In 2008, stored mature Picea pungens seeds from five families, representing diverse origins from New Brunswick (Family 113 and 8285450, gifted from New Brunswick Department of Natural Resources and National Tree Seed Center, Natural Resources Canada, respectively), Nova Scotia (200301, purchased from Nova Tree Seed Company, Inc., Truro, Nova Scotia), and Ontario (OSC, purchased from Ontario Seed Center, Waterloo, Ontario) in Canada and New York, USA (10910, purchased from TreeHelp.com, Buffalo, New York), were used as materials. Prior to the experiments, all seeds were stored at low temperature for two or more years.
Prior to embryo extraction, all seeds were submerged in 75% ethanol for 1 min, rinsed three times in sterile water, soaked in 10% sodium hypochlorite with a small amount of Tween-20 for 30 min, rinsed five times in sterile water, and then kept in sterile water for 4 h to soften the seed coat. Mature zygotic embryos were extracted from seeds under a stereomicroscope. Experiments for key SE steps were then carried out, and details for the respective medium supplements and PGR concentrations are presented in Table 1 and for the experimental materials in Table 2.
Exp. 1: Initiation of embryogenic tissue
The initiation medium was half-strength Litvay’s medium18 (½ LM) supplemented with sucrose, l-glutamine, gelrite, and a (standard) PGR concentration combination containing 10.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 μM benzyl aminopurine (BA) (referred to as the standard PGR concentration; Table 1). To investigate the effects of PGR concentration, two PGR concentration treatments, low and high, were also tested. The low treatment included two concentration combinations containing (le ) 5.0 μM 2,4-D and (le ) 2.5 μM BA, and the high treatment had four concentration combinations containing (ge ) 12.5 μM 2,4-D and (ge ) 7.5 μM BA (Table 1). The pH of the medium was adjusted to 5.8 prior to sterilization.
All five families, with an average of 79 (range 75–94) mature zygotic embryos per family, were cultured on the initiation media in 100 × 15-mm Petri dishes containing approximately 25 ml of medium (approximately 10–15 embryos per dish and approximately 10 dishes per family × PGR combination) in darkness at 24 (pm ) 1 °C. One month later, SE initiation was assessed by recording the success or failure of each cultured embryo. Success was defined for initiation of an embryo if embryogenic tissue (ET) was established. The initiation frequency (= number of embryos with successful initiation/number of cultured embryos) was then calculated to indicate initiation efficiency. Once an embryo had developed ET, it was given a clone identification number. Note that a clone (embryogenic line) in this study refers to embryogenic tissues initiated from a single embryo.
Exp. 2: Proliferation of embryogenic tissue
Once an ET clone was identified, a rapid proliferation of ET for cryopreservation and/or production was needed. This experiment used ET from 12 clones representing three families (113, 8285450 and OCS) obtained in Exp. 1. ET was proliferated via a method of two steps: liquid suspension culture on liquid proliferation medium, followed by culture on solid proliferation medium. The base proliferation medium was the same as the initiation medium, but the PGR combination varied, being 3.75 µM BA and 6.25 µM 2,4-D for liquid and 7.5 µM BA and 12.5 µM 2,4-D for solid cultures (Table 1).
Before transferring to the proliferation medium, ET was cultured on medium without BA and 2,4-D for seven days. Approximately 30–40 mg ET per clone was subcultured in flasks (10–15 mg ET per flask) with 50 ml liquid proliferation medium for seven days. During this period, the flasks were shaken vigorously (121 RPM) to break up the tissue pieces into a fine suspension. The suspension in a flask was poured onto filter paper to remove liquid, and the filter paper with attached cells was placed onto the surface of fresh, solid proliferation medium. The tissues were cultured in darkness at approximately 24°(pm ) 1 °C for two weeks and then subcultured every 14 days on the solid medium. ET weights for each flash were recorded before and after the liquid culture period, and the percentage increase (= (frac{after-before}{before}times 100)) was used as the response variable for proliferation efficiency. Due to its difficult measurement, proliferation in solid culture was not recorded.
Exp. 3: Maturation of somatic embryos
This experiment was conducted using ET from 38 clones initiated from three families (113, 8285450 and OCS) in Exp. 1 and 2. The maturation medium was 1/2 LM containing 6% sucrose, 0.6% gelrite, 0.05% glutamine, and 60 (upmu )M abscisic acid (ABA) (Table 1).
Before transferring to the maturation medium, ET was subcultured on solid proliferation medium for two periods, each period being 7 days, and then cultured on proliferation medium without BA and 2,4-D for another seven days. ET clumps that grew vigorously were selected for maturation. 3–8 Petri dishes each with 6–8 clumps (approximately 0.5 g) were prepared for each clone. Cultures were kept at approximately 24 °C, low light intensity (5 mol/(upmu )m2/s, cool white, fluorescent lamps GE F72T12/CW), and a 16-h photoperiod for six weeks without subculturing onto fresh medium. By the end of the culture period, the total number of normal embryos (1.5–2 mm in length, having 2–5 distinct cotyledons, embryonal root caps, and smooth hypocotyls) and abnormal embryos were counted and expanded on a per gram basis. The number of normally matured embryos per gram was used as the indicator of maturation efficiency.
Exp. 4: Germination and rooting
This experiment involved 13 clones representing two different families (8285450 and OCS) from Exp. 3. The germination medium was a modified ½ LM paired with 2% sugar, 0.6% Gelrite, 0.05% l-Glutamine and no PGRs (Table 1).
Matured embryos at the cotyledonary stage in Exp. 3 were isolated and placed onto germination medium (20 mature embryos per dish and approximately three dishes per clone) for culture under similar environmental conditions as for maturation. After 1 week of culture in darkness at 24 (pm ) 1 °C, cotyledonary and small radicles appeared. After culture for the next four weeks in the light, normal emblings (plantlets germinated from somatic embryos with young hypocotyls of 1–2 cm and radicles of 2–3 cm) had developed. The total number of embryos planted and the number of emblings developed after the culture were recorded. The frequency (= number of normal emblings/number of embryos planted) was calculated to indicate germination efficiency.
Exp. 5: Acclimation
This experiment used six clones representing one family (8285450) and one clone from family OCS in Exp. 4. On average, 44 emblings per clone with properly developed radicles and hypocotyls were directly transferred into pots (filled with Jiffy peat pellets). For the first two weeks in the greenhouse, the emblings were kept under a 16-h photoperiod at 25 (pm ) 5 °C and each embling plantlet was kept under a glass beaker to protect against dehydration. The emblings were grown until they reached a height of 10–15 cm (20–25 weeks). The number of emblings that successfully developed was counted, and the survival rate (= number of successfully developed emblings/total number of planted emblings) was calculated as the indicator of acclimation efficiency.
Data analysis
In Exp 1, the response variable for initiation, the number of embryos with successful initiation out of the total number of embryos cultured (data in ‘event/trial’ type), was assumed to follow a binomial distribution and analyzed using the following models:
$$ y_{ijk} = mu + F_{i} + T_{j} + FT_{ij} + P_{kleft( j right)} + varepsilon_{ijk} , $$
(1)
where ({y}_{ijk}) was the event/trial in initiation of the kth petri dish of the ith family and jth PGR treatment and kth PGR concentration combination within the jth PGR treatment. (mu ) was the overall mean, ({F}_{i}), ({T}_{i}), ({P}_{kleft(jright)}), and ({FT}_{ij}) were the fixed effects of the family, PGA treatment, PGR combination within a treatment, and the interaction between F and T, respectively, and ({varepsilon }_{ijkl}) was the random error.
The response variable of Exp 2, the percent ET increase of the kth dish of the jth clone within the ith family (({y}_{ijk})) is continuous data and, was, therefore, analyzed using analysis of variance following the model below:
$$ y_{ijk} = mu + F_{i} + C_{jleft( i right)} + varepsilon_{ijk} , $$
(2)
where ({C}_{jleft(iright)}) was the fixed effect of the jth clone within the ith family, and the others remained the same as explained previously.
In Exp. 3, the total number of normally matured embryos per gram ET cultured is count data and can be modeled using a Poisson distribution. The data were analyzed using Model [2] but paired with a negative binomial distribution, which is similar to the Poisson distribution but can handle the over-dispersion issue.
In Exp. 4 and 5, the response variable was the ‘event/trial’ type. The numbers of emblings over the embryos cultured on germination medium and the normal emblings developed over the total emblings transferred in acclimation were analyzed using Model [2], where all terms remained the same as previously described.
SAS programs19 were used in the analyses (i.e., logistic procedure for the ‘event/trial’ data, GLM procedure for the continuous data, and GENMOD procedure for the count data). Means and their standard errors (SE) were computed either on an original scale directly or transformed to the original scale via a link, depending on the data type. Multiple comparisons among levels of a factor were made using the Tukey method. Pearson correlations at the clonal level among efficiencies of proliferation, maturation, germination, and acclimation were tested for significance. Unless otherwise stated, the significance level in this paper refers to (alpha )=0.05.
Research involving plants
The authors declare that this study on plants in this study, including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation.
