Packaging and Storage of samples for testing under National Certification System for Tissue Culture Plants (NCS-TCP/8)

 

As you might already be aware, testing and certification is an important activity under NCS-TCP. Sample submission is a crucial step therefore; proper packaging and labelling must be done prior to submission of samples for testing under NCS-TCP.

 

Packing/Labelling/Forwardal of samples for testing:

The sample collected should be blotted dry to remove excess moisture before packing.

The sample is placed in between paper towels, packed in self-sealing/zip-lock polythene bags of appropriate size. The sample is affixed with a label (Annexure-3C as available on NCS-TCP website) and  kept  in  a  ventilated  card  board  box  and  /or  thermocool  box  for  forwardal  to Accredited Test Laboratory within specified time under cool conditions.

 

The packing box is marked on top of the box with the address of Accredited Test Laboratory  with  appropriate  instructions  such  as  “Handle with  care/Tissue Culture Plants/Rush Delivery” and  either  couriered  or  delivered  in  person  to  the  concerned Accredited Test Laboratory within 24 hrs period under cool conditions.

 

Receipt /storage of sample at Accredited Test Laboratory:

 

Samples are either stored in vacuum desicator with anhydrous calcium chloride (CaCl₂) or freeze dry or store at -80 ⁰C in a deep freezer at the Accredited Test Laboratory.

Source - Standard Operating Procedure of NCS-TCP

 

Significance of virus indexing of tissue culture raised Bamboo plants (NCS-TCP/7)

Bamboos are economically important species for their broad applications in both agriculture and industry. Bamboo belongs to family Poaceae and grows in tropical, sub-tropical and temperate regions with an uneven distribution based on annual precipitation, altitude, soil conditions and temperature.

Large tracts of natural bamboo forest occur in tropical Asian countries including India, Myanmar, Thailand and China. In India, bamboo is found growing naturally in almost all parts of the country except Kashmir (Bamboo Resource of the Country, Forest Survey of India, 2017). However, bamboo crops are under the threat of a major pathogen, Bamboo mosaic virus (BaMV), a member of the genus Potexvirus in the family Flexiviridae (Lin et al., 1992).

Symptoms for infected plants include foliar mosaic and stripe, brown internal streaking of the shoots and culms, and culm abortion (Lin and Chen, 1991; Elliot and Zettler, 1996). Culms are poorly developed and have shortened internodes, also the new shoots are hard in texture, hence, deplete the quality for eating and canning (Lin and Chen, 1991).

Bamboos are normally vegetatively propagated and the virus is probably transmitted through vegetative propagation of infected, non-indexed planting material and mechanically by the unclean practices employed in the routine cutting of bamboo shoots (P. Awasthi, A. Sood and V. Hallan, 2016).

Testing of mother plant/stock culture and tissue culture raised plants for Bamboo Mosaic Virus is possible at Accredited Test Laboratory (ATL) at University of Agricultural Sciences, GKVK. , Bangalore

(Typical symptoms of bamboo mosaic caused by Bamboo mosaic virus (BaMV) include distinctive interveinal chlorotic mosaic patterns and striping on the leaf surfaces)

Reference:

P. Awasthi, A. Sood and V. Hallan,  2016, Viral Diseases of Bamboos, Bamboos in India, ENVIS RP on Forestry and Forest Related Livelihoods

Scot Nelson and Wayne Borth, September (2011) Plant Disease, Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawai‘i-Mänoa.

Type of various viral infection in Banana and significance of certification to overcome its spread

India is the largest producer of Banana in the world and accounts for about 21 percent of the world banana produce, producing over 31 million metric tonnes of banana every year. However, banana continues to face pests and disease infestations affecting the production and productivity in India. Viral diseases are a major constraint to production since they affect the yield and quality.

In India, Banana bunchy top virus (Babuvirus), Banana streak virus (Badnavirus), Banana bract mosaic virus (Potyvirus) and Cucumber mosaic virus (Cucumovirus) are the major viral diseases infecting the banana growing regions of the country.

1.      Banana bunchy top disease

Symptoms:

-        Initial symptoms of Banana Bunchy Top Disease consist of appearance of dark green streak in the veins of lower portion of the leaf midrib and leaf stem.

-        The symptom of the prominent green streaks in veins of lamina is referred to as ‘Morse code streaking’.

-        Plants with advanced stage of infection show rosette appearance with progressively shorter, narrow, brittle textured leaves giving rise to the common name of ‘bunchy top’.

-        Infected banana plants usually will not fruit, however if fruit is produced the banana hands and fingers are likely to be distorted and twisted.

-        Banana is transmitted by infected suckers and banana aphid.

Fig: Morse code streaking on the leaves

Pcr, ® & Chen, Yan & Hu, Xiaoping. (2013). High-throughput detection of banana bunchy top virus in banana plants and aphids using real-time TaqMan. Journal of virological methods. 193. 177-183. 10.1016/j.jviromet.2013.06.013.

Fig: Bunchy top disease-late stage infection

http://www.musarama.org/en/image/bbtd-late-stage-infection-165.html

Transmission:

-        Primary mode of transmission is through infected suckers, corms/bits and also through tissue propagated material.

-        Secondary transmission is by banana black aphid Pentalonia nigronervosa in a semi persistent manner

2.      Banana bract mosaic disease

Symptoms:

-        Banana bract mosaic disease is characterized by presence of spindle shaped pinkish to reddish streaks on pseudostem, midrib and peduncle.

-        Typical mosaic and spindle shaped mild mosaic streaks on bracts, peduncle and fingers are also observed.

-        Suckers exhibit unusual reddish brown streaks at emergence and separation of leaf sheath from central axis.

-        Clustering of leaves at crown with a travelers palm appearance, elongated peduncle and half filled hands are its characteristic symptom.

Transmission:

-          The disease is caused by a virus belonging to potyvirus group. The virions are flexuous filamentous

-          The virus is primarily spread through infected suckers.

-          The virus might be transmitted through aphid vectors. In the field, aphids vectors such as Aphis goosypii, Pentolonia nigronervosa and Rhopalosiphum maidis transmit the disease.

http://agritech.tnau.ac.in/crop_protection/banana_diseases/crop_prot_crop%20diseases_fruits_banana_2.html

3.      Banana streak disease

Symptoms:

-          Symptom for streak virus disease can vary from an inconspicuous flecking to lethal necrosis depending on the isolate of the pathogen, the host cultivar and the environment.

-          Symptoms mostly include narrow, discontinuous and sometimes continuous chlorotic or yellow streaks that run from the leaf midrib to the margin.

-          Spindle or eye-shaped patterns are present in some cases. Yellow blotches have also been associated with banana streak.

-          Sometimes the lamina can be distorted. Streaks later darken to orange and often become brown or black.

-          Necrosis has also been seen on the midrib and petiole. Necrosis occurs more under low temperature, short-day conditions.

-          The intensity of symptoms has been associated with the concentration of virus in the tissue; the higher the virus concentration, the more severe the symptoms.

-          Other symptoms associated with disease are stunting, cigar leaf necrosis, internal necrosis of the pseudostem, a reduction in bunch size, incomplete emergence of bunches and bunches emerging through the side of the pseudostem. Occasionally, dark streak symptoms may be visible on the pseudostem and fingers may be distorted

Transmission:

-          Banana streak viruses are pararetroviruses belonging to the genus Badnavirus, in the family Caulimoviridae.

-          Natural field spread occurs by either mealybugs or use of infected planting material, such as suckers.

-          Vegetative transmission is the primary mode of transmission.

Fig: Symptoms of Banana streak virus showing lines of yellow in large blocks from midrib to leaf edge, turning brown or black with age.

http://www.pestnet.org/fact_sheets/banana_streak_disease_215.htm

4.      Cucumber mosaic disease

Symptoms:

-          Diffused mosaic/ line pattern/ ring spots symptoms appear in affected leaf lamina.

-          Deformation and curling of leaves are also reported.

-          Some other symptoms include rosette appearance of leaf arrangement and conspicuous inter-veinal chlorosis.

-          Symptoms are usually more severe when temperature falls below 24˚C.

-          Affected plants are stunted and will throw small bunches with malformed fingers.

-          Sometimes plants may die if severe strain of the virus affects the plant.

Transmission:

-          The virus is transmitted through infected suckers and it is also acquired from a wide range of host plants growing near banana fields through aphid vectors.

-          The most common aphid are: Aphis gossypii and Rhopalosipum maidi.

-          The virus is most frequently transmitted mechanically.

Fig: Cucumber Mosaic Virus (CMV) infected Banana plant.

Dheepa, R. and Subramanian Paranjothi. “TRANSMISSION OF CUCUMBER MOSAIC VIRUS (CMV) INFECTING BANANA BY APHID AND MECHANICAL METHODS.” (2010).

With use of quality tissue culture banana planting material it is ensured that virus free and genetically uniform plants are provided to the farmers. Under NCS-TCP, batch certification is done prior to dispatch of plants to check that the plants are virus free.

Suggested References for disease symptoms:

R. Selvarajan and M. M. Mustaffa, Viral Diseases of Banana, Technical Bulletin, NCRB-ICAR.

Sampling strategy of testing and certification of tissue culture raised plants under NCS-TCP (NCS-TCP/5

A. Plant Tissue/Stock Culture

Mother Plant/Stock Culture is the source for propagation of tissue culture plants. It is important to test that they are free of any viruses to ensure success of tissue culture operation and production of quality tissue culture plants. Virus free mother plant/stock culture is pre-requisite for maintenance of appropriate tissue culture standards. A list of all known viruses for tissue culture raised plants is available on NCS-TCP website (www.dbtncstcp.nic.in).

Sampling of mother plant/stock culture

Each sample should ideally have at least 0.5 gm of tissue per virus per test for all known viruses to be tested.

Although, all mother plant tissue/stock cultures should be tested. The cost of testing for huge number of samples would affect the overall cost of the plantlet. Therefore, if number of mother plant tissue/stock culture is large, the samples from batches consisting of a maximum of 10 mother plants/stock cultures may be pooled for testing.   In such cases –

(i)      The tissue culture unit must maintain proper record of individual  mother plants/stock cultures of each batch, so that individual mother plants/stock cultures or smaller batches could be tested, in cases where  the pooled samples are found positive for infection, so that only the cultures from infected mother plant/stock culture are discarded.

(ii)    If testing is not done as envisaged above, all the cultures generated from the infected mother plants/stock cultures will have to be discarded.

Facilities seeking recognition/renewal of recognition under NCS-TCP need to test the starter material/ stock culture prior to its large scale multiplication 

B. Tissue Culture Raised Plants

Once the tissue culture raised plants are grown, it is verified that these plants are virus-free and also genetically uniform. This is done in order to ensure that no infected plants are transferred to the fields and only quality planting material reaches to the farmers.

Sampling of tissue culture plants

Similar to sampling of mother plant/stock culture, each sample should have at least 0.5gm of tissue per virus per test for all known viruses to be tested. An additional 1.0gm of tissue should be provided for genetic fidelity/uniformity testing.

In case of sampling for quality (genetic fidelity) testing, sample from mother plant (clone) from which tissue culture plants produced will also be collected and forwarded for testing.

The following scale of sampling should be used for virus and/or quality (genetic fidelity/ uniformity) testing, in case of tissue culture raised plants (ex-agar plants/hardened plants). The scale is defined depending  on  the  lot  (batch)  size  of  tissue  culture  plants  produced,  just  prior  to dispatch/shipment of consignment.

Lot size	Number of tissue culture plants to be sampled
Up to 1000 Nos	1% plants subject to a minimum of 10 Nos
1001 to 10000 Nos	0.5% of plants subject to a minimum of 10 Nos
10001 to 100000 Nos	0.1% of plants subject to a minimum of 50 Nos

(Reference: Guidelines and SOPs of NCS-TCP) 

Designing the commercial plant tissue culture laboratory as per the NCS-TCP Standards (NCSTCP/4)

Plant Tissue culture technology require specifically designed laboratory in order to ensure maintenance of tissue culture standards in producing quality plant cultures.

An ideal plant tissue culture laboratory must contain the following exclusive functional areas:

            1. Washing Room (s)

2. Media Preparation Room (s)

3. Media Storage Room (s) maintained under positive pressure           

4. Inoculation Room (s) maintained under positive pressure

5. Growth Room (s) maintained under positive pressure

6. Transfer/ grading Room (s) 

7. Insect proof greenhouse/ poly house with double door entry fitted with humidity control for primary hardening area      

           8. Insect proof Nursery/Shade house Area (s) with double door entry covered with

               appropriate mesh to provide partial shade for secondary hardening area

Laboratory areas can be classified into sterile area and non-sterile area. The areas under positive pressure such as media storage room, inoculation room, growth room and passage area which are considered as sterile area must have separate entry in order to maintain their sterility. Entry into these areas should have facility for hand and foot washing, air curtain/ air shower along with cubicle for dress change and dress storage space. It may also be noted that class 100,000 sterility level should be maintained through pressure module/ AHU/ HVAC/ etc in media storage room, inoculation room and incubation/growth room.

It is important that the layout of laboratory building is planned to restrict free movement of human and materials between sterile and non-sterile area. A basic layout is given below which might be referred by potential entrepreneur aspiring to establish a new laboratory (Figure 1). 

Pass box(s) fitted with UV and see through glass and or/other suitable mechanism should be available. Pass box(s) would enable transfer of autoclaved media into media storage room immediate after autoclaving and transferring plants from growth room to transfer area without letting humans enter into other area.

A well-maintained fire-fighting system with emergency exit, path showing fluorescent strip for guiding the emergency exit, fire alarm/ smoke alarm and fire extinguisher are a safety requisite for the laboratory. Uninterrupted power supply should also be available to support the power requirement.

In addition to above basic equipment including electronic weighing balance, pH meter, conductivity meter, microwave oven, de ionizer/distillation unit/RO water facility, autoclave etc, should be available within the laboratory.

Figure 1: Schematic layout describing the different activities and man and material movement. This may vary depending upon crop, capacity, topography etc. Corridor and firefighting system including emergency exit should also be suitably planed while constructing the facility.