Criteria for assessment of tissue culture units
Potato - tissue culture - raised minituber - (PTCMT) standards for certification
National Consultation on Certification of Tissue Culture Raised Plants
19 April 2005, The Ambassador Hotel, New Delhi

Executive Summary
| Agenda | List of Participants


Introduction
In India, several plant tissue culture laboratories and commercial facilities have been setup recently, and they have been generating large number of tissue culture raised plants of commercial crops and forest trees. However, the country lacks organized testing of quality of regenerants and their freedom from viruses.

The most deleterious variants in tissue culture raised plants are those that effect yields, quality and carry infection of viruses which are difficult to diagnose. To bridge the gap, the Department of Biotechnology, Govt. of India has established recognized test centres for testing of tissue culture raised plants for quality and freedom from viruses.

The participating institutions and contact persons are:

Indian Agricultural Research Institute, New Delhi
Dr Y S Ahlawat (yahlawat@yahoo.com), Dr V G Malathi

Indian Institute of Horticultural Research, Bangalore
Dr M K Reddy (mkreddy@vsnl.com), Mr Duleep Kumar Samuel

Institute of Himalayan Bioresource Technology, Palampur
Dr A A Zaidi (Zaidi_aijaz@yahoo.com), Dr Raja Ram

National Chemical Laboratory, Pune
Dr P K Ranjekar (pkr@ems.ncl.res.in)

SPIC Science Foundation, Chennai
Dr T S Lokeswari (tsloki@yahoo.com), Dr George Thomas

The Energy and Resources Institute, New Delhi
Dr Sanjay Saxena (sanjays@teri.res.in), Dr Shashi Bhushan Tripathi

Goal
To ensure production of virus-free plants of assured quality raised through tissue culture.

Objectives
To develop protocols for virus diagnosis and quality testing
To test tissue culture raised plants for assured quality and freedom from viruses.

Testing for Virus and Quality
A large number of viruses infect horticultural, plantation and forest plants which are being micropropagated by the tissue culture industry. Most important of these are aphid-transmitted poty-, cucumo- and luteoviruses, whitefly transmitted geminiviruses, thrip-transmitted tospoviruses, mealy bug transmitted ilarviruses.

Generation of virus-free planting material is an ideal strategy to confine these viruses and also to facilitate the movement of materials across the domestic and international boundaries. Tissue culture is an useful approach for generating virus-free planting material. In order to minimise the risk of inadvertent propagation of virus infected plants and introduction of somaclonal variability, the tissue culture raised plants need to be thoroughly indexed for freedom from viruses and checked for quality. Careful indexing based on recent biotechnological methods such as imunoprobes, nucleic acid probes and polymerase chain reaction (PCR) would ensure phytosanitary safety during the movement of planting materials. Similarly, molecular testing will ensure quality control.

The National Facility for Virus Diagnosis and Quality Control of Tissue Culture Raised Plants (NFVD & QC), is aimed at providing virus testing and quality testing support to the tissue culture industry to ensure that only (a) virus-free mother plants are used for micropropagation, and (b) virus-free and quality tested tissue culture plants are supplied to the growers. Hence, not only the risk of inadvertent introduction and subsequent spread of viruses would be minimised but also the quality of plants would be assured. This will be achieved by developing a biophysical, immunological and molecular technique based virus diagnosis programme and molecular based quality control programme of the plant species of interest. The testing protocols will not only detect the known viruses, but also the unknown viruses.

The test for virus and quality will have direct impact on developing a vibrant tissue culture industry as the demand for certified tissue culture raised plants will grow exponentially.

Virus testing
The country has expertise to undertake diagnosis of plant viral diseases through the application of biophysical, immunological and molecular techniques. Immunodiagnosis is the most useful technique using polyclonal and monoclonal antibodies raised against viruses. Polyclonal antibodies are now available in the country for various groups of viruses like potyviruses, tospoviruses, tobamoviruses, potexviruses, luteoviruses, badnaviruses, closteroviruses, etc. (Varma and Ramachandran, 1996). These polyclonal antibodies have been very useful in the specific identification of viruses and their strains, and for plant virus diagnosis. The technology for the production of monoclonal antibodies is also available in the country, but their production has not caught up to the desired level for plant viruses due the lack of adequate facilities.

Various immunological and molecular techniques like ELISA (Malathi et al, 1989; Jain et al, 1998), immuno-electron-microscopy (Ahlawat and Varma, 1997), DIBA, Western blotting (Bhat et al, 1996, 1997), immunofluorescence (Varma et al, 1993), nucleic acid hybridization using radio-labeled probes (Raj and Singh, 1996; Mandal et al, 1997) and PCR (Varma et al, 1999) have been used successfully for the diagnosis of virus infections in a variety of plant species. Of these techniques, ELISA, immuno electron microscopy, dot-blot and PCR are the most sensitive and reliable techniques which can be very effectively used for the purpose of diagnostic services to tissue culture industry in the country.

For nucleic acid hybridization, c-DNA probes are also available for the gemini-, poty-, tobamo-, and badnaviruses (Varma and Ramachandran, 1996). Electrophoresis and direct electron-microscopy (Ramachandran et al, 1992; Viswanathan et al, 1998) are also being commonly used particularly for detecting viroids and unknown viruses.

The viruses for which diagnostic probes (antibodies and cDNA probes) have been developed are known to infect a variety of plant species like: banana, brinjal, cassava, citrus, Frenchbean, garlic, gladioli, groundnut, lilies, mungbean, onion, rice, soybean, sugarcane, tobacco, tomato, urdbean, ornamental plants etc.

Citrus (Citrus spp.) is the third largest fruits crop grown in India, with a great potential for export if raised from desirable virus free planting material to increase production and maintain quality. Its cultivation in India extends to over 0.37 million hectares with an annual production of 2.98 million tonnes. The productivity of citrus fruits in India is comparatively low owing to many biotic stresses of which viruses play a significant role. In India, more than 15 virus and virus like diseases occur on citrus (Singh, 1996). Of these, three viral diseases namely tristeza, ringspot and mosaic, and citrus greening disease caused by a BLO are widely distributed in the country. While tristeza and greening present worldwide the ringspot and mosaic diseases are of specific concern in India. Tristeza disease is caused by a closterovirus which has been detected from all parts of the country. The severe strain of CTV is most commonly distributed and result in tree decline (Chakraborthy et al., 1993). Mild strains of the virus are also reported, but they were not found useful for cross protection (Chakraborthy et al., 1993). capillovirus is associated with the ringspot disease, and a double stranded DNA virus belonging to badnavirus group is associated with the mosaic disease (Byadagi et al., 1993; Byadagi and Ahlawat, 1995; Ahlawat et al, 1996). Citrus greening disease, caused by a fastidious bacterium, Liberobacter asiaticum (LA) is present throughout the country and has been the major cause in nearly wiping out citrus orchards in Coorg, Karnataka (Varma et al., 1993). Often these pathogens are found in mixed infections (Ahlawat, 1997).

Banana is another important commercial fruit being grown in all parts of the country. The estimated area under banana is 3.92 m ha with production of 10.4 m tonnes/annum. This crop is adversely affected by the bunchy top disease which has spread throughout the country. The causal virus has been cloned for developing c-DNA diagnostic probes (Bhat and Varma, 1996). Another important viral disease of banana is infectious chlorosis which is caused by cucumber mosaic virus. This disease is also widely distributed throughout the country causing serious economic losses (Summanwar and Marathe, 1982). Other two viral diseases namely the bract mosaic (Rodoni et al., 1997) and streak mosaic (Selvarajan et al., 1996) are localized and are of minor importance, but these are spreading fast in the absence of appropriate diagnostic systems and healthy planting material certification programmes.

With the discovery of viroids as potential plant pathogens several diseases which were earlier attributed to viral infections are now known to be caused by viroids. Viroids are single stranded circular RNA molecules with low molecular weight, devoid of proteins. Since they are free infectious RNAs it is not possible to develop protein based diagnostic reagents. To date about 43 of them were found in vegetatively propagated crops like citrus, potato, grapes, hops, apple and avocado etc. In India, research on viroids started only recently and the diseases caused by viroids have been recorded in tomato (Mishra et al., 1991), potato (Khurana et al., 1989), citrus (Ramachandran et al., 1993), and coleus (Ramachandran et al., 1992).

In India, viral diseases of ornamental plants are common. Most of these plants are infected by the viruses belonging to cucumovirus (Sang and Varma, 1975), potyvirus (Singh et al., 1970; Roy and Ramachandran, 1988), and tobamovirus (Varma and Gibbs, 1977). In orchids, infection by CyMV is very common (Varma and Ramachandran, 1994).

Quality control
With the advent of the PCR technique and DNA based molecular markers, some laboratories in India have developed expertise in these areas and are using them for assessment of genetic diversity, phylo-genetic analysis, verietal typing and establishing true to type of tissue cultured plants.

Somaclonal variants of pigeonpea were analysed using RAPD (Prasannalatha et al., 1999) and the polymorphic fragment scored for an identifiable locus like white seed, high seed mass, low Helicoverpa incidence etc. RFLPs were reported in studies on regenerated maize, rice, potato, coffee, populus and others (Rani et al., 1998; Mohan et al., 1997).

Microsatellite primed-Polymerase Chain Reactions (MP-PCR) were used to study genetic fidelity of regenerated shoots of Eucalyptus, populus and coffee species (Rani et al., 1995; Rani et al., 1998). Polymorphisms could not be detected in bread wheat genotypes using 23 simple sequence repeats indicating the inadequacy of the molecular analysis (Varshney et al., 1998). In Populus deltoides six of the off-types were reported through RAPD analysis of 32 randomly selected plants from a population of 500 individual plants. The plants were derived from nodal segments (Rani et al., 1995). Similarly, in sugar beet Munthali et al. (1996) reported 0.05% somaclonal polymorphism. Existence of distinct genetic differences among several variants of peach was reported by Hashmi et al. (1997). Rani and Raina (1998) reported minor variation in RAPD banding pattern among the regenerated plants of Eucalyptus tereticornis and Eucalyptus camadulensis derived from enhanced axillary branching.

From the contrasting reports on the use of molecular marker techniques for analysis of micropropagated plants it is evident that the marker system chosen for analysis needs to be evaluated for its suitability for the application before it can be further used (Gupta and Varshney, 1999). TERI is a leading group to standardize the technique of AFLP in India and utilize it for studying the genetic diversity in Azadirachta indica (Singh et al., 1999). Attempts have also been made to compare the various techniques of genetic typing such as RAPD, RFLP and AFLP. Results have demonstrated that even though RFLP and RAPD markers are useful in studying both intra and inter-specific variation, the extent of polymorphism generated by AFLP makers is several times higher. Hence, AFLPs may be the method of choice for discriminating very closely related genotypes (such as the micropropagated plants) as compared to other molecular markers.

Importance of the work
The banana viruses primarily spread through the use of infected suckers but the secondary spread also occurs through aphid vectors. Field spread is much faster if virus free suckers are not used as initial planting material (Sharma, 1988). An annual loss of about Rs. 40 million has been reported due to BBTV in the Kerala state alone (Mehta et al., 1964). In the hills of lower Palanis, BBTV destroyed 60% of the banana crop (Keshavamurthy, 1980). BBTV is also spreading fast in Maharashtra and Karnataka causing serious economic losses. Infectious chlorosis has been reported to be severe in Maharashtra and South India causing heavy losses in yields (Summanwar and Marathe, 1982). The incidence of infectious chlorosis in Tamil Nadu has been reported to be 13.4% (Elongovan et al., 1990). BBTV results in a yield reduction of about 40 per cent of bunch weight in cultivars Nendran, Poovan, Robusta, Red Banana, Bluggoe and Pisang Awak resulting in upto 68% reduction in yield (Selvarajan and Singh, 1997).

In India, banana streak virus severely infects 'Mysore' and Cavendish group of bananas. Many plants remain under symptomless normal condition, but, under stress like high temperature, nutrients they develop severe symptoms. The yield loss due to severe infection of BSV is reported to be more than 48 per cent (Annual Report NRCB, Trichy). The BSV infected plants have poorly filled fingers, sometimes seediness has also been observed. Seeded fruits are unfit for marketing. The disease is assuming a major threat to banana industry owing to its fast spread, the tissue cultured plants supplied by some of the firms were found to have cent per cent infection in Maharashtra (Cavendish) and Tamil Nadu (Poovan) (Selvarajan and Singh, 1997). Sensitive and reliable techniques for the detection of banana viruses in planting material are not available. It is necessary to develop virus specific diagnostic kits for the reliable and sensitive detection of these most common viruses so that tissue culture raised plants could be tested.

Like banana, other plant species, which are being micropropagated by the tissue culture industry in the country, are also affected by a number of viruses (Table 1). For value addition, it is important that all the tissue culture raised plants are certified for freedom from viruses. Apart from the plant species listed in Table 1, plants like citrus and oil palm are also being propagated through tissue culture intervention. Gradually, more and more plant species will be taken up for micropropagation through tissue culture.

Several plants are produced annually through tissue culture techniques. These plants are planted in large numbers in the fields either for evaluation purposes or for routine plantation. In either case it is important to maintain the genetic fidelity of the plants. Since sophisticated techniques like the AFLP have been developed and found useful, in this study we propose to use them to maintain a strict quality check of the plants produced at the MTP. This service will be made available not only to R & D centers developing tissue culture protocols but also to the established tissue culture industries for certification of planting material. Establishing clonal uniformity for horticultural species is very important as for most species it takes 3-4 years to bear fruits and certification in terms of disease free nature and expected yields are very important for the grower.

Table 1 Plants identified for testing viruses

Plant species Virus
Fruit crops
Banana Banana bunchy top nanovirus
  Cucumber mosaic cucumovirus
  Banana bract mosaic potyvirus
  Banana streak badnavirus
Papaya Papaya ring spot potyvirus
  Papaya leaf curl geminivirus
  Papaya mosaic potexvirus
Strawberry Raspberry ring spot virus
  Strawberry latent ring spot virus
  Tomato black ring virus
Plantation crops
Black pepper Little leaf
Clove  
Large cardamom Chirkey potyvirus
Small cardamom Cardamom mosaic potyvirus
  Nilgiri necrosis virus
  Kokke kandu virus
  Foorkey virus
Vanilla Vanilla mosaic potyvirus
  Vanilla necrosis potyvirus
  Cymbidium mosaic potexvirus
  Odontoglossum ring spot tobamovirus
  Uncharacterised potyvirus
  Uncharacterised rhabdovirus
Coffee  
Tea  

Ornamentals

Anglaonema Dasheen mosaic potyvirus
Alstomeria Potyvirus
Alocasia Potyvirus
Anthurium Anthurium mosaic virus
Asparagus Asparagus 1 potyvirus
  Asparagus 2 ilarvirus
  Asparagus 3 potexvirus
Begonia Cucumber mosaic cucumovirus
  Impatiens necrotic spot tospovirus
  Tomato spotted wilt tospovirus
Caladium Dasheen mosaic potyvirus
Calathia  
Carnation Carnation 1 and 2 alphacryptovirus
  Carnation latent carlavirus
  Carnation ringspot dianthovirus
  Carnation etched ring caulimovirus
  Carnation mottle carmovirus
  Carnation necrotic fleck closterovirus
  Carnation Italian ringspot tombusvirus
  Carnation rhabdovirus
  Carnation vein mottle potyvirus
  Carnation yellow stripe necrovirus
Chrysanthemum Chrysanthemum B carlavirus
  Chrysanthemum spot potyvirus
  Chrysanthemum vein chlorosis rhabdovirus
  Chrysanthemum aspermy cumovirus
  Cucumber mosaic cucumovirus
  Impatiens necrotic spot tospovirus
  Tomato ring spot nepovirus
  Tomato spotted wilt tospovirus
Colocasia Dasheen mosaic potyvirus
Dieffenbachia Dieffenbachia mosaic virus
  Dasheen mosaic potyvirus
Ficus  
Fittonia Potyvirus
Freesia Freesia mosaic potyvirus
  Freesia leaf necrosis varicosavirus
Gerbera Tobacco rattle tobravirus
  Gerbera mosaic virus
  Gerbera symptomless rhabdovirus
Gladiolus Arabis mosaic mepovirus
  Bean yellow mosaic potyvirus
  Cucumber mosaic cucumovirus
  Narcissus latent macluravirus
  Impatiens necrotic spot tospovirus
  Strawberry latent ring spot nepovirus
  Tobacco mosaic tobamovirus
  Tomato spotted wilt tospovirus
Iris Iris germanica leaf stripe rhabdovirus
  Iris mild mosaic potyvirus
  Iris severe mosaic potyvirus
  Iris Japanese necrotic ring virus
  Iris fulva mosaic potyvirus
Kalanchoe Kalanchoe mosaic potyvirus
  Kalanchoe isometric virus
  Kalanchoe latent carlavirus
  Kalanchoe topspotting tospovirus
Lily (Asiatic, Oriental, Longifolium) Cucumber mosaic cucumovirus
  Chrysanthemum aspermy cucumovirus
  Impatience necrotic spot tospovirus
  Lily symptomless carlavirus
  Lily mottle potyvirus
  Lily X potexvirus
  Tomato spotted wilt tospovirus
  Tulip breading potyvirus
Limonium Potyvirus
Orchids (Cymbidium) Cymbidium mosaic potexvirus
  Cymbidium ring spot tombusvirus
  Impatiens necrotic spot tospovirus
  Odontoglossum ring spot
  Tobamovirus
  Orchid fleck rhabdovirus
  Tomato spotted wilt tospovirus
Phalaenopsis Orchid fleck rhabdovirus
Philodendron Dasheen mosaic potyvirus
  Konjak mosaic ......virus
Rose Apple mosaic ilarvirus
  Arabis mosaic virus
  Prunus necrotic ring spot ilarvirus
  Rose rosette
  Rose streak virus
  Rose Tobamovirus
  Strawberry latent ring spot virus
  Tobacco mosaic virus
  Tobacco streak virus
Spathiphyllum Dasheen mosaic virus
Syngonium  
Xanthosoma Dasheen mosaic potyvirus
Zantedeschia (Calla lily) Cucumber mosaic virus
  Dasheen mosaic potyvirus
  Tomato spotted wilt virus
Forest trees
Bamboo Bamboo mosaic potexvirus
Eucalyptus Little Leaf
Poplar Poplar decline potyvirus
  Poplar mosaic carlavirus
  Poplar vein yellowing mucleorhabdovirus
Teak  
Neem