Annals of Sri Lanka Department of Agriculture. 2007.9:81-87.
INTROGRESSION OF ECONOMICALLY IMPORTANT GENES FROM WILD RICE Oryza eichingeri TO CULTIVATED RICE Oryza sativa H.M.P.S. KUMARI, I.W.M.K.N.K. YATAWARA, U.N.D. SILVA, S.M.M. SAMARAKOON, Y.M.S.K. SAMARAKOON, D.S.de Z. ABEYSIRIWARDENA and T. RAJAPAKSHA Rice Research and Development Institute, Batalagoga, Ibbagamuwa
ABSTRACT This study involves the improvement of rice with introgression of resistance genes for biotic stresses from wild rice to cultivated rice through wide hybridization. Rice Research and Development Institute (RRDI), Batalagoda, has already started to produce wide hybrids using embryo rescue method. One accession of wild rice species Oryza eichingeri reported as rich source of important genes was collected from Dambulla, Sri Lanka. It was tested for diseases and pests using conventional screening methods and was ranked high (0) in showing resistance to brown plant hopper (BPH), blast and bacterial leaf blight. Cultivated rice varieties, Bg 750, Bg 300, Bg 94-1, Pachchaperumal, E.A.J Samba and Seeraga samba were used as crossable varieties to make wide hybrids with O. eichingeri. Cultivated O. sativa (AA) and distance wild species O. eichingeri (CC) produced F1 of AC hybrids with 99% to 99.5% grain sterility. Highest fertility was observed in Bg 750 x O. eichingeri (1%) combination. All the F1 and F2 plants showed resistance to BPH and Blast as in wild species and BC1F1, BC1F2 and BC1F3 plants showed segregation of such resistant character. These results suggest the polygenic behavior of relevant genes. Increasing the level of seed setting was observed with back crossed plants. From 5770 advanced back crossed plants prepared with recurrent parents, 250 plants showed introgression of resistant traits in high rank (0). Although the level of introgression was low as 4.3%, results showed the possibility and feasibility of transferring useful genes from distant local wild rice species to cultivated varieties. KEYWORDS: Oryza eichingeri, Oryza sativa, Rice, Wide hybridization.
INTRODUCTION Wild species of Oryza are rich sources of useful genes for the improvement of cultivated rice. However, efforts to transfer useful traits from wild species to cultivated rice have been very limited. Few examples are the transfer of the gene for grassy stunt virus resistance from Oryza nivara (Khush et al., 1977) and transfer of cytoplasm from O. perenis to develop cytoplasmic male sterile lines for hybrid seed production. Brar et al. (1996) reported transfer of genes for tungro resistance from O. officinalis (CC), O. latifolia (CCDD) and Yellow stemborer tolerance from O. ridleyi, brown plant hopper (BPH) and bacterial leaf blight resistance from O. rhizomatis and O. eichingeri. Jena and Kush (1990) transferred brown plant hopper and white backed plant hopper (WBPH) resistance genes from O. officinalis (CC) to O. sativa. Some studies with O. eichingeri showed that preparation of cross
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combination with AA genomic species was difficult due to low pairing and various irregularities and complete sterility. In recent years, disease and insect outbreaks on resistant rice have become a serious problem. Germplasm resources of rice, including wild species, have been evaluated to identify donors and genes highly resistant to known biotypes of BPH, WBPH and green leaf hopper were found (Heinrichs et al., 1985). O. sativa and its closely related wild species O. nivara, O. perenis and O. longistaminata share the AA genome. These wild species can easily be crossed with O. sativa and genes from them transferred to cultivated rice by conventional crossing and back crossing procedures. However, wild species with other genomes are difficult to cross with O. sativa because those crosses produce completely sterile male plants. Scientists have investigated the hybrids of AA genomic and CC genomic groups (Katayama, 1965) and found bivalent formation of AC with a low frequency in pollen mother cells. Previous finding of Kumari et al. (2005) reveals that attempts to produce interspecific hybrids between cultivated varieties and O. eichingeri produced through embryo rescue was a success. In Sri Lanka, utilization of potentially important wild rice species in the rice improvement program has not been reported. Improved rice varieties have been developed in Sri Lanka using pest and disease resistant traits transferred from wild rice species into cultivated rice of other countries. Hence, development of rice varieties with introgression of resistant genes for biotic stresses from wild rice to cultivated rice through wide hybridization is urgent and important. In the present study, the objective was to screen selected accession of O. eichingeri for biotic stresses like BPH, blast and bacterial leaf blight and then produce wide hybrids between O. eichingeri and compatible varieties of O. sativa. MATERIALS AND METHODS One accession of O. eichingeri collected from Dambulla was used as a wild rice species in the study. Although O. eichingeri is reported to be resistant to BPH, selected accession was further screened for BPH, bacterial leaf blight and blast diseases using conventional greenhouse tests according to the standard evaluation system of rice 1980 (Heinrichs et al., 1985). Fifty seedlings of wild rice species were tested for BPH resistance using the resistant (PTB 33) and susceptible check (Bg 380) varieties. Blast resistance was tested with the 50 seedlings of the wild rice in the normal blast nursery using Pachchaperumal as the susceptible check variety. Reaction in Bacterial Leaf blight was tested in the greenhouse inoculating the bacterial suspension in cut leaves of 5 plants of the selected wild rice species.
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Wide hybrids were made using embryo rescue technique with cross compatible six O. sativa varieties (Seeraga Samba, E.A.J. Samba, Pachchaperumal, Bg 300, Bg 94-1 and Bg 750) identified from the previous study (Kumari et al., 2005) and O. eichingeri. In making wide hybrids, maternal spikelets of O. sativa were emasculated by hand before anthesis and pollination was performed twice with fresh pollen of the wild species at their anthesis. Cross pollinated spikelets were covered with an oil bag and developing embryos were collected before degeneration at 7-11 days after pollination. Detached hybrid embryos were cleaned with Teepol â„˘ using tap water. At the clean air bench, they were surface sterilized with 70% ethanol for 2 minutes, 20% sodium hypo chlorite for 10-15 minutes and by rinsing with sterile distilled water three times 1 minute each and cultured on improved MS agar medium (25% MS and 0.005 GA3) and incubated at 25Â°C with photoperiod of 16h. The germinated plants were transferred to sterilized soil in the greenhouse. The growing hybrids were tested for pollen fertility (iodine test), seed setting, presence of awn, reactions to BPH and blast. Number of chromosomes and plant height of hybrids were also measured. F2 plants also produced through embryo rescue technique using F1 seeds showed very low level of germination. To improve seed setting and other characters, recurrent backcrossing was performed with F1, F2 and F3. All F1, F2 and BC1F1, BC1F2, BC1F3 plants were screened for BPH and blast to make sure the resistant genes in breeding populations were present. Probability in the transfer of pest and disease resistance was measured as a percentage. All F1 F2 and BC1 generations were maintained in the greenhouse and therefore conventional statistical designs were not applied. Pedigree selection methods were followed for the selection of resistant varieties. In chromosome counting, root tips were fixed in 1:3 Acetic acid:Alcohol solution. Number of chromosomes in the samples was observed using microscopic acetocarmine squash method. To improve those lines as advanced breeding lines, BC1 plants were crossed with improved varieties like Bg 300, Bg 357, Bg 359, and Pokurusamba. Plants were established in the field for selection and evaluation of improved breeding lines. The evaluation of about 250 lines having resistance to BPH, blast and BLB are in progress. RESULTS AND DISCUSSION Screening of wild rice Wild rice accession of O. eichingeri was successfully screened against pests and diseases like BPH, blast and BLB (Table 1). In the BPH test,
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all the O. eichingeri plants remained without any damage and showed a high level of resistance (scale 0) even surviving after the death of resistant check PTB 33. In the blast nursery, O. eichingeri plants grew comparatively longer without even a single patch of blast infection. In the BLB test no BLB symptom were observed on O. eichingeri. Results revealed the selected wild rice accession of the species is having some resistant genes for pest and diseases. Plant regeneration All collected premature embryos could be germinated in improved embryo rescue medium (25% MS and 0.005 GA3) and the efficiency of germination was close to 100%. After culturing for 6 weeks complete development of plants could be observed. Total of 16 F1 hybrids were obtained through embryo rescue technique (Table 2). Highest number of F1 plants (5) was observed with E.A.J. samba x O. eichingeri cross. Lowest number of F1 plants (1) was observed in Seeraga samba x O. eichingeri cross combination. All the F1 plants showed intermediate height between parents with awn, large grain type, profuse tillering and transferred resistance to BPH, blast and BLB. Characteristics of progenies All the F1 plants had intermediate heights between wild rice and O. sativa parents with the presence of awn, regular flowering and tillering habit. Plant height of Bg 750 x O. eichingeri and Bg 300 x O. eichingeri was 70cm, 94- 1 x O. eichingeri and E.A.J. samba x O. eichingeri was 80cm, Pachchaperumal x O. eichingeri was 75cm and Seeraga samba x O. eichingeri was 72cm. Rate of seed setting was low in inter species cross combination. More than 50% pollen sterility and 1 to 0.5% seed setting was observed with F1 progenies. Highest seed setting was observed with Bg 750 x O. eichingeri (1%) and weakest plant type was observed with Seeraga samba x O. eichingeri. As a result of very low seed setting and germination ability, F2 plants were also produced through embryo rescue technique. Segregating populations of F2 were similar to the morphology of F1 of wide hybrids (Table 2). Highest seed setting rate of F2 was observed in cross combination of E.A.J. samba x O. eichingeri (50%). Varieties Pachchaperumal, Bg 94-1, and Seeraga samba being highly susceptible to many diseases and good products of AC combination are highly resistant to tested diseases, showing inheritance of those genes. Inheritance of disease resistance characters showed up to BCF1 generation without any change suggests such resistant genes are pollygenic. Back crossing was performed with recurrent parent with F1 and F2 plants.
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BC1 plants showed change of some characters like plant height seed setting, absence of awn, shattering and age. Table 1. Important traits present in O.sativa varieties and O. eichingeri. Variety
Reaction to BPH
Reaction to blast
Reaction to BLB
O. eichingeri Bg 750 Bg 300 Bg 94-1 Pachchaperumal EAJ Samba Seeraga samba
Resistant Susceptible Moderately resistant Susceptible Susceptible Susceptible Moderately resistant
Resistant Moderately resistant Moderately resistant Susceptible Susceptible Susceptible Moderately resistant
Resistant Moderately resistant Susceptible Susceptible Susceptible Susceptible Susceptible
Table 2. Morphological characteristics of F1 and F2 from different cross combinations.
Bg 750 x O. eichingeri Bg 300 x O. eichingeri Bg 94-1 x O. eichingeri Pachchaperumal x O. eichingeri EA J samba x O. eichingerri Seeraga samba x O. eichingeri
F1 No of plant
Plant height (cm)
% Seed setting
Presence of awn
F2 No of plant
3 2 2
70 70 80
1 0.6 0.7
Yes Yes Yes
Perennial Perennial Perennial
Plant height (cm)
% Seed setting
Presence of awn
6 5 4
70 70 85
25 25 20
Yes No Yes (50%)
Annual Annual Annual
Perennial (50%) Perennial
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Through back crossing with recurrent improved parent, 35 plants were obtained in BCF1 from the cross combination of Pachchaperumal x O eichingeri, 2 plants from Bg 300 x O. eichingeri, 5 plants from Bg 94-1 x O. eichingeri, 10 plants from EAJ Samba x O. eichingeri and 4 plants from Bg 750 x O. eichingeri. Single plant selection pedigree method was performed with BCF1 plants, planted in pots in the greenhouse. Some BC1F3 plants obtained from the above crosses closely resembled O. sativa parent with additional characters like resistance to BPH and blast. However, all differed slightly from each other and from the O. sativa parent in leaf length, leaf width, flag leaf angle, plant height, seed type, number of tillers and harvesting time. The most notable features of these progenies was the complete absence of undesirable traits of the wild species, such as grain shattering, spreading growth habit, weak stems and long awns. Some BC1F1 plants of Bg 300 x O. eichingeri showed weak plant type with no seed setting. Bg 750 x O. eichingeri showed 50% seed setting and plant type was closer to the wild type. Number of chromosomes present in each generation were different and varying number of chromosomes were always present in all the BC1 segregating populations (n+1, n+2, n) indicating nonpairing of some chromosomes. Plants from Pachchaperumal performed well up to BCF3 having important characters like disease resistance to BPH and blast. Selected plants with favorable economically important characters were forwarded to prepare another cross combination with improved breeding lines such as Bg 360, Bg357, Bg 359 and Pokuru samba to improve as best lines for the rice breeding program. Regular inheritance of disease resistance genes for many generations reveal polygenic or linkage between many genes. From 5770 BC3 plants, 250 plants were selected to forward as highly resistance lines for biotic stresses and having improved morphological characters (Table 3). As a first attempt present generations show success with introgression of important genes which can be used in rice improvement. Table 3. Introgression of disease and pest resistant genes in back crossed plants. Segregating BC1F1 BC1F2 BC1F3 population (No. of (No. of (No. of Traits plants 57) plants 144) plants 5770) BPH Blast
CONCLUSIONS Selected O. eichingeri (CC) accession was rich with important genes for resistant to pest and diseases such as BPH, blast and BLB and their successful introgression was observed with O. sativa rice varieties such as Bg 750, Bg 300, Bg 94-1, Pachchaperumal, E.A.J. Samba and Seeraga samba.
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With introgression of important traits, improvement of plant type was observed with 250 back crossed plants. ACKNOWLEDGEMENTS The authors wish to acknowledge Ms A.P.D.P. Wimalasiri for the assistance rendered during this study. REFERENCES Brar, D.S., R. Dalmacio, R. Elloran, R. Aggarwal, R. Angeles and G.S. Khush. 1996. Gene transfer and molecular characterization of introgression from wild Oryza species into rice. In: Rice Genetics III. G.S. Khush Ed. International Rice Research Institute, Mannila, Philippines. Heinrichs, E.A., F.G. Medrano and H.R. Rapusas. 1985. Genetic evaluation for insect resistance in rice. International Rice Research Institute, Manila, Philippines. Jena, K.K. and G.S. Khush. 1990. Introgression of gene from O. officinalis Well ex Watt to cultivated rice, O. sativa, Ther Appl Genet 80: 737-745, IRRI. Katayama, T. 1965. Cytological studies on genes O. sativa. I. Chromosome pairing of intrspecific hybrid O. sativa x O. officinalis under different temperature conditions. Jpn. J. Genet 40: 307-313. Khush, G.S., K.C. Ling, Aquino and R.C. Aguiero. 1977. Breeding for resistance to grassy stunt in rice. In: Proc. 3 rd Int. Congr. SABARAO, Canberra, Australia. Plant breeding papers I (4):39. Kumari, H.M.P.S., W.M. Abayarathne and D.S. de Z. Abeysiriwardana. 2005. Development of protocol for embryo rescue. Annals of the Sri Lanka Department of Agriculture 7: 157-164.