Introduction
Agaricus bisporus is one of the widely consumed mushrooms in the world because of good taste and flavor [1]. The production of A. bisporus has increased in China, the United States, Poland and the Netherlands [2]. In Korea, A. bisporus has been one of the most popular edible mushrooms and the total Korean production of A. bisporus was 11,493 MT (79 billion won) in 2014 [3]. The high productivity of valuable mushrooms in Korea was mainly attained by improving cultivation techniques developed from 1960s. Since the new cultivar ‘Sae-Ah’ for A. bisporus was developed in 2010, many trials were carried out to breed better cultivars.
A. bisporus has two haploids in the basdiospore as the multinucleate. This mushroom shows secondary homothallism that is capable of making a sexually reproducing colony from a single spore when it propagates in a complete isolation [4]. Whereas other mushrooms are heterothallism that reproduces sexually among individuals having compatible mating types [5]. Although A. bisporus mostly generates two spores, around 10~15% of basidia actually produce four spores having haploid nuclei that are compatible with the other haploid nuclei (homokaryons)[6]. Because of this low selection of spores based on homothallic nature, the breeding of A. bisporus is more difficult than that of other mushrooms. The usual methods for breeding of A. bisporus are mutation [7], mating [8-10], isolation and regeneration of protoplast [11], and transformation [12-14].
It is known that the mycelium of heterokaryons grows faster than putative homokaryons [15]. Because A. bisporus do not have clamp connections [16], putative homokaryons are distinguished from hetrokaryons by comparing the speed of mycelial growth of A. bisporus. This kind of selection was usually done for obtaining mother strains and checking the mating compatibility. Recently, molecular analyses such as restriction fragment length polymorphism (RFLP) [17], Random Amplification of Polymorphic DNA (RAPD) [18, 19] and Inter Simple Sequence Repeats (ISSR) [20, 21] are also used for selection of homokaryons instead of measuring mycelial growth.
In order to develop improved cultivars giving high yield and good quality for farmers, breeders often utilize morphological and agronomic traits [22]. The cultural, morphological and agronomic traits of mushrooms are useful characteristics for the total yield and the quality of mushroom production. For instance, optimal temperature of mycelia growth and fruiting formation is known to affect the total yield. Also, when the cap color is whiter, the price of mushrooms is more expensive in the market.
The new cultivar ‘Saedo’ is the most popular cultivar in Korea. In this study, those morphological and agronomic characteristics of the new cultivar were investigated and compared to the reference control ‘Saejeong’ to classify two varieties and introduce the ‘Saedo’ cultivar.
Materials and Methods
Breeding procedure
We selected two strains, ASI 1038 and ASI 1346 among two hundreds of collections in mushroom department of National Institute Horticultural and Herbal Science. Over one thousands of single spore isolates (SSIs) were collected from spores of the selected two strains. SSIs were analyzed with a RAPD marker to select homokaryons (S1038- 211, S1346-20). The S1038-211 and S1346-20 strains were crossed and resulted in a new crossed line (A175). A175 was crossed again with the homokaryon, S1346-15. From the second crossing, we developed a new cultivar, B397 that is designated as ‘Saedo’ (Fig. 1). The compatibility between homokaryons was identified by the RAPD analysis.
Measurement of cultural characteristics
The ‘Saedo’ cultivar cultured on CDA (4% dried compost, 0.7% Malt extract, 1% sucrose, 2% agar) media in various temperatures (15, 20, 25 and 30oC) for 14 days with three replicas. ‘Sajeong’ cultivar was used for the reference control. The mycelial growth was measured with vernier calipers on day 7 and 14 after inoculation.
Investigation of agronomic and morphological characteristics
The ‘Saedo’ and the reference control, ‘Sajeong’, were cultivated with three replicas to investigate morphological and agronomic characteristics at Mushroom Cultivation Center in mushroom department of National Institute Horticultural and Herbal Science, Eumseong, Korea. The mother spawns of two cultivars were made with wheat media (20 kg wheat, 0.6 kg gypsum, 0.3 kg calcium carbonate/100 bottles) and cultured for 14 days and cultured at 25oC, 65% humidity for 20 days. The 500 g of cultured spawns were inoculated in boxes (534 × 378 × 168 mm) with 10 kg phase II compost. The phase II compost was prepared at 65oC for 6 hrs and 55 for 7 days, respectively. The mycelium grows at 25, 80% humidity for 15 days. When approximately four fifths of the mycelium cultured in the compost, casing soil was put on the compost. Fifteen days after the casing soil was put, the growing temperature and moisture was changed into 16, 85% humidity to generate pin-heading. After harvesting mushrooms during the 1st and the 2nd flush, the total yield and the yield-related traits such as the number of the fruiting bodies and the mushroom weight was investigated [23]. As for the agronomic traits, the mycelia cultural period, the earliness and the harvesting period were analyzed. Lastly, the diameter and the length of the pileus, the thickness and the length of the stipe, the whiteness (CR-400; Konica Minolta, Tokyo, Japan) and the hardness (TA1 Texture Analyzer; LLOYD, West Sussex, UK) of both the pileus and the stipe were measured as the morphological traits.
Field experiment
Two cultivars, ‘Saedo’ cultivar and ‘Saejeong’, were tested at eight mushroom farms in at Geoncheon-eup, Gyeonjusi in March 2015. The ‘Saedo’ cultivar was cultivated in two 215 m2-scale farms and two 264 m2-scale farms. The ‘Saejeong’ cultivar was cultivated in two 231 m2-scale farms and two 264 m2-scale farms. The yield was investigated from the 1st to the 5th flush and the fruiting bodies harvested in the 1st flush were measured in the diameter and length of the pileus, the thickness and the length of the stipe and the whiteness (CR-400; Konica Minolta) and the hardness (TA1 Texture Analyzer; LLOYD) of both the pileus and the stipe.
Results and Discussion
Breeding of ‘Saedo’ cultivar
The homokaryons have one meiotic nucleus from the tetrasporic basidia and thus give rise to self-sterile [24]. Appressed colony morphology and slow growth are morphological markers to identify homokaryons [25]. But it is time-consuming and difficult. Kavousi et al. [26] reported that homokaryons have significantly less bands than those of heterokaryons in RAPD analysis. In this study, over one thousands of SSIs were analyzed with a RAPD marker which is a selected marker from the OPA series (data not shown). The selected homokaryons from each strain (ASI 1038 and ASI 1346) were crossed. The compatibility of the crossed lines was confirmed by the selected RAPD marker, OPA12 primer (Fig. 2). Finally, the crossed line, B397, was selected as a superior line through investigation of productivity, agronomic and morphological traits.
Characteristics of mycelial growth
The mycelial growth was measured on MMP (2.09 g/L MOPS, 10 g/L malt extract, 5 g/L mycological peptone, 17.5 g/L agar, adjusted to pH 7.0 with KOH) agar [27] and CDA media in A. bisporus. In this study, we cultured on CDA media on day 7 and 14 at 15, 20, 25 and 30oC, respectively. The mycelial growth of the ‘Saedo’ cultivar was compared to the reference control, ‘Saejeong’. In the early mycelia growth phase, the ‘Saejeong’ cultivar grew significantly faster than ‘Saedo’ at 15, 20 and 30oC, cultural temperature (p < 0.05), respectively. On day 14, the mycelium of the ‘Saedo’ cultivar grew as much as ‘Saejeong’ at 25oC (Fig. 3). In the mushroom farms the mycelium of spawn grows well when the compost temperature is maintained at 24~27oC [28], and thus it was identified that the ‘Saedo’ cultivar has an optimal mycelial growth in the farm.
Agronomic and morphological characteristics
The ‘Saedo’ cultivar and the reference control, ‘Saejeong’ were cultivated in 10 kg compost (per box) during the 1st and 2nd flush. The total production yield of the ‘Saedo’ cultivar, 937.4 (417.4) g, increased significantly with 1.89 folds more than the reference control, 496.1 (247.4) g. As for the morphological traits, the ‘Saedo’ cultivar was substantially thicker than the reference control in pileus, 26.6 (0.5) mm and stipe, 22.3 (1.2) mm (t-test, p < 0.05) (Table 1). Originally, A. bisporus has a pileus with broad, flat scales. But customers mostly see a hemispherical shape in supermarkets before it flattens out due to maturity [29]. From the mushroom’s standpoint, the hemispherical shape is an early stage in their development, so the shelf life of a hemispherical shape mushroom is much longer than a broad shape mushroom. For this reason, the shape of the mushroom is important to its shelf life. That is why cultivars should have a large and thick pileus with a hemispherical shape and thick and short stipe to keep a long shelf life. With these findings, the ‘Saedo’ cultivar was identified as a cultivar with high productivity and good quality.
Field experiment
In this field experiment, the total production yield of the ‘Saedo’ cultivar, 12.8 (2.5) kg/m2, was generally higher than the reference control, ‘Saejeong’ cultivar, 10.2 (3.3) kg/m2. During the 2nd flush, the production yield of the ‘Saedo’ cultivar, 4.0 (0.8) kg/3.3 m2 was considerably higher than the reference control, 2.76 (0.3) kg/3.3 m2 (t-test, p < 0.05). During the 5th flush, only ‘Saejeong’ cultivated. Most farmers cultivate A. bisporus during the 1st until 3rd flush due to low productivity. As this point, ‘Saedo’ was proved as a high productive cultivar in average 10.1 kg/m2 during the 1st and 2nd flush (Fig. 4). In morphological traits, the diameter of the pileus, 48.51 (2.2) mm, and the length of the stipe, 41.7 (4.0) mm, of the ‘Saedo’ cultivar was longer than the reference control, 42.6 (1.7), 35.5 (4.1) mm. The pileus, 27.3 (1.3) mm, and the stipe, 19.7 (2.0) mm, of the ‘Saedo’ cultivar is thicker than the reference control, 22.6 (2.0), 24.1 (5.5) mm (Fig. 5). But the hardness of the pileus of the ‘Saedo’ cultivar, 4.3 (0.6) N, was lower than the reference control, 5.7 (0.3) N. The above morphological traits were statistically significantly different between the ‘Saedo’ cultivar and the ‘Saejeong’ cultivar (t-test, p < 0.01 or 0.05) (Table 2). From these results, we identified that the ‘Saedo’ cultivar had a better shape and higher productivity than the ‘Saejeong’ cultivar. The ‘Saedo’ cultivar occupies half of the distribution rate of Korean varieties, because of that reason. Also, 37% of the Korean farmers have cultivated these varieties last year.
Table 2. Morphological characteristics of the ‘Saedo’ cultivar and the reference control ‘Saejeong’ in a field experiment in March 2015
Significant differences (t-test, p < 0.05 and < 0.01) between treatments for each time point are indicated by * and **.
