Inte rnatio nal Jo urnal o f Sc ie ntific & Eng inee ring Re se arc h, Vo lume 3, Issue 3, Marc h-2012 1
ISS N 2229-5518
Efficient regeneration of Psidium spp. For in vitro
screening of wilt resistant rootstock
Pallavi srivastava, Pravesh Chandra srivastava
Abs tract- Guava w ilt is a serious problem in its cultivation. Wilting of young and adult trees has caused enor mous damage to the standing crop and orchards. The source of resistance to the particular disease is not reported w ithin the commercial varieties. How ever, some o f its w ild relatives like P. molle, P. chinensis, P. friedrichshthalianum, P. cattlianum and P. guineense have been recorded escaping the disease. Thus, an easy strategy w as evolved to screen and develop w ilt resistant plants, w hich can be used as eff icient rootstocks. The possibility to perf orm su ch a selection strategy under in vitro conditions depends largely upon availability of eff icient regeneration and clonal propagation system. No earlier reports are available on the mi- cropropagation technique f or these Psidium spp. Our study has indicated the importance of seasonal inf luence and media constituents on in vitro prolif e- ration of the nodal buds. Rooting of the micro shoots w as attempted and good success w as achieved.
Index Terms—. guava species, wilt, micropropagation, growth regulators, BA, NAA
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Guava ( Psidium guajava L.) is the fourth major fruit crop of the India and popularly known as poor man’s fruit or apple of the tropics ( Yadava, 1996). Guava wilt disease ( GWD) is causing heavy damage, depending upon various agro-climatic and soil conditions. Large numbers of pathogens have been reported to be associated with the disease. Efforts have been made over past few decades to widen its narrow genetic base for wilt resistance/ tolerance by conventional breeding me- thods. However, control measures other than the eradication of disease tree do not exist so far ( Vos et al., 2000 ). Identifica- tion and development of wilt resistant rootstock, is the only strategy to combat the disease. Some of the wild guava species viz. Psidium molle, Psidium chinensis, Psidium guineense and Psidium cattleianum were found promising as wilt resistant / tolerant rootstock for cultivable guava (Edward, 1960; Edward and Gaurishankar, 1964; Singh etal. 1977). These species are propagated by conventional method of stooling as well as through seedling raised from open pollinated seed but the success rate is quite low. Micropropagation of these guava species would be useful in mass multiplication of genetically uniform plants and ensure availability of plant material for conventional methods of grafting. Shoot bud culture of these species would also aid in in vitro screening of shootlets against fungal toxins, so as to ascertain the exact nature of the species as far as reaction to the disease pathogen is concern .
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Pravesh Chandra Srivastava is currently pursuing Ph.D. in Dravidian
University,Kuppam, PH-+919415485286. E-mail: praveshchan-
2 MATERIALS AND METHODS
The trees of guava species viz. Psidium molle, Psidium ch i- nensis, Psidium guineense and Psidium cattleianum were s e- verely headed back. The new sprouts were sprayed regularly with fungicide and antibiotics (0.1%) regularly. Nodal buds were collected in October and April to study the seasonal re- sponse. Nodal buds ( 1-2 cm) from the new vegetative growth were excised and brought to the laboratory in Polyvinylpyrro- lidone (PVP) 100mg/l + Ascorbic acid ( 100 mg/l), Gen- tamycine sulphate ( 50 mg/l), Bavistin (100 mg/l) solution. The buds were washed thoroughly under tap water. The washed material was agitated for one hour at 120 rpm in anti- oxidant solution ( PVP + Ascorbic acid 100 mg/l) for removal and control of excessive phenol leaching. Surface sterilization was carried out with 10 % H2O2 for all the species followed by
0.1% Hgcl2 for 6 minutes. Explants were washed with autoc- laved water at least five times. Explants were inoculated on Murashige and Skoog (MS) media ( Murashige and Skoog,
1962) fortified with plant growth regulators. The MS media
was prepared as per standard procedure. Explants were inc u- bated vertically in culture tube. The cultures were incubated in the dark for 8 hours initially and a llowed to grow umnder
5000 lux at 25ºC + 2ºC with 16 hour photoperiod. Observa- tions were taken on percent bud induction and number of mi- croshoots / explant in each media. All the treatments were replicated three times and data was subjected to statistical analysis in CRD.
3 RESULTS AND DISCU SSION
Explants of all the species of guava under study were collected throughout the year to study the most favorable season. Oc- tober- November was found most suitable season for culture. Good proliferation and growth was recorded in explants cu l- tured during this period. Thus the seasonal response was not in confirmation with Amin and Jaiswal’s ( 1987) reports,
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Inte rnatio nal Jo urnal o f Sc ie ntific & Eng inee ring Re se arc h Vo lume 3, Issue 3, Marc h-2012 2
ISSN 2229-5518
where April – June was reported most suitable season for cul- ture initiation. Results revealed ( Table –1) that shoots of P. molle. and P. chinensis ( Fig. 1)responded well for in vitro bud induction and subsequent growth of microshoots. Obser- vation were taken on various traits and found high ly signifi- cant when subjected to CRD ana lysis. Critical differences were given at 1% and 5% level of significance. The perusal of data clearly indicate that BA at 3.0 mg/l along with 0.1 mg/l NAA registered early and maximum bud induction in P. molle. and P. chinensis whereas GA3 in the same combination gave early and maximum bud induction in P.cattleianum and P.guineense. Role of BA and NAA in micropropagation has already been discussed in Psidium guajava L. (Papadatou et. al. 1990). A proliferation rate of 1-5 microshoots ( Table – 1) was achieved within 8 weeks of incubation. The shoots were kept in state of active growth by serial subcultures. The mu l- tiplication via nodal buds was the favoured strategy because of absence of basal callus and fairly uniform shoot prolifera- tion. Generally genotype dependent multiplication via buds is the preference so as to maintaining the genetic stability ( George, 1993).
Our experiments have indicated that pre treating with anti- oxidants alone was effective for combating problems ass o- ciated with phenol leaching and its toxicity to growing cu l- tures. Only P. molle presented some problems, which was overcome by pretreatment of antioxidant and subculturing. Thus the guava species under study differ from the culture and pre-conditioning requirements of P. guajava as reported earlier by Siddiqui and Farooqui ( 1997) and other workers.
Resistance to the soil born pathogens is the only permanent option for managing the wilt disease. In vitro screening is rel i- able and safe alternative to sick plot technique. Our experi- ments on shoot bud proliferation via nodal bud cultures are being used for developing efficient in vitro screening methods of guava wilt resistance. The reliable protocol for micropropag a- tion of these wilt res istant/ tolerant species of guava and its utiliza- tion as roots tock for cultivable guava will go a long way in comba t- ing the menace of guava wilt dis eas e. Alternatively it would be help- ful in formulating in vitro micrografting s trategies of commercial cultivar viz. Allahabad Safeda and Sardar on these roots tocks .
al., 1977). These species are propagated by conventional method
of s tooling as well as through s eedling rais ed from open po llinated s eed but the success rate is quite low. Micropropagation of these guava s pecies would be useful in mass multiplication of g enetically uniform plants and ens ure availability of plant material for conven- tional methods of grafting. Shoot bud culture of these sp ecies would also aid in in vitro screening of shootlets against fungal
toxins, so as to ascertain the exact nature of the species as far as reaction to the disease pathogen is concern.
The authors are grateful to Prof. R.K. Pathak, Director, CISH, Lucknow for providing facilities to carry out the work. The work was supported by the grant of UPDASP/ UPCAR - sponsored networking project on guava wilt ( Biotechnology component).
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[7] Siddiqui, Z.M. and Farooq, S.A. 1997. Tissue culture studies on nodal explants of guava. Indian J. Hort. 54 (4): 276-279.
[8] Singh, U.R., Dhar, L. and Singh, G. 1977. Note on performance of guava cultivars and Psidium species against wilt disease u n- der natural field condition. Haryana J. Hort. Sci. 6: 149-150.
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Inte rnatio nal Jo urnal o f Sc ie ntific & Eng inee ring Re se arc h Vo lume 3, Issue 3, Marc h-2012 3
ISSN 2229-5518
Media | P. molle | P. chinens is | P. cattleianum | P. guineese | |||||
Media | % Bud induc- tion | Number of micros hoots | % Bud in- duction | Number of micros hoots | % Bud in- duction | Number of micro- shoots | % Bud in- duction | Number of micros hoots | |
MS+ BA 1.0 mg/l | S1 | 10.00 | 1.3 | 12.00 | 1.0 | 12.00 | 1.0 | 10.00 | 1.00 |
MS + BA 3.0 mg/l NAA 0.1 mg/l | S2 | 80.00 | 3.6 | 63.00 | 5.0 | 43.00 | 3.00 | 12.00 | 1.2 |
MS + BA 3.0 mg/l NAA 0.1 mg/l GA 1.0 mg/l | S4 | 55.00 | 2.1 | 30.00 | 2.1 | 55.00 | 4.00 | 60.00 | 3.8 |
MS + BA 3.0 mg/l GA 2.0 mg/l | S5 | 30.00 | 1.0 | 12.00 | 1.8 | 10.00 | 1.7 | 5.00 | 2.0 |
MS + BA 0.5 mg/l | S7 | 20.00 | 1.0 | 40.00 | 3.5 | 15.00 | 1.0 | 20.00 | 2.00 |
CD % | 1 % 5 % | 2.4 1.6 | 0.4 0.3 | 3.0 2.1 | 0.4 0.3 | 1.01 0.8 | 0.2 0.1 | 0.4 0.2 | 0.4 0.3 |
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