their male fertile (B) as well as their male sterile (cms; A) form. The genotype was found to exert a pronounced effect on the regeneration frequency (Table I), the best responding line being H A 300. Presence or absence of the cms trait had no visible influence on the regeneration rate. The two lines, H A 89 and H A 401, are genetically relatively closely related and also showed similar frequencies for shoot induction as well as for the proportion o f explants responding to the treatment. Furthermore, Mirasol, a hybrid obtained from H A 274 and H A 89, showed a shoot regeneration frequency (number o f shoots per cotyledon) intermediate between the two parents. However, the percentage of responding explants was higher than for either parent, which might be explained by heterosis. Our observations made so far with a limited number of genotypes are consistent with the idea that amenability to tissue culture is a genetically determined and transmissible trait, as has been previously described for, e.g., melon [12], wheat [13] or orchard grass [14]. This genotype-related regeneration ability may even be enriched by breeding, as shown for alfalfa [15].

and shoots reached a size sufficient for excision only after one or more subcultures on a medium with a reduced (1/10) concentration of both hormones. These shoots were often vitrified. A small percentage of them, nevertheless, developed into normal shoots and root formation was observed in some cases. All regenerated plants showed a phenotype atypical for sunflower (Figs. If and lg). They were generally stunted, often branched with a flower on each branch and the main axis was frequently shorter than the side shoots. The inflorescences were small and carried a varying proportion of sterile flowers, but seeds (3--30) have been harvested from each plant after selfing (at present, more than 20 plants). Analysis of the offspring is currently in progress. The atypical phenotype of the regenerated plants can be related to the premature flowering in vitro that is almost unavoidable. Similar observations have been made with plants regenerated from immature embryos [1--3,16,17]. In these cases, the subsequent generations obtained by selfing were found to be completely normal and indistinguishable from normal seed-grown plants.

Transfer to greenhouse

Sunflower cotyledons carry a high potential for direct shoot regeneration. This phenomenon has recently been reported in a short note [18] and is not unlike the situation known from other plants [8--10]. Power et al. [19] described a system for

The fast-growing shoots (type 1) presented no major difficulty either for rooting or for subsequent transfer to the greenhouse. Type 2 and type 3 shoots were more problematic. They grew slowly

Conclusion

226

the regeneration of adventitious shoots from mature and immature embryos. In this case, morphogenesis occurred along the cut edges together with callus formation and the morphogenic potential was rapidly lost after germination. We have shown here in a different system that the use of sunflower cotyledon obtained from a young plantlet permitted fast and efficient regeneration of fertile plants without an intervening callus phase. A histological study is under way, but from preliminary observations and by analogy to cucumber [20], we have the impression that even the fastgrowing shoots (type 1) are formed de novo and not from pre-existing meristems. This system should be useful for clonal multiplication of individual genotypes and has a potential for transformation experiments.

Acknowledgments We gratefully acknowledge the gift of seed material from Sanofi Elf BioRecherches (Toulouse, France), as well as from Cargill (Sauzet, France). A.S.E. was supported by a stipend from CONICET, Argentina.

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