International Training Workshop on
The Conservation and Utilization of
Tropical/Subtropical Plant Genetic
Resources
January 9-11, 2007
Held at SOFRI, Vietnam
Published by
The Conservation and Utilization
of Tropical/Subtropical Plant
Genetic Resources
Proceedings of International Training Workshop
Edited by
Sheng-Chung Huang Dr. Hui-Lung, Chiu Ms. Chih-Yu, Wang Chien-Yih Lin Director Assistant Research Assistant Research Director General Plant Germplasm Divison, TARI Plant Germplasm Divison, TARI Plant Germplasm Divison, TARI TARI, Council of Agriculture
Published by
Taiwan Agricultural Research Institute
Council of Agriculture
Taiwan Agricultural Research Institute (TARI) Council of Agriculture (COA)
189 Chung-Cheng Road, Wufeng, Taichung Hsien 41301 Taiwan, ROC
Tel: +886-4-2330-2301 Fax: +886-4-2333-8162 Web site: www.tari.gov.tw
Copyright©2007 Taiwan Agricultural Research Institute
All Right Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copying owner.
Organizing Committee
Food & Fertilizer Technology Center for the Asian & Pacific Region (FFTC) Southern Fruit Research Institute, Viet Nam (SOFRI)
Taiwan Agricultural Research Institute (TARI)
Sponsored By
Council of Agriculture
ISBN-13: 978-986-00-9220-2 GPN: 1009600775
Taiwan Agricultural Research Institute, Spec. Publ. No.128 Printed in Taiwan, ROC, by Asia United Advertising Ltd. February 2007
Preface
The “International Training Workshop on the Conservation and Utilization of Tropical/Subtropical Plant Genetic Resources,” sponsored by the Council of Agriculture (COA), Taiwan and the Food and Fertilizer Technology Center for the Asian and Pacific Region (FFTC), was successfully held on January 9-11, 2007. The Workshop was hosted by the Taiwan Agricultural Research Institute (TARI) and taken place at the Southern Fruit Research Institute (SOFRI) at My Tho, Tien Giang, Viet Nam.
Plant genetic resources (PGR) are the basis of crop improvement. Genetic variations and diversity of plant are the motivation force for continuous progress in breeding. In recent years, agricultural production and management have adopted various elite cultivars and a more specialized economic production model. The demand of new agricultural products is on the increase in almost all the countries due to population increase so that PGR is the basis for developing, producing and marketing of new food, healthcare and pharmaceutical products. In the tropical and subtropical countries of the South Asian and Pacific region, there are abundant PGRs which require good practices of genebank management. In other words, the conservation, development and sustainable utilization of PGR should further be improved. An alarmingly high number of stored accessions are in need of regeneration as a significant proportion of the materials collected in the past is now under the endangered situation. Therefore, it is very meaningful to hold this Training Workshop so as to promote the collaboration on the exchange and sharing of plant germplasm materials and practice experiences.
The Training Workshop focused predominantly on the conservation and utilization of tropical/subtropical PGRs. The major topics included (1) the information, knowledge, and practices regarding the conservation and utilization on PGRs; (2) new technology and efficient management of PGRs; (3) particular problems, constraints and solutions of the conservation and utilization of PGR; (4) decisions on appropriate tropical/subtropical plant germplasm management strategies.
The delegates, researchers or experts on PGR from Indonesia, Malaysia, Myanmar, Philippines, Taiwan, Thailand, and Vietnam were invited to attend and requested to deliver technical or country reports in the Workshop. More than 10 PGR coordinators or experts from research institutes, universities, and related agencies in Vietnam submitted scientific posters for exhibition in the Workshop. This proceedings cover all the scientific papers presented by the invited speakers, which should be of significant value to the agricultural scientists and industries dealing with the conservation, utilization, and management of PGRs. I would like to take this opportunity to express my gratitude to all participants whose attendance contributed to the very success of the Training Workshop. Special thanks are given to all the speakers for their prompt preparation of manuscripts and presentation materials. The Workshop and Proceedings would not have been possible without the financial support from the Council of Agriculture (COA), the superior assistance from FFTC, SOFRI, and the excellent assistance from the staff members of SOFRI.
Chien-Yih Lin, Ph.D.
Director General
Taiwan Agricultural Research Institute Council of Agriculture
Welcome Address
Dr. Nguyen Minh Chau
Director, Southern Fruit Research Institute (SOFRI)
Tien Giang – Viet Nam
Respected Professor Hong-Ji Su, Food and Fertilizer Technology Center for the Asian and Pacific Region (FFTC); Dr. Sheng-Chung Huang, Director of National Plant Genetic Resources Center, Taiwan Agriculture Research Institute (TARI); Mr. Nguyen Van Khang, Director of Service of Agriculture and Rural Development of Tien Giang Province. Ladies and Gentlemen,
It is my great pleasure to welcome all of you on behalf of all the staff of the Southern Fruit Research Institute (SOFRI). I wish all of you in good health and having very good time in Vietnam.
Dear friends,
Plant Genetic Resources are very important to us, as with the intensive agriculture those valuable resources were and being disappeared. Hence, a joint efforts and actions of all of us to save those resources are very necessary before these plant resources disappeared, particularly those of medicine plants, and disease resistant genes in wild plants.
I am happy that the Taiwan Agriculture Research Institute (TARI), and the Food and Fertilizer Technology Center for the Asian and Pacific Region (FFTC) have decided to start their efforts in saving those plants with this workshop. I think we all hope this workshop will soon lead to
a project which aiming at collection, conservation, evaluation and utilization in improving our crop plants. With a joint efforts, we can share our methodology in screening those valuable resources against insect and diseases, and donate those valuable resources to others international centers or countries who can conserve them before they are disappeared. As a result, we can know these plants characteristics faster than doing alone in each country.
Dear friends,
FFTC has many experiences in running international projects like the citrus greening, and improving banana projects for some Southeast Asian countries, Vietnam had been selected by FFTC to cooperate with since 1996. Southern Fruit Research Institute has started to work with FFTC, Taiwan University, Taiwan Banana Res. Institute since 1996. With my experiences with FFTC, I strongly believe that with new project, together we will be able to save lot resources and be able to screen our plants resources for diseases resistant faster.
Last but not the least; I would like to thank FFTC who has chosen Southern Fruit Research Institute as the co-organizer of this important meeting and to thank to all the speakers for coming. I would like all of you have a pleasant an enjoy stay in Vietnam. If you need any support from the local co-organizer, please do not hesitate to talk to us.
For the study tour, Dr Sheng-Chung Huang and I would like to show you not only the Mekong Delta but also the highland of South Eastern part where we have a lot of rubber trees, durian, jackfruit and rambutan trees..…
Thank you.
Opening Address
Dr. Chien-Yih Lin
Director General, Taiwan Agricultural Research Institute, COA
(Addressed by: Dr. Sheng-Chung Huang, Director, PGRC, TARI)
Honorable Director Nguyen Van Khang, Service of Agriculture and Rural Development; Ms. Nguyen Thi Thanh Mai, Principal Specialist, Department of Agronomy – MARD; Dr. Nguyen Minh Chau, Director, Southern Fruit Research Institute (SOFRI); Dr. Hong-Ji Su, FFTC representative, distinguished speakers, honorable guests, dear participants, ladies and gentlemen:
First of all, on behalf of one of the Committee Organizers, the Taiwan Agricultural Research Institute (TARI), I would like to thank FFTC and SOFRI for taking part in co-organizing this important workshop. Please also accept my sincere apology for not being able to be present at this event because of prior commitments. I would also like to express my sincere appreciation to Dr. Chau and his colleagues for holding this timely and meaningful International Training Workshop on "The Conservation and Utilization of Tropical/Subtropical Plant Genetic Resources".
The importance of plant genetic resources has been well recognized worldwide. The greatness of agricultural improvement depends on not only the advancement of scientific research but also the utilization of abundant and diversified plant genetic resources. The resulted improvement in both the yield and quality of agricultural products has exerted impacts on almost every aspect of our life.
Due to the ideal geographical location, Taiwan has been benefited by the abundance of tropical and subtropical plant genetic resources in the development of agricultural industry. We also have established core facilities of gene bank and integrated information system of management. As one of the Southeast Asian countries, we realize the importance of multilateral cooperation of exchanging and sharing technology and materials on the collection, identification, and conservation of plant genetic resources. We believe that the continuous advancement of agricultural production in the whole Southeast Asian region can be achieved only through such kind of cooperation. During the past decades, Taiwan has been cooperating with many neighboring countries in this aspect. One very significant example is that by sending agricultural missions abroad, our specialists offer and share our learning and experiences in plant genetic resources with local scientists, farmers, and agricultural industries.
In this workshop, we are honored and pleased to invite distinguished speakers from Indonesia, Kampuchea, Malaysia, Myanmar, Philippines, Taiwan, Thailand, and Vietnam to present their specialties in plant genetic resources. In addition, many experts of Vietnam contribute posters for exhibition and offer their experiences and expectations to a new and more advanced agricultural production. On this special occasion, I foresee opportunities of international cooperation in the resolution of general and particular problems and constraints in gene bank management and utilization of germplasm.
Finally, I wish for every success of this international training workshop. I also wish to extend my sincerest welcome and blessings to all of you. To our friends, I wish all of you a very pleasant stay in this lovely and friendly country – Viet Nam.
Special Remarks
Mr. Sing-Hwa Hu
Director, Food and Fertilizer Technology Center (FFTC)
(Delivered by: Dr. Hong-Ji Su, Professor Emeritus, Dept. of Plant Pathology,
National Taiwan University/Consultant, FFTC)
Dear Participants and Distinguished Guests:
On behalf of FFTC, it is such a great pleasure for me to express my warmest welcome to all the participants from eight countries of Southeast Asia, attending this international workshop on Conservation and Utilization of Tropical and Subtropical Plant Genetic Resources. While our FFTC Director, Mr. Hu, is unable to join us in this activity due to a very important prior commitment, I would like to convey on his behalf our heartfelt gratitude to the organizers, especially to Dr. Chau, Director of SOFRI, and co-organizers Dr. Hong also of SOFRI and Dr. Huang, Director of the Taiwan National Genebank, TARI. Without their utmost efforts, the success and smooth conduct of this present workshop would not have been possible. The primary goal of this workshop is to promote the full utilization and effective conservation of plant genetic resources through international exchange of new information, technical experience, and useful germplasm exchange. We are sure that these activities will be much beneficial to each participating country in Southeast Asia.
Through the full utilization of genetic resources, agro-industries will be able to better produce enough foods and grow better living environments for the people. Likewise, in the face of a looming global energy crisis and the depletion of energy sources such as oil/gas, bioenergy from plant genetic resources (PGR) will be our only hope for the future. Bioenergy can be generated from PGR via the photosynthesis of plants from the solar energy. Most importantly, PGR is the basic foundation of crop improvement. Genetic variation and diversity of plants are the motivating force for the continuous progress in breeding and crop
improvement, in particular, and in the attainment of sustainable agriculture in general.
We deeply congratulate all the scientists and experts in the area of plant genetic resources, for your valuable contributions to this scientific field. We likewise encourage each and every one of you to continue this scientific and moral mission to preserve and conserve, as well as to efficiently and fully utilize the plant genetic resources of the world. We hope that the present workshop will be most successful, particularly in promoting future development and international collaboration in this area.
Again, our sincerest thanks to our participants for your important presence in this workshop, and we wish all of you good health and happiness.
Congratulatory Address
Mr. Nguyen Van Khang
Director, Service of Agriculture and rural Development
Tien Giang – Viet Nam
Honorable Dr. Hong-Ji Su, FFTC Representative; Dr. Sheng-Chung Huang, Director, Plant Genetic Resources Center, Taiwan Agricultural Research Institute (TARI); Dr. Nguyen Minh Chau, Director, Southern Fruit Research Institute (SOFRI); Ms. Nguyen Thi Thanh Mai, Principal Specialist, Department of Agronomy - MARD; distinguished speakers and guests. Ladies and gentlemen! Good morning!
First of all, I would like to express my special thanks to the organizers who have given me this opportunity to give my congratulatory address. It is my great pleasure to warmly welcome all of you to our Tien Giang province for the International Workshop on "The Conservation and Utilization of Tropical/Subtropical Plant Genetic Resources".
I understand that all we have now about 25 persons coming from 8 countries including Viet Nam. The issues of the topic and the objectives of the workshop that you are going to work for are considerably important, particularly with the developing countries like Viet Nam. In the speedy change of social and economical condition we do many mistakes leading to the disappearance of valuable genetic resource of traditional herbs, fruit species, and many other natural variations of plants.
It is estimated that Viet Nam has about 12,000 species of higher plants but only 7,000 have been identified. In a recent assessment, Viet Nam was rated as the 16th most biologically
diverse country in the world; over a thousand medicinal plants are recognized. The conservation and evaluation work in our country do not provide an adequate counter measure to this pressure on resources. And therefore, we need more support for these jobs.
It is evident that fast economic growth with a lack of tight economic and environmental controls and also some parameters such as air pollution, acid rain, water flow, soil erosion and climate change are being affecting much on the conservation and utilization of our plant genetic resources. Therefore, I do hope that the workshop will bring to all of you some new idea of research, investigations, and management for better conservation and utilization of plant genetics resource for the sake of people in long term.
Distinguished guests,
Taking this opportunity, I would like to introduce you some information of Tien Giang province. Tien Giang is one of the major agricultural provinces of the Mekong Delta Viet Nam, particularly; it has biggest areas of fruit growing areas where Southern Fruit Research Institute exists. Population of Tien Giang is 1,698,851 people; GDP is 12,772 billion VND, of which Agriculture occupies 48%. Agronomy is of importance for instance in 2005 crops brought about 6,277 billion VND with 73.8% of total agriculture value of the province. Annual crops: 3,615 billion VND; Perennial trees: 2,603 billion VND; Food plant: 2,814 billion VND and particularly fruit tree 2,480 billion VND.
I would like to express our hospitality to the international guests and other our Vietnamese friends as well. I believe Southern Fruit Research Institute will take care of you very well and once again wish you enjoy your stay in Viet Nam. Finally, on behalf of Tien Giang Agricuture sector, I wish you a very happy and reproductive new year of 2007 and have your pleasant time with impressive experience in Viet Nam.
Conservation, Disease-indexing and Utilization of
Pathogen-free Citrus and Banana Genetic Resources
in Taiwan
By
Hong-Ji Su
1, A-Shiarn Hwang
2, Su-Yin Lee
3, and Chih-Ping Chao
3Abstract
Virus and virus-like diseases (greening) of citrus and banana have been causing considerable damage to fruit production worldwide due to their systemic invasion and common spread through infected budwoods, vegetatively propagated seedlings, and transmission by insect vectors. The heat-tolerant form of greening locally known as Huanglungbin (HLB) in China has seriously affected citrus trees in the tropical/ subtropical regions of Asia, and recently in the North and South America. The HLB caused great damage to the citrus industry by shortening tree lifespan and lowering fruit quality. Citrus tristeza virus (CTV) is the most common and destructive virus in the western hemisphere, and has a worldwide distribution. Citrus tatter leaf capillovirus (CTLV) has a potential to become prevalent when trifoliate orange and its hybrids are used as rootstocks. The citrus exocortis viroid (CEV) occurs in many countries, and its commercial effects are primarily on trees grafted on trifoliate orange and some its hybrids, and Rangpur lime. The bunchy-top virus (BBTV) disease has been the common and destructive virus disease of banana since the beginning of this century in Asian and Pacific regions. The banana mosaic caused by CMV has become epidemic after common cultivation of tissue-culture plantlets. Banana streak badnavirus and bract mosaic potyvirus have newly spread to many banana areas in the Pacific regions in recent years. These systemic diseases have become one of the serious constraints for the industry of citrus and banana in the Asian and Pacific region.
These diseases are generally controlled by the integrated control measures including cultivation of pathogen-free seedlings, elimination of inoculum sources and prevention of secondary spread by vector insects. In view point of germplasm utilization, establishment of pathogen-free nursery system is primarily important for preventing prevalence of these diseases. National scheme of citrus pathogen-free nursery system is consist of PF citrus
1. Department of Plant Pathology & Microbiology, National Taiwan University, Taipei, Taiwan; e-mail: [email protected]
2. Chia-Yi Agricultural Experiment Station, TARI, Chia-Yi, Taiwan 3. Taiwan Banana Research Institute, Pingtung, Taiwan
foundation in Chia-Yi Agriculture Experimental Station repository and the approved nurseries in private sector and fruit cooperative. The production and utilization of PF-citrus seedlings has been performed under citrus sapling certificate regulation. The virus-free banana cultivars of economic importance obtained through virus-indexing and tissue culture under heat-therapy temperature have been kept in the foundation screen-house for mass production of PF banana TC-plantlets in Taiwan Banana Research Institute. The PF citrus and banana seedlings are commonly cultivated for improving the agro-industry in Taiwan. The precise and rapid indexing techniques are indispensable for management of pathogen-free nursery system and health management of citrus and banana plants in the field. Development and application of molecular diagnostic probes including monoclonal antibodies, DNA probes, and primer pairs for polymerase chain reaction (PCR) were made for formulating indexing techniques including ELISA, rapid diagnostic strips, PCR and RT-PCR to be discussed in this presentation . The establishment of virus-free nursery system for controlling citrus and banana virus diseases has been properly performed through shoot-tip micro-grafting, and tissue culture via virus-free stock indexing and heat-therapy with tissue culture in Taiwan.
Introduction
Citrus and Banana are the most important fruits for local consumption and export in Taiwan and the other Asian countries. Citrus production with so many cultivars, are the most important industries for fresh fruit consumption, and juice processing world-wide particularly in the tropic and subtropical areas. Banana and plantain (Musa spp) are the important crops for local consumption as fresh fruit and staple food, and export in the Asian, African and American countries. Some systemic virus and virus-like diseases have been causing considerable damage to fruit yield and quality, and have became one of the serious constraint for the production of citrus and banana world wild.
Citrus greening (Huanglungbin = HLB) was first reported in 1947 from South Africa. The psyllid borne virus-like disease caused by Liberibacter asiaticus has been devastation citrus industry in Taiwan since 1951. This disease has been prevalent in tropical and subtropical citrus areas in Asia and South Africa, and appeared in North and South America recently. This disease caused great damage to the citrus industry by shortening tree lifespan less than 10 years. Most citrus cultivars, except pummelo, were already susceptible to the Asian form of HLB before the 1970s. However, pummelo became infected by a new GO strain in Taiwan and Southeast Asia since 1971. Citrus tristeza closterovirus (CTV) was assumed originated in China quite a long time ago. The CTV caused a great damage to citrus industry in South America and Africa in the 1920s and has a worldwide distribution. Some new strains of CTV have evolved in South America and Asia in recent years, and have been causing severe
stem pitting, stunting and affecting fruit yield and quality. Citrus tatter leaf capillovirus (CTLV)
was first found in 1962 in Mayer lemon introduced to California from China. Several lines of Satsuma mandarin grown in Japan have been found to be infected by CTLV since 1975 (Miyakawa, 1980). The virus has a potential to become prevalent when trifoliate orange and its hybrids are used as rootstocks. The CTLV has been commonly in most citrus cultivars without significant symptom in Taiwan. Exocortis is a bark-scaling and tree-stunting disease caused by citrus exocortis viroid (CEV). The disease was first described as scaly butt in 1948 in Australia. The casual agent occurs in many countries, and its commercial effects are primarily on trees grafted on trifoliate orange and some its hybrids, and Rangpur lime. The CEV was scarcely found in sweet orange and Eureka lemon trees rooted on Rangpur lime in Taiwan. The greening disease commonly with mix-infection of citrus tristeza and tatter leaf viruses, causes sever yellowing and decline, and ultimately death of citrus trees.
Banana bunchy-top babuvirus disease has been the common and destructive virus disease of banana since the beginning of this century in Asian and Pacific regions. Several occurrence of outbreak become the limiting factor for banana industry in Taiwan during the past decades. Banana mosaic caused by cucumber mosaic cucumovirus (CMV) was first reported in New South Wales in 1929 and in Central American in 1957. This disease had become prominent in Taiwan in 1974, and severe outbreaks occurred recently after common cultivation of tissue-culture plantlets. Banana streak badnavirus disease (BSV) was first described in Mysore (AAB) from Ivory Coast in 1974. The BSV was found worldwide in most of the banana-growing areas in recent years, and had potential for spreading and becoming epidemic. There is the potential for serious yield losses with some virus isolates. Plant death was reported in Africa. Disease incidence varies between countries and this may be related to strain differences and vector activity. The virus infection was recently (1994) found in exotic Mysore and Latundan, and native wild banana in Taiwan. Banana bract mosaic disease was first noted in 1979 in Mindanao, the Philippines. It has subsequently been widespread throughout the Philippines. The disease has also been found in India, Sri Lanka, Vietnam, and Western Samoa. A potyvirus, banana bract mosaic virus (BBrMV) isolated from infected plants, has been assumed to be the causal agent. Yield losses of up to 40in Cardaba (ABB)
and Lakatan (AA) have been recorded in the Philippines. Particularly, the most important cultivar, Saba (ABB) is commonly infected. The virus has been found in dwarf Cavendish (AAA) seldom in Taiwan recently.
These systemic diseases are transmitted by vegetative propagation of seedlings such as sucker, cutting, tissue culture plantlets and bud-scion for grafting as well as insect transmission. These diseases are generally controlled by the integrated control measures including cultivation of pathogen-free seedlings, elimination of inoculum sources and prevention of secondary spread by vector insects. Establishment of pathogen-free nursery
system is primarily important for preventing prevalence of these diseases (Su and Chen, 1991). Shoot-tip micro-grafting (STG) of citrus and STG combined with heat therapy have been performed for obtaining virus-free foundation stock of citrus in Taiwan (Su and Chu, 1984).The virus-free nursery system for controlling banana virus diseases has been performed by tissue culture via with virus-free stocks indexing and heat-therapy with tissue culture in Taiwan. Pathogen-free citrus and banana germplasm foundation have been established for production and cultivation of pathogen-free citrus seedling and banana TC-plantlets.
The precise and rapid indexing techniques are indispensable for management of pathogen-free nursery system. Development and application of molecular diagnostic probes including monoclonal antibodies, DNA probes and primer pairs, were made for formulating indexing techniques (Tsai and Su, 1991; Hung, Wu and Su, 1999). As a rule, a virus disease and virus-like disease are diagnosed by characteristic symptoms and bioassay with indicator plants. Nevertheless, disease indexing and detection of the causal agents are precisely and rapidly performed by enzymelinked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and reverse-transcription (RT) PCR with molecular diagnostic probes.
Disease Indexing of Citrus Greening and Virus Diseases
1. Citrus greening pathogen is commonly detected by polymerase chain reaction (PCR) analysis
Detection of HLB in symptomless citrus plants is accomplished by PCR test which has been commonly applied to indexing citrus foundation stocks and pathogen-free seedlings (Su, Hung, Wu, 1995). The sensitivity of HLB detection is uplifted by development and application of PCR analysis with adequate primer pairs (F: CAC CGA AGA TAT GGA CAA CA; R: GAG GTT CTT GTG GTT TTT CTG) (Huang, Wu and Su, 1999). The PCR-amplified DNA products are analyzed by ordinary electrophoresis (Fig. 1). (Protocol referring to the following chapter on the detection of banana viruses).
2. Citrus tristeza virus is commonly detected effectively by ELISA with polyclone and monoclonal antibodies. Development and application of monoclonal antibodies through hybridoma technology was made for differentiation certain strains of CTV (Tsai and Su, 1991). Rapid diagnostic strip has developed with monoclonal antibody for CTV detection. The globulins labeled with colloidal gold, are used for preparing the strips. The virus antibodies-secondary anti-mouse antibodies are fixed in upper part of chromatographic paper strip.(ELISA protocol referring to the following chapter on the detection of banana viruses).
Protocol of diagnostic strips:
Collect 0.2~0.3 grams of tissue specimen from several leaves by cutting into pieces into an eppendorf tube.
(1) Grind the tissue pieces within 0.8ml of extraction buffer in an eppendorf tube by using bamboo/ wooden stick.
(2) Remove the test strip from its protective pouch (bring the test to room temperature before opening the pouch to avoid condensation of moisture on the membrane). (3) Immerse a strip in the extraction buffer solution in eppendorf tube with arrow end
pointing toward the solution not over labeled antibody pad. Do not immerse the strip over the MAX line.
(4) Wait for pink-colored bands to appear. Depending on the concentration of CMV, positive results may be observed within 2~3 minutes. However, to confirm negative results, the complete reaction time of 20 minutes is required.
Interpretation of results:
(1) POSITIVE: Two distinct pink-colored bands will appear, one in the plant test region and one in the control region.
(2) NEGATIVE: Only one pink-colored band appears in the control region. No apparent pink band appears in the plat test regions.
(3) INVALID: A total absence of pink-colored bands in both regions. If neither a test band nor a control band appears on the membrane, the test should be considered void. Improper testing procedures or deterioration of reagents probably occurred.
3. Citrus tatter leaf virus detection: ELISA with monoclonal antibodies has been developed, however its sensitivity of detecting the virus is low. Reverse transcription PCR (RT-PCR) was formulated by using cDNA primer pair (F: GGA AGA CTC ACA TAG ACC CG; R: TAC TCT
CCG AAC CTG CCT C) derived from the sequence of CTLV (Ohira et. al., 1995) for detecting the virus more rapidly. (RT-PCR protocol referring to the following chapter on the detection of BBrMV virus).
4. Citrus exocortis viroid is detected by RT-PCR with cDNA primer pair (F: GCT CCA CAT CCG ATC GTC; R: TGG ACG CCA GTG ATC CGC). (RT-PCR protocol referring to the following chapter on the detection of BBrMV virus).
Establishment of Pathogen-free Citrus Foundation Stock and Nursery
System
In order to control the systemic virus and greening disease of citrus, the national scheme of virus-free nursery system was initiated, and conducted under joint program of R and D including NTU, TARI, and TBRI in Taiwan since 1983.
1. Micro-grafting for Obtaining Pathogen-free Citrus Foundation Stocks.
An improved method of shoot-tip micro-grafting (STG) was developed by using triangle-hole cut (Su and Chu, 1984) instead of inverted T cut described by Murashige et al. (1972). The STG program and health indexing were conducted in the Plant Virology Laboratory, National Taiwan University. The outline of STG schedule is as follows (Fig. 2):
Etiolated stock seedling grown on solid media (Troyer citrange or other rootstock, 2 week-old in vitro) Micro-grafting shoot-tip into rectangular triangular hole,
Or V-cleavage of decapitated seedling
Culture on liquid medium, incubate in the dark for 2 days. Growing in growth chamber or green house (28-30oC)
Under cheese cloth shading for 2 weeks. Growing under uncovered condition for 2-3 weeks.
Double grafting to potted rootstock seedlings
(Rough lemon, or other rootstock), and cultivate in green house The solid and liquid media were prepared following Murashige et al. (1972).
Preparation of Citrus STG Medium (pH 5.7)
(1) Solid medium for growing STG seedling: MS salt mixture (Gibco BRL), 4g; Sucrose, 20g; ddH2O, 1L; Agar, 1% (10g)
(2) Liquid medium for growing STG seedling: MS salts, 4.2g; Sucrose, 40g; growth factors (1L/100x stock: i-inositol, 100mg; thiamine-HCl, 0.2mg; pyridoxine-HCl, 1mg; nicotine acid, 1mg)
2. National scheme of citrus pathogen-free nursery system
Pathogen-free citrus foundation stocks. The stocks are produced by means of improved method of shoot-tip grafting and heat-therapy in the Plant Virus Laboratory, National Taiwan University (PVL/NTU) and the Chia-Yi Agricultural Experimental Station of Taiwan Agricultural Research Institute (CAES/TARI) (Fig. 3). The STG-seedlings indexed as pathogen free by PVL/NTU, are used as foundation stock, and are transferred to the National Pathogen-free Citrus Foundation Stock Collection in screen-houses in CAES/TARI.
Scion-Propagation parent trees. Pathogen-free bud-woods are multiplied in scion-propagation trees derived from foundation stocks in insect-proof screen-house of CAES/TARI, the Taiwan Fruit Marketing Cooperative (TFMC) or approved nurserymen.
Pathogen-free citrus nurseries. Pathogen-free seedlings are produced by using pathogen-free scions derived from the parent trees in the Healthy Nursery Screen-houses operated by each branch office of the Taiwan Fruit Marketing Cooperative (TFMC) and approved nurserymen.
Citrus growers. The healthy certificated citrus seedlings are transplanted to the orchards, and protected by integrated control measures.
Note: The act of citrus bud-wood certificate has been established the production of pathogen-free citrus seedling in Taiwan since December of 2004.
3. Production of Pathogen-free Banana Seedling
These diseased might readily prevail if the mother stocks are infected latently with the causal viruses. Production of virus-free seedlings including suckers and tissue-culture (TC) plantlets through pathogen-free nursery system has to be managed by strict implementation of health indexing. The virus-free nursery system for controlling banana virus diseases has been properly performed through tissue culture via virus-free stock indexing and heat-therapy with tissue culture in Taiwan. The virus-free TC-seedlings of banana are widely grown for controlling banana virus in Taiwan.
Conservation of pathogen-free citrus germplasm
The citrus germplasm collection has been kept in Citrus gerplasm repository in Chia-Yi Agricultural Experiment Station, Agricultural Research Institute, Chia-Yi, Taiwan (Table 1). Table 1. Citrus germplasm collection in Taiwan
Horticultural
classification Cultivars free from specific-pathogens Cultivars not indexed
1. Mandarin
Common mandarin Bower, Dancy, Clementine, Encore, Esparta,
Fremont, Genshokan, Guapile, Kara, Kinnow, Parson's special, Pixie, Ponkan, Early Ponkan, Sunburst, Sun Keim Woon
Drafted Ponkan
Satsuma Okitsu No.3, Okitsu Wase, Miho Wase,
Miyamoto, Nanka No.20, Emoto, Kowa, Nichinan-1
Yonezawa, Miyagawa Wase, Yamakawa, Kinho 1, Kinho 2, Kinho 3, Ohzu, Imamura, Aochima, Toman
Small fruit mandarin Cleopatra, Depressa, Sunki, Calamondin, Rangpur lime
2. Sweet orange
Common Fukuhara, Hong-Jian, Jaffa, Joppa,
Liu-cheng, N. Hamlin, Suenaga, Parson Brown, Pera, Pineapple, Shamouti, Selectra, Sugar, Sun Wickom, White Siletta,
Fukumatobeni
Seedless Liu-cheng, Mars early, Red Siletta, Tarocco, Trovita,
Valencia Olinda, Cutter, Campell, Frost, Liu Gim Gong Rhode Red
Blood Moro, Sanguinella
Navel Dream, Frost, Morita, Ogawa, Fukumoto,
Shirayanagi, Washington
Cara cara, Omishima, Yoshita
3. Tangor Hai-Li, Iyo, King, Kiyomi, Murcott, Tankan, June-Tankan, Nova
Temple, Ortanique
4. Tangelo Minneola Orlando
5. Grapefruit Duncan, O P Davis, Marsh, Frost Marsh, N. JBC 430 Marsh, Shamber, Star Ruby, Orablanco
Little River, Red Mexican, Thompson Pink, Red Blush
6. Pummelo Kao Pan, MatoWentan, Thai White, Thaired pummelo,
Chandler, Mitsuyu, Red Wentan, Toyu, Wanpaiyu, Webber, Kaopan Yoshikawa Wentan,
7. Lemon Eureka, Lisbon, Monachello, Teresa, Villafranca
8. Lime Bearss, Mexican
9. Kumquat Hong Kong, Oval, Meiwa, Chang-shou
10. Citron Buddda’s hand, Etrog
12. Sour orange Australia, Chinotto, Common, Varigated sour,
Bergamote
13. Sweet lime Palestine
14. Trifoliate and hybrid
Common trifoliate, Rusk, Swingle, Troyer Carrizo, Flying Dragon, Pomeroy
15. Miscellaneous Excelsa, Meyer, Kawano, Shiranui, Natsudaidai, us 119
Beni-Hassaku, Excelsa, Hassaku, Hystrix, Hyuganaysu, Kimikan, Kinkoji, Konijimi, Kotokan, Kabuchi, Yuzu
Germplasm free from greening pathogen, citrus tristeza virus and citrus tatter leaf virus.
The pathogen-free citrus cultivars obtained through shoot-tip micro-grafting and disease-indexing made in Virology Laboratory, Dept of Plant Pathology, NTU and CAES, Have been conserved in the Pathogen-free Citrus Foundation Screen-house of CAES. Pathogen-free citrus seedlings are produced by using the PF-scions supplied from the Foundation Center.
The pathogen-free nursery system has been properly performed through improved techniques of shoot-tip micro-grafting for obtaining virus/HLB-free citrus foundation stocks, and then production of healthy citrus seedlings under sapling certificate regulation in Taiwan.
Disease Indexing of Banana Virus Diseases
The reliable, sensitive and rapid indexing techniques are indispensable for management of pathogen-free nursery system and health management of banana plants in the field. Development and application of molecular diagnostic probes including monoclonal antibodies, DNA probes, and primer pairs for polymerase chain reaction (PCR) were made for formulating indexing techniques (Wu & Su, 1990; Su et al., 1997).
1. ELISA with monoclonal antibodies against BBTV and CMV.
It is difficult to purify a virus from virus-infected banana plant tissues due to high content of phenolic latex (Wu & Su, 1990; Wu et al., 1997). Therefore, it is better to produce monoclonal antibodies (McAb) of unique quality against banana viruses through hybridoma technique with impure virus preparation as immnogen. High specific monoclonal antibodies with high titer were prepared against BBTV and CMV respectively. Enzyme-linked immunosorbent assay (ELISA) with different McAbs was developed for virus detection and differentiation of the virus strains (Su et al., 2003). Adequate buffer solution has to be selected for virus extraction in ELISA test in order to eliminate interfering substances in banana latex
and stabilize virus extract. The adequate buffer for extracting BBTV is 0.5M Tris buffer (pH 7.5) containing 0.1% Na-DIECA, 5% sucrose and 0.5% skin milk, and for extracting CMV with 0.2M potassium-phosphate buffer (pH=7.4) containing 0.5% Na2SO3. Direct DAS-ELISA with
alkaline phosphatase (AP)-conjugate of McAb (2H6) is used for BBTV detection, and CMV detection with AP-conjugate of McAb (7-1).
Protocol of virus detection by direct DAS-ELISA with AP-conjugate of McAb
(1) Micro-plates are coated with purified IgG of monoclonal antibody (2H6) against BBTV. Add 100ul of McAb (2ppm) in coating buffer to each well, and incubate the plates in a moist box at 37 oC for 2 hours.
(2) Wash the plates with PBS-T, 3~5 times.
(3) Add 100 µl/well of virus extracts from banana samples in virus extraction buffer at 1/5~1/20 dilution (W/V), then keep the micro-plates in a moist box at 37 oC for 2 hours or at 4 oC overnight.
(4) Wash the plates with PBS-T, 3~5 times.
(5) Add 100 µl/well of alkaline phosphatase (AP)-conjugate in PBS-T at 200x (0.5ppm) dilution. Incubate plates in a moist box at 37 oC for 2 hours.
(6) Wash the plates with PBS-T, 3~5 times.
(7) Add 100 µl/well of p-nitrophenyl phosphate (1mg/ml) in substrate buffer, and incubate plates in a moist box at 37 oC for 30~60 minutes.
(8) Read ELISA values of absorption at 405 nm.
Rapid Diagnostic Strips for BBTV and CMV
Monoclonal antibodies against banana bunchy-top virus (BBTV) and, cucumber mosaic virus (CMV) were developed by Laboratory of Plant Virology, National Taiwan University (e-mail: [email protected]). The rapid diagnostic strips have been developed through cooperation by PVL of NUT and Meikang Biotechnology Company (e-mail: [email protected]). The globulins labeled with colloidal gold, are used for preparing the strips. The virus antibodies-secondary anti-mouse antibody are fixed in upper part of chromatographic paper strip (Protocol referring to the chapter on the detection of citrus virus).
Buffers(Store at 4-6oC) PBST (pH 7.4) NaCl 8.0 g KH2PO4 0.2 g Na2HPO412H2O 2.9 g KCl 0.2 g NaN3 0.2 g Tween 20 0.05 % dd H2O 1 L Coating buffer (pH 9.6) Na2CO3 1.5 g NaHCO3 2.93 g dd H2O 1 L
Substrate buffer (pH 9.6) Diethanol amine 97 ml
H2O 800 ml
MgCl2 12H2O 100mg
dd H2O 1 L
2. Detection of banana bunchy-top virus (BBTV) and banana streak badnavirus (BSV) with polymerase chain reaction (PCR) followed by electrophoresis analysis
In order to increase sensitivity of virus detection and specificity of virus-strains differentiation, the primer pairs specific to BBTV strains were derived from the nucleotide sequences of BBTV-DNA through cloning and sequencing, and applied to formulation of the following protocol of PCR amplification followed by electrophoresis analysis. The primer pair specific to BSV quoted from J. Thomas’ sequence, was applied to PCR analysis; the ordinary schedule for viral DNA/RNA extraction formulated through comparative trials, was commonly adapted for obtaining constant detection results. More recently, tissue-soak DNA extraction method mentioned below was developed for establishing rapid and inexpensive detection method of BBTV and BSV in PCR analysis.
PCR primer pairs:
BBTV primer pairs:
C1-CR:
F: 5’-GGA AGA AGC CTC TCA TCT GCT TCA GAG AGC-3’ R: 5’-CAG GCG CAC ACC TTG AGA AAC GAA AGG GAA-3’
S-CR:
F: 5’-GGG GCT TAT TAT TAC CCC CAG C-3’
R: 5’-AGC GCT TAC GTG GCG CAG CAC TAA CT-3’ SR-CR:
F: 5’-TGT CGT CGG CGA CGA AGT CG-3’
R: 5’-GGA CAT CCT CCT CCT TCA GAA GAG AGA-3’
BSV primer pairs:
F: 5’-CAA CTC AAG AGC CTA GTA TGC-3’ R: 5’-TAC CTC CGA CCG TAT TTC CAG-3’
PCR cycles for BBTV:
C-1 primer:
Step 1: 94 oC 4 min; 50 oC 1 min; 72 oC 2 min; 1 cycle.
Step 2: 94 oC 1 min; 50 oC 1 min; 72 oC 2 min; 30 cycles.
Step 3: 72 oC 10 min.
Step 4: 4 oC soaking. S-CR, SR-CR primer:
Step 1: 94 oC 4 min; 60 oC 1 min; 72 oC 2 min; 1 cycle.
Step 2: 94 oC 1 min; 60 oC 1 min; 72 oC 2 min; 30 cycles.
Step 3: 72 oC 10 min.
Step 4: 4 oC soaking.
PCR cycles for BSV:
Step 1: 94 oC 4 min; 50 oC 1 min; 72 oC 2 min; 1 cycle.
Step 2: 94 oC 1 min; 50 oC 1 min; 72 oC 2 min; 30 cycles
Step 3: 72 oC 10 min.
Step 4: 4 oC soaking
Ordinary extraction of viral DNA (total nucleic acid):
1. Collect the leaf samples from banana plants infected with BBTV or BSV.
2. Froze the chopped tissue, 0.5g in liquid nitrogen and grind to fine powder in coffee blender or a mortar and pestle, or grind the fresh tissue directly.
3. Add 3 ml of DNA-extraction buffer* +1 % Sarkosyl (add 2.7 ml of DNA extraction buffer and 0.3 ml 10% Sarkosyl). Stir the tissue-powder suspension.
4. Transfer the suspension to a 1.5 ml eppendorf tube and incubate at 55 oC / 1hr in water bath.
5. Centrifuge the tube at 6,000 rpm / 5min.
6. Save sap supernatant (800 µl), add 100 µl of 5 M NaCl and 100 µl of CTAB / NaCl (10 % CTAB in 0.7 M NaCl), incubate at 65 oC / 10min.
7. Add equal volume of chloroform / isoamyl alcohol (24:1), mix thoroughly and spin at 11,000 rpm / 5 min (for BSV) or 6,000 rpm/ 5 min (for BBTV), save aqueous suspension.
8. Add equal volume of phenol / chloroform / isoamyl alcohol (25:24:1). Mix thoroughly and spin at 11,000 rpm / 5 min. (for BSV) or 6,000 rpm / 5 min (for BBTV), save aqueous suspension.
9. Add 0.6 volume of isopropanol to precipitate the nucleic acid. Incubate at -20 oC / 30 min. spin at 12,000 rpm / 20 min.
10. Wash pellet with 70% ethanol to remove CTAB residual. Briefly dry pellet and re-suspend in 100 µl of TE buffer. Store at -20 oC.
* DNA extraction buffer for BBTV and BSV in PCR analysis : 100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl, 100 µg / ml proteinase K (may be omitted); pH8.0 .
* DNA extraction reagents : 10% Sarkosyl (N-Lauroyl sarcosine); 5M NaCl; 10% CTAB (Hexadecryltrimethylammonium Bromide) in 0.5 M NaCl; chloroform: isoamyl alcohol (24:1); phenol: chloroform: isoamyl alcohol (25:24:1); isopropanol; 70% alcohol (ETOH); TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0).
3. Detection of banana bract mosaic potyvirus (BBrMV) by reverse-transcription PCR (RT-PCR) analysis
The formulated RT-PCR-electrophoresis analysis was applied to detection of BBrMV. The RT-PCR was developed by using the c-DNA primer pairs specific to BBrMV derived from B.C. Rodoni’s sequence.
Extraction of viral RNA (total nucleic acids) from banana leaf tissues:
The present extraction procedure is almost identical with those for BBTV and BSV extraction.
cDNA primer pair for RT-PCR of BBrMV:
F: 5’-AAC GCT CAG CCT ACT TTT CG-3’ R: 5’-CAT ATC ACG CTT CAC ATC TTC A-3’
Thermocycles for RT-PCR of BBrMV:
Step 1: 50 oC 35 min; 94 oC 2 min.
Step 2: 94 oC 30 sec; 56 oC 30 sec; 68 oC 45 sec; 10 cycles.
Step 3: 94 oC 30 sec; 56 oC 30 sec; 68 oC 45 sec*; 25 cycles.
(*: 5 sec increasing ramp) Step 4: 68 oC 7min.
Reaction mixture:
PCR reagents amount / tube
ddH2O 10µl
5x Superscript II buffer 2.5µl 10x Taq buffer 2.5µl 100 mM DTT 1.25µl 10 mM dNTPs 2µl Taq polymerase (BRL or BerTaq) 0.2µl Superscript II polymerase (BRL) 0.25µl BBrMV primer pair (10 pmol /µl each) 1µl RNA template 0.25µl
Establishment of Virus-free Banana-cultivar Foundation and
Production of Pathogen-free TC-plantlets
The systemic diseases including virus diseases and Fusarial wilt might readily prevail if the mother stocks are infected latently with the causal viruses. Production of virus-free seedlings including suckers and tissue-culture (TC) plantlets through pathogen-free nursery
system has to be managed by strict implementation of health indexing. The pathogen-free (PF) banana cultivars of economic importance were obtained through virus-indexing and tissue culture under heat-therapy temperature (35 oC/3 months) (Wu and Su, 1991). The PF banana
cultivars have been kept in the foundation screen-house of Taiwan Banana Research Institute (TBRI) (Fig. 4). The mass production of PF banana plantlets through tissue culture with the suckers from the foundation stocks (Fig. 6) has been properly performed in TBRI (Table 2).
Table 2. Pathogen-free foundation stocks in TBRI
Genome type Cultivars
AA Kluai Khai
AAA
Taiwan Cavendish (Peichiao, Senrenchiao), Cavendish B. F (Africa), Grande Naine (Honduros), Lacatan (Jamaca), Poyo, Valery
(Philippines), Vietnam Cavendish, Lacatan (Philippines), Gros Michel, William (Dwarf), Cavendish mutants resistant to Fusarial wilt
(Formosana, Taichiao No. 1) AAB Latundan, Mas, Raja
Cultivars free from BBTV, BSV, CMV, BBrMV, and Fusarial wilt pathogens
The virus-free banana TC-seedlings through hardening in screen-house 2 months (Fig. 7), are widely grown by farmers for controlling banana virus in Taiwan (Fig. 8).
Conservation of Banana Germplasm and Pathogen-free Banana
Cultivars
The banana production was the most important agro-industry for local consumption and export during 1960~80s. Therefore, 242 cultivars of banana germplasms collection has been obtained from the domestic and foreign 34 banana-growing areas would-wild (Table 3). The cultivar collections have been kept within test tubes in germplasm bank (Fig. 5), screen-house and field conditions in Chia-Yi Agricultural Experiment Station, TARI, Chia-Yi, and TBRI, Pintung.
Banana streak badnavirus is commonly detection banana cultivars in symptom express form and virus-genome integrated form showing chloratic symptom or symptomless (Fig. 9). The cultivars introduced from INIBAP germplasm collection in Belgium, were found free from BBTV, CMV and BBrMV, but commonly infected by BSV of genome-integrated form (Table 4).
Table 3. Cultivar number of banana germplasm collection in TBRI (242 cultivars)
Local Musa spp. Foreign Musa spp. Unknown origin Genome type Test
tube Screen-house Field Test tube Screen-house Field Test tube Screen-house Field
AAAA 0 0 0 10 10 6 0 0 0 AAAB 0 0 0 5 5 0 0 0 0 AABB 0 0 0 2 2 0 0 0 0 ABBB 0 0 0 1 1 0 0 0 0 AAA 11 11 7 87 87 70 4 4 4 AAB 1 1 0 27 27 20 16 16 16 ABB 5 5 2 14 14 9 8 8 8 BBB 0 0 0 4 4 2 0 0 0 AA 1 1 2 19 19 13 0 0 0 AB 0 0 0 2 2 2 0 0 0 BB 1 1 1 3 3 3 1 1 1 Abaca 2 2 2 4 4 4 0 0 0 Unknown 5 5 5 9 9 1 0 0 0 Total 26 26 19 187 187 130 29 29 29
Table 4. Virus detection of cultivars from Belgium (INIBAP)
Virus detection Cultivar name Genome type
BBTV CMV BSV BBrMV
-1 Pisang Jari Buaya AA - - - -
-2 FHIA-01 AAAB - - ++ -
-3 FHIA-02 AAAB - - + -
-4 FHIA-03 AABB - - +++ -
-5 Williams (Bell, South Johnstone) AAA - - - -
-6 Cachaco ABB - - - -
-7 Gros-Michel AAA - - +++ -
-8 Yangambi KM5 AAA - - - -
-9 FHIA-17 AAAA - - - -
-11 SH 3436-9 AAAA - - - - -12 TMB × 1378 ABBB - - - - -13 TMB × 5295-1 AAAB - - +++ - -14 SH-3640 Hybrid - - +++ - -15 FHIA 18 Hybrid - - +++ - -16 FHIA 21 (#68) AAAB - - +++ - -17 CRBP 39 - - +++ - -18 FHIA-25 Hybrid - - + - -19 Pisang Ceylan AAB - - ++ -
Discussion and Conclusion
(1) The production of citrus and banana fruit crops are the most important argoindustry world-wild for fresh-fruit consumption, staple food, processing and export as cash crops. (2) Virus and virus-like diseases of citrus and banana have been causing commonly causing
great damage to fruit production in fruit yield and quality due to their systemic invasion and epidemic spread through infected budwoods, vegetatively propagated seedlings, and transmission by insect vectors.
(3) In view of gremplasm utilization, establishment of pathogen-free nursery system is primarily important for preventing the prevalence of these systemic diseases.
(4) Development and application of precise and rapid indexing techniques are indispensable for management of pathogen-free nursery system and health management of PF seedling cultivation in the field.
(5) The conservation and effective utilization of citrus and banana genetic resources must be concerned to be free from the systemic virus, virus-like pathogens through disease indexing and pathogen elimination in germplasm (STG, and heat-therapy tissue culture).
References
1. Diekman, M. and Putter, C.A.J. 1996. Musa spp. FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm. No. 15.
2. Hung, T.H., S.C. Hung, C.N. Chen, M.H. Hsu and H.J. Su. 2004. Detection by PCR of
Candidatus Liberibacter asiaticus, the fastidious bacterium causing citrus hunaglongbing, in vector psyllids: application to the study of vector-pathogen relationships. Plant Pathology; 53: 96-102.
3. Hung, T.H., Wu, M.L., and Su, H.J. 1999. Detection of fastidious bacteria causing citrus greening disease by non-radioactive DNA probes. Ann. Phytopathol. Soc. Jpn. 65: 140-146.
4. Hung, T. H., Wu, M.L., and Su, H.J. 1999. Development of a rapid method for the diagnosis of citrus greening disease using the polymerase chain reaction. J. Physopathology 147: 599-640.
5. Lockhart, B.E.L. 1986. Purification and serology of a bacilliform virus associated with banana streak disease. Phytopathology 76: 995-999.
6. Lee, S.W. 2003. Micro-propagation of Cavendish banana in Taiwan. FFTC Technical bulletin 163: 1-7.
7. Matsumato, T., Wang, M.C. and Su, H. J. 1961. Studies on Likubin. In: Proc. 2nd Conf. IOCV, pp. 121-125.
8. Miyakawa, T. 1980. Occurrence and varietal distribution of tatter leaf-citrange virus and its effects on Japanese citrus. pp. Proc. 8th Conf. IOCV, IOCV Riverside.
9. Murashige, T. et al. 1972. A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones. Hort Science 7: 118-119.
10. Musharam, A. and Whittle, A.M. 1991. Stem pitting strains of citrus tristeza virus in Indonesia. PP. 150-155. Proc. 11th. Conf, IOCV, IOCV Riverside.
11. Nakahara, K. et al. 1998. An improved procedure for extracting nucleic acids from citrus tissues for diagnosis of citrus viroids. Ann. Phtopathol. Soc. Jpn. 64: 532-538.
12. Roistacher, C.N. and Morens, P. 1991. The worldwide threat from destructive isolated of citrus tristeza virus – a review. In: Proc. 11th Conf. IOCV, IOCV Riverside, pp. 7-19. 13. Ryu, I.H. and H.J. Su 1999. Vectorship of mealybugs transmitting banana streak
badnavirus. Plant Pathol. Bull. 8: 19-20.
14. Su, H.J. 1981. A tristeza virus strain causing dwarfing of pummelo and grapefruit. In: Proc. Intern. Soc. Citriculture Vol. 1, pp. 423-430.
15. Su, H.J. and Chen, C.N. 1991. Implementation of IPM of citrus virus and greening (likubin) diseases. In: Integrated Control of Plant Virus Diseases. FFTC Supplement No. 1, pp. 3-11.
16. Su, H.J. and Chu, J.Y. 1984. Modified technique of citrus shoot-tip grafting and rapid propagation method to obtain citrus budwoods free of citrus viruses and likubin organism.
In: Proc. Int. Soc. Citriculture. Vol. 2. pp. 332-334.
17. Su, H. J., Hung, T.H. and Tsai, M.C. 1991. Recent developments on the detection of citrus greening disease. In: Proc. 6th Inter. Asia Pacific Workshop on “Integrated citrus
health management”, Kuala Lumpur, Malaysia, p89.
18. Su, H.J., Hung, T.H. and Wu, M.L. 1997. First report of banana streak virus infecting banana cultivars (Musa spp.) in Taiwan. Plant Disease 81: 550.
19. Su, H.J., L.Y. Tsao, M.L. Wu and T.H. Hung. 2003. Biological and molecular categorization of the strains of Banana bunchy top virus. J. Phytopathology 151: 290-296.
20. Tsai, M.C. and Su, H.J. 1991. Development and characterization of monoclonal antibodies to citrus tristeza virus (CTV.) strain in Taiwan. In: Proc. 11th Conf. IOCV, IOCV
Riverside, Taiwan, pp. 46-50.
21. Tsai, M.C., Su, H.J. and Garnsey, S.M. 1993. Comparative study on stem-pitting strains of CTV in Asian countries. In: the Proc. 12th Conf. IOCV, IOCV Riverside, Taiwan, pp.
16-19.
22. Wu, M.L., Hung, T.H. and Su, H.J. 1997. Strain differentiation of cucumber mosaic virus associated with banana mosaic disease in Taiwan. Ann. Phytopathol. Soc. Jap. 63: 176-178.
23. Wu, R.Y. and Su, H.J. 1990. Production of monoclonal antibodies against banana bunchy top virus and their use in enzyme-linked immunosorbent assay. J. of Phytopathology 128: 203-208.
24. Wu, R.Y. and Su, H.J. 1990. Purification and characterization of banana bunchy top virus. J. Phytopathol. 128: 153-160.
25. Wu, R.Y. and Su, H.J. 1991. Regeneration of healthy banana plantlets from banana bunchy top virus-infected tissues cultured at high temperature. Plant Pathology 40: 4-7. 26. Yeh, S.H., Su, H.J. and Chao, Y.C. 1994. Genome characterization and identification of
Figures 1~4: Taiwan GO isolates 5: Okinawa, Japan 6: China 7: Malaysia 8: Vietnam 9: Thailand 10: Saudi Arabia 11: Healthy CK
Fig. 1. Detection of HLB pathogen by polymerase chain reaction (PCR)/ electrophoresis analysis.
Fig. 2. Shoot-tip micro-grafting for obtaining pathogen-free citrus cultivar to be used for foundation stock.
Fig. 3. (Left) National Pathogen-free Citrus Foundation Stocks Collection Center, Chia-Yi Agricultural Experimental Station of Taiwan Agricultural Research Institute.
(Right) Pathogen-free citrus foundation stocks kept in the insect-proof screen-house.
Fig. 5. Banana germplasm collection in test tube kept at 17 oC (TBRI)
Fig. 6. (Left) Meristem culture from growing point of pathogen-free banana suckers (Right) Multiplication of adventitious buds through tissue culture via 5~6 cuts.
Fig. 7. Mass production of PF banana TC-plantlets in tissue culture room (TBRI)
Fig. 9. Banana plantation with PF TC plantlets
Fig. 10. BSV detection in various banana cultivars of different genome types by PCR test. 1.Cavendish (AAA)- BS severe; 2.Latundan (AAB)- chlorosis; 3.Awak (ABB)- symptomless 4.M.balbisiana (BB)- symptomless; 5.Abaca (M. textilis) - symptomless
In-situ Conservation of Plant Genetic Resources in
Vietnam: Achievements and Lessons Learned
By
Nguyen Thi Ngoc Hue
2, Luu Ngoc Trinh
1I. Role of in-situ approach in the conservation of plant genetic resources
in Vietnam
Genetic resources, among which plant genetic resources share a relevant contribution, are the most important of the whole biodiversity resources. Plant genetic resources play a determinant role of ensure the sustainable agricultural development, the sustainable food production and environment protection, that is why their conservation has been calling for an increasing attention.
There are two approaches of plant genetic resources conservation, the ex-situ and the in-situ. The ex-situ conservation, professionally is a synonym of the seed genebank, had principally developed in developed countries and CGIAR research centers in 1960's, 1970's and 1980's. In-situ conservation is beneficial in that it can maintain genetic resources in natural evolution. Only in-situ conservation can truly preserve the ecological system. Promoting in-situ conservation is the primal measure towards reestablishment of the lost biodiversity.
In situ conservation is concerned with maintaining species’ populations in the natural habitats where they occur, whether as uncultivated plant communities or in farmers’ fields as part of existing agro-ecosystems (Brush, 1995; Bellon et al., 1997). In situ conservation of crop plants involves the conservation on-farm of local crop landraces with the active participation of farmers. The goal of in situ conservation is to encourage farmers to select and maintain local crop diversity for the benefit of humankind, at the same time benefiting from it for their livelihood needs. The objective of on-farm conservation may be diverse as diverse stakeholders are involved.
In situ conservation has the potential to
(1) conserve the processes of local adaptation of crops to their environments,
1. Director, Plant Resources Center, VNAAS. e-mail: [email protected]
2. Deputy Director, Plant Resources Center, Vietnam Academy of Agricultural Science. (VNAAS ) e-mail: [email protected]
(2) conserve diversity at all levels – the ecosystem, the species, and the genetic diversity within species,
(3) improve the livelihood for farmers,
(4) maintain or increase control and access of farmers over their genetic resources,
(5) integrate farmers into the national PGR system and involve farmers directly in value addition process, and
(6) link farming community to genebank for conservation and utilization.
The importance of conservation of agro-biodiversity for future of global food security lies in its potential to supply required germplasm to crop breeders and other users. On-farm conservation will provide public and private benefits (socio-economic, ecological and genetic benefits). However, in comparison with the ex-situ, the in-situ conservation is remarkably more complicated, not only for performance, but also for conducting research activities because it closely links with agricultural development tendency, mainly the market orientation. To conduct in practical the in-situ conservation plant genetic resources demands a lot of studies on both socio-economical and biological fields of sciences
As any developing country, Vietnam needs firstly to develop the ex-situ conservation to safely conserve the important and valuable, but actually severely eroding genetic resources. Paralleling to the ex-situ, have also developed the in-situ conservation in a strategy of safeguarding the genetic resources for food and agriculture in both aspects, inter-specific and intra-specific or genetic diversity.
II. Achievements on in-situ conservation of plant genetic resources in
Vietnam in the past period
1. Forest genetic resources and wild relatives of crop genetic resources
Vietnam government has marked the boundaries of special used forests system in nationwide. Up to now, there are 102 special used forests that were established by government under control of Ministry of Agriculture and Rural Development. Based on IUCN classification system for reserve management, Vietnam classified the special used forests system as 4 levels the following:
Level 1: National Park Level 2: Nature Reserve
Level 3: Species/ habitat management protected area Level 4: Protected landscape or seascape
Forest genetic resources and wild relatives of crop genetic resources have been protecting management protected areas which were set up by the Government. Forestry tree genetic resources, wild relative plants and wildlife are being preserved and exploited at 12 National Parks, 72 Nature Reserves, Species/ habitat management protected areas, 18 Protected Landscapes and Seascapes (See the Appendix 1).
2. Projects on in-situ conservation of plant genetic resources in Vietnam:
-2-1. Strengthening scientific basis of in-situ conservation of agricultural
biodiversity on-farm
The project was implemented during 1999-2003. The Project has the three main objectives:
1. To support the development of a framework of knowledge on farmer decision-making processes that influence in situ conservation of agricultural biodiversity
2. To strengthen national institutions for the planning and implementation of conservation programs for agricultural biodiversity, and
3. To broaden the use of agricultural biodiversity and the participation in its conservation by farming communities and other groups.
The partners of Project were Hanoi Agricultural University, Tay Nguyen University and Hue University, Cantho University and PGRC-VASI. The national project coordinator (NPC) was Director of VASI, with support from Plant Genetic Resource Center (PGRC). The following activities and achievements were obtained:
Crops and Site selection
Step1: Crops such as Rice, taro and cardamon were selected for the study. The major criteria for priority crop selection were native to East Asian Center of Origin, rich intra-specific diversity and importance to farming community. Site characterizations, diversity fairs, landrace characterizations, preference ranking of landraces and baseline survey were the major activities accomplished during the project period.
Based on the results of survey, balancing all the criteria and indicators and with those agro-biological, seven macrosites were selected:
Mountain: Da bac district of Hoa binh province and Sapa of Laocai Highland: Nho quan district of Ninh binh province and Krongno of Daklac Lowland, Red River Delta: Nghia hung district, Nam dinh province
Lowland, Mekong River Delta: Tra cu district, Tra vinh province Lowland central coast: Phuvang of Thuathien-Hue
Site selection for in situ conservation: in Vietnam based on following criteria: Environmental (natural and human managed) diversity
High species and intra-species diversity High cultural diversity
Sustainability of on-farm conservation Opportunity for improvement
Accessibility
The sites differ for several characteristics and represent the ecological zones and are most appropriate for the study Example: Tracu (Mekong delta; managed by Can Tho); Nghiahung (red river delta, lowland <1.5m; high yield potential, landraces are still maintained, traditional Kinh ethnicity); Nhoquan highland (60-90m; intermediate intervention; intermediate diversity of taro and rice) and Dabac (mountainous areas; 400-700 m; no intervention; high diversity of upland rice and taro crops)
Step 2: After finishing the selection of districts, which is called as "macro-sites", it forwarded to the selection of micro-sites or selection of communes within district and villages within commune. Selection of village is the basic step of the micro-site selection. The village in Vietnam is not only an administrative unit but traditionally also a unit of culture. Cultural features such as religion, tradition, custom, occupation... vary from one to the other village, but are usually homogenous within a determined one. Selection of village is mainly based on surveyed data in company with consultation of district and commune officers and utilization of existed statistical data play a very important role.
There are following microsites were selected:
- Tat village, Tanminh commune, and Cang village, Doanket commune, these two villages belong to Dabac district, Hoabinh province;
- Yenminh village, Yenquang commune, and Quangmao village, Thachbinh commune. These two villages belong to Nhoquan district, Ninhbinh province
- Donglac vilage, Nghialac commune and Kienthanh village, Nghialoi commune belong to Nghiahung district, NamdÞnh province.
- Tra kha and Rong lon villages, Daian commune belong to Tracu district, Travinh province.
- Bankhoang commune in Sapa district, Laocai province,
- Phuda commune in Phuvang district, Thuathien - Hue province, - Krong No district, Daclac province
