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Medium optimization · Regeneration. Abbreviation 2,4-D: 2,4-Dichlorophenoxyacetic acid. Introduction. The first regeneration systems developed for oat (Avena.

Plant Cell Rep (2002) 20:1156–1161 DOI 10.1007/s00299-002-0467-7

CELL BIOLOGY AND MORPHOGENESIS

A.M. Nuutila · C. Villiger · K.-M. Oksman-Caldentey

Embryogenesis and regeneration of green plantlets from oat (Avena sativa L.) leaf-base segments: influence of nitrogen balance, sugar and auxin Received: 7 January 2002 / Revised: 21 March 2002 / Accepted: 25 March 2002 / Published online: 14 May 2002 © Springer-Verlag 2002

Abstract The nitrogen composition and sugar and auxin concentrations of callus induction medium were optimized in order to improve the regeneration of green plants from two elite oat cultivars, Aslak and Veli. For both cultivars, the production of green plantlets was doubled by optimization. However, the results obtained also clearly demonstrated that cultivars of the same species may differ drastically in their requirements for essential media components. Veli clearly required higher total amounts of nitrogen (67.8 mM) than Aslak (44.9 mM) but less maltose and 2,4-dichlorophenoxyacetic acid (28 g l–1 and 0.6 mg l–1) than Aslak (38 g l–1 and 2 mg l–1). This result indicates that the optimal production of green plantlets through embryogenesis requires that media be optimized for each cultivar separately. Keywords Oat · Avena sativa · Mathematical models · Medium optimization · Regeneration Abbreviation 2,4-D: 2,4-Dichlorophenoxyacetic acid

Introduction The first regeneration systems developed for oat (Avena sativa L.) were based on embryogenic cultures initiated from immature (Cunmings et al. 1976; Rines and McCoy 1981) or mature embryos (Carter et al. 1967; Lörz et al. 1976), thus utilizing the capacity of the embryogenic tissues. However, other explants have also been successfully used as sources of totipotent cell lines. These explants include seedling mesocotyls (Heyser and Nabors 1982), apical meristems (Schütze et al. 1984a; Zhang et al. 1996), leaf segments (Schütze et al. 1984b), axillary tiller buds (Shewkhawat et al. 1984) and leaf-base seg-

ments (Chen H et al. 1995; Chen Z et al. 1995; Gless et al. 1998a). Embryogenic cultures from immature and mature embryos (Somers et al. 1992; Torbert et al. 1998) and from leaf-base segments (Gless et al. 1998b) have also been used as a target for gene transfer by particle bombardment. The use of leaf-base segments as a target in gene transfer experiments has gained interest due to the short cultivation time required in vitro, which decreases the risk of somaclonal variation (Gless et al. 1998b). Both MS medium (Murashige and Skoog 1962) and L3 medium (Jähne et al. 1991) have been used for leaf-base cultures (Chen H et al. 1995; Chen Z et al. 1995; Gless et al. 1998a). Gless et al. (1998a) found that L3 medium enhanced regeneration in vitro. While various hormone combinations have been tested for oat leaf-base cultures (Chen Z et al. 1995; Gless et al. 1998a), the composition of the media with respect to nitrogen source, sugar and growth regulators has not otherwise been optimized systemically even though previous studies have clearly demonstrated that the choice of culture medium affects regeneration efficiency. The nitrogen balance of the culture medium has a major effect on the somatic embryogenesis of several species. In particular, the ratio of nitrate to ammonium and the addition of reduced nitrogen in the form of amino acids affect somatic embryogenesis (Reinert et al. 1967; Grimes and Hodges 1990; Shetty and Asano 1991; Nuutila et al. 2000). It has also been demonstrated that the nitrogen requirements vary between different stages of embryogenesis (Higashi et al. 1996). In the investigation reported here we were able to enhance the regeneration efficiency of oat leaf-base cultures by optimizing the nitrogen composition and sugar and auxin concentrations of the culture media.

Communicated by H. Lörz

Materials and methods

A.M. Nuutila (✉) · C. Villiger · K.-M. Oksman-Caldentey VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland e-mail: [email protected] Tel.: +358-9-4564454, Fax: +358-9-4552103

Plant material Mature seeds of two oat (Avena sativa L.) cultivars, Aslak and Veli, were used as starting material. The seeds were dehulled and

1157 Table 1 Optimization of nitrogen concentrations of media: the experimental design and its independent variables: X1 = nitrate concentration (in millimoles), X2 = ammonium concentration (in millimoles) and X3 = organic nitrogen concentration (in millimoles)

Table 2 Optimization of auxin and sugar concentrations of media: the experimental design and its independent variables: X1 = auxin (2,4-D) concentration (in milligrams per liter) and X2 = maltose concentration (in grams per liter)

Experiment Coded values

Experiment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Real values (mM)

x1

x2

x3

X1

X2

X3

–1 1 –1 1 –1 1 –1 1 –1.682 1.682 0 0 0 0 0 0 0 0 0 0

–1 –1 1 1 –1 –1 1 1 0 0 –1.682 1.682 0 0 0 0 0 0 0 0

–1 –1 –1 –1 1 1 1 1 0 0 0 0 –1.682 1.682 0 0 0 0 0 0

20.0 40.0 20.0 40.0 20.0 40.0 20.0 40.0 13.2 46.8 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0

8.0 8.0 17.0 17.0 8.0 8.0 17.0 17.0 12.5 12.5 4.9 20.1 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5

3.0 3.0 3.0 3.0 9.0 9.0 9.0 9.0 6.0 6.0 6.0 6.0 0.95 11.1 6.0 6.0 6.0 6.0 6.0 6.0

surface-sterilized with 70% ethanol for 5 min and with sodium hypochlorite (4% available chlorine) for 10 min and then thoroughly rinsed with sterile water. Seeds were germinated under light conditions on MS medium solidified with 4 g/l Phytagel (Sigma, St. Louis, Mo.). Leaf-base segments, 1 mm in length, were dissected from 3- to 4-day-old seedlings. Segments 1–6 were compared for their embryogenic callus induction efficiency. Only callus containing distinct embryogenic structures under the stereomicroscope was considered to be embryogenic and is hereafter in all later experiments referred to as embryogenic callus. Other types of calli (e.g. watery, translucent callus) were discarded. On the basis of this comparison, only segments 1–3 were used in the medium optimization experiments. Comparison of callus induction media A preliminary experiment indicated that the L3 medium was more efficient than the MS medium in producing green regenerants from the Finnish elite cultivars, Aslak and Veli. Consequently, the L3 medium was chosen for further optimization studies. Regeneration medium After the segments had been cultured for 3 weeks on callus induction medium, the calli that had formed were transferred onto MS medium supplemented with 0.2 mg/l kinetin (regeneration medium) and incubated for 1 week in the dark at 22°C followed by culture under light conditions at 22°C. Optimization of nitrogen Ammonium, nitrate and organic nitrogen concentrations were chosen as independent variables for optimization of L3 medium, employing a 23-factorial experimental design with six star points and six replicates at the center point (Table 1) (Box and Wilson 1951; Cochran and Cox 1957). Ninety-six leaf-base segments were used for each experimental point. After the segments had been cultured for 3 weeks on the optimization medium, the calli

1 2 3 4 5 6 7 8 9 10 11 12

Coded values

Real values

x1

x2

X1 (mg l–1)

X2 (g l–1)

–1 1 –1 1 –1.414 1.414 –1 1 0 0 0 0

–1 –1 1 1 0 0 –1.414 1.414 0 0 0 0

1.0 3.0 1.0 3.0 0.6 3.4 2.0 2.0 2.0 2.0 2.0 2.0

15.0 15.0 45.0 45.0 30.0 30.0 8.8 51.2 30.0 30.0 30.0 30.0

that had formed were transferred to regeneration medium as described earlier. A total of 20 experiments were carried out. Second-order polynomial mathematical models describing the effects of selected variables on the number of green plantlets regenerated were derived from the experimental results. The medium with the optimized nitrogen composition was compared with the original medium in two independent experiments (five replicates each). Optimization of auxin and sugar Auxin (2,4-D) and maltose concentrations were chosen as independent variables for the second optimization of the L3 medium. These were optimized together, since interactions of sugar and auxin have been reported in the literature (Lazzerri et al. 1988). A 22factorial experimental design with four star points and four replicates at the center point was employed (Table 2) (Box and Wilson 1951; Cochran and Cox 1957). Ninety-six leaf-base segments were used for each experimental point. After the leaf-base segments had been cultured for 3 weeks on the optimization medium, the calli that had formed were transferred to regeneration medium as described earlier. A total of 12 experiments were carried out. Second-order polynomial mathematical models describing the effects of selected variables on the number of green plantlets regenerated were derived from the experimental results. The medium with optimized auxin and sugar concentrations was compared with the original medium in two independent experiments (five replicates each).

Results Embryogenic callus induction from leaf-base segments Leaf-base segments 1–6 were compared for their capability to produce embryogenic callus on MS medium. For both cultivars, the first three segments produced embryogenic callus, whereas segments 4–6 produced very little or no embryogenic callus (Fig. 1). On the basis of this result, only segments 1–3 were used in further optimization studies. Comparison of MS and L3 medium Both MS and L3 medium were tested for their efficiency of embryogenic callus induction and plantlet production.

1158

Fig. 1 Production of embryogenic callus on MS medium from leaf-base segments 1–6 by two Finnish elite oat cultivars, Aslak and Veli. Different letters denote statistically significant differences (P