In Vitro Propagation of Morchella esculenta and Study of its Life Cycle

Among the edible mushrooms, Morchella esculenta (Pers.) is a highly prized mushroom which consists of a short stalk and a conical, pitted, hollow pileus. It is commonly known as a morel or sponge mushroom, or gucchi, etc. It belongs to the class Ascomycetes, order Pezizales and family Morchellaceae . It is found throughout the world. In Pakistan, it is found in northern areas from Murree, Patriata, and Gilgit under pine vegetation. It is therapeutic in nature; therefore its medicinal and biochemical properties are under investigation throughout the world, especially in regard to treating the hazardous diseases such as cancer, tumors, etc. The present in vitro study was conducted for the first time in Pakistan. The three media soil and soil extracts Agar and Luria Agar were used. Mycellial growth was obtained on all these media, but the LA medium showed better growth. Microscopic studies had shown pigmentation is most likely due to the secondary mycelium. Among these mycelium ascogenous hyphae are produced that may lead to the formation of ascogonium and antheridium with the dilated tips. The compact mass of prosenchymatous and pseudoparenchyma tissues was observed.


Introduction:
Morchella are heterotrophs (Panday, 2000). Some species of Morchella form symbiotic associations with plants by connecting mycelium with roots. This symbiotic association is called the mycorhizal association. Morchella esculenta is an example. It is considered a saprotroph (Kuo, 2008;Frank et al., 2010).
Morchella esculenta is a proteinacious edible mushroom also known as gucchi in Punjab (Repin, 1901;Panday, 2000). It has great importance as it is used as a vegetable and is equivalent to or better than meat (Abel et al., 1993). It is delicious. Medically, it is very important, containing interferon which are anticancerous. The generic name Morchella is derived from the German word "Morel" meaning "Mushroom," while the specific epithet esculenta is Latin meaning "edible". So it is considered an edible fungus (Reilly, 2011). Morphologically, the Morchella species is commonly known as the common morel, true morel, sponge morel, Black morel, yellow morel, M. esculenta morel mushroom, haystack, dry land fish, etc. (Dörfelt, 2001;Roody, 2003). It is also known as Phallus esculentus (Fries, 1753). It is also known as a sponge mushroom because its fruiting body (cap) has honey-comb like depressions. Morchella vary in color, shape and size. Its fruit bodies are polymorphic (Masaphy et al., 2010). It is classified in the kingdom fungi, division ascomycota, class pezizomycetes, order pezizales, family Morchellaceae, genus Morchella and species esculenta (Persoon, 1801).
Morchella are commonly found under trees. They grow in temperate forests, and appear as the frost melts. They are also found on burnt soil and trees burnt by fire (Wurtz et al., 2005). The Morchella is native to Pakistan and Pakistan also exports it. In Pakistan, it is found in the Northern areas starting in Murree, Patriata, and the Gilgit pine forest, as well as Azad Jamu Kashmir (Kaul, 1975). It is also found in Britain, Ireland, Europe (Reilly, 2011), Brazil (Cortez et al., 2004), Turkey (Taskina et al., 2010, Israel (Masaphy et al., 2009) and the Himalayas (Kanwal et al., 2010). Morchellas are also found in Asian countries and from sites throughout most of North America. This edible mushroom is very rarely found in Australia, where several other members of the genus Morchella are known to occur. It is found in the same season in different countries.
Mycelium is always found in the form of masses of hyphae. It consists of inconspicuous, branched, filamentous and septate hyphae that grows in the humus soil. This septate hypha contains many nuclei. Apothecia (fruiting body) consist of asci containing ascospores. The ascocarp consists of Stipe, Pileus, Hymenium and Sub-hyminum. Stipe consists of a fleshy stalklike structure. Its height is around 3 inches and its diameter is one inch. This stalk-like structure bears a sponge-like cap which is also termed as pileus (Ower, 1982;Vashista et al., 1939). Pileus is about 7-5cm long and 4-5cm wide. The presence of pits and ridges is because of the growing structure of hymenium. Basically hyminial layer grow unequally. The depression and ridges are brown in colour (Negi, 2006). This hyminial layer later consists of elongated, cylindrical cells called asci. Asci contain ascospores; a single asci contains 8 ascospores, which later are released from it (Ammirati, 1985). The asci are surrounded by protected structures, called paraphysis (Sharma, 1988). The sub-hymenium layer consists of pseudoparenchymatous tissue that is present beneath the hymenium layer. These tissues are present in the depression region of the pileus, termed the hypothecium (Vashista et al., 1939;Pandey, 2000).
There are different views about the germination of ascospores. After the dispersal of ascospores, they fall in soil. In the presence of the required condition, e.g. moist humus soil, they germinate and produce mycelium (Harvey et al., 1978). They germinate at a low temperature (2ºC), and after one year they do not remain viable in this soil surface (Schmidt, 1983).

MATERIALS AND METHODS
The sample was collected from the Kashmir Valley in the spring season of 2012. The dried sample of Morchella esculenta was processed for cultivation. To grow M. esculenta, soil was also brought from Azad and Jammu Kashmir.

COMPOSITION OF FIRST MEDIUM (SOIL)
Soil and water

COMPOSITION OF 2 ND MEDIUM (L.A)
Distilled Water 100 mL Tryptone 1 g Yeast extract 0.5 g NaCl 1 g Agar 1 g One hundred millilitres of L.A medium was prepared by weighing 1g of tryptone, 0.5g of yeast extract and 1g of NaCl. Distilled water was added up to the mark. These were mixed thoroughly and pH 6.4 was maintained. Agar (1g) was added at the end. Media was then autoclaved at 120 o C and 15 psi.

Soil 2g
Water 100g Agar 2% 2 g of soil was weighed and added to 100 ml of water. It was mixed thoroughly and the homogenized solution was filtered. 2% agar was added into this filtrate. The pH was observed before and after filtration by the pH meter. Observed pH was 6.6. Media was then autoclaved at 120 o C and 15 psi.

PREPARATION OF SOIL
The soil had been sterilized in the autoclave at 120 o C and 15 psi. Autoclaved soil was transferred to a new polar box. Soil was watered according to requirement.

PREPARATION OF INOCULUM
Sample was ground. The prepared material was termed the inoculum.

INOCULATION UNDER ASEPTIC CONDITION
Inoculum was inoculated into the soil box containing the prepared soil. A thermometer was set with it. This medium was then placed in at 4ºC. The pouring and inoculation of the 2nd (L.A) and 3rd media (soil extract) was done in a laminar flow bench. Roxithromycin (antibiotic) was also added to these media before inoculation. After inoculation in both media (L.A and soil extract agar), plates were placed at 4 ºC. Pileus of Morchella esculenta was examined under the microscope. The spore size and diameter was taken by a standardized ocular micrometer and stage micrometer.

STUDY OF CULTURE
The growth in the first media, i.e. soil media, was observed after 1 st , 2 nd , 4 th and 6 th week of inoculation. The growing material was examined under the microscope. The staining procedure that was followed was: Place a drop of waste water sample on a slide. Add one, or at most, two drops of the Lactophenol cotton blue (Nagamani et al., 2005) and observe the slide under the microscope.
For the 2 nd and 3 rd medium, some material from the media had been picked and the slide was prepared in water and examined under the microscope. The measurements of hyphae were taken with a stage and ocular micrometer for 1 st , 2 nd , 4 th and 6 th week.

RESULTS
The aim of in vitro propagation of Morchella esculenta is to proceed for cultivation commercially. If successful, it could help meet the protein needs of the growing world population, With commercial cultivation, we may able to provide it to people at lower cost than wild harvested Morchella esculenta.
The section of pileus was examined in Lactophenol. It bears the asci and paraphysis in layers. The paraphysis are septate, and may or may not be swollen at their tips. The 8 spored asci were cylindrical, and measured about 240 -280 x 18 -21. The ascospores were 16 -21 x 7.8 -18 ellipsoidal, slightly grained at the ends, smooth and yellowish.

Macroscopic studies
The mycelia growth on the SEA is light brown and milky white on the L.A and soil media (Table  1).

MICROSCOPIC STUDY OF CULTURE
The cultural studies of soil medium showed different hyphal structures like coil form hyphae, rhyziomorph, ( fig 1 a,b) compact mass of prosenchyma, (fig1 c ) and pseudoparenchyma tissue (fig 1d) after one, four and six week respectively.
The mycelial colony on the soil extract agar showed no growth after the first week, but it showed pigmented healthy mycelium. The highly pigmented mycelial growth increased in thickness after four and six weeks respectively (fig 1e and 1f) On the L.A media, comparatively better growth of mycelium was obtained with slightly compact patches. With the germination of spore and actively spreading hyphae (fig 1g), healthy mycelium formed on this media as compared to first media (soil). The compact form of hyphae developed, and was termed the plectenchyma (fig 1 h). Among the compact hyphae, differentiated hyphae with curved swollen tips can be seen, and might be representing the ascogonium and antheridium of the culture (fig1 i).  Figure 1: a-hyphae grew parallel and other spreading outwards trying to form rhizomorph; bhyphae grew in coil form; c-thin mat of mycelium where single hyphae can be observe easily termed as prosenchyma or prosoplectenchyma; d-comparatively thick but single hyphae cannot be distinghuished termed as pseudoparenchyma; e-pigmented healty mycelium developed was consinting of interwoven hyphae; f-highly pigmented thick growth mycelium developed comparatively; g-Actively growing hyphae from the grounded inoculums; h-Formation of healthy mycelium tending to becoming parallel; i-convolution structure of hyphae might be representing ascogonium and antheridium.

Discussion:
In the present study, the original soil of the same locality was used, which may have all the required substances such as sugars, minerals etc., where they were already growing. As in vitro culturing was carried out, well mycelial growth was observed on all these media at the temperature 2.2°C (Schmidth, 1983). Powdered form inoculum was used. The growth of mycelium was abundant and thick on soil, soil extract agar and the L.A medium at 2.2°C (Schmidth, 1983;Guler et al., 2005;Guler, Winder, 2006;Gilbert, 1960). However, Gilbert (1960) had used the semi broth media. Microscopic studies showed ascospore germination at 2.2°C (Winder, 2006;Schmidth, 1983). Mycelial formation occurred after the first week on soil media in scattered and rhizomorphic form (Guler et al., 2005). Guler et al (2005) obtained the same structure by using malt extract agar. Thick and Compact mycelium formation developed after 4-6 weeks in three media (Goldway et al., 2000).
Convoluted and compact mycelium were observed in the L.A media. The compact mycelial growth seems to be showing a tendency to the formation of ascogonium and antheridium.

Conclusion:
In the present study, the mycelial growth of M. esculenta was obtained on the following media: Soil, Soil extract agar and L.A media. The L.A medium showed better growth than the other two. Temperature and pH parameters were considered. Microscopic studies showed different hyphal structures, like rhizomorph and the compact mass of prosenchyma and pseudoparenchyma tissue, were obtained. The pigmented formation of mycelium observed most are probably the secondary mycelium. Among these, ascogenous hyphae are produced that lead to the formation of ascogonium and antheridium with dilated tips. The media used in the present study could be better if it could be supplemented with other natural substances of rich protein like wheat bran, rice bran etc.