is a self-pollinating diploid species that has become a model species for the commercial strawberry (F. X ananassa
Duch.) and other members of the Rosaceae family because it has a small genome (240 Mb), a short generation time, and an available full genome sequence [1
]. In order to facilitate research in ameliorating the qualitative (i.e., flavonoid biosynthesis and other polyphenols) and quantitative characters of the plant with F. vesca
], a large number of replicate clonal plants are required for individual experiments. Most Fragaria
species produce clonal plants on stolons (commonly called runners) that develop from axillary buds [5
], but there are some important non-runnering genotypes within F. vesca
], notably the F. vesca semperflorens
, that flower constantly, with progeny that are primarily seed-derived and do not produce any runners (stolon, vegetative self-propagating unit).
Studies of in vitro micropropagation of F. vesca
have successfully regenerated shoots from leaf and petiole explants using combinations of benzyladenine (BA) and indolebutryic acid (IBA) following transformation by Agrobacterium
]. F. vesca
leaf explants placed abaxial side up regenerated shoots more rapidly than those placed adaxial side up after an Agrobacterium
transformation treatment [11
]. No difference was observed in the regeneration response of leaf explants for F. vesca
versus F. vesca semperflorens
, runnering versus non-runnering phenotypes, respectively, although differences in petiole responses did differ as more shoots were produced by the latter form at comparable levels of BA plus IBA [10
]. However, genotypic variation in post-transformation shoot regeneration was noticed in F. vesca
]. Thidiazuron (TDZ) was shown to replace BA and promote callus and shoot initiation from F. vesca
leaf explants [12
], although a F. vesca
× F. vesca semperflorens
interspecific hybrid produced fewer shoots than F. vesca
in response to TDZ [14
], suggesting some genotype sensitivity to TDZ. TDZ may also reduce subsequent shoot elongation [2
]. Within the majority of these studies, it was not clear how the transformation and subsequent selection protocols may have determined shoot regeneration potential separately from the inherent capacity for such potential within each genotype. Regeneration of plants after callus formation, as is common in transformation studies, leads to much greater somaclonal variation among progeny than after meristem micropropagation [15
], and this lack of uniformity is a problem for subsequent genetic studies.
Even though F. vesca
may be self-pollinated, seed-derived populations of genotypes that do not produce stolons exhibited some level of variability [2
], necessitating lengthy periods of controlled intraspecific pollination of each genotype of interest to create reasonably uniform homozygous populations for subsequent research. Even without transformation, reliable and rapid in vitro propagation techniques for substantially and rapidly increasing the number of clonal plants from non-runnering genotypes for physiological and molecular studies are desirable. To date, there have been no protocols for shoot regeneration from shoot explants (i.e., a shoot with an axillary meristem) of non-runnering F. vesca
genotypes. Thus, the present study was performed to establish such a protocol by (1) comparing the rates of new shoot production from seedling shoot axillary bud explants versus leaf explants, (2) determining the effective concentrations of BA, TDZ, and IBA for in vitro shoot regeneration from both leaf and shoot explants of Fragaria vesca
, (3) determining if genotype has an effect on the responses, and (4) assessing if adaxial versus abaxial placement affects leaf explant response.
2. Materials and Methods
2.1. Plant Material
Seeds of four F. vesca genotypes, the non-runnering Baron Solemacher (F. vesca semperflorens), rarely runnering Pineapple Crush, and runnering types Ivory and Yellow Wonder, were collected from several self-pollinating, individual plants of each genotype grown in a greenhouse at the University of Kentucky, Lexington, KY, USA, washed under tap water, and air-dried.
2.2. Seed Germination and Culture
Seeds were dipped into 30% Clorox bleach (v
2.5% sodium hypochlorite) plus 10% sodium dodecyl sulfate (SDS) (v
) solution for 20 min. Seed was then rinsed with sterilized water 3 times under a laminar flow hood. After surface sterilization, 20 seeds were placed in sterile 20 mL Petri dishes containing 4.4 g L−1
Murashige and Skoog’s Basal Salt (MS) (Sigma®
], 30 g L−1
sucrose, and 7 g L−1
Bacto agar (BD-Difco®
) for germination. The medium was prepared by adjusting the pH to 5.7 prior to autoclaving at 121 °C and 105 kPa for 70 min. After 14 days of germination, 3 seedlings with little growth were transferred to each of the 50 mL jars containing the same medium to allow for better growth. Seed germinated in two to three weeks.
2.3. Explant Culture
Leaf lamina (36 mm2) and shoot (6–8 mm) explants were excised from 5 week-old sterile, in vitro Baron Solemacher seedlings in order to regenerate shoots. The shoot explant consisted of a piece of the main stem, a petiole base, and an axillary meristem at the petiole base. To assess the effect of different combinations and concentrations of plant growth regulators (PGRs) on shoot regeneration, two excised shoot explants were placed in each sterile 20 mL Petri plate containing MS Basal Salt with the following treatments: benzyladenine (BA) at 2 or 4 mg L−1 or thidiazuron (TDZ) at 1 or 1.5 mg L−1, each with indole-3-butyric acid (IBA) at 0.125, 0.25, or 0.50 mg L−1. Thus, there were 12 treatment combinations (BA+IBA or TDZ+IBA) in total and each was replicated 4 times. Possible genotypic variation was compared using four shoot explants from each of the 4 genotypes on 2 mg L−1 BA versus 1.5 mg L−1 TDZ, each combined with 0.25 mg L−1 IBA. There were 4 replicates of each genotype by treatment combination. In a third experiment, leaf explants were placed adaxial side up versus abaxial side up using the set of BA+IBA or TDZ+IBA treatments described above. With leaf explant placement as an additional treatment, there were 24 total treatments, each replicated 4 times. The Petri dishes were held in a laboratory at 22 °C temperature under fluorescent lighting with an 18 h daylength.
After 6 and 9 weeks of culture, callus production by each explant was rated as: 0 = no callus, 1 = low quantity of callus, 2 = medium quantity of callus, or 3 = high quantity of callus. After 10 weeks, regenerated shoots were transferred to Petri plates containing half-strength MS medium without growth regulator for rooting. The mean value of each set consisting of two explants in each Petri plate was considered as a replication. All the experiments were conducted in a completely randomized design (CRD).
Shoot number per shoot explant and the number of shoot explants producing new shoots were recorded after 6 and/or 9 weeks of culture. Shoot explants did not produce visible callus. With leaf explants, relative callus production and the number of new shoots per explant were recorded after 6 and 9 weeks of culture, and the % of explants producing callus was calculated. Analyses of variance (ANOVA) were performed (SigmaPlot 12.0, Systat Software, Inc., San Jose, CA, USA), and means were compared using the Student–Newman–Keuls method at P < 0.05. Results were expressed as least squares means ± standard error of the mean.
An effective in vitro shoot regeneration protocol for Fragaria vesca
was demonstrated from both shoot axillary bud and leaf explants. For shoot explants, BA at 2 and 4 mg L−1
and TDZ at 1.5 mg L−1
were the best treatments for shoot regeneration. However, there were no effects of IBA concentration on shoot regeneration. Only Baron Solemacher among the four F. vesca
genotypes (including Pineapple Crush, Ivory, and Yellow Wonder) showed a difference between use of TDZ versus BA. From leaf explants, 4 mg L-1
BA plus 0.5 mg L−1
IBA resulted in the maximum callus production and number of shoots per explant, and the IBA effect increased from 0.125 to 0.5 mg L−1
. In contrast, TDZ promoted high callus production but resulted in reduced numbers of shoots, and IBA concentration had no effect on the production of callus and shoots. Leaf explants placed abaxial side up produced more callus but no more shoots than those placed adaxial side up. After 9 weeks of culture, Baron Solemacher leaf explants had generated 4.6 shoots per explant with the best treatment (Table 3
), while shoot explants from Baron Solemacher had generated 30.8 shoots with the best treatment (Table 2
). Thus, the protocol for using shoot axillary bud explants is a better alternative to leaf explants for generating high numbers of clonal shoots from a single seedling plant in vitro, avoiding callus production and with the possibility of a large homozygous population of plants for the non-runnering F. vesca