Introduction Use of quality planting materials - prerequisite for increasing production and productivity of banana

Transcription

1 A novel molecular technique for mass indexing of tissue cultured banana plants for Banana Bunchy Top Virus (BBTV) W.A.R.T. Wickramaarachchi, K.T. Ragnaswamy and K.S. Shankaraappa Horticultural Crops Research and Development Institute, Department of Agriculture, Sri Lanka

2 Introduction Use of quality planting materials - prerequisite for increasing production and productivity of banana

3 BBTV Among four major viral diseases known to infect banana in Sri Lanka, BBTV is the most devastating virus heavy yield losses if planting materials have been pre infected Regarded as a quarantine pest of high importance Listed as one among 100 of the world s worst invasive alien species Practical management options for BBTV Phytosanitation Virus free planting materials

4 Tissue culture Rapid mass multiplication through tissue culture has been extensively and productively used in banana industry TC process eliminates weeds, nematodes, bacterial and fungal pathogens; but not virus Procedures have been established to avoid infected sources through virus indexing

5 Tissue culture vs virus indexing An effective indexing depends on reliable diagnostic methods Specificity Sensitivity PCR and its variants are increasingly adopted for banana virus indexing Virus indexing Test every mother plant Subsequent indexing of a representative portion of plants (1-10%) 10%) randomly selected

6 Necessity of techniques for mass indexing Indexing large number of samples is required It is extremely cumbersome, costly and time taking for indexing large number of samples with presently available molecular techniques Scenario is fast changing with the emergence of a number of domestic TC industries Not volunteered to get their TC plants virus indexed Demanding for quick & sensitive mass detection techniques need requirement

7 Combinatorial arrangement of original samples, pooled row and pooled column samples Column PCR Methodology 12 X 12 combinatorial arrangements of original tissue culture banana samples Row PCR R R R R R R R R R R R R12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 Large number of samples could be assayed with a relatively small number of PCR reactions

8 Row & column PCR Equal amount of banana tissue from 12 samples (50 mg from each sample) arranged as a row were pooled (composite sample) for DNA extraction. subjected to row PCR with BBTV specific primers to identify the row(s) accommodating BBTV infected sample(s) Similarly DNA extraction from column composite sample Column PCR to identify the column(s) accommodating infected sample(s) Primers used for detection of BBTV Primer Sequence (5-3 ) Amplicon size ( bp) BBTV-DNA1-F BBTV-DNA1-R GGA AGA AGC CTC TCA TCT GCT TCA GAG AGC CAG GCG CAC ACC TTG AGA AAC GAA AGG GAA 1110 bp Reference Harding et al., 1993

9 PCR ingredients and conditions PCR reaction mixture - 25 μl reaction volume The mixture was subjected to initial denaturation at 94 0 C for 4 min followed by 30 cycles of denaturation at 94 0 C for 1 min, annealing at 58 0 C for 30 S, extension at 72 0 C for 1.5 min and a final extension at 72 0 C for 10 min

10 Results

11 Spatial arrangements of tissue culture samples for pool DNA extraction and amplification of row PCR and column PCR

12 Secondary screening of samples through PCR for exact location of BBTV infected samples in combinatorial screening. S-28, S-33, S-76 and S-81 indicate original samples suspected to be BBTV infected

13 Cost effectiveness of the technique Indexing 144 samples using routine method (144 PCR reactions ) Indexing 144 samples using newly developed method (28 PCR reactions) Cost involved indexing 144 samples Average cost of indexing a sample LKR 500 X 144 = 72,000 LKR 500 X 28 = 14,000 LKR 500 LKR 14,000/144 = 100 Cut down the direct cost by 80%

14 Advantages Further testing of individual sample in a group is not required. Cost of testing per sample can be further reduced as the technique can even detect the virus even in the pooled DNA samples of 100 Indexing 10,000 samples with 100 row PCR and 100 column PCR (98% saving on cost). Testing reports are available to TC industry within limited time period

15 Conclusions PCR Large number of samples including mother plants, in vitro cultures and hardening plants can be indexed for BBTV using relatively less number of reactions BBTV free quality banana tissue culture plants can be ensured by using this technique. Any number of samples less than 144 and arranged in a combinatorial format can also be indexed by this technique The technique developed by us for mass indexing of banana samples for BBTV is the first such attempt and has a number of advantages over the conventional PCR techniques currently used

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