Mushroom Culture (0+1)

Genetic Improvement of Mushrooms (Contd..)

3. HYBRIDIZATION :
It consists of mating of self sterile and compatible homokaryotic lines that results in creation and selection of desired traits, but it is the assembling of the best combination of genes into one individual variety so as to produce higher yield with best quality mushroom which is the ultimate objective of a mushroom breeding programme. Application of biotechnology in the genetic improvement programme of mushroom has introduced new technique such as DNA based markers which has provided much needed boost to the on going breeding efforts in case of button mushroom. Rafalski and Tingey, 1993 have described the use of DNA based technology in breeding programme as Molecular Breeding. The DNA based marker like RFLP (Restriction Fragment Length Polymorphisms ), Allozymes, RAPDS (Random Amplified Polymorphic DNAs) , ITS ( Internal Transcribed Spacing) etc: are being utilized under Molecular Breeding programmes for isolation of homokaryons, confirmation of hybridization, assessment of diversity in Agaricus breeding programme. The most commercially cultivated mushrooms in the world . Horst-U1, Horst-U2 , S-11 , S-791 strains of A.bisporus are other examples of the breeding successes .The greatest advantage of hybridization programme is that the hybrids generated are known to give maximum yield performance and show phenotypic stability under stress.

Following are the steps involved in hybrid development in mushrooms :

• Selection of parent lines
• Isolation of homokaryons
• Crossing of compatible homokaryons
• Identification of hybrids.

A. Selection of parent lines :
Lines possessing wider adaptability, genes resistant to diseases and insect-pests, better yield performance and morphological characters, better shelf life, suitability of processing etc; are chosen as parent for hybridization. In India the high yielding and better quality germplasm lines like S-11 , S -44 , NCS-6 , ARP -215 , ARP-217 , 224 , 225 ; P-1 , ITCC-1924 , S-56 etc; have been identified as parents for single spore selection and hybridization. RAPD markers have successfully been used for assessment of diversity in A.bisporus germplasm at DNA nucleotide level and to fingerprint each genotype for strain protection.

B. Isolation of Homokaryons:
For hybrid breeding , isolation of homokaryons from heterokaryotic parental lines is required which is very difficult in case of A.bisporus due to its unusual life cycle termed as secondary homothallism or Intramixing where majority of spores are binucleate and self fertile heterokaryons as compared with the other heterothallic species such as A bitorquis., A campestris , A arvensis etc ; where each basidiospore gives rise to self –sterile mycelium and hence single spore isolate in these mushrooms are homokaryotic and are cross fertile. Moreover, A . bisporus also lacks clamp connections, the morphological markers which facilitate easy distinction of homokaryons from heterokaryons. These factors have affected the mushroom improvement programme in A.bisporus.

The traditional method of homokaryon isolation technique involves the identification of naturally occurring monokaryon spores which are at a frequency of 0.1 to 40 per cent in A.bisporus. These homokaryons are isolated either by dilution plate technique or the micro manipulation technique. In dilution plate technique the basidiospores are serially diluted in sterilized distilled water and plated on a suitable agar medium so that it may have 8-10 spores only in a plate.. The spores are stimulated for quick germination by placing growing mycelium in the lid of the Petri dish which are placed upside down and incubated at 25-28º C . The plates are critically examined after 4-5 days of incubation and the single spore colonies are marked under microscope and aseptically transferred to the slants containing suitable agar medium like MEA , PDA , Compost-agar medium etc. The micromanipulator method consists of picking the spores directly from aberrant three and four basidia aseptically. The germinating single spore can also be picked up and transferred separately by using this method.

The following criteria are used alone or in combination for the identification of homokaryons in A.bisporus.

• Colony morphology
• Slow mycelial growth
• Non- fruiting
• Source of spores ( 3 and 4 -spored basidia )

The mycelium derived from infertile single spore isolate (homokaryons ) in A.bisporus is mostly of slow growing and appressed type, whereas the homokaryons of hetrerothallic spp. like A .bitorquis also grow slowly but it exhibits a dense matted type of mycelial growth.The criteria of colony morphology, slow mycelial growth and non-fruiting characters used for identification of homokaryons are cumbersome and affected by the environmental factors and also the frequent reports of homokaryotic fruiting in several cultivated mushrooms by different mushroom workers making these criteria unreliable and ambiguous. With the availability and use of modern tools of molecular biology viz; Allozymes , Restriction Fragmented Length Polymorphism ( RFLP) , Random Amplified Polymorphic DNAs ( RAPD ) etc; for homokaryon isolation in A.bisporus, genetic improvement of button mushroom has become easy. These techniques have been found to be quick and reliable as these are based on DNA markers and are not influenced by the environmental factors.

C. Crossing of compatible homokaryons :
The known compatible homokaryons are anastomosed ( mated ) by growing them side by side on sterilized agar medium and incubation at favourable temperature as the method devised by Eliott ( 1978 ). The formation of fluffy growth at the hyphal confrontation or junction zone is indicative of development of a hybrid. A big piece of the mycelial growth from this junction zone is cut and shifted aseptically to a new agar medium for getting pure hybrid culture which is further used for preparing spawn for its testing.

D. Confirmation of hybrid testing :
In case of widely cultivated mushrooms , A.bisporus and A.bitorquis , the fertile heterokaryon or hybrid formed by mating of two compatible homokaryons forms no clamp connection and secondly the microscopic observation of only heavier growth at the zone of confrontation between the two compatible homokaryons. But following the latest development, formation of hybrids in these mushrooms can be confirmed by a number of tests like the fructification test, auxotrophic markers, resistance markers, allozyme markers, DNA markers – RFLP’s and RAPDs as described below :

Evaluation: After the identification / confirmation of new hybrid formation, these are evaluated through Initial Evaluation Trial ( IET ) for the yield, quality, resistance against diseases and pests and for other traits. The hybrids found superior and better are further put to multilocational trials and those found successful are again put in On-farm trials in farmers’ field and only then one with best performance on all aspects , is released for commercial production / cultivation.

Hybridization has only been found to be the most reliable and sustainable strain improvement method in case of mushrooms.The hybrid U-1 and U-3 of A.bisporus developed by Dr.Gerda Fritsche in Holland are still performing good since 1981 which possess characters like high production with good canning quality. Similarly hybrids K- 32, K -26 and K -46 of A.bitorquis also performing very well .

4. MUTAGENESIS :

Abrupt change on the genes of DNA of some mushrooms may result in development of variability and a new strain, though it is very rare. The common white button mushroom is also the result of mutagenic change that occurred in some cream strain of A.bisporus during 1927 (Kligman,1950 ). Now-a-days mutagenesis is attempted to get new variable strains for selection and hybridization programmes. Here hyphal fragments. protoplast and basidiospores are used for mutagenesis to develop strains with desired traits like resistance against fungicides and diseases, higher yields, tolerance for high temperature, sporeless strain etc.

5. PROTOPLAST FUSION :

It is a non-conventional method of gene transfer as it involves breaking down of the natural barrier of gene exchange as found in conventional system of breeding. The following technology is involved in protoplast fusion:

• Use of cell wall digestive enzymes , Novozyme 234 with osmotic stabilizer (0.6 M Sucrose or 0.5 M MgSo4 )
• Fusion of the protoplast with CaCl₂ and polyethylene glycol.
• The regeneration of evaluation of somatic hybrids.The technique of rising electrical pulsation (Electrofusion) for the protoplast fusion is also being used now-a-days.

6. GENETIC ENGINEERING:

• The technique is based on the systematic practice of isolation, cloning and insertion of desirable gene (s) onto the genome of target organism. For successful delivery of transferring DNA into mushroom genome , Electroporatin and Biolistic methods have been recently used ( Moore et al,1995 and Mooi Brock et al, 1996 )
• Other approaches like Marker Assisted Selection ( MAS ) , Back crossing and the use of Quantitative Trait Loci (QTLS) have also been considered new approaches for button mushroom breeding and recently Agrobacterium tumifaciens has successfully been used for the genetic transformation of gill tissue and germinating spores in A.bisporus .