Space-breeding and nuclear techniques to improve cassava as an energy crop

The Spring issue of Universitas Helsingiensis has a feature on how the latest biotech and nuclear techniques are being used to improve cassava, identified as a promising biofuel crop. The text is an abriged version of a lecture delivered by S. Mohan Jain at the Plenary Session of the 1st International Conference on Cassava Breeding, organised by Brazil's Ministry of the Environment and the University of Brasilia (1–5 December 2006, Brasilia, Brazil).

The author works at the Plant Breeding and Genetics division of the International Atomic Energy Agency (IAEA) and was awarded a 2005 Nobel Peace Prize certificate for his work as a member of staff of the International Atomic Energy Agency, the winner of the 2005 Nobel Peace Prize.

Mohan Jain outlines different breeding techniques - from classic plant tissue culture and somatic embryogenesis over innovative breeding techniques in space to nuclear techniques for inducing useful mutations - and indicates why cassava makes for an ideal bioenergy feedstock. Earlier we already referred to a program by the U.S. Department of Energy's Joint Genome Institute, where Norman Borlaug, father of the 'Green Revolution' is sequencing the crop's genome, in order to improve it with an eye on bioenergy production (earlier post). Improved cassava is set to make vast parts of the developing world prime biofuel producers.

A tropical root crop
Cassava (Manihot esculenta) is a perennial root crop, cultivated all over the tropics for its starchy tuberous roots as a valuable source of calories, and is planted on about 16 million hectares of land. The crop adapts well to a variety of soil and climatic conditions, is drought tolerant and has the ability to be grown on depleted and marginal soil.

The total annual cassava root production worldwide is 184 million tonnes, out of which 50% production is in Africa, 30% in Asia and 20% in Latin America. The average yield varies widely, e.g. 7–10 tonnes/ha in Ghana, which is far below, for example, that of India (26 tonnes/ha) and Thailand (37 tonnes/ha). The low yield in cassava-growing countries is due to poor fertilisation, drought, severe infection of the planting material (stem cuttings) with African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and the newly identified virus named South African cassava mosaic virus (SACMV), diseases, poor-quality cultivars and the short shelf-life of tuber roots. Yields in these regions can be substantially improved. Cassava is an important source of carbohydrate in adverse climatic conditions. The crop is valued in many areas as a food staple.

In Accra, Ghana, the President launched the President’s Special Initiative (PSI) under which the government will promote cassava starch. The cassava project will create a ready market for 25,000 farmers, and about 70,000 jobs would be created. In Nigeria, a similar project is underway, also called the Presidential Cassava Initiative (earlier post).

In addition, the tuberous roots of cassava can be left in the ground for several years prior to harvesting, providing security against famine. Cassava also has the highest rate of CO2 assimilation into sucrose of any plant measured, and has great potential for enhancing carbohydrate allocation to sink tissues. It is also increasingly being used in processed food and fodder products and by the chemical, pharmaceutical, paper and textile industries.

Cassava nutrition
Cassava is poor in providing sufficient nutrition to its consumers. The tubers are the main source of carbohydrates (35%), and provide a negligible amount of proteins. Fresh leaves have a much higher amount of proteins (7%) than tuber flesh (0.5–1.5%). Starch is the main carbohydrate source in root tubers, and is present in very low levels in fresh leaves. Future efforts are needed to improve cassava nutrition both in root tubers and fresh leaves by using mutagenesis and the latest biological tools, such as molecular biology. The selection of appropriate genetic material should be made from the natural and induced germplasm for the development of new cassava varieties high in nutritional values so that malnutrition and related diseases, e.g. Konzo, could be addressed. Konzo is a neuro-logical disorder and leads to spastic paralysis of the legs; and is attributed to high levels of dietary cyanide in cassava.

Cassava as a biofuel crop
In Brazil, sugar cane is a major bioenergy crop and has made this country a world leader in bio-ethanol production. Cassava has the potential to become another major bio-energy crop together with sugar cane. It is an attractive fuel crop because it can give high yields of starch and total dry matter in spite of drought conditions and poor soil. Energy requirements of cassava represent only 5–6% of the final energy content of the total biomass, showing an energy profit of 95%, assuming complete utilisation of the energy content of the total biomass:
biomass :: biofuels :: energy :: sustainability :: cassava :: ethanol :: bioenergy :: plant breeding :: space breeding :: genomics :: nuclear :: IAEA ::

Alcohol production from cassava has an overall efficiency of 32%. Cassava could become an industrial crop by developing cultivars with different starch compositions. Useful variations in native starch quality – altering the proportion of amylase to amylopectin, for instance, which changes the physiochemical properties of the polymer – could open new market niches at better prices. Molecular tools would be of great value in identifying the genes responsible for starch synthesis.

Dr. Li’s research group, Beijing, China, has developed a new sweet sorghum mutant variety Yuantian No. 1 by seed irradiation with gamma radiation. This variety has 20% more sugar than the parental lines and is an excellent source both as a feed and as a bio-energy crop, or a bio-ethanol producer.

In Thailand, a research group reported the official release of a new Thai cassava cultivar Rayong 9 with improved starch and ethanol yields. This cultivar is a successful plant type, producing good-quality stakes with a high rate of germination, as well as a large number of stakes from each plant. In Brazil, a new class of cassava (Manihot esculenta Crantz) has been identified and their storage roots show unusual free sugar accumulation and novel starch, and accumulate over 100 times more free sugar (mainly glucose) than commercial varieties.

A group in the USA suggested that transportation biofuels such as synfuel hydrocarbons or cellulosic ethanol, if produced from low-input biomass grown on agriculturally marginal land or from waste biomass, could provide much greater supplies and environmental benefits than food-based biofuels. They found that ethanol, produced from corn, yields 25% more energy than the energy invested in its production, whereas bio-diesel produced from soybeans, yields 93% more. Cassava can grow under harsher climatic conditions, and would be ideal for transport biofuel.

Biotechnology
Plant tissue culture refers to the growing and multiplication of cells, tissues and organs of plants on defined solid or liquid media under aseptic and controlled conditions. The micro-propagation technique for rapid shoot proliferation can be achieved from any part of the plant such as the shoot tip, tiny stem cuttings, roots, and auxiliary buds. Normally, commercial companies use micro-propagation extensively in large-scale plant multiplication. However, the high cost of in vitro plant production, the low volumes produced, the degree of labour intensiveness, and tissue-culture-derived plant variations all hinder the rise in profits of commercial enterprises, and therefore it is highly desirable to modify the techniques to overcome these problems for the supply of high-quality planting material to small and large commercial cassava growers.

Somatic embryogenesis
Somatic embryogenesis is an ideal technique for the clonal propagation of woody and fruit plants and genetic gain can now be achieved through it. The formation of somatic embryos from somatic cells by a process resembling zygotic embryogenesis is one of the most useful features of plants and offers a potentially large-scale propagation system for superior clones. Normally, the initiation of embryogenic cultures is done by culturing immature zygotic embryos, sometimes with mature zygotic embryos, and offshoots. The maintenance of embryogenic cultures is critical for preventing tissue-culture-derived variation. Also, it is critical to cryopreserve immediately after embryogenic cultures are initiated to prevent variation and preservation of elite germplasm. Well-developed somatic embryos are germinated to regenerate plants (somatic seedlings), which are acclimatised, and then finally transferred to the field. Somatic embryogenesis is highly genotypic dependent, and it would be useful to modify the culture medium accordingly. For large-scale production of somatic embryos, a ‘bioreactor’ system works well, e.g. the ‘temporary immersion system’ (RITA bioreactor). The low cost of production of somatic embryos and the high germination rate are highly desirable for large-scale production in a bioreactor. This system has yet to be tried in cassava.

Nuclear techniques for mutagenesis
Nuclear applications in food and agriculture have contributed greatly to enhancing agricultural production of seed and vegetative propagated crops (see IAEA). Even though nuclear technology has greatly benefited agriculture, it still has immense potential in the genetic improvement of cassava and other crops. More than 2300 mutant varieties have officially been released in many countries (see the joint FAO/IAEA database on mutant varieties.).

Both chemical and physical mutagens are used to induce mutations. Among them, gamma rays and ethyl-methane sulphonate (EMS) are widely used for mutation induction. Fine embryogenic cell suspension cultures are most suitable for inducing mutations by transferring the cultures onto filter paper and then plating them on agar-solidified culture medium for gamma irradiation. Initially the LD50 (lethal dose) dose is determined, which is used as an optimal dose for mutation induction. Irradiated cells are further cultured in the fresh medium for the development, maturation, and germination of mutated somatic embryos. This approach provides mutated somatic seedlings in a short period of time and also prevents chimeras, which otherwise requires the plants to be multiplied up to the M1V4 generation for chimera dissociation. Alternatively, shoot tip or bud wood can be irradiated and the plants multiplied up to the M1V4 generation to produce pure mutants by dissociation of chimeras.

Sung and Somerville (USA), working on Arabidopsis thaliana, have discovered a mutation, called “pickle”, in plants that mimics what happens in seeds, which typically is the accumulation and storing of proteins and oils. This mutation in plants causes the accumulation of large amounts of oils, proteins, and starch in the taproot. This finding could also make possible the creation of more nutritious root crops with a better balance of oil, protein, and starch, e.g. in cassava and other root crops.

Space-breeding concept
Space conditions can induce mutations of plant seeds, and can be helpful in accelerating crop breeding. It may be possible to obtain rare mutants that may make a significant breakthrough in important economic characteristics of crops, such as yield and quality, which are difficult to get using other breeding methods on the ground. The plant seeds are sent into space in a space rocket, and when the rocket is back on earth, the plant seeds or in vitro shoot cultures or microspores are studied to ascertain the influence of cosmic rays on the generation of new mutants.

There are only a few countries involved in this type of work, and China is one of them. Since 1987, 13 recoverable satellites have been used by Chinese scientists and researchers to carry more than 80 kilograms of plant seeds belonging to over 70 species, involving their main cereal, fibre, oil, vegetable, and melon and fruit crops. Through ground planting and selecting experiments by breeders in more than 50 research units covering more than 20 provinces, cities and regions in China, good results have been achieved. More than 20 mutant varieties have been developed and officially released. In rice, a new variety EYH No. 1 has been released that gave a total yield 14.5 tonnes/ha.

Space breeding involves a big investment and good technological support. The opportunities for conducting a space experiment are very limited. It is important to simulate on the ground the conditions of space in order to conduct research work which would reveal how space-induced mutations occur and then to apply the mechanism to plant breeding.

Future prospects
Cassava mutants could be developed to produce value-added biomass for cost-effective production of bio-ethanol. The use of this crop as a source of bio-energy would generate employment, enhance the economic status of its growers, protect the environment, and most likely cut the consumption of fossil fuel. Arable land for growing cassava may have to be increased for bio-energy production, as would the export of bio-ethanol to energy-hungry countries such as China and India. Brazil has already started producing bio-ethanol from cassava. African countries should also follow Brazil and they could become a major source of bio-ethanol production. This can be achieved through biotechnology and mutation, and also the exploitation of natural cassava germplasm/genetic variation for breeding. Biotechnology is an additional tool to assist plant breeders, and can be helpful in reducing the time to develop a cultivar.

To date, a lack of communication between plant breeders and biotechnologists has hindered crop improvement; however, as growers are now faced with maintaining sustainable crop production under climate change conditions and an ever-growing human population such cooperation becomes essential.