eChapters

Procedure

Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.

Heat the tube in a boiling water bath for 1 hour.

Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.

Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.

Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding.

Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours.

Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid.

After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

DNA Migration in Agarose and Polyacrylamide Gels

Recommended Gel Percentages for Separation of Linear DNA

Mole (mol)

1 mole of any substance contains 6.02 x 10²³ units. The number of moles in a given weight of substance can be calculated using following equation.

1. pH Meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

2. UV Spectrophotometer

Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Search for genetic material—nucleic acid or protein/DNA or RNA?

• Griffith’s Transformation Experiment
• Avery’s Transformation Experiment
• Hershey-Chase Bacteriophage Experiment
• Tobacco Mosaic Virus (TMV) Experiment

Timeline of Events

• 1890 Weismann - substance in the cell nuclei controls development.
• 1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.
• 1928 Griffith’s Transformation Experiment
• 1944 Avery’s Transformation Experiment
• 1953 Hershey-Chase Bacteriophage Experiment
• 1953 Watson & Crick propose double-helix model of DNA
• 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material

BC

1750 The Sumerians brew beer.

500 The Chinese use moldy soybean curds as an antibiotic to treat boils.

250 The Greeks practice crop rotation to maximize soil fertility.

100 Powdered chrysanthemum is used in China as an insecticide.

AD: Before the 20^th Century

1590 The microscope is invented by Janssen.

1663 Cells are first described by Robert Hooke.

1675 Leeuwenhoek discovers protozoa and bacteria.

1745 Maupertuis proposes an adaptationist account of organic design

1797 Jenner inoculates a child with a viral vaccine to protect him from smallpox.

1802 The word “biology” first appears.

The Laboratory Worker is Surrounded by Many Dangers such as from..........

Handling of infectious materials.

Handling of broken glassware.

Accidental spill of corrosive reagents.

Swallowing of corrosive reagents such as concentrated Sulphuric acid,

Hydrochloric acid, sodium hydroxide, trichloroacetic acid etc.

Swallowing of infectious specimen.

Inhalation of poisonous fumes.

Infection in the conduct of necropsies.

Potential hazards in the form of inflammable chemicals and gas leakages.

Maize (Zea mays L.) has tropical origin and is traditionally grown in monsoon. The crop is grown in diverse geographical and climate conditions around the world and in India maize is grown in kharif (rainy season), rabi (winter season) and spring seasons where more than 70 per cent of the production happen in kharif alone. Of late, the area under rabi maize has increased because of higher productivity and ever increasing demand.

Insect pests of maize

Compared to most cereals, maize faces fewer biotic and abiotic constraints in production. Among biotic stresses, insect pests are major constraints in the production and productivity of maize crop. The earlier literature cites over 160 insect and mite species which attack maize crop (Fletcher, 1914, 1917; Ayyar, 1940; Bhutani, 1961; Pant and Kalode, 1964) but later Mathur (1987) observed over 250 species of pests associated with maize in field and storage conditions. Dick and Guthrie (1988) identified 87 species that directly or indirectly exert severe stress on corn culture in tropical and temperate regions throughout the world. Excluding stored grain insects, Luckman (1978) lists 34 pests or pest groups for which chemical controls are recommended on corn in the United States. There have been more than 130 insect pests reported to cause damage to maize in India but only about a dozen cause economic losses (Sarup et al., 1987).

Shrinking land resources, vulnerable market of cash crops along with climatic aberrations have adversely affected the Indian agriculture and as a result, small farmers are increasingly alienating from this profession. To sustain their livelihood, farmers are increasingly adopting the alternative sources and dairy farming is one of the best-suited businesses, which can rightly fit at this stage. It is fast emerging as a potential business in rural India, particularly in the peri-urban belt. Among the major inputs required to set up a dairy unit, round the years availability of good quality fodder is the foremost requirement. Green forages are rich and cheapest source of carbohydrates, protein, vitamins and minerals for dairy animals. The importance of forages in our country is well recognized since feeding forages alone accounts for over 70% of the cost of milk production. Hence by providing sufficient quantities of green fodder instead of costly concentrates and feeds to the milch animals, the economics of dairy unit can easily be maintained. Milch animals need around 40 kg of green fodder daily for better milk production and for maintaining good health of the animal. There is a large number of fodder crops grown in our country, the majority being dominated by pearl millet, sorghum, maize and oats in the non-leguminous and berseem and lucerne in the leguminous category. Pearl millet and sorghum are extensively grown as cereals in the southern and dryer regions of the country and the stover remained is used as animal fodder. However, the nutritional quality of the stover is very poor due to higher content of indigestible lignin and low levels of cellulose, whereas, the cultivated green fodders are mostly confined to the North-Western plain zones of Punjab, Haryana and Western Uttar Pradesh. The availability of round the years quality green fodder, therefore, seems to be a tough task. At present, the country faces a net deficit of 61.1% green fodder, 21.9% dry crop residues and 64% feeds (IGFRI Vision, 2030).

Foodgrains are a fine example of diversity of nature with many varieties and varied features. They are storable food commodities and lend themselves to numerous processing manipulations. Nutritionally foodgrains are rich source of almost all known nutrients and several phyto-chemicals. The increasing incidence of metabolic diseases are attributed to unbalanced energy rich diets lacking in fiber and protective bioactive compounds such as micronutrients and phyto-chemicals (Prakash, 2013). Cereals along with pulse combinations constitute an important part of human diet in many parts of the world because of easy availability, low cost, long shelf life and nutritional balance. During recent past, India has made significant progress in improving the food security of its masses. The green revolution of 60’s helped the country in achieving the food security by improving the availability and access components (Kaul et al., 2016). However, surplus quantity of food produced is not uniformly distributed and the quality component is not addressed properly which has a direct impact on nutrition. In spite of surplus production of foodgrains still malnutrition persists. However, it can suitably be overcome by combining cereals with good quality pulses and other value addition techniques which will go long way in solving the problem of malnutrition. Even though country is producing large quantity of coarse grains particularly maize but its utilization for direct consumption is very low which is mainly due to poor quality of protein. Although maize contains higher proportion of protein (10-12 %), its quality is poor as compared to that of protein in rice due to presence of high concentration of alcohol soluble protein fraction ‘prolamine’ also known as ‘Zein’ in the endosperm. Zein is very low in some essential amino acids, mainly lysine and tryptophan which contribute to more than 50% of the total protein. On the other hand, very high amount of leucine and imbalanced proportion of isoleucine contribute to poor quality of protein in normal maize. To overcome such deficiencies, scientists developed Quality Protein Maize (QPM) where the quality of protein is not only higher than the common maize, but also significantly higher than that of other cereal grains. In the recent past QPM has got special distinction among the cereals due to presence of high amount of essential amino acids like lysine and tryptophan. QPM can be utilized for diversified purposes in food and nutritional security as infant food, health food, convenience food, specialty food and emergency ration. In India maize is consumed mainly in the form of boiled corn or roasted as pop corn. In order to increase the proportion of maize for direct consumption, several traditional food-processing and preparation methods can be used at household level to enhance the bioavailability of micronutrients in maize based diets. These methods include alkali cooking, thermal processing, mechanical processing, soaking, germination/ malting and fermentation. In countries like Central America and Africa, it is converted into food products by grinding, alkali processing, boiling, cooking and fermentation (Srilakshmi, 2008).

Maize or corn (Zea mays L.), which was first domesticated in Mexico, is the most widely grown cereal crop in the world. Maize is consumed as feed and food, and also has myriads industrial applications, including the production of bioethanol and starch. Due to its highest genetic potential, production and productivity among the cereal crops, maize is called as “Queen of the Cereals”. Noble Laureate Dr. Norman E. Borlaug, the Father of Green revolution stated 15 years ago that “The past two decades saw the revolution in rice and wheat, the next few decades will be known as maize era”. In India, maize is the third largest grain crop and its production and demand is increasing rapidly. In India, it is primarily a non-food cereal crop, with only 10% being consumed as direct human food, while the rest being used for feed, industrial and export purposes. The effective deployment of various frontier technologies viz. genetic engineering, doubled haploids, molecular markers, phenomics etc.has greatly helped in raising productivity of maize crop worldwide in the last decade.The present chapter provides an overview on role of frontier technologies in maize crop improvement.

Seed in agriculture recognized as one of the most vital and crucial input in agriculture ever since the human kind has started tilling the land to produce the food for his existences. Seed technologist defined seed as matured fertilized ovule consisting of an intact embryo, endosperm and or cotyledon with protective covering (seed coat). A quality hybrid maize seed production system ensures design of the production technology which enables to maintain the healthy embryo, endosperm and /or cotyledon with protective covering. In fact the search of quality seed had acted as a major force to evolve the science of plant breeding. Ever since the farmers understood the importance of seed, they started trading the quality seed within community and now between geographies. Self-pollinated crops possess the natural mechanisms to maintain the intactness of genetic constitution of a seed during multiplication as there is very minimal chance of natural crossings. On the contrary, cross pollinated crops like maize requires special techniques in seed multiplication to maintain the genetic constitution of a F₁ hybrid. Hybrid Maize seed production techniques are well defined and are used by public and private sectors to produce the required hybrid seed in India. In this chapter, I will be highlighting the process, potential agro-climatic zones, hybrid maize seed production capacity and its future perspective in India. In addition, it also reviews the current system and possible strategic move in order to support the maize seed production system and steps required to meet future demand.

Maize is a miracle crop and is popularly known as “Queen of Cereals” due to its very high yield potential. It’s a leading crop worldwide in terms of area, production and productivity and is suitable to diverse agro-climates. Its extent of adaptability is unmatched by any other crop. Due to this adaptability to diverse climates, maize had been grown by many generations in different parts of the world. Earlier, the primary utilisation of maize was focused towards human consumption and even today it is a staple food of many underprivileged societies especially in African countries and certain tribal pockets in India. It is playing an important role as a cheap source of carbohydrate in daily meals due to high productivity and wider adaptability. With the passage of time the productivity increased due to conscious selection towards high yield by farmers and researchers. This enhanced production and productivity opened new doors of utilisation of this wonderful crop. Maize found its place as a feed crop for economically important anima ls like chicken and swine. This shift towards feed industry got encouragement by change in the food habits of people. Looking at the recent consumption pattern of maize as a food crop displays declining trend. As per a NSSO survey (2014) maize consumption in rural areas in India has seen a decline of around 35% from 3.7 kg/year in 2004-05 to 2.4 kg per year in 2009-10, and further declined to 1.56 kg during the year 2011-12. Continued efforts towards yield enhancement and decline in human consumption made its way to utilise surplus maize in economically viable industrial products. Maize is a crop where every part of its plant can be utilised to produce food and non-food products. Oil, starch, glucose, dextrose and sorbitol are few important products produced by processing of maize grains. Apart from these, maize has been very widely used for production of ethanol which is used as a fuel in many developed countries. Direct utilisation of maize grains in poultry and other feed industries is on the rise with increasing demand of such foods. Maize oil is of high quality and is being widely used in developed countries. Maize is a major source of industrial starch which finds its way to diverse utilization such as food and beverage, pharmaceutical, textile, paper and other industries. Processing of maize is done by two ways- Dry Milling and Wet Milling. Wet milling produces starch, gluten and husk and Dry Milling produces corn meal, germ, grits and animal feed. Apart from these processed products, maize is also being developed to be utilised as a biopharmaceutical crop. Maize crop is being bioengineered to extract products of therapeutic and medicinal importance. Such a diverse utilisation of maize has generated a necessity to improve morpho-physiological traits which are important with respect to its industrial utilisation. In the present chapter, we will discuss various aspects of maize improvement for industrially important traits which will include oil, starch, methionine, biofuel and biopharmaceuticals.

The ever mounting population in 21^st century is a great challenge for the researchers to provide ample food and that has to be achieved within the context of existing challenges such as deteriorated arable land along with increasing adverse affects of changing climate. There is demand to increase food production to the tune of 70% by 2050 (Wani and Sah, 2014). Although major crops have achieved good progress, vulneRability to climatic variability emerged as a serious concern in crop improvement which could be attributed to higher sowing densities that in turn lead to more competition for water and nutrients. Abiotic stresses can be considered as major yield restraining factors among different crops. The projected estimations for changes in abiotic stresses such as drought, water logging and heat reveals far serious concerns for crop productivity (Qin et al., 2011; Bailey-Serres et al., 2012). Therefore, the concern of global food security in the era of climate change has compelled plant scientists in different crops to shift to breeding for climate resilient genotypes. Maize, being a hardy crop has an inherent potential to perform well under sub-optimal environments with minimum crop management (Kole et al., 2015). Abiotic stresses such as drought, heat and water logging affects maize yield adversely.

Although, conventional breeding approaches have exploited its fullest potential for production of elite maize hybrids but still little success attained for breeding climate resilient genotypes or varieties owing to the fact that stress tolerance is multi-genic or quantitative in nature. Therefore, the best option is to utilize the molecular breeding along with the conventional approaches for development of climate smart varieties.

Maize occupies an important position in the world economy, and serves as an important source of food and feed (Gupta et al., 2015a). Together with rice and wheat, it provides at least 30% of the food calories to more than 4.5 billion people in 94 developing countries (Shiferaw et al., 2011). In India, maize is an important cereal too, and is grown on an area of 9.2 million hectares with production of 24.2 million tonnes (www.indiastat.com). The demand for cereals will continue to increase as a consequence of the expanding human population. The world will have around 7.7 billion people by 2020, and it will reach up to 9.3 billion by 2050, and the demand for maize between now and 2050 will double in the developing world (Rosegrant et al., 2009). By 2025, India too would require to double the production (50 million tonnes) of maize grain to meet the domestic demand (Yadav et al., 2016).

Malnutrition caused by inadequate consumption of balanced diet has emerged as one of the alarming problems especially in the under-developed and developing world. Mostly the resource-poor suffer from ‘hidden hunger’, a term often used to describe malnutrition caused due to micronutrient deficiencies in staple food diet (Bouis and Welch, 2010).

An estimated two billion people suffer from micronutrient deficiency, while 795 million people are undernourished (IFPRI, 2016a). Of the 667 million children under the age of five worldwide, 159 million are stunted, while 50 million do not weigh enough for their height (IFPRI, 2016b). An estimated 45% of deaths of children under age five are linked to malnutrition (Black et al., 2013). Malnutrition contributes to global burden of disease, and loss in annual gross domestic product (GDP) in Asia and Africa to extent of 11%. South Asia is home to more than 35% of the world’s poor, and 21.9% of the population of India lives in poverty, and thus vulnerable to various health problems (IFPRI, 2016a). 15.2% of the people in India are undernourished, and more than one third of the world’s underweight malnourished children live in India, posing severe socio-economic loss to the country.

The ability to fast-track development and delivery of improved maize cultivars with capacity to provide climate-resilience (high and stable yields in stress-prone environments), coupled with improved nutritional quality, will be critical for sustainable intensification and diversification of maize-based cropping systems, and for providing enhanced income opportunities to the smallholders. This warrants coordinated multi-institutional efforts and implementation of innovative strategies to further increase genetic gains through breeding, and to translate these gains in the farmers’ fields. This article will focus only on the novel tools/ technologies/strategies that are being employed by CIMMYT for increasing genetic gains, especially for grain yield under stress-prone environments in the tropical/sub-tropical regions of sub-Saharan Africa, Latin America and Asia.

Novel tools/techniques/strategies for increasing genetic gains in the tropics

“Genetic gain” (also called as selection response) refers to the difference in the mean value of the selection criterion between original generation and next generation (formed from only the selected individuals), when they were compared in the same environment. The selection criterion is the trait(s) on which selection is based. While conventional breeding has been successful in improving maize yields over the decades, and especially in the last one decade in India through single-cross hybrids, the significantly rising demand for maize in the tropical regions of sub-Saharan Africa and Asia warrants sharp increases in genetic gains. Thus, the major challenge for the present generation of product development teams is to double the genetic gains for grain yield under stressed environments, i.e., from the present <1% to at least 1.5% within the next 5-6 years.

Maize (Zea mays L.) occupies an important place in world agriculture due to its high yield potential and greater demand. Because of its divergent character, maize is grown over a wide range of climatic conditions, ranging from near sea-level to several thousand meters above sea-level (2,700 msl). It can also be grown in all types of soils ranging from sandy to heavy clay. Among the factors adversely affecting productivity, incidence of diseases is prominent. In India Maize is prone to a number of biotic stresses like foliar diseases, downy mildews, banded leaf and sheath blight, stalk rots and ear rot caused by fungi and bacteria, incidence of which are variable in time, space, and genotype. Disease may be minimized by the reduction of the pathogen’s inoculum, inhibition of its virulence mechanisms, and promotion of genetic diversity in the crop (Richard et al., 2005). Conventional plant breeding for resistance has an important role to play that can now be facilitated by marker-assisted selection.

Chemical control measures for some of the diseases are helpful in reducing losses, however for stabilizing production it is necessary to build level of genetic resistance. Germplasm improvement programme undertaken in sixties and early seventies by All India Co-ordinated Maize Improvement Project provided an opportunity for combining resistance against important diseases with other desirable agronomic traits. Thus, breeding for resistance had been an important objective of the overall crop improvement programme and it had been a cooperative venture of the plant breeders and plant pathologists. Consequently, it was recognized that there should be an efficient technique for the mass multiplication of inoculums, artificial inoculation technique and scoring/rating of plant disease as a pre-requisite for the successful identification of resistant sources in such improvement programme. With these objectives in to consideration, a six days seminar on the “Techniques of scoring for resistance to diseases of maize in India” was held at Andhra Pradesh Agriculture University, Hyderabad during 15-20 March, 1982 (Payak and Sharma, 1983).

Maize (Zea mays L., 2n=20) is a cereal crop that is grown widely in more than 165 countries in a range of agro-ecological environments. Even in Asia, the rapid increase in maize acreage in the last decade is an indication that it is winning the battle as compared with rice. The Asian region will see itself as a major global player in the near term. In the past it was mainly confined to food in India and many other countries however, now it has become the industrial crop. Maize has several industrial applications and more than 1000 products are being developed from maize in India and around 3500 products in USA and other countries. It is a raw material for many of the important industries viz. pharma, textile, paper, film, tyre food, processing, packing and biofuel, etc. The growth rate of poultry, livestock, fish, wet and dry milling industries are very high therefore, maize demand will continue to increase in year to come.

Global Maize Scenario

Globally, maize is primarily used as feed livestock and has many industrial uses; however in many developing countries it is still used as a food crop. Small-scale farmers in both sub-Saharan Africa and Mesoamerica generally grow maize as a food crop for household consumption and for saline urban markets. Maize is particularly important in the diets of the rural and urban poor in sub-Saharan Africa and Latin America. Globally, maize is grown in 165 countries in a total of 185.1 million hectares area with 1018.1 million tones of production and 5.52 tonnes/ha productivity (Anonymous 2015-16). Almost 70% of the total maize production in the developing world is coming from low and lower middle income countries. In Asia, maize has recorded the fastest annual growth (around 4%), as compared to other cereals. Maximum maize production was in USA (353.7 million tonnes) followed by China (218.6 million tonnes) and Brazil (80.3 million tonnes) (Fig. 1), while the area under maize cultivation was in China (36.3 million hectare) followed by USA (35.5 million hectare) and Brazil (15.3 million hectare) (Fig. 2). The average world maize productivity is 5500 kg/ha, but the highest productivity of maize was achieved in USA (9970 kg/ha) followed by Canada (9591 kg/ha) and France (8137 kg/ha) (Fig. 3).