Breeding for Nutrient Efficiency: A Model for the Future

K.K.Vinod

Having identified a plethora of putative QTLs that contribute to nutrient use efficiency, the logical next step is to design appropriate selection strategies for improving genotype efficiency against low nutrient stress. However, not-so-significant effects of the identified QTLs are a serious concern in designing robust MAS strategies, since QTL-by-environment interaction may jeopardize the selection process. Further, the arcane effects of the identified QTLs confound the scientific soundness of the selection programmes. It is unlikely that many major-effect QTLs may emerge for nutrient use efficiency in future. This reduces us to the option of using small effect QTLs for future rice improvement.

One of most encouraging observations in the abiotic stress breeding so far is the interoperability of abiotic stress resistance across the causative factors. Armed with strict phenotyping tools, it is likely that an integrated approach of marker assisted selection may help us in improving abiotic stresses together, rather than the individualistic approach for each causative factor. I prefer to propose a model of recurrent selection for improving nutrient use efficiency, by channelizing the genotypes that has been selected for drought, salinity, submergence, and for nutrient use through an integrated breeding programme that also select for N and P use efficiency together. This process offer the advantage of including any new genotype in the breeding chain at any time, while a new genotype can emerge at any stage that are suitable for various target environments. This program will be more resource abundant, including beneficial gene flow, which is otherwise not the case in targeted breeding programmes for individual stress resistance.

Marker Assisted Selection

K K Vinod

Primary objective of any plant breeding programme is selection of the best genotype from an array of breeding lines (genotypes). Conventionally, selection involves various traits that are expressed in plants (phenotypes) and the selection tools employed by the breeders involve qualitative (visual or perceivable traits) and/ or biometrical (quantitative) traits. However, since gene expression is always modified by the environment due to several adaptive reasons, selection of traits is not always as successful as it should be. This led to the thinking of selecting directly for the genes themselves, as the genes are coded on the DNA molecules that are free of environmental interference. However, selection of genes was not an easy job as it appeared to be, because of their obscure locations on the genome. Hence, this is achieved indirectly by selecting detectable DNA variations in the individual genomes, which are either associated or closely linked to the target genes, as detected by the co-segregation of these fragments with phenotype. Such detectable DNA fragments are called markers. Use of these molecular markers has opened a novel way for selection known as marker assisted selection or marker aided selection (MAS). Extensive use of molecular markers by the present day breeders has opened up a separate field of study, the molecular breeding.

Principles of MAS

As the name indicates, MAS is not a breeding method but markers are used as a selection aid. We consider that traits may be typically controlled by single or many genes. Although in strict sense no trait is controlled by a single gene but a group of genes, the practical consideration of mono, oligo and polygenes revolves around the number of genes that produce the perceivable quantum effect of the trait. Any detectable DNA fragment lying around or within these genes can report the presence of this gene in its carrier. This forms the fundamental idea behind MAS. MAS therefore can involve in selection for both qualitative and quantitative characters. However, MAS is not generally advocated for the selection of easily selectable traits, especially those qualitative traits with high heritability and penetrance, while for traits with low heritability and low expressivity MAS may be a good option.

Since markers tag for genes, MAS is useful mostly in transfer of genes from a donor to a recipient. Therefore the donor should have a distinguishable marker allele that is linked to the target gene. This marker allele should be distinctly different from the allele produced by the same marker in the recipient genotype. Now while crossing both donor and recipient it is easy to determine which progeny carry the gene by identifying the linked marker allele. The target allele of the gene may be either a dominant one or recessive. Most commonly used procedure in transfer of single gene from donor to recipient is backcross breeding. Since the recipient genotype is used repeatedly in crossing steps in the backcross process it is also called recurrent parent (RP). Using markers, the backcross breeding proceeds by selecting progenies that are carriers of the target allele until a stable homozygous genotype is obtained which has almost entirely of the recipient genome carrying the target allele. This is called marker assisted backcross breeding (MABB). Depending on the transfer of dominant or recessive allele backcross procedures may suitably vary.

Conservation of plant genetic resources

K.K.Vinod

Maintenance of biodiversity is an essential prerequisite for the continued production of new cultivars of current crops, for the development and exploitation of newer crops.

In situ conservation is the conservation in any habitat where the germplasm normally occurs, not only in natural habitats, but also in farms, gardens and other man-made habitats of relevant germplasm. Ex situ conservation on the other hand refers to any collection maintained outside the normal habitat of the germplasm including seed collections and in vitro tissue cul­ture, and also living collections in botanic gardens and collections of spe­cies with recalcitrant seed.

The objective of in situ conservation, at least for agricultural purposes, is to conserve the maximum possible number of alleles and/ or maximum possible diversity of genotypes whilst permitting continued evolution. This is of importance in generating new genes or genotypes, particularly: (i) in response to changing environments, e.g. genes for resistance to newly evolved strains of pathogens; and (ii) by continued selection of landraces by farmers or gardeners (at least where law still permits). Additional ben­efits include conservation of much more biodiversity - entire ecosystems - than just the targeted crop germplasm. Against this is the disadvantage that the germplasm cannot be efficiently utilized because characterized genotypes cannot be readily tracked.

The objective of ex situ conservation is to maintain a collection contain­ing as many alleles as possible, and/ or as diverse a range of gene combina­tions (i.e. genotypes) as possible, in a form that can be readily utilized for breeding and research. For efficient utilization, genetic variation within the collection must be appropriately characterized. For efficient conservation, ideally the collection should be as small as possible commensurate with conserving maximum diversity. In practice, in the face of rapid genetic ero­sion it is often necessary to collect germplasm before it can be ascertained that it contains genes or genotypes not already present in the collection: most collections are therefore considerably larger than strictly necessary for the diversity they contain.

There are three groups of implica­tions for efficient conservation.

First, efficient collection of diverse germplasm for ex situ conserva­tion depends on having good knowledge of the spatial distribution of genetic diversity. Inevitably it is not possible to know the exact location of every genotype. Instead, a good understanding of the factors that control the distribution of genetic diversity is necessary to devise a collecting strat­egy that maximizes the diversity sampled.

Second, in addition to efficient construction of an ex situ collection, effi­cient maintenance of the same also depends on good understanding of the factors that control the distribution of genetic diversity - in this case to control the genetic shifts that occur whenever a population is sampled, subsampled or regenerated.

Third, efficient in situ conservation depends on good knowledge of the distribution of genetic diversity in space and time, and of the factors that control its distribution. In particular, an in situ conservation area must have a size, heterogeneity and structure that maximize genetic variance maintained by evolution.

The efficient achievement of both the primary objectives requires a knowledge of the genetic nature of variability, population structure, the distribution of diver­sity and the factors that control them.

Genetics of Sugarcane Quality Traits and Breeding for Quality Improvement

K.K.Vinod

The quality traits in sugarcane depend on the end use of the cane in the industry rather than a common list of quality traits.  The main parameters are listed in the table below.

Major quality traits in sugarcane

Industry	Quality traits
Sugar Industry	Sugar recovery Sucrose content in juice Purity coefficient of juice
Distillery	Higher juice volume Higher total sugars
Paper Industry	Higher cellulose Lower Ash content Lower Lignin content Lower Pith content or higher Fibre/pith ratio Lower colouring matter in bagasse Higher L/D Ratio of ultimate fibres

The novel idea of using the wild species Saccharum spontaneum (as male with S. officinarum as female) in sugarcane breeding with the objectives of incorporating gene complexes for biotic and abiotic stresses and for high biomass production resulted in a tremendous level of useful genetic variability including that of better cane quality, sugar content and incorporation of rationing ability. This paved the way for revolutionising sugarcane agriculture and sugar industry not only in India, but also in most of the sugarcane growing countries of the world. Most, if not all, of the present day varieties world-over have the Coimbatore bred canes in their genealogy.

In India, Co 419 released in 1933 became the most popular variety in tropical India and was rightly hailed as the 'wonder cane' the world-over. Co 997 and Co 1148, released in 1950s, became the ruling varieties in Andhra Pradesh and North India respectively. Co 1148 remained the most predominant variety for over four decades in sub-tropical belt and was hailed the 'Wonder Cane of North India'. Co 62175 became the most sought after variety by jaggery farmers owing to its heavy yield. Varietal evaluation conducted across seasons and different months of juice analysis helped in the identification of varieties with high sucrose levels such as Co 7204, Co 7704, CoA 7601, CoC 671, Co 8336, Co 8338 etc.

In the USA, evaluation of F₁ crosses between commercial hybrids and Miscanthus and Erianthus (as male parents) indicated that thin stalk and low sucrose content were strongly influenced by Miscanthus and Erianthus. However, mean sucrose content and purity of F₂ and BC₁ seedlings of crosses between commercial hybrids and Miscanthus spp., Miscanthidium spp. and Erianthus spp. were markedly improved over that of F₁ hybrids, but mean stalk diameter was still very small.

In Taiwan, sugar content in a Saccharum × Miscanthus hybrid showed a steady increase in successive nobilized generations. Clones selected from F₁ Saccharum × Miscanthus hybrids were productive in fibre yields, with better pulp sheet as compared to a commercial control. Some clones from BC₁ crosses involving sugarcane and sorghum had sucrose content equivalent to commercial controls and proved to be early maturing (TSRI, 1995-96).

In sugarcane breeding, the available evidences indicates that selection of parents based on their juice quality would be successful in increasing the juice quality of the succeeding generation, but selection of parents followed by progeny testing would seem to be necessary for increasing cane yield. Studies on milling characters showed that starch content in juice, percentage of reducing sugars, fibre percent fresh weight has high heritabilities and hence both parental selection and individual selection would be effective for these characters. Roach (1968) found highly significant male × female interaction effects for yield and sucrose in S. officinarum × S. spontaneum crosses. Heterosis was observed for early growth, stalk height, cane yield, flowering, pollen production and level of reducing sugars.

Inbreeding depression for components of cane yield viz., stalk diameter, stalks per plant, and stalk length was reported but there was no apparent depression for brix. This suggested that most of the genetic variance for brix be additive in nature.  However, it is important that non-additive genetic variance for cane yield and its components is required in sugarcane. This indication is supported by the few studies that have provided estimates of additive and non-additive genetic variances (Hogarth, 1987).

Studies on 14 crosses between commercial hybrids (female) and S. spontaneum (male) and found families to differ significantly for stalk number, stalk diameter and cane quality characters. Narrow-­sense heritability estimated from the partitioning of variances between and within family ranged from relatively low for dry matter % cane (Brix % cane + fibre % cane) (0.17) to moderately low for stalk number and fibre % cane (0.39 and 0.38, respectively) and moderately high for cane quality characters (0.67-0.90) and stalk diameter (0.75). These estimates were generally higher than those obtained from commercial × commercial crosses. The distribution of Brix, sucrose content, juice purity and fibre content in crosses between commercial hybrids and S. spontaneum clones were continuous and controlled by polygenes, and additive genetic variance was more important than dominance genetic variance for all four characters.

In experiments using diallel crosses, specific combining ability (indicating non-additive genetic variance) has generally been greater than general combining ability for both cane yield and sugar content as measured by brix. However, due to the possibility of self-pollination in such crosses, these results should be interpreted cautiously.

Estimates of heritability and clonal repeatability on an individual basis for three milling characters were reported in sugarcane.

Heritability and clonal repeatability of milling characters in sugarcane

Character	Heritability	Clonal Repeatability
Starch (log ppm)	0.53 +/- 0.14	0.58+/-0.04
Reducing sugars % extract	0.31 +/- 0.13	0.57+/-0.04
Fibre % fresh weight	0.34 +/- 0.15	0.72+/-0.03

This indicated that progress from selection of parents should have some value, particularly for starch content. Progress from clonal selection should be very good for all characters even on an individual basis. Studies on the inheritance of ash in juice showed that for ash percent in juice, 95% of the genetic variance was additive. Thus, there should be no difficulty in breeding for this character, if it is deemed desirable. However, the authors pointed out that selection based on ash content might reject many varieties with good agronomic characters.

Increasing fibre content of commercial varieties but still maintaining an acceptable sugar content was thought to be an attractive economic option that could cut down on imported coal and also reduce CO₂ emissions. In the case of fibre percent fresh weight. 80% of the genetic variance is additive, and the character is moderately heritable on an individual basis (h,² = 0.45), indicating that selection of parents for low or high fibre content should be effective in producing the progeny with desirable fibre content.

Evaluation of S. spontaneum, S. robustum, S. sinense, S. officinarum, commercial hybrids and Erianthus spp. for cane quality components, Brix % cane (total solids of juice), pol % cane (apparent sucrose), fibre % cane and dry matter % cane, by the method described by Saint Antoine and Froberville (1964). A ratio of pol : fibre (PF) was derived from the pol % dry matter and fibre % dry matter. Juice purity was also derived from the ratio of Brix % cane and pol % cane. These studies revealed high levels of fibre and dry matter content in S. spontaneum clones as compared with S. officinarum. Commercial hybrids ranked very close to S. officinarum, and the Erianthus group ranked very close to S. robustum. Brix % cane and pol % cane were the highest in S. officinarum and commercial hybrids whereas sucrose content of the Erianthus group was even lower than that of S. spontaneum. The PF ratio showed equal partitioning of dry matter between pol and fibre in the S. officinarum and commercial clones.

Many correlations between agronomic and milling characters were found in sugarcane. The genotypic correlation between cane yield and fibre percent was 0.57 and for sucrose percent and fibre was –0.76. Significant positive correlations between fibre content and brix was also reported. A very weak correlation between fibre content and brix in juice as well as negative correlations between brix and ash were also reported in sugarcane. This indicated that selection for brix might produce varieties with lower ash content.

With further developments, microsatellite markers could soon become a tool for genetic fingerprinting, genetic mapping and to assist in selecting specific, genetically diverse parents for use in introgression. The development of marker assisted selection would have a major potential use in introgressing a single quantitative trait locus (QTL) or multiple QTL from an ancestral species into commercial cane. The search for molecular markers for sucrose genes to assist in breeding and selection could reduce the number of backcrosses necessary to recover the recipient genotype near to the donor target genes.

UV-B Radiation Stress in Plants

K.K.Vinod

Our nearest star, the sun, emits short wavelength radiation that is incident on the earth’s atmosphere. Most of the radiation in the atmosphere is infrared radiation (700-3000 nm, 67% of the photons) and visible light (400-700 nm, 28%; Nobel, 1983). Ultraviolet radiation (UV, 200-400 nm), on the other hand, reaches the atmosphere in smaller amounts (5% of the photons). The biologically most hazardous part of UV radiation, i.e. UV-C (200-280 nm) and UV-B (280-320 nm) below 290 nm, are completely absorbed by the stratospheric ozone (O₃) layer and by other oxygen molecules in the atmosphere (Frederick, 1993). In addition, the ozone layer absorbs some longer-wave UV-B and UV-A radiation (320-400 nm) (Fig. 1). Consequently, of the photons at the earth’s surface, only about 2% are in the ultraviolet range (Nobel, 1983). However, of the total solar energy reaching the earth’s surface, UV-B radiation comprises about 1.5% and UV-A radiation about 6.4% (Frederick et al., 1989). The intensity of UV-B radiation, in particular, is affected by the thickness of the ozone layer, which in turn varies periodically as a consequence of natural processes such as seasons, winds and solar cycles. In addition, latitude, time of year and time of day determine the length of the path of a UV-B photon through the absorptive ozone layer (Caldwell et al., 1980). 

Fig 1. Ultraviolet spectrum 

On average, the ozone concentration in the stratosphere is low, i.e. about ten ozone molecules per million molecules of air; and it is highly dynamic because the ozone molecules are created and destroyed continuously. However, since the 1970’s, human activities have disrupted the natural balance between synthesis and breakdown of ozone. Depletion of the ozone layer has repeatedly been reported to occur over Antarctica, but in the 1990’s there were also frequent occurrences of major spring-time ozone depletion over the Arctic. It has been found that the main man-made compounds responsible for enhancing ozone breakdown are the chlorofluorocarbons (CFC) (Fig. 2) and nitrogen oxides. Recently, it was also found that the increasing concentrations of greenhouse gases result in stratospheric cooling, thus creating suitable conditions for breakdown of ozone molecules. Therefore, the most recent predictions based on stratospheric chemistry and climate-change models estimate that in the northern areas (60-90° N), compared with the long-term means, the maximum springtime UV-B radiation will increase up to 50-60% in 2010-2020. 

Fig 2. Chlorine atoms induce the decomposition of two ozone molecules into three oxygen molecules in a net chain reaction in which the chlorine atoms are regenerated so that decomposition of ozone continues. 

Effect of UV-B radiation in plants

Elevated levels of UV-B radiation will have many direct and indirect effects on plants (Fig. 3). Even present-day levels of UV-B radiation affect the growth and development of plants. The direct effects of UV-B radiation on plant cells are mostly damaging, because UV-B photons have enough energy to create lesions in important UV-B-absorbing biomolecules such as nucleic acids and proteins. It is known that the photoproducts of DNA formed by UV-B radiation, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone and (6-4) photoproducts, are all toxic and mutagenic. In addition, the altered DNA and RNA structures may interfere with transcription and replication; and therefore protein synthesis may be slowed down during UV-B stress. In order to avoid the effects of DNA damage, plants have efficient systems for DNA repair, including photoreactivation and excision repair, which are involved in restoring the structure of genetic material during exposure to UV-B radiation. However, the indirect effects of UV-B on plant cells can also be damaging: UV-B radiation may cause oxidative damage in chlorophylls and polyunsaturated lipids by increasing the formation of free radicals and peroxides (Jordan, 1996). To prevent oxidative damage, cells contain antioxidants, e.g., phenolic compounds, that scavenge the free radicals. Phenolic compounds have variable antioxidant properties; and several studies have shown that during UV-B exposure, the production of compounds with efficient antioxidant structures, such as additional hydroxyl groups on ring B of the flavonoid skeleton, is favoured. Plant cells also contain enzymes, e.g., superoxide dismutase (SOD) and catalase, which scavenge superoxide radicals and protect the cells against H₂O₂, respectively. 

Fig. 3. Effects of UV-B radiation on plant system.

Signal transduction and gene expression 

In addition to damaging plant cell components, UV-B radiation often exerts its effects through altered patterns of gene activity; e.g., the effects of UV-B radiation on photosynthesis, UV-B-screening phenolics, growth, reproductive processes, plant form and timing of life phases, are all caused by altered gene action. The mechanisms by which plants perceive UV-B radiation are not fully understood, but it has been suggested that direct absorption of UV-B by DNA could result in the formation of a “signal” that regulates the transcription of genes.

Signal transduction pathway in relation to UV-B radiation stress. Reactive oxygen species (ROS) increase in response to UV-B and are an important component in the regulation of both up-regulated and down-regulated genes. The nature and origin of the ROS involved in the early part of UV-B induced signalling pathways have been investigated in Arabidopsis thaliana. The increase in PR-1 transcript and decrease in light harvesting chlorophyll binding gene (Lhcb) transcript in response to UV-B exposure was shown to be mediated through pathways involving hydrogen peroxide (H₂O₂) derived from superoxide (O₂^•-). In contrast, the up-regulation of PDF1.2 transcript was mediated through a pathway involving O₂^•-- directly. The origins of the ROS were also shown to be distinct and to involve NADPH oxidase and peroxidase(s). The upregulation of CHS by UV-B was not affected by ROS scavengers, but was reduced by inhibitors of nitric oxide synthase (NOS) or NO scavengers. Together these results suggest that UV-B exposure leads to the generation of ROS, from multiple sources, and NO, through increased NOS activity, giving rise to parallel signalling pathways mediating responses of specific genes to UV-B radiation (Fig. 4).

In addition, it has been hypothesized that in plant cells, specific UV-B photoreceptor-mediated signalling processes regulate gene expression. However, the characteristics of a UV-B photoreceptor and how the signals are transduced after UV-B perception, are not yet known.

In addition to the increase in ROS other known signal transduction intermediates increase their levels. These include salicyclic acid (SA), jasmonic acid (JA) and ethylene. Using Arabidopsis mutants that are insensitive to SA, JA and ethylene (NahG, jar 1 and etr 1-1 respectively), clear differences in gene activity response have been demonstrated. For instance, an increase in expression of the two pathogen-related genes PR-1 and PDF1.2 are depended on SA and ethylene or JA and ethylene respectively. In contrast, down-regulation of RNA transcripts for photosynthetic proteins was independent of all three compounds. Furthermore, although ROS is involved in down-regulation of RNA for photosynthetic proteins, the chloroplast signal may not be involved.There are at least three separate signal transduction pathways involved in UV-B gene regulation and substantial “cross-talk” must take place.

A unique response of plants to UV-B radiation relates to the property of high photosynthetically active radiation (PAR) to ameliorate its damaging impact. The interaction of UV-B and PAR was initially discovered at the physiological level, but Jordan et al., (1992) demonstrated that high PAR also reduced down-regulation of gene expression. This ‘protection’ against UV-B damage did not involve synthesis of protective pigments, but was related to the function of the photosynthetic apparatus itself. The photosynthetic system can act as a photoreceptor and specific wavelengths can change chloroplast gene expression. Research conducted by Jordan et al., (1994) on etiolated tissue is also indicative of a strong link between the development of the photosynthetic apparatus and UV-B-induced gene expression. The connections between UV-B radiation and photosynthesis, and the signal transduction pathways that lead to modification of gene expression are yet to be comprehended.

Fig.4.  Schematic illustration of signal transduction pathways induced by UV-B radiation

It was found that in genetically modified tobacco (Nicotiana sylvestris) increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin indicating that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.  

References

Caldwell, C.R. 1993. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro. Plant Physiology 101:947-953.

Frederick, J.E. 1993. Ultraviolet sunlight reaching the Earth’s surface: a review of recent research. Photochemistry and Photobiology 57:175-178.

Nobel, P.S. 1983. Biophysical Plant Physiology and Ecology. Freeman & Co., New York, pp.185-238.

Jordan, B.R. 1996. The effects of ultraviolet-B radiation on plants: a molecular perspective. In: Callow, J.A. (editor). Advances in Botanical Research, Vol 22. Academic Press, New York, pp. 97-162.

Jordan, B.R., P. James and S.A-H. Mackerness. (1998). Factors affecting UV-B induced changes in Arabidopsis thaliana gene expression: role of development, protective pigments and the chloroplast signal. Plant & Cell Physiolog, 39: 769-778.

Jordan, B.R., J. He, W.S. Chow and J.M. Anderson. Changes in mRNA levels and polypeptide subunits of ribulose bisphosphate carboxylase in response to supplemental UVB radiation. Plant, Cell and Environment 15: 91-98, 1992.

Why I am proud to be a Plant Breeder?

Plant breeding was the science which existed as long as the humans did... It probably started when the first man was hungry.. and when he selected what to eat... and when he probably thought to have his favourite food near his home.... He did exploration, collection and conservation of the grass and grains he needed... he grew the fruits he was fond of... He did selection of the best among the natural variability...Later, he started agriculture...and finally, selecting those types which yielded more than the one he was growing earlier.

PLANT BREEDING was continuous process...Earlier man never knew how variation occurred...and never knew how it was occurring... and how it perpetuated.

The science of GENETICS opened up a whole new area of knowledge.. the knowledge which catered the needs of ever growing global population.. The knowledge which is going to sustain the generations to come..

COME...LET US PARTAKE THAT GREAT KNOWLEDGE