Plant biotechnology and Molecular Markers in Nepal

Introduction

Molecular markers generally used are restriction fragment; southern hybridization; restriction fragment length polymorphism; polymerase chain reaction based markers such as DNA amplification fingerprinting, random amplified fragment length polymorphism, simple sequence repeats, cleaved amplified fragment length polymorphism. The isozyme based protein gel electrophoresis technique which has been exploited in the breeding and genetic study of our economically important crop species is one of the most popular biochemical markers.

History of molecular markers in Nepal

Plant biotechnology also includes regeneration of plants from cells, or tissue or organ, or embryo or other parts through the test tube culture in laboratories. The cell may have foreign gene transferred in the laboratory which is known as transgenic cells. The plants regenerated from the transgenic cells are known as transgenic plants. Plant biotechnology also includes technology of production of new plants or plants products using technology. The plant biotechnology also includes use of extraordinary use of DNA for a variety of purposes including plant breeding.

First time, the plant tissue cultured was started in Nepal in 1976 at the Nepal government’s National Herbarium and Plant Research Laboratory in Godawari. This laboratory basically has been running clonal propagation of hard wood trees, horticulture crops, medicinal plants, ornamental plants and over 30 kinds of orchids. The scientists in the laboratories have been working in some anther culture as well as some chromosome works in their high interests.

One private laboratory called as RLABB (Research Laboratory for Agriculture Biotechnology and Biochemistry) funded by Rockfeller Foundation of the USA was established in 1986 in Kathmandu, Nepal. The Scientist V. N. Agrawal pioneered a work in regeneration of cold tolerant rice from Jumli Marshy through anther culture technology. He also worked in agriculture crop species and forest tree species using some molecular markers. This lab had also worked in the area of sex-determination of dioecious plant Lapsi and micropropagration of many plants like pinus, Autocarpous, Rice, orchids, etc. Some other private enterprises (Botanical Enterprise, Nepal Biotech Nursery, Himalayan Floratech, Microplant Nepal) have also working in rapid propagation of orchids using tissue culture techniques.

Potato Development Centre at Khumaltar has been working on tissue culture of potato to regenerate and multiply virus free clone of potato. Basic research on tissue culture was also initiated at Central Department of Botany, Kritipur mainly of medicinal activities. The graduate students have also working in some agricultural, medicinal, ornamental and forestry crops and trees under the supervision of senior level professors in Department of Botany of Kirtipur Tribhuvan University, Kathmandu, Nepal.

A new biotechnology laboratory Green Research Technology was established which has been working in the regeneration of virus free plants of Citrus, Cardamom and potato plants using meristem culture in Kathmandu, Nepal. Laboratory for Agriculture Biotechnology and Biochemistry, Sanepa, Potato Development Centre at NARC, and Central Laboratory of Rampur Campus of Institute of Agriculture and Animal Science, Rampur (IAAS, Rampur, Chitwan, Nepal) of Tribhuvan University have  some basic facilities to conduct scientific molecular marker based works.

Micro plant Nepal, Nepal Biotech Nursery, Himalayan Floratech, Nepal Biotech Nursery, Himalayan Floratech, Botanical Enterprise, Potato Development Center, Plant Research Division, Green Research and Technologies, and Central Department of Botany, Kirtipur are existing and working in in vitro plant culture for different species of crop species of economic importance and poverty alleviation.

Nepal Biotechnological Association (NBA) and Nepal Molecular Biology Society (NEMBIS) are organizations working in the promotional developmental and commercial applications of research findings in the field of plant biotechnology.

Molecular markers in Plant Breeding in Nepal

No routine and regular works are in the process of molecular map fingerprinting in any government and private owned laboratories in Nepal. Ministries of Education, Science, Technology, and Agriculture are not interested to allocate enough and regular funds to the government laboratories for exploitation of molecular markers based plant breeding. Some irregular works are going on sporadically in some crops.

Utilization of Molecular Markers in Plant Breeding

Utilization of molecular markers in plant breeding takes place in two step works. First, construction of high density of molecular markers of our crop species and then; utilization of the molecular markers in marker based selection and breeding of our crops. Nepal cannot allocate for construction of high density chromosome map of the genome of a few very important economically important crops such as rice, wheat, maize, potato. Plant biotechnology laboratories in Nepal can exploit the information of the high density genome mapping of the crops worked in developed countries and published in the international plant genome related publishing journals.

Molecular Markers Technology in Nepal

Molecular markers technology consists of creation of a variety of restriction enzymes; construction of cloning vectors such as plasmid, phage, yeast artificial chromosomes (YAC); cloning technology; DNA x DNA hybridization; DNA x RNA hybridization; DNA amplification through polymerase chain reaction (PCR); DNA sequencing; primer construction technology; labeling and detection technology. High density map construction of our crops is very sophisticated and expensive molecular marker development technology which we cannot do in the neglected field in the laboratories of Nepal. But, developed resource rich countries have been working in the construction of high density chromosome mapping. We can get sequence information and buy primers to amplify the target DNA region to develop marker based selection breeding for PCR based marker assisted selection and breeding works of our crops. High density mapping of wheat, rice, maize, sorghum have been constructed in the resource rich countries like the USA, Japan, UK, France in coordinated projects.

Isozyme and DNA Fractionation as molecular markers in Nepal

Plant protein analysis is performed through the protein gel electrophoresis technique. DNA Amplification and DNA fractionation has been performed in few laboratories in Nepal. But the such works are not regular in the laboratories since the government has not been regular to allocate funds for such works.

DNA hybridization fingerprinting in Nepal 

Scientists are interested to do works using DNA hybridization fingerprinting technique; but they are not getting enough funds and regular electric supply to do such works in Nepal. Still some sporadic works are going on in some private and government laboratories.

DNA amplification fingerprinting in Nepal

DNA amplification fingerprinting have been done through PCR (polymerase chain reaction) in some laboratories in some crop species such as rice, cardamom; but the works are not regular in the few laboratories in Nepal. Biotechnology Laboratory of Nepal Agriculture Research Council, Khumaltar-Lalitpur, Nepal has become a leading laboratory in Nepal in recent days (2012).

Marker assisted selection based breeding

No any crop has been developed through the marker assisted selection breeding techniques in Nepal yet.

Use of molecular markers in Nepal

Professor Dr. R.C. Sharma worked to develop blast resistant rice for Nepalese farmers. For it, he got some funds from Rockfeller Foundation of the USA to work in collaboration with IRRI scientists (International Rice Research Institute, Los Banos, Philippines). Dr RC Sharma worked on blast resistance of popular Nepalese rice Laxmi and introduce pyramid blast resistance genes into popular Nepalese rice: Masuli. Post-doctorate plant pathologist, Dr. Tika B. Adhikari also worked in bacterial blight pathogen of rice using RFLP, PCR based DNA fingerprinting techniques  in Nepal and Kansas University of the USA during 1990s in the funding of Rockfeller Foundation. He did some part of it at IAAS, Rampur-Chitwan and some part his research at Kansas University in the USA. He used both radio-active isotopes and multi-color color staining detection techniques.

Breeding of Rice 

Few scientists are working in characterization of submergence genes in wild rice of Nepal in Biotechnology Lab of Nepal Agricultural Research Council (Personal communication with B. Tamang, 2012)

Breeding of Maize 

No any molecular markers have been used for the improvement of the maize crop. Conventional breeding and evaluation of the local and exotic germ plasm have been going on the crop of the maize in Nepal.

Breeding of Wheat 

Few scientists have been working in exploration of leaf rust, stem rust and yellow rust resistance genes in Nepalese bread wheat at Biotechnology Lab of Nepal Agricultural Research Council (Personal communication with S. Acharya Sharma, 2012). Sharma worked on previously identified gene sequence based primer information of the rust resistance of the wheat.

Breeding of Lentil (Lens culinaris)

Few scientists have been working in the study of genetic and morpho-physiological diversity exploration of lentil genotypes of Nepal and ICARDA through Simple Sequence Repeat (SSR) markers and natural field based morpho-physiological phenotypes at the laboratory of plant biotechnology of Nepal Agricultural Research Council (Personal communication with U. Kushwaha, 2012).

Breeding of Arahar Cajanus cajan 

No molecular markers based works have been applied in any department in Nepal to study and exploit the science of Cajanus cajan in Nepal. Most of the works are selection activities of the crop to breed promising cultivars of arahar.

Breeding of Potato

Plant Biotechnology laboratory of Nepal Agriculture Research Council (NARC) have also worked to regenerate in vitro disease free plants and multiply planting materials of potato.

Breeding of Sugarcane

Few scientists are working in generation and rapid multiplication of virus free sugarcane through tissue culture based somaclonal variation creation technique in Nepal in Biotechnology Lab of Nepal Agricultural Research Council (Personal communication, 2012)

Breeding of Cardamom

Few scientists are working in generation and rapid multiplication of virus free cardamom in Nepal in Biotechnology Lab of Nepal Agricultural Research Council (Personal communication, 2012). They have been using ELISA test too to identify whether the regenerated plants have viral pathogen still existing.

Breeding of Strawberries

Few laboratories are regenerating disease free planting materials of exotic strawberry species in Kathmandu and Kakani, Nepal.

Breeding of Papaya:

No laboratory is working using molecular markers to study and exploit the science of the crop of papaya in Nepal. Horticulturists can apply sex determining isozyme marker to differentiate female papaya seedlings from male in early age.

Breeding of Kiwi:

Private nurseries are working to regenerate and multiply disease resistance planting materials of Kiwi fruit in Nepal. Some innovative farmers and ICMOD are also producing some planting materials of grafted type.

Breeding of dwarf orange

Dwarf orange scion has been introduced from Japan and grafted onto Trifoliate orange rootstock in Kirtipur Horticulture Farm of the government in Kathmandu, Nepal. Its multiplication in the government farm as well as private is going on.

Cooperation from the people of Japan for fruit development in Nepal

Breeding of lemon have introduced some high yielding orange, kiwi fruit, pear, germplasms into Nepal through JICA (Japan International Cooperation Association). Japanese horticultural scientists worked in Kirtipur Horticulture Farm of Government of Nepal in Kathmandu. They sometimes conducted Fruit Fair in Kathmandu with Awards to the farmers who have best fruits of apple and orange. They worked in the exchange of the fruits between Nepal and Japan.

Outside the boundary of molecular markers

Different field level farms of NARC have been working in the breeding, selection and evaluation of native and exotic genotypes of different crops by  conventional breeding. They could not adopt molecular techniques to study the science of the crops to breed them for Nepalese farmers. Some of the crops are water melons, pumpkin, cucumber, chillies-capsicum, brinjal, black mung, Masyang, cowpea, Mung bean, kidney bean, colorful beans, pea, tomato, okara, cauliflower, broccoli, mustard, rapeseed, sunflower, some more are outside the boundary of plant biotechnology and plant molecular markers. In other words, they are inside fence of conventional breeding and selection.   

Conclusion

Plant molecular markers and plant biotechnology have a variety of uses that help solve many problems of crop species to alleviate poverty of our Nepalese farmers. In Nepal, the agricultural yield is very low in comparison to developing and developed countries. Farmers are facing many problems like crop losses due to insects, disease, weeds, and droughts, low fertility of soil, other abiotic and biotic stresses. Also, climate change and growing population are most burning issues which must be handled to solve the problems. Food insecurity, and poor nutrition must be practically must be addressed for Nepal people.

This is prepared in June of 2012.